supplemental file for : dimov, d., vuchkov, a. (2020). sheep genetic resources in bulgaria with focus on breeds with coloured wool. genetic resources 2 (3), 11–24. doi: 10.46265/genresj.hxsv9592. ressources génétiques des moutons en bulgarie avec focus sur les races à la laine de couleur doytcho dimov département des sciences animales, université agricole de plovdiv, bulgarie https://orcid.org/0000-0003-4069-4214 e-mail: doytcho.dimov@gmail.com atanas vuchkov département des sciences animales, université agricole de plovdiv, bulgarie https://orcid.org/0000-0002-5129-0277 e-mail: a_vu@abv.bg corresponding author: doytcho.dimov@gmail.com (d.dimov) résumé le but de cette étude était de décrire les ressources génétiques des ovins à laine pigmentée en bulgarie et d'évaluer le potentiel de production de laine pigmentée les races de moutons indigènes en bulgarie peuvent être divisées en trois groupes en fonction de la couleur de la toison : races avec toison entièrement pigmentée; races dans lesquelles se trouvent des animaux à toison entièrement pigmentée et des animaux à toison entièrement blanche; races dans lesquelles se trouvent des animaux à toison tachetée. dans les populations de races ovines bulgares indigènes, l'expression phénotypique de plusieurs allèles a été trouvée, ce qui provoque la coloration de la laine extensiond, agoutia, pigmented headt. dans les populations de certaines races, la couleur blanche est due à l'allèle agoutiwt le locus agouti. le potentiel de production de laine pigmentée du pays a été estimé à 135 358 kg de laine en suint. les tendances « bio » dans les modes de vie augmentent progressivement l'intérêt pour des produits en laine naturellement colorés. l'industrie de la mode commence tout juste à prêter attention aux tendances de la mode en matière de protection de l'environnement. mots clés: races de moutons, laine colorée, locus, allèles https://doi.org/10.46265/genresj.hxsv9592 mailto:doytcho.dimov@gmail.com supplemental information for: habimana, r., ngeno, k., shyaka, a., ntawubizi, m., mahoro, j., hirwa, c. d., ingabire, a., kiptui, l., gafarasi, i. m., otieno, t. o. (2020). growth performance and immune response to newcastle disease in four gene pools of indigenous chicken in rwanda. genetic resources 1 (2), 42–50. doi: 10.46265/genresj.lpjs9396. résumé au rwanda, l'élevage de volailles est l'activité la plus prédominante pour améliorer les moyens de subsistance dans les zones rurales. la poule locale est appréciée pour sa production en œufs et en viande. malgré son importance économique, peu de considération a été accordée à cette population par les programmes d'amélioration genetique au rwanda. pour cette raison, il existe peu de connaissance sur sa production et ses performances de résistance aux maladies. cette étude avait pour objectif d’évaluer les performances de croissance et la réponse immunologique de la poule locale sur le vaccin contre la maladie de newcastle dans quatre pools génétiques retrouvées au rwanda. cent quatre-vingt-neuf (189) poules locales provenant de quatre pools génétiques ont été élevées en station en utilisant un dispositif expérimental randomisé. par la suite, elles ont été vaccinées avec un virus commercial vivant contre la maladie de newcastle. les données sur le poids corporel ont été collectées chaque semaine pendant 20 semaines tandis que les échantillons de sérum ont été collectés après 35 jours. les anticorps ont été détectés en utilisant un test elisa indirect. l'analyse de la variance a été effectuée en utilisant la procédure de modèle linéaire generalisé de sas. les courbes de croissance ont été modelisées à l'aide de la fonction logistique. les résultats ont révélé des différences significatives (p <0,001) et importantes entre les quatre pools pour les performances de croissance et la réponse en anticorps au vaccine. le pool de gènes a était le plus lourd (1,6 kg) en 20 semaines et le pool de gènes c a exprimé la réponse immunitaire la plus élevée (8 161 titres d'anticorps). les résultats ont indiqué que la sélection du pool génique a pouvait produire une poule avec un bon caractère de performance de croissance, tandis que le pool génétique c pouvait être sélectionné pour son immunité plus élevée contre le virus de la maladie de newcastle. mots-clés: poule local, poids corporel, anticorps, maladie de newcastle https://doi.org/10.46265/genresj.lpjs9396 short communication genetic resources (2020), 1 (1) 49–52 doi: 10.46265/genresj.2020.1.49-52 https://www.genresj.org issn: 2708-3764 replenishment and rationalization of seed collections of pumpkin, vegetable marrow and summer squash for ex situ conservation and use for breeding in armenia alvina avagyan *, gayane sargsyan, raya balayan and laura tadevosyan scientific center of vegetable and industrial crops of the ministry of economy of the republic of armenia, v. darakert, ararat marz, republic of armenia abstract: the conservation and sustainable use of crop genetic resources are crucial for food security and economic development, and diverse genetic resources are a critical input in the agricultural production process. to conserve the gene pool of vegetable crops of armenia, the scientific centre of vegetable and industrial crops of the ministry of economy of the republic of armenia carried out activities to upgrade storage facilities and optimize the existing seed collections. newly created long-term and medium-term seed collections of pumpkin, vegetable marrow and summer squash reliably conserve genetic resources of these crops and create a base for undertaking genetic enhancement and base-broadening efforts. a database and application of barcodes and qr-code labels facilitate the search of required accessions, making the stored genetic materials more accessible for users and promoting their wider use in breeding. keywords: cucurbitaceae, base collection, active collection, breeding citation: avagyan, a., sargsyan, g., balayan, r., tadevosyan, l. (2020). replenishment and rationalization of seed collections of pumpkin, vegetable marrow and summer squash for ex situ conservation and use for breeding in armenia. genetic resources 1 (1), 49-52. doi: 10.46265/genresj.2020.1.49-52. © copyright 2020 the authors. this is an open access article distributed under the terms of the creative commons attribution license (cc by 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. introduction vegetable production is one of the leading sectors in armenian agriculture. variation in agro-ecological conditions, climate and altitude in armenia, as well as longtime traditions in multipurpose use of vegetables have led to a large diversity of vegetables grown by farmers. among vegetables species of the cucurbitaceae family, the following are of specific interest due to their palatability, nutritional value and dietary qualities: (1) pumpkin (cucurbita maxima duchesne, cucurbita maxima var. turbaniformis (m.roem.) l.h.bailey, cucurbita moschata duchesne, cucurbita pepo l.); (2) vegetable marrow (cucurbita pepo l. subsp. pepo var. pepo l.); and (3) summer squash (cucurbita pepo l. subsp. ovifera ∗corresponding author: alvina avagyan (alvinaav@hotmail.com) (l.) d.s.decker). dietary benefits of pumpkin and vegetable marrow are associated with a high content of vitamins c, b1, b2, b6, pp, e and carotenoids (αand β-carotene, lutein and zeaxanthin), a favorable ratio of potassium and sodium, and low calorie content. these crops are given a great importance in the production of baby food (piskunova and muteva, 2016). summer squash is an excellent source of manganese, copper, folate, magnesium, potassium, and fiber, whereas vitamins c and a act as antioxidants, which may help to protect against hardening of blood vessels (hashash et al, 2017). the nutritional value of pumpkin, vegetable marrow and summer squash makes these crops attractive both for large farms providing vegetables for canneries and for small landholders producing these crops in response to market demand. moreover, ongoing state programmes on subsidizing agriculture and providing preferential loans and investments that are being put into the modernization of armenia’s greenhouse sector received: 2020 04 20 accepted: 2020 07 16 published online: 2020 08 31 https://www.genresj.org https://www.dx.doi.org/10.46265/genresj.2020.1.49-52 https://www.genresj.org https://www.dx.doi.org/10.46265/genresj.2020.1.49-52 mailto:alvinaav@hotmail.com 50 avagyan et al genetic resources (2020), 1 (1) 49–52 create an enabling environment for the introduction and cultivation of different vegetables, both in open fields and in greenhouses. the scientific centre of vegetable and industrial crops (scvic) has engaged in breeding and seed production of vegetable crops and provides growers with seeds of different varieties of pumpkin, vegetable marrow and summer squash that have been bred at the centre and released in the country. so far, the numbers of locally bred varieties of the above-mentioned crops are not many: three varieties of vegetable marrow, four of pumpkin and one of summer squash. to meet grower and consumer demands in both production level and varietal diversity of cucurbitaceous vegetable crops there is a need for strengthening breeding programmes and undertaking genetic enhancement efforts. for this purpose, genetic resources of cucurbitaceous species are required as inputs into the continuing process of enhancement through selective breeding. to conserve and provide breeders with germplasm for further breeding programmes, the seed collection that over many years has been created and maintained at the scvic under room conditions was significantly improved by the purchase of freezers, the acquisition of new and regeneration of old accessions, and the rationalization of the existing ex situ collection (sackvillehamilton et al, 2003) . materials and methods base and active collections were established both from newly introduced and regenerated germplasm as well as from accessions of working/breeding collections that were collected and maintained in paper bags over a period of about twenty years under room conditions without the possibility to control temperature and humidity. accessions of foreign varieties have been received in the frameworks of collaboration projects with other countries with the purpose of testing under local conditions. accessions of research material were received from the world vegetable center with the aim of multiplication and use in breeding programmes. seeds of landraces were collected from local markets and farms. the process of acquisition of germplasm, seed drying, regeneration and storage of base and active seed collections was carried out in line with internationally accepted standards (fao, 2014). to maintain the genetic integrity of original accessions during regeneration in open fields and avoid risk of outcrossing, plants were planted in blocks, not in rows, and 1,000 m isolation distance between different varieties was maintained. seeds were collected from plants in the center of each block. in addition, samples with limited numbers of seeds were regenerated in individual polyethylene film greenhouses using handpollination. selected seed samples were dried to appropriate moisture content using a drying chamber. before storing, initial seed viability tests were conducted. dried seeds were placed in laminated aluminum foil bags of 11 µm thickness and hermetically sealed. all seed packages were labeled with printed barcode and qr-code, which includes information on accession name, acquisition date, origin and biological status, reducing the possibility of errors and facilitating search of requested accessions. accessions of the base collection are stored under long-term conditions at a temperature of –18◦c. material placed under mediumterm conditions (i.e. active collection) is stored under refrigeration at 5◦c. passport data for each accession are fully documented according to the fao/bioversity multi-crop passport descriptors (alercia et al, 2015). the database for accession records is developed in excel format compatible with eurisco. results and discussion purchasing the appropriate seed storage deep freezers and refrigerators, drying chamber, germination boxes and aluminum foil bags provided the possibility for establishing base and active seed collections, each with a specific target and coverage. the active collection of pumpkin, vegetable marrow and summer squash was created to provide breeders, farmers, and researchers with seed material for study and use in breeding, research and educational programmes, as well as for seed material exchange. all armenian varieties of pumpkin, vegetable marrow and summer squash released in the country since 1991 are included in the active collection regardless of their present cultivation area and market demand. the active collection includes 11 foreign varieties, which are in demand on the local market due to their economically important traits, such as early maturing and high productivity. research material that, based on preliminary studies, has potential interest for breeders is also included in the active collection. the base collection serves as safety material for the active collection and mainly includes varieties bred in armenia, traditional farmers’ varieties, as well as the most valuable breeding/research material of both local and foreign origin. accessions were incorporated into the base collection based on the following criteria defined by a scientific council of the scvic: • strategic importance of a locally bred variety for the country’s food production and sustainable agriculture.widely cultivated varieties contributing to a country’s food security are included in the base collection. • prevalence of local farmers’ variety in the country. landraces that are disappearing from markets and farmers’ fields as a result of the spread of more productive modern varieties are placed under long-term storage conditions. • significance of a foreign variety for food security. although the majority of the varieties of cucurbitaceae cultivated in armenia are of armenian origin, in specific agro-climatic zones some foreign varieties ensure higher yields compared genetic resources (2020), 1 (1) 49–52 rationalization of seed collection of cucurbitaceae species in armenia 51 figure 1. structure of the base and active seed collections of the cucurbitaceae family at the scientific centre of vegetable and industrial crops, armenia. with local ones, and such varieties were added to the base collection. • value of less common variety in terms of a potential for a niche market. varieties that are of specific interest for farms providing products for restaurants or high-end specialty stores were included in the base collection. • high mean value for major breeding traits. considering priority areas of breeding, research materials that could serve as a source of important traits, such as resistance to powdery mildew or early ripeness were incorporated into the base collection. the scvic maintains a breeding collection of 88 accessions of pumpkin, vegetable marrow and summer squash, which is stored for a short period (from one to three years, depending on the species) under room conditions. such collections are intended for breeding research conducted in the short term. some accessions of the breeding collection have low initial germination or insufficient number of seeds, and must be regenerated, processed and stored in priority order. the existing ex situ collections of pumpkin, vegetable marrow and summer squash were expanded by including obsolete varieties, landraces, research lines, samples obtained from partners in the framework of collaborative projects and cooperation with the world vegetable center. accessions were requested according to the top priority areas of breeding of cucurbitaceae species in the country, in particular high productivity, early ripeness, bushy growth habit and resistance to powdery mildew. at the same time, duplicate accessions and accessions with low seed variability were eliminated. the enlarged seed collections of pumpkin, vegetable marrow and summer squash are not big compared with the collections of other species of the cucurbitaceae family (figure 1): they are new and contain valuable accessions of varieties and hybrids selected or bred in armenia with viable seeds. in total, the collection (base and active) includes 117 accessions of six species and covers all available varieties, hybrids, breeding lines and landraces of armenian origin. the ratios of breeding varieties, research material and landraces vary depending on the crop. thus, about 72% of the seed collection of pumpkin are represented by modern varieties. among accessions of vegetable marrow, the research material represented by breeder’s lines, hybrids, and lines of individual selections from hybrid populations exceeds the number of accessions of modern breeding varieties (figure 2). the summer squash collection is represented in equal quantity by varieties and research/breeding material. in the base collections of pumpkin, vegetable marrow and summer squash, breeding varieties prevail, while most of the accessions of the active collections are represented by breeding and research material (figure 2). although there is a limited number of landraces of the gourd family existing in the country, the inventory indicated gaps in the collection in terms of farmers’ varieties of pumpkin and vegetable marrow, which are being gradually displaced by commercially developed modern varieties. as for summer squash, this crop is a relatively new one in cultivation, so landraces do not exist. varieties of armenian origin prevail over foreignintroduced varieties of pumpkin, vegetable marrow and summer squash in both collections. the number of accessions of introduced breeding/research material exceeds those of local origin, triggered by the need for diverse germplasm from outside armenia in order to minimize genetic uniformity of new varieties. conclusion as a result of establishing base and active collections of pumpkin, vegetable marrow and summer squash at the scvic the conservation of the genetic resources of these crops is ensured under conditions that meet internationally accepted standards. due to the enlargement and rationalization of the ex situ collection, a resource base for genetic enhancement of these crops to produce substantial economic benefits was created. it can serve for breeding new varieties that meet requirements of local agricultural production, such as early maturity, high yield, resistance to pests and diseases, tolerance to abiotic stresses and other characteristics dictated by producers and consumers. the conserved genetic resources are available for users and can be provided upon request. as soon as the work on upgrading the collection of the remaining species of cucurbitaceae, particularly bottle gourd (lagenaria siceraria (molina) standl.) and loofah (luffa aegyptiaca (l.) roxb.) is finished, the passport data on all cucurbitaceae accessions will be recorded in eurisco to make the information on available accessions accessible for users at national and international levels. the work on inventory of the collections, seeds viability tests and placing for long-term and midterm storage has started with crop species of the cucurbitaceae family. this is the first step in upgrading the vegetable crops genetic resources collection and establishing properly managed seed collections. the future activities will be focused on further enlargement 52 avagyan et al genetic resources (2020), 1 (1) 49–52 figure 2. numbers of accessions of local and foreign origin in base and active collections of pumpkin, summer squash and vegetable marrow at the scientific centre of vegetable and industrial crops, armenia. of the collection through acquisition of genetic resources with genes linked to important agronomic traits such as disease resistance, stress tolerance and high nutritional value to expand the genetic spectrum of germplasm used in pre-breeding and breeding, and on efficient management of germplasm collections and relevant data through installation of an open-source program. the work of upgrading and rationalizing seed collections will continue for tomato, pepper and eggplant collections. it is planned to initiate the process of safety duplication of varieties of armenian origin in the svalbard global seed vault upon finishing the upgrading and rationalization process for the entire vegetables collection. acknowledgements authors wish to thank the technical staff of the scientific centre for vegetable and industrial crops (scvic) for assistance in seed drying, sorting and packaging. the scvic expresses its gratitude to rijk zwaan breeding b.v. (the netherlands) for providing laminated aluminum foil bags. supplemental data supplemental file 1: abstract in russian author contributions aa established base and active collections, analyzed database information, wrote the manuscript; gs supervised the project on ex situ conservation, contributed to acquisition of germplasm; rb performed viability tests, selected seeds for ex situ conservation; tl collected seeds for ex situ conservation. conflict of interest statement the authors declare that no conflict of interest exists. references alercia, a., diulgherof, s., and mackay, m. (2015). fao/bioversity multi-crop passport descriptors v.2.1 [mcpd v.2.1]. url: https://www.bioversityinternational.org/e-library/ publications/detail/faobioversity-multi-croppassport-descriptors-v21-mcpd-v21/. fao (2014). genebank standards for orthodox seeds. in and others, in genebank standards for plant genetic resources for food and agriculture rev. ed., fao, rome, italy, 17-61. hashash, m. m., el-sayed, m. m., abdel-hady, a. a., hady, h. a., and morsi, e. a. (2017). nutritional potential, mineral composition and antioxidant activity squash (cucurbita pepo l.) fruits grown in egypt. european journal of biomedical and pharmaceutical sciences 4(03), 5–12. piskunova, t. m. and muteva, z. f. (2016). the vir collection – a source of initial breeding material for the perspective directions of breeding of a vegetable marrow and pumpkin . vegetable crops of russia 3, 18– 23. (in russ.). url: https://doi.org/10.18619/20729146-2016-3-18-23. sackville-hamilton, r., engels, j. m. m., and van hintum, t. (2003). rationalization of genebank management. in a guide to effective management of germplasm collections, ed. engels, j. and visser, l., (rome, italy: ipgri), volume 6 of ipgri handbooks for genebanks, 80-92. https://www.bioversityinternational.org/e-library/publications/detail/faobioversity-multi-crop-passport-descriptors-v21-mcpd-v21/ https://www.bioversityinternational.org/e-library/publications/detail/faobioversity-multi-crop-passport-descriptors-v21-mcpd-v21/ https://www.bioversityinternational.org/e-library/publications/detail/faobioversity-multi-crop-passport-descriptors-v21-mcpd-v21/ https://doi.org/10.18619/2072-9146-2016-3-18-23 https://doi.org/10.18619/2072-9146-2016-3-18-23 https://www.genresj.org/index.php/grj/article/view/16/suppfile1 introduction materials and methods results and discussion conclusion supplemental data author contributions conflict of interest statement short communication genetic resources (2021), 2 (4), 66–71 doi: 10.46265/genresj.pybz4246 https://www.genresj.org issn: 2708-3764 estimation of genetic erosion on ethiopian tetraploid wheat landraces using different approaches alemayehu zemede lemma a, firew mekbib b, kebebew assefa a and zewdie bishaw c a ethiopian institute of agricultural research (eiar), addis ababa, ethiopia b faculty of agriculture and environmental sciences, school of plant sciences, haramaya university, diredawa, ethiopia c international center for agricultural research in dry area (icarda), addis ababa, ethiopia abstract: the demand and use of improved crop varieties by farmers has increased in the central highlands of ethiopia, where continuous loss of local traditional varieties has been occurring in the last two to three decades. the objectives of the study were to assess the extent of genetic erosion and perception of farmers and associated causes for the reduction of traditional farmers’ varieties. direct field assessment covering 56 wheat farms and a survey in which 149 farmers participated were carried out in three districts of central ethiopia. based on data collected during direct farm assessment, the loss of genotypes was found to be 88% in ada followed by 80% and 60% in lume and gimbichu districts, respectively. the farmer survey indicated an even greater loss of diversity of100% in ada followed by lume (93%) and gimbichu (67%). diseases and pests as well as shorter growing seasons associated with climate change were identified as main causes for farmers to switch to modern varieties. the expansion of high yielding improved bread and durum wheat varieties also contributed to gradually replace local durum wheat varieties by local farmers of these districts. overall, genetic erosion of tetraploid wheat varied among the three districts of central ethiopia. reductions in the number of farmers and area coverage in the study districts could be used as good indicators for the existence of genetic erosion. keywords: direct farm assessment, genetic erosion indicators, genetic integrity, landraces, tetraploid wheat citation: zemede lemma, a., mekbib, f., assefa, k., bishaw, z. (2021). estimation of genetic erosion on ethiopian tetraploid wheat landraces using different approaches. genetic resources 2 (4), 66–71. doi: 10.46265/genresj.pybz4246. © copyright 2021 the authors. this is an open access article distributed under the terms of the creative commons attribution license (cc by 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. introduction ethiopia is the largest wheat producer in sub-saharan africa with about 1.6 million ha of durum and bread wheat (central statistical agency, 2017). wheat is one of the major cereal crops in the ethiopian highlands, which range between 6 and 16 n latitude, 35 and 42 e longitudes, and from 1500 m to 2800 m above sea level (masl). ethiopia has already been identified as an important center of diversity for different crops. durum wheat is a tetraploid wheat variety traditionally grown on heavy black clay soils (vertisols) of the ethiopian highlands between 1800-2700 masl, where it is mainly produced by small scale farmers. durum wheat is suitable for manufacturing pasta products (e.g. macaroni, spaghetti); however, in ethiopia it is also used for making “injera”, a white leavened ethiopian bread made from grain flour, and other local foods. durum wheat grown in ethiopia is constituted by a few improved varieties and a large number of traditional farmers varieties, commonly referred to as landraces (bechere et al, 2000; eticha et al, 2006; bishaw et al, 2014).durum wheat is extensively cultivated and grown by a large number of farmers and a significant number of traditional farmers’ varieties have been recorded in the central highlands of ethiopia in the past 20 to 30 years (negatu et al, 1992). statistics on durum wheat production in ethiopia from the past decades are difficult to obtain, they are usually lumped with bread wheat in reporting. the total area under wheat production in 1983was reported to be 625,590 ha of which 60-70% was estimated to be used forthe production of durum (tesema, 1991). the following received: 19.05.2021 accepted: 18.08.2021 published online: 17.12.2021 https://www.genresj.org https://www.dx.doi.org/10.46265/genresj.pybz4246 https://www.genresj.org https://www.dx.doi.org/10.46265/genresj.pybz4246 genetic resources (2021), 2 (4), 66–71 estimating genetic erosion on tetraploid wheat 67 decades saw a drastic reduction in area coverage for durum wheat which dropped to around 50% of total wheat production area (gashawbeza et al, 2003). recent reports indicated that the area coverage for durum has further declined to not more than 15-20% from a total 1.6 million ha of lands (mengistu et al, 2016). introduction of modern varieties, improved production practices and problems associated with biotic stresses like crop pests and diseases as well as abiotic stresses such as droughts in traditional farming systems were among the major problems faced by the farmers leading to gradual replacement of traditional farmers’ varieties. genetic erosion is defined as “the loss of genetic diversity, in a particular location and over a particular period of time, including the loss of individual genes and the loss of particular combinations of genes such as those manifested in landraces or varieties” (fao/ipgri, 2002). it could be considered as a consequence of the loss of diversity in the farmer’s field and will have a major effect on the future crop productions and productivity for indigenous crop species of ethiopia. the problem has been increasing and is expected to be more aggravated for durum wheat than other cereal crops. several research reports from survey studies and farmers’ discussion indicated loss of diversity on tetraploid wheat has increased faster than expected in different environments of ethiopia. the reduction in number of farmers, traditional farmer varieties or landraces and area coverage confirmed the danger and extent of genetic erosion on tetraploid wheat, as reported in multiple studies. teklu and hammer (2006) reported genetic erosion of 88%, 100% and 78% for the durum wheat species triticum durum, t. turgidum and t. dicoccon, respectively. in harar zuria, the same authors detected genetic erosion of 88.9 % for t. durum and 100% for both t. turgidum and t. dicoccon. in the western shoa zone of ethiopia, genetic erosion reached 75% and 62% for tetraploid wheat species in ambo and dandi districts, respectively (geleta and gausgruber, 2013). in a similar study farmers identified 26 tetraploid wheat land races (21 from akaki and 17 from ejere), which were once widely grown in the area and of which only six were currently available; the average loss of diversity was thus estimated to be 77% (tsegaye and berg, 2007). besides primary sources collected from a survey using semi-structured questionnaires and farmers’ group discussions, this study also used direct field assessment to identify recently cultivated landraces and compare them to previously available landraces in the similar districts, calculating levels of genetic integrity and erosion based on the procedures of hammer et al (1996). the reduction in numbers of farmers growing landraces and area of cultivation were identified as useful indicators for the existence of genetic erosion (brown and hodgkin, 2015). the objectives of this present study were to calculate and estimate on farm genetic erosion and identify associated causes in three districts of central ethiopia using different approaches. results obtained from direct field assessments and observations as well as information collected from farmers’ interviews and discussions were used to investigate the current status of durum landraces and to determine trends based on the number of farmers using landraces and the area of cultivation of the landraces. materials and methods study areas the study was conducted in three districts of the central highlands of ethiopia based on direct observation, a semi-structured survey questionnaire and group discussions. direct observation was conducted across three different routes which covered gimbichu, ada, and lume districts during the 2017 cropping season (figure 1). a survey questionnaire and group discussions were conducted in ada, lume and gimbichu districts of the central highlands of ethiopia in 2018. the three districts were purposely selected to represent the durum wheat agro-ecology environment of the east shoa zone: 1) ada represents a midland wheat growing agroecology area,where modern crop varieties are assumed to be dominantly cultivated; 2) lume represents a midland area where both landraces and modern wheat cultivars are believed to exist due to interventions by the ethiopian biodiversity institute to recover the status of local in situ conservation; and 3) gimbichu district represents wheat growing agro-ecology at high elevation and is an area where durum landraces are still extensively cultivated. research methodology direct observations direct wheat field assessment and identification of species and varieties of species was conducted in november 2017 along three routes passing through major wheat producing districts in the central highlands of ethiopia (figure 1). the survey was made on 56 wheat growing farms at approximately three to fivekilometer intervals when the crop was observed. the different wheat species were classified into modern bread or durum and tetraploid landraces based on their spike characteristics and uniformity. the number of landraces available in 2017 compared to the landraces recorded in 1992 was the basis for calculating genetic integrity and genetic erosion. modern wheat (bread and durum) and landraces area coverage was calculated as percentage of each wheat species area to the total wheat area of the 56 farms visited during direct farm assessment. survey study a survey on perception on use of landraces and genetic erosion was carried out to collect data from149 farmers in three villages of the ada, gimbichu and lume districts 68 zemede lemma et al genetic resources (2021), 2 (4), 66–71 figure 1. travel routes for direct farm assessment. using semi-structured questionnaires between10 and 25 december, 2018. group discussions farmers’ group discussions were held in ada, gimbichu and lume districts between 20 and 25 december 2018 to collect information about the past and current status of landraces and to identify causes why farmers stopped growing landraces in the area. three group discussions in each district consisted of 12-15 systematically selected farmers from surrounding villages, also taking into consideration gender representation. data collection and summary genetic erosion was calculated according to the formula of hammer et al (1996): ge = 100% gi, where ge is genetic erosion and gi is genetic integrity, which is given as: gi = n2/n1 x 100. the number of farmers cultivating landraces and their relative area coverage were determined. the number of landraces cultivated recently as compared to previous number was the basis for calculation of genetic erosion. in the present study, the number of landraces grown by the farmers 25 years ago (negatu et al, 1992) was considered as n1 and the number of landraces recorded in 2017 represented n2. table 1. genetic erosion on tetraploid wheat since 1992 in three districts of the central highlands of ethiopia districts landraces cultivated in 1992 landraces cultivated in 2017 genetic integrity (%) estimated genetic erosion (%) gimbichu 15 6 40 60 lume 15 2 20 80 ada 16 2 12.5 87.5 results direct observation genetic erosion occurred on tetraploid wheat after 25 years based on direct farm assessment (table 1). the variation was significant on loss of diversity on tetraploid wheat at 0.01%. the average genetic erosion across districts was 75.8%, which is lower compared to previous reports (geleta and gausgruber, 2013). the loss of diversity on tetraploid wheat was found to be the highest in ada (87.5%) and lowest in gimbichu (60%) based on direct field assessment (table 1). the proportion of landraces compared to modern wheat varieties was lower in lower altitude than high altitude growing environments (figure 2). in 2017 the proportion of landraces in altitudes above 2400m were 70% while below 2400m this dropped to about 30% compared to modern wheat. number of farmers and area of cultivation modern bread and durum wheat as well as tetraploid landraces area coverage were estimated based on random field visits and identification of wheat species found in the field and calculated as percentage of each wheat species area to the total wheat area in a given district. in 2017 only 8.3% of farmers in lume, about 23.5% in ada and 40.7% in gimbichu were growing local varieties. when the three districts were considered, only figure 2. the number of tetraploid wheat (dw), local landraces (lr) and modern wheat varieties (bw) grown in different altitude zones based on direct field observations in 2017. genetic resources (2021), 2 (4), 66–71 estimating genetic erosion on tetraploid wheat 69 figure 3. the proportion of landraces (lr), modern bread wheat (bw) and durum wheat (dw) varieties by area coverage and number of farmers in three ethiopian districts in 2017 based on direct field observations on 56 farms in the districts. 24.2% of farmers were growing local durum wheat varieties. on average 24.5% of wheat growing area was occupied by landraces across the three districts compared to modern wheat varieties. the highest area coverage was in gimbichu (52.3%) while the lowest was 2.5% in lume (figure 3). farmers’ survey in order to obtain a more pronounced picture of the estimated loss of genetic diversity in central ethiopia, semi-structured interviews were conducted with 149 farmers from three communities in the three districts. based on interviews with farmers, significant genetic erosion was observed in tetraploid wheat after 25 years (table 2). the loss of diversity in tetraploid wheat reached up to 100% in ada and was lowest in gimbichu (67.7%), with an average of 87%, based on the survey. in the 2018 growing season only 7.3% of farmers in the lume district but 68.4% in gimbichu were maintaining landraces of wheat. none of the farmers interviewed in ada district were growing wheat landrace varieties. a similar trend was observed in cultivation area of landraces compared to modern varieties. the sample area coverage was 13.5% in gimbichu and only table 2. genetic erosion of tetraploid wheat since 1992 in three districts of the central highlands of ethiopia based on semi-structured interviews with 149 farmers. districts durum wheat varieties cultivated in 1992 durum wheat varieties cultivated in 2017 genetic integrity (%) estimated genetic erosion (%) gimbichu 15 5 33.3 67.7 lume 15 1 6.7 93.3 ada 16 100.0 figure 4. percentage of landraces (lr) and modern bread wheat (bw) and durum wheat (dw) varieties by area coverage and number of farmers in three districts based on interviews with 149 farmers in 2018. 1.5% in lume. no farms were cultivating landraces in ada district (figure 4). group discussions the reasons given by farmers on why they are currently not growing local varieties are presented in table 3. the most common reason accounting for high losses in all three districts were identified as diseases and pests, mainly stem rust, followed by terminal drought associated with a short growing season. the data also indicated that the availability and supply of better yielding improved bread and durum varieties gradually replaced local varieties inducing farmers of these districts to stop or reduce growing local durum wheat varieties (table 3). the expansion of high value crops like tef and chickpea was also mentioned as cause for losses of local varieties in ada and lume districts, whereas in gimbichu water logging and the difficulty of using landraces for making local injera and bread due to its hard seed were raised as reasons for replacing landraces with modern wheat varieties. similar findings and reasons were reported by tsegaye and berg (2007) in studies conducted in lume and akaki districts of the central highlands of ethiopia. on the contrary, the existence of a short growing season resulted in change in crop variety from farmers’ to modern varieties that could be suited to early planting. use of high amounts of fertilizer associated with growing modern varieties have resulted in shifting from landraces to modern varieties particularly in ada and lume districts where high genetic erosion was observed. 70 zemede lemma et al genetic resources (2021), 2 (4), 66–71 table 3. reasons for not growing local durum wheat landraces in 2018 in ada, lume, and gimbichu districts, provided by farmers during the group discussions. causes of genetic erosion ada (n=37) lume (n=55) gimbichu (n=57) all (n=149) diseases and pests affecting landraces 15 48 36 99 shorter growing season associated with climate change 13 27 21 61 improved bread wheat 6 23 13 42 improved durum wheat 4 15 20 39 water logging 18 18 unavailability of seed for the landraces 7 2 2 11 utilization (durum landraces not suited for production of injera and bread) 6 6 shortage of land and urbanization 5 5 expansion of high value crops (tef and chickpea) 4 4 discussion the results indicated that on average 75.8% and 87% genetic erosion was observed on tetraploid wheat after 25 years based on direct farm assessment and survey study, respectively. the levels of loss observed from both approaches appear to be similar to that reported in western ethiopia (geleta and gausgruber, 2013). the loss observed was higher in ada and lume, situated at lower altitude, than in the high altitude zone above 2400 masl of gimbichu district, where significant areas of land are still used to grow landraces. this difference can be attributed to the fact that both ada and lume districts have similar agro-ecology, access to big market centers and are also near to the debre-zeit agricultural research center where a lot of improved wheat cultivars are developed, demonstrated and distributed. modern wheat and tef crops are more expanded (assefa et al, 2015) and could also be major causes for loss of diversity in the ada and lume districts. the presence of higher diversity in gimbichu district might be associated to the merits of landrace adaptation and yield stability (berg, 2009). similarly, tsegaye and berg (2007) reported 77% genetic erosion and relative diversity for tetraploid wheat in ejere, a high elevation district of central ethiopia. our study demonstrated a correlation between the extent of genetic erosion and altitude. this indicates the relative importance of focusing further landrace collection and conservation efforts on altitudes above 2400 masl to capture more diversity. the study further confirmed the existence of better occupation in terms of area coverage and maintenance of landraces by large numbers of farmers in gimbichu as compared to ada and lume. this could be due to the expansion of modern wheat varieties at the expense of the landraces in the latter districts as illustrated in figure 3 and figure 4 and confirmed by farmers’ group discussions. negatu et al (1992) reported that 64% of sample farmers grew local durum wheat varieties in the same districts of central ethiopia. the low percentage of farmers currently growing local varieties in the three districts are likely the result of commendable expansion of well performing modern varieties. the results found in the current survey and previous research reports were comparable to those obtained in direct farm assessment. the different approaches followed may account for the large variations observed on the extent of genetic erosion although the trends were similar between the altitudes. the findings of the study have a significant impact for in situ conservation of landraces for future use in breeding to combat the challenges of climate change. farmers’ opinions gathered in group discussions indicated that drought contributed to high loss of landraces in ada and lume and the result contradicts the existing theory that landraces are well adapted and coevolved in the environment they originated. the results contribute to the setting of targeted strategies in high altitude zones for in situ conservation and exploitation of landraces in future breeding programs. future studies should take into account direct observations as alternative approaches to assess and estimate genetic erosion of tetraploid wheat and other indigenous crop species. acknowledgments the authors would like to thank the ethiopian institute of agricultural research (eiar), agricultural growth program ii, (agp ii) and the international center for agricultural research in the dry areas (icarda) for providing technical and financial support for the execution of the field experiments. the authors also acknowledge the support provided by wondogenet research center and debre-zeit research center. research and technical staff members of the crop research process of debre-zeit research center are highly acknowledged for assisting us in field activities. author contributions a. zemede proposed, designed, and conducted the trial, analyzed data, drafted and finalized the manuscript. f. genetic resources (2021), 2 (4), 66–71 estimating genetic erosion on tetraploid wheat 71 mekbib, k. assefa and z. bishaw made valuable contributions to and commented on the final manuscript. conflict of interest statement the authors declare that they have no competing interest. references assefa, k., cannarozzi, g., girma, d., kamies, r., chanyalew, s., plaza-wüthrich, s., blösch, r., rindisbacher, a., rafudeen, s., and tadele, z. (2015). genetic diversity in tef [eragrostis tef (zucc.) trotter]. frontiers in plant science 6, 177. doi: https: //doi.org/10.3389/fpls.2015.00177 bechere, e., kebede, h., and belay, g. (2000). durum wheat in ethiopia: an old crop in an ancient land (addis ababa: institute of biodiversity conservation and research). berg, t. (2009). landraces and folk varieties: a conceptual reappraisal of terminology. euphytica 166, 423–430. doi: https://doi.org/10.1007/s10681-0089829-8 bishaw, z., struik, p. c., and van gastel, a. j. g. (2014). assessment of on-farm diversity of wheat varieties and landraces: evidence from farmer’s fields in ethiopia. african journal of agricultural research 9(39), 2948– 2963. doi: https://doi.org/10.5897/ajar2013.7574 brown, a. h. d. and hodgkin, t. 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(2003). fertilizer n effects on yield and grain quality of durum wheat. tropical agriculture 80, 146–151. geleta, n. and gausgruber, h. (2013). on-farm diversity and genetic erosion of tetraploid wheat landraces in ambo and dandi districts, west shewa, ethiopia. science, technology and arts research journal 2(1), 1–9. hammer, k., knüpffer, h., xhuveli, l., and perrino, p. (1996). estimating genetic erosion in landraces two case studies. genet resour crop evol 43, 329–336. doi: https://doi.org/10.1007/bf00132952 mengistu, d. k., kidane, y. g., fadda, c., and pè, m. e. (2016). genetic diversity in ethiopian durum wheat (triticum turgidum var durum) inferred from phenotypic variations. plant genetic resources: characterization and utilization 16(1), 39–49. doi: https://doi.org/10.1017/s1479262116000393 negatu, w., mwangi, w., and tesema, t. (1992). farmers’ varietal preferences for durum wheat in ada, lume and gimbichu woredas. ethiopian journal of agricultural science (ethiopia) 13, 89–100. teklu, y. and hammer, k. (2006). farmers’ perception and genetic erosion of tetraploid wheat landraces in ethiopia. genet resour crop evol 53, 1099–1113. doi: https://doi.org/10.1007/s10722-005-1145-8 tesema, t. (1991). research recommendations and future strategies of the ethiopian durum wheat improvement program. in wheat research in ethiopia a historical perspective, ed. gebre-mariam, h., tanner, d. g., and hulluka, m. m., (addis ababa: iar/cimmyt), 164-170. tsegaye, b. and berg, t. (2007). genetic erosion of ethiopian tetraploid wheat landraces in eastern shewa, central ethiopia. genet resour crop evol 54, 715–726. doi: https://doi.org/10.1007/s10722-0060016-2 https://doi.org/10.3389/fpls.2015.00177 https://doi.org/10.3389/fpls.2015.00177 https://doi.org/10.1007/s10681-008-9829-8 https://doi.org/10.1007/s10681-008-9829-8 https://doi.org/10.5897/ajar2013.7574 https://doi.org/10.1007/s10722-004-6095-z https://doi.org/10.1007/s10722-004-6095-z https://doi.org/10.1007/bf00132952 https://doi.org/10.1017/s1479262116000393 https://doi.org/10.1007/s10722-005-1145-8 https://doi.org/10.1007/s10722-006-0016-2 https://doi.org/10.1007/s10722-006-0016-2 introduction materials and methods study areas research methodology direct observations survey study group discussions data collection and summary results direct observation number of farmers and area of cultivation farmers' survey group discussions discussion author contributions conflict of interest statement short communication genetic resources (2022), 3 (6), 15–21 doi: 10.46265/genresj.hjeh3830 https://www.genresj.org issn: 2708-3764 farmers using local livestock biodiversity share more than animal genetic resources: indications from a workshop with farmers who use local breeds anne lauvie *,a, nathalie couix b and jean-michel sorba c a umr selmet, université de montpellier, cirad, inrae, institut agro, montpellier, france b inrae umr agir, castanet tolosan, france c inrae lrde, corte, france abstract: recognizing the products from farms that use local breeds is key to in situ conservation of local animal biodiversity. recognition often focuses on a small number of specific breeds or products but could be expanded to include multiple local breeds and products. this paper shows that several farmers who use local breeds can share principles among the multiple dimensions of their farming systems. we analyzed the exchanges among nine farmers who use local breeds on the different dimensions of their farming systems at a workshop held in november 2017. we present the principles they shared and discuss (i) the fact that bringing the principles to the fore requires a collective participatory approach, (ii) the fact that shared principles may also concern dimensions often neglected in livestock farming systems approaches, and (iii) how a collective participatory approach can help recognize the products and activities of farmers who use local breeds. keywords: local breeds, livestock farming systems, value, recognition, practices citation: lauvie, a., couix, n., sorba, j. (2022). farmers using local livestock biodiversity share more than animal genetic resources: indications from a workshop with farmers who use local breeds. genetic resources 3 (6), 15–21. doi: 10.46265/genresj.hjeh3830. © copyright 2022 the authors. this is an open access article distributed under the terms of the creative commons attribution license (cc by 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. introduction as reported by the food and agriculture organization of the un (fao, 2015), local livestock biodiversity is under threat. as livestock and livestock diversity contribute to livelihoods, food security and rural development, as well as having cultural and environmental dimensions (fao, 2015), it is important to maintain the diversity of and add value to local breeds, through their sustainable use and development. to this end, several examples of using geographical indications to support the products of these breeds have been reported (mathias et al, 2010). these labels often focus on a specific product associated with a single breed. beyond labels focused on local breed products, it is hypothesized that, if different civil society stakeholders have a positive image of farmers who use local breeds, this will contribute ∗corresponding author: anne lauvie (anne.lauvie@inrae.fr) to their conservation (gandini et al, 2010). in this line of thought, it is important to raise public awareness of farming activities and products linked to local breeds. general recognition of products and activities of farmers who use local breeds should not necessarily focus on a single breed and product, but could, for instance, apply to a whole territory. recognition at this scale would then concern a wide range of products and could include several local breeds of different species. it is recognized that the choice of a breed and the way the breed is managed are an integral part of the logic of a whole livestock farming system (lauvie et al, 2015), and that breeds and the types of farming systems within which they are used are linked (sturaro et al, 2013). however, the same breed can be used in different farming systems (perucho et al, 2020). despite this diversity, to better recognize the activities and products of farms at the territory scale, it is important to identify what they have in common. indeed, recognition often received: 25.11.2021 accepted: 04.04.2022 published online: 27.07.2022 https://www.genresj.org https://www.dx.doi.org/10.46265/genresj.hjeh3830 https://www.genresj.org https://www.dx.doi.org/10.46265/genresj.hjeh3830 mailto:anne.lauvie@inrae.fr 16 lauvie et al genetic resources (2022), 3 (6), 15–21 depends on putting the spotlight on elements that justify recognition. these elements could for instance be included in a product specification or charter, or through communication campaigns. a prerequisite condition for recognition is thus to identify shared elements that could be highlighted. we therefore questioned if certain elements are shared by the different farming systems that use local breeds. to answer this question, we aimed to identify the characteristics shared by several farming systems that use local breeds. we refer to these common underpinnings of a system as shared ’principles’. in this definition, shared principles can be associated with an existing diversity of practices. this paper aims to characterize the nature of common principles identified as shared by several farming systems that use local breeds. the paper is organized in three sections: first, we present the methodology chosen to analyze the exchanges between local breed farmers on the different dimensions of their farming systems during a workshop on production systems and territory. we then identify the shared principles, and show how farmers who use local breeds may share farming system practices or values that go beyond technical or genetic aspects. finally, we discuss (i) the fact that bringing these principles to light requires a specific participatory approach and the difficulties in applying this approach to a larger group of farmers, (ii) the fact that these principles concern dimensions often neglected in livestock farming systems approaches, and (iii) how to recognize the specificities of farming systems that use local breeds while supporting diversity. materials and methods this paper analyzes the content of the exchanges that took place during a workshop held in november 2017. the workshop was one of several organized during an action research project conducted in partnership between a research team of the french national research institute for agriculture, food and environment, and the fédération des races de bretagne (federation of local breeds in the region of brittany, federation hereafter). the action research aimed to promote recognition of the products and activities linked with local breeds in brittany. the project was organized by the federation, which gathers local breed associations in the region. participation was voluntary and open to farmers who are members of the federation. as a consequence, a variety of farmers using local breeds attended, and several different species were concerned. in this paper, we analyze the content of a workshop on the theme: production systems and territory. the workshop brought together nine farmers, four researchers (three participating in the action research and one involved in another part of the project, as an observer), and one facilitator from the federation. among the breeds raised by the nine farmers, those concerned by the federation were nantaise, armoricaine, bretonne pie noir, and froment du léon cattle breeds, chèvre des fossés goats, and landes de bretagne sheep, all of which were included in this study. animals of other breeds are also raised on some farms, but these breeds were not among those covered by the federation. six of the farming systems produced meat, three mainly produced dairy products, and one produced both wool and meat. at the time of the workshop, three farmers mentioned they had another activity combined with raising livestock – one grew vegetables, and the other two had off-farm activities. the four participants from the research institute came from three different fields: one from management sciences, one from sociology and two (including the observer) from livestock farming systems. the exchanges were organized under three topics: (1) livestock feeding system and land use, (2) breeding practices, and (3) processing and sale of products and other activities such as opening the farm to visitors. the discussion on each topic was divided into two parts: (1) a roundtable to present each farmer’s practices related to the topic, and (2) a collective discussion to agree on what they had in common about the topic. the exchanges were audio-recorded for internal report writing, and used for further analysis. we analyzed the characteristics shared by the farming systems identified by the workshop participants, using the audio recordings and report to stay close to the collective expression of principles shared among the workshop participants. we analyzed the exchanges concerning individual practices to capture their diversity in more detail. we conducted a thematic analysis of the notes reporting the exchanges. the diversity of practices described for each topic was reported with the aim of illustrating the diversity within a shared principle. we used this empirical data to better characterize the nature of the shared principles. results elements shared by farmers referred on different dimensions of the livestock farming systems. table 1 lists the results of the collective discussion aimed at reaching an agreement on what the farmers’ systems had in common. the three dimensions of the farming systems reported in the table are the same as those used to organize the workshop, and correspond to the three topics. the shared elements listed in table 1 are phrased in a way that is as close as possible to the way they were expressed by the group of farmers. “grazingbased systems” and “free-range (quasi integral)” refers directly to the characteristics of the livestock farming systems. “orientation toward autonomy” refers to the shared desire to move towards livestock feed autonomy, even if the farmers underlined this is more difficult genetic resources (2022), 3 (6), 15–21 farmers using local livestock biodiversity share more than animal genetic resources 17 table 1. what the participant farmers considered they had in common in their farming systems dimensions of farming systems what farmers considered they had in common livestock feeding systems and land use grazing-based systems free-range (quasi integral) orientation toward autonomy (principle to be modulated depending on the species) use of hedges purchased feed is not industrially processed floristic diversity match between the choice of environment and the choice of breed attention to animal health and animal wellbeing animal breeding and genetics management of a genepool: “farmers are gene passers” product processing and sales and other activities farmers make their animals and activities visible farmers establish direct links with their customers trust is based on mutual knowledge and not on a label the taste of products is co-built and shared with consumers (and farmers consume their own products) association between market relations and friendship proximity can be interpersonal and/or geographical everything is used/no waste farmers seek balance and not expansion with some species than with others. alongside their aim to achieve feed autonomy, they pointed out that if they had to buy feed, they avoided buying industrially processed feed. some of the shared elements concerned the farm ecosystem and how it is valorized: “use of hedges”, “floristic diversity” of the pastured areas was considered important. more generally, they underlined the consistency between the breed and the farm agroecosystem: “match between the choice of environment and the choice of breed”. they also mentioned they all pay attention to “animal health and animal wellbeing”. the only principle linked with genetic management they share is that they consider themselves responsible for the “management of a genepool”. several shared principles refer to what farm gate sales enable: “farmers can make their animals and activities visible”, there is a “direct link with the customer”, there may be an “association between market relations and friendship”, “trust [is] based on mutual knowledge and not on a label”, “the taste of products is co-build and shared with the consumers”. another principle linked with farm gate sales is that “proximity can be interpersonal and/or geographical”, as in some cases farmers sell their products at a distance from the farm but where they know the customers. finally, the last two principles are values shared by the farmers: “everything is used”, is a declaration of their desire to avoid waste, and “farmers seek equilibrium balance and not expansion”. shared principles refer to both practices and values the elements the farmers consider as shared are expressed in different ways, and may be of a different nature. some are formulated as generic practices (e.g. “when feed is purchased it is not industrially processed”). when principles are expressed as a shared generic practice, they may include different ways of expressing the practice. some are not expressed as practices (e.g. “farmers seek balance and not expansion”) but as values, i.e. they refer to elements that are important in the farmers’ views, something they care about. when principles are expressed as values, the values can also be expressed through different practices. shared principles associated with a diverse range of practices we inventoried the diversity of practices expressed during the roundtables. linking them to the corresponding shared principle revealed the diversity of farmers’ practices as illustrated in table 2. several shared principles refer to relational values and practices all farmers who attended the workshop practised direct sales (farm gate sales), but when talking about what they have in common, the farmers did not mention this practice per se as a shared principle; instead, they mentioned the values associated with the practices that were not focused on economic but on relational values such as “direct link with the client” and “proximity that can be interpersonal and/or geographical”. the shared principle “the taste of products is co-built and shared with the consumers” also refers to relational values and interactions. indeed, the interactions with consumers lead to better mutual knowledge: farmers learn about consumers’ preferences, while the customers learn about the farming activity and the animals. for instance, one farmer mentioned that customers became accustomed to watching the animals they will eat in 18 lauvie et al genetic resources (2022), 3 (6), 15–21 table 2. examples of the range of practices associated with a shared principle shared principle illustration of the diversity of associated practices “floristic diversity” soil preparation practices (e.g. new flora are observed after subsoiling, or the use of limestone to treat acid soil) plan to sow pasture (given the difficulties encountered in spontaneously obtaining floristic diversity due to local conditions) leaving sowed multispecies grasslands to evolve in the long term (i.e. to become permanent grassland) and improve “by themselves”. “farmers are gene passers” the future of young female animals: when referring to their conservation objective, the farmers pointed out that they do everything possible to keep young female livestock, while other farmers mention situations where it is difficult to keep a young cow (a cow whose behaviour is dangerous, for instance) several farmers said they follow the advice on the genetic management of rare breeds, where there are differences in organization among breeds. “direct link with customer” all farmers used farm gate sales (some farmers were also part of a short supply chain with one intermediary) farmers chose to sell individual cuts of meat. this requires a dedicated place and time farmers chose to sell several different cuts of meat in boxes weighing several kilos. for these purchases, farmers use a dedicated booking organization, and define a sales period linked with the slaughtering and processing period farmers may sell both individual cuts and mixed cuts in boxes. the end, grow, and two farmers mentioned people who had given up eating meat and started eating meat again. several farmers mentioned receiving feedback about their products, and two farmers who process milk explained that when they develop a new product, they have some customers taste it, and they take the feedback they receive into account when adjusting the recipes. the shared principle “farmers make their animals and activities visible” refers to a diversity of relational practices and to the description of individual practices illustrating how farmers interact with the public: organizing barbecues or dinners and shows on the farm with the possibility to visit the farm, opening their farm to visits on demand, receiving a helping hand from customers who have become friends, holding art exhibitions on the farm through the intermediary of an association. discussion identifying shared principles underlying livestock farming systems through collective approaches the approach we present in this paper consisted of identifying shared principles that underpin livestock farming systems. we show that these principles can be expressed both as practices and values. agroecology is a scientific field in which the principles underpinning farming systems are widely acknowledged. wezel et al (2020) reviewed the different agroecological principles reported in the literature with the aim of proposing consolidated principles. these authors defined principles as actionable statements that contain both normative aspects (that assert values) and causative aspects (that explain relationships) (wezel et al, 2020). agroecological principles are generally defined by scientists and experts to provide a permanent generic scope that can be used to guide analysis, support transition and evaluate systems. the principles are generically formulated but can be applied locally through a range of practices suited to local conditions (wezel et al, 2020). in this context, some agroecological studies aim to establish links between generic agroecological principles and the diversity of practices and ways of acting on agroecosystems (toffolini et al, 2018). the principles we identified in this study are actionable statements that can be expressed through a diversity of practices depending on the farm. however, these principles are formulated as statements shared by a group of farmers, to support work to achieve better recognition of their activities and products. the shared practices and values identified are consequently the result of bringing together diverse views – the product of a collective process within a group, the contours of which may change. therefore, in contrast to generic agroecological principles, these principles may change over time. as the identification of shared principles necessarily results from the views of a group, a participatory approach is required. the need for a collective process leads to a methodological challenge. the workshop reported in this paper involved only a small number of farmers. although this study confirms that this group of farmers who use local breeds share more than genetic resources, it is impossible to conclude what all farmers using local breeds share at the federation scale. the federation has many members, and it would be impossible to include all the farmers of the member associations in a face-to-face collective reflection. one possible way to proceed would be to design a collective form of governance to enable each farmer to monitor the process and give their view, genetic resources (2022), 3 (6), 15–21 19 even if they do not participate actively in the workshop discussions. the shared principles of livestock farming systems concern several dimensions. this study showed that farmers who use local breeds not only share the genetic resources they use but also other elements of their farming systems. those elements are expressed as values and practices, and are interrelated, as values are related to “how farmers ‘make sense’ of their practices” (darnhofer et al, 2012). this calls for further developments in the field of genetic resources management to better understand what farmers who use local breeds do share. we have shown that principles shared by all the farmers are reflected in practices that vary with the farm. diversity among farming systems is indeed a key research object of livestock farming systems, and is often tackled through the use of different kinds of typologies depending on the aim of the study (alvarez et al, 2018). here, in accordance with the aim of our study, our approach was more focused on the identification of common principles than on the characterization of the diversity of possible practices that each principle covers. our work also shows that shared principles of livestock farming systems using local breeds are not only linked with breeding and genetics but with other dimensions, including feeding and sanitary systems. the systemic approach to livestock farming considers several dimensions and their associated practices in a given farming system. although feeding, sanitary, reproduction and renewal practices are at the core of the livestock farming systems approach, what landais (1994) termed the “valorization practices” of animal products (processing, sales, etc.) are also an integral part. our study confirms the importance of the processing and sale of farm products in the overall functioning of the system. few studies of livestock farming systems pay sufficient attention to the sale of farm products (nozières, 2014), even though it may play an important role. indeed, here we have shown the key role of farm gate sales or short supply chains. although direct sales strategies are important in terms of the farm’s overall economic strategy as well as work organization, our results show that the relations established with consumers are also very important and interact with other dimensions of the system. milk and meat processing, for instance, can be influenced by direct sales, as the range of products and the recipes can evolve in response to consumer feedback about the products. the relational dimension of short supply chains is studied in social and economic sciences (chiffoleau et al, 2019), and livestock farming system approaches also consider this relational dimension increasingly relevant, as underlined by darnhofer et al (2012). more generally, this work underlines the importance of the different relationships created by the farmers around their farms, not only through direct sale, but also farm visits or other activities. these different interactions could be the subject of further studies in livestock farming system approaches. challenges: how to recognize specifics of farming systems that use local breeds without excluding diversity? gaining recognition for the products and activities of farms using local breeds is a challenge. this is fully consistent with the wider challenge of adding value to local breeds and helping ensure their continued viability (ligda and casabianca, 2013). several studies focused on products made from local breeds, with or without specific labelling, showing the advantages of adding value to breeds by adding value to products, particularly in an approach focused on economic value. (verrier et al, 2005; mathias et al, 2010). however, other kinds of value may also be at play, and an approach focused on products and activities could broaden the perception of adding value to one or several local breeds. this would include the interrelated strategies identified by ligda and casabianca (2013): ”1. linkage of local breeds to traditional products and/or tourism/agritourism; 2. promotion of local breeds in specific farming systems, such as organic production, conservation grazing, sylvopastoral systems and small-scale low-input farms and hobby farms; and 3. general strategies focusing on the promotion of local breeds (marketing, legislation, organizational issues and raising public awareness) (papachristoforou et al, 2013).” the desire to better recognize the products and activities of farms that use local breeds also reflects a general need to raise awareness of local animal biodiversity. in a previous study on the motives for buying products, conducted with short supply chain consumers who buy products from five french rare local breeds, it appeared that the breed was not spontaneously mentioned as a reason for the purchase. this was considered interesting from a global perspective for consumers to better understand the origin of the food they eat (couzy et al, 2017). although the conclusion of this study cannot be extrapolated, it illustrates the dual need to improve awareness of local biodiversity and provide information about the breeds raised, along with additional information on the production process. identifying the principles shared by farming systems based on the use of local breeds is a first step towards better recognition of the activities and products of farms using local breeds. achieving recognition could be inspired by existing strategies (e.g. labels on products, labels on farms, labels on sales outlets, logos, charters, specifications) or designed ad hoc. in a study of participatory guarantee systems for organic agriculture, lemeilleur and allaire (2018) provide farmers using local livestock biodiversity share more than animal genetic resources 20 lauvie et al genetic resources (2022), 3 (6), 15–21 insights into the different dimensions of recognition setups. they distinguished three types of shared resources in such setups: ideas (shared principles, shared knowledge about practices etc.), equipment (frames of reference, e.g. specifications or charters, guarantee mechanisms, e.g.participatory guarantees or third party certification, etc.) and artefacts (logos, denominations, etc.) (lemeilleur and allaire, 2018). the results of the present study offer more possible ideas for such a setup. however, as we explained earlier, it is a challenge to develop a collective governance that would allow each farmer, who cannot be directly involved in a discussion held to define common principles, to nonetheless be able to follow the process and give their views. diversity is a key notion for farmers who use local breeds. as shown in a previous study (lauvie et al, 2014), diversity is considered not only in terms of genetic resources but also in terms of the farmers involved, the different farming systems, farm products, etc. identifying the activities and products of farmers who use local breeds to make them easier to recognize without reducing the existing diversity is a challenge. finding a balance between diversity inclusion and differentiation and/or protection, depending on the final aim of such a setup, questions the degree to which products and activities should be specified. conclusion this article presents the results of an action research project aimed at identifying the principles shared by farming systems that use local livestock breeds. we observed that farmers who use local breeds share more than just a breed: they also share principles concerning livestock feeding systems and the sale of products, for instance. we also observed that the shared principles are expressed both as practices and values. we identified the methodological challenges associated with identifying common principles shared by livestock farming systems using local breeds. we finally discussed what can be done to increase recognition of the activities and products produced by farmers using local breeds – a process that can both maintain and develop the use of local breeds and, consequently, favour local livestock biodiversity. acknowledgements this research was conducted in the framework of a project led by the federation des races de bretagne, and funded by fondation carasso (under the auspices of the fondation de france). we are grateful to all the participants who attended the workshop. authors contributions anne lauvie: study conception and design, data collection, analysis and interpretation of results, draft manuscript preparation and manuscript revision. nathalie couix and jean michel sorba: study conception and design, data collection, analysis and interpretation of results, 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(2020). agroecological principles and elements and their implications for transitioning to sustainable food systems. a review. agronomy for sustainable development 40, 40– 40. doi: https://doi.org/10.1007/s13593-020-00646-z farmers using local livestock biodiversity share more than animal genetic resources https://doi.org/10.1017/s2078633614000502 https://doi.org/10.4000/economierurale.5813 https://doi.org/10.4000/economierurale.5813 https://doi.org/10.1017/s207863361300026x https://doi.org/10.1017/s207863361300026x https://doi.org/10.1017/s2078633610001001 https://doi.org/10.1017/s2078633610001001 https://doi.org/10.1017/s2078633612000495 https://doi.org/10.1017/s2078633612000495 https://doi.org/10.1080/1828051x.2020.1713027 https://doi.org/10.1080/1828051x.2020.1713027 https://doi.org/10.1016/j.livsci.2013.09.011 https://doi.org/10.1080/21683565.2018.1514677 https://doi.org/10.1080/21683565.2018.1514677 https://doi.org/10.1017/s1014233900005538 https://doi.org/10.1017/s1014233900005538 https://doi.org/10.1007/s13593-020-00646-z https://doi.org/10.1007/s13593-020-00646-z introduction materials and methods results elements shared by farmers referred on different dimensions of the livestock farming systems. shared principles refer to both practices and values shared principles associated with a diverse range of practices several shared principles refer to relational values and practices discussion identifying shared principles underlying livestock farming systems through collective approaches the shared principles of livestock farming systems concern several dimensions. challenges: how to recognize specifics of farming systems that use local breeds without excluding diversity? conclusion acknowledgements authors contributions conflict of interest statement short communication genetic resources (2022), 3 (5), 51–58 doi: 10.46265/genresj.usts1801 https://www.genresj.org issn: 2708-3764 survey and conservation of crop landraces in northwest syria munzer al darvish *,a, anas al kaddour b, akram bourgol c, yasser ramazand, yousef hallak e and shelagh kell f a aleppo university and agriculture research centre, syria b aleppo university and general organization for seed multiplication, syria c general commission for scientific agricultural research, syria d plant protection directorate, ministry of agriculture, syria e ministry of agriculture, syria f school of biosciences, university of birmingham, edgbaston, b15 2tt, birmingham abstract: syria lies at the heart of the fertile crescent – one of the centres of diversity of staple crops such as wheat, barley, chickpea and lentil. the country has historically been rich in agrobiodiversity, including crop landraces valued for their nutritional and culinary qualities, as well as for their resilience. with their cultivation already in decline before the start of the syrian crisis in 2011, this study was undertaken to assess the current status of crop landraces in northwest syria, and to initiate an ex situ conservation programme. we found a significant decline in the number of landraces in cultivation, indicating a loss of locally adapted genetic diversity. influencing factors include insufficient seed supply, competition with commercial hybrids, falling market demand and neglect by relevant government authorities. despite not seeing conservation as their responsibility, the participating farmers were supportive of landrace conservation and willingly contributed seeds for ex situ conservation. keywords: plant genetic resources, genetic erosion, on-farm conservation, landraces citation: al darvish, m., al kaddour, a., bourgol, a., ramazan, y., hallak, y., kell, s. (2022). survey and conservation of crop landraces in northwest syria. genetic resources 3 (5), 51–58. doi: 10.46265/genresj.usts1801. © copyright 2022 the authors. this is an open access article distributed under the terms of the creative commons attribution license (cc by 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. introduction crop landraces have been defined as “dynamic populations” of cultivated plant species that have distinct identities and historical origins, and which lack formal crop improvement, and are typically “genetically diverse, locally adapted and associated with traditional farming systems” (camacho-villa et al, 2005). they are “closely associated with the uses, knowledge, habits, dialects, and celebrations of the people who developed and continue to grow” them (veteläinen et al, 2008), and their continued existence traditionally relies on repeated cycles of seed selection and sowing (almekinders et al, 1994; maxted et al, 1997). these populations character∗corresponding author: munzer al darvish (drgeneral2015@yahoo.com) istically exhibit high levels of adaptation to local environmental conditions, including abiotic and biotic stress tolerances, through a combined process of human and natural selection (almekinders et al, 1994). crop landraces are valued not only for their adaptation and resilience to local climatic conditions and resistance to pests and diseases, but also for their nutritional and culinary qualities – such as flavour, colour and texture – and their cultural value (fao, 2019b). furthermore, they constitute a valuable pool of genetic diversity for crop improvement – particularly in the development of varieties with abiotic and biotic stress tolerance and for incorporating farmer-preferred traits (fao, 2019b). however, the socioeconomic change and transformation of production systems that occurred worldwide in the twentieth century, has resulted in the substitution of landraces with modern cultivars and a received: 11.12.2021 accepted: 03.03.2022 published online: 04.05.2022 https://www.genresj.org https://www.dx.doi.org/10.46265/genresj.usts1801 https://www.genresj.org https://www.dx.doi.org/10.46265/genresj.usts1801 mailto:drgeneral2015@yahoo.com genetic resources (2022), 3 (5), 51–58 conservation of crop landraces in syria 52 widespread reliance on crop monocultures (veteläinen et al, 2008; van de wouw et al, 2010; frison et al, 2011; maxted et al, 2011; dwivedi et al, 2016; fao, 2019b; petropoulos et al, 2019). this homogenization, and associated loss of landrace diversity, has rendered agriculture, food security and livelihoods vulnerable to the impacts of climate change, such as drought, heatwaves, storms, floods and frosts, as well as unexpected pest and disease outbreaks (genres bridge project consortium, ecpgr, erfp and euforgen, 2021). under the current climate crisis, diversification in farming is needed, including the use of more genetically diverse crop varieties resilient to the increasingly extreme and uncertain impacts of climate change (fao, 2008, 2019a; genres bridge project consortium, ecpgr, erfp and euforgen, 2021). the conservation of crop landraces is therefore an essential component of sustainable agricultural development (dwivedi et al, 2016; ficiciyan et al, 2018; fao, 2019b), and this includes incentives to retain the cultivation of landraces on farm, as well as to ensure complementary ex situ conservation in genebanks as a backup for potential losses in farmers’ fields, and for access for research and crop improvement (maxted et al, 1997, 2011; veteläinen et al, 2008; fao, 2011, 2019b). syria lies in the fertile crescent – a region of considerable topographic and climatic diversity (msea, 2009) and in the historical centre of origin of many world food crop staples, such as cereals (e.g. wheat, barley and rye), legumes (e.g. lentil, chickpea and broad bean) and fruits and nuts (e.g. peach, pear, almond and pistachio), as well as animal feed (vavilov, 1926; damania, 1994; fao, 1995; willcox, 2012; mazid et al, 2014; jaradat, 2017). syrian crop landrace diversity has contributed to crop improvement for increased agricultural production (e.g. in barley (ceccarelli and grando, 2000)). the same authors concluded that “securing the continuity of the evolutionary processes within landrace populations is of vital importance for future generations.” the threat to syrian crop landraces was already recognized in the 1990s, particularly in relation to wheat, vegetables and fruit trees, as a result of the adoption of genetically uniform modern varieties and the lack of policies to protect landraces from competing commercial varieties (fao, 1995). although landraces remain in cultivation in syria, their use has declined over time due to the push towards new higheryielding varieties, when fertilizers and agrochemicals are available (fao, 1995). in addition, the younger generation is moving away from agriculture, either as a profession or for subsistence purposes (veteläinen et al, 2008; maxted et al, 2011; mazid et al, 2014), and while crop landraces are often valued for their unique qualities, some do not produce sufficiently high yields for adequate income generation (veteläinen et al, 2008). organizations active in plant genetic resources conservation and crop research in syria before the onset of the crisis in 2011 included the arab centre for the study of arid zones and dry lands (acsad), founded in damascus in 1968, the syrian ministry of agriculture and agrarian reform (smaar) seedbank in douma, and the international centre for agricultural research in the dry areas (icarda), established in tel hadya in 1975. the majority of icarda’s international staff fled syria in 2012, with its genebank operations ceasing in 2015 (alkiswany, 2012; gewin, 2015). following damage to its facilities, the general organization for seed multiplication (gosm), the national source of seed multiplication and supply for commercial varieties, also ceased its operations in idleb governorate, northwest (nw) syria (crs, 2015). in 2013, the syrian interim government (sig) established a parallel seedmultiplication entity in opposition-controlled areas of idleb and aleppo governorates to try to meet local needs in nw syria through the import and export of commercial/hybrid seeds for cereals, legumes, potatoes, and vegetables, with its seed-multiplication activities limited to commercial wheat varieties. since the beginning of the syrian crisis, agricultural production has fallen considerably due to drought, migration, insecurity, loss of government subsidies and the rising prices of inputs, such as fuels and fertilizers, as well as limited access to markets (kelley et al, 2015; mohammed et al, 2019; al-ghazi, 2021). for example, by 2017, only 18 out of 33 commercial wheat varieties were still in use by farmers in nw syria, 9 for durum and 9 for soft wheat (gosm, 2017). at the same time, the conservation of syria’s crop landraces has not been a priority for local authorities and humanitarian actors, and initiatives to conserve local genetic resources have been limited by insecurity and loss of expertise. at national level, acu (2016) reported that syria’s farmers were still producing 32% of their own wheat seeds, while being reliant on traders for 50%, and other sources (including donated and subsidized seed) for the remaining 18%. however, in nw syria, farmers were reported to source 90–95% of their seed through informal channels (crs, 2015). further, the primary focus of icarda and smaar on cereals and cash crops has resulted in an absence of data on landrace vegetables, legumes (e.g. common bean and cowpea) and forage crop varieties (icarda, 2017) it was within this context that in 2018, the authors initiated a study of the status of crop landraces of vegetables, legumes, cereals, forages and cash crops in nw syria, with the aim of ensuring the availability of this diversity for future generations, and in the longer term, the multiplication and provision of good quality seeds for small-scale farmers in the region. specifically, the objectives were to: (a) understand the current use of crop landraces in nw syria and assess whether there has been any loss of diversity, (b) reveal the factors influencing their continued cultivation as well as the potential causes for declining use and (c) inform the process of collecting landrace seeds to initiate ex situ conservation. 53 al darvish et al genetic resources (2022), 3 (5), 51–58 materials and methods the study was conducted between december 2018 and november 2019 in idleb governorate in the districts of maaret tamisrin, saraqeb, ma’arrat an’numan and the idleb subdistrict (figure 1). this region is located within the second and third agroecological zones, incorporating both mountains and plains. agroecological zone 2 covers 2,473,000 ha (13.4% of the country) with an annual rainfall of 250 to 350mm and no less than 250mm across two-thirds of the monitored years. agroecological zone 3 comprises 1,306,000 ha (7.1% of the country) with an annual rainfall of 250 to 350mm and no less than 250mm over half of the monitored years (fao, 2003). in december 2018, prior to starting the data collection, preliminary meetings were held with local council and community representatives in each of the four districts to explain the information on the study, its methods, objectives and importance, as well as to help identify suitable study participants from the local areas (i.e. individuals with considerable experience and a good knowledge of agriculture and crop diversity within the study area). the study combined 75 one-to-one face-to-face interviews and 25 focus groups (fgs), each of which involved 5 to 10 participants. the research participants included established farmers (67), agricultural engineers (15) and agricultural researchers (18) – the latter including former local researchers from icarda and the general commission for scientific agricultural research (gcsar). an interview questionnaire (see supplemental data) was developed and shared with both interviewees and fg participants, requiring them to answer questions as to which landraces had fallen out of use and why they believed this had happened, as well as to provide related timeframes. in the fgs, each of the questions was discussed collectively, in order to reach consensus on the answer. the recruitment of participants with good local background knowledge was central to obtaining a full and accurate picture and to achieving the research objectives, as was the willingness of study participants to share that local knowledge. the majority of the farmers involved (89%) had farms of 1,000m2 plus, so of sufficient size to be cultivating a range of crops, with 22 of those interviewed aged 55 years or over. the age and experience of this group provided the study with good historical knowledge of the crop landraces that had been cultivated in the area, as well as changes in their use. due to local social norms, all study participants were male. however, some farmers’ wives participated in the interviews and shared their knowledge. the one-to-one interviews were conducted from january to march 2019 using hard copies of the questionnaire to allow interviewees to provide answers in writing. as an introduction to the interviews, the interviewer shared information on the study, its methods, objectives and anticipated duration. participants were then invited to sign a consent form. if the participant was happy to proceed, the interviewer then proceeded to the interview, reading out each question in sequence. where requested, the interviewer provided further clarification to avoid any misunderstanding and ensure accurate responses to the questions. answers were provided by the interviewees in writing on the questionnaire form. in the case of illiteracy amongst some of the farmers, the interviewer wrote interviewee responses on their behalf. in the case of the fgs, the interviewer captured the groups’ answers in writing once consensus had been reached. all interview and fg data were transferred to ms excel and anonymized by the team prior to analysis, to ensure confidentiality. the research team discussed the characteristics of each landrace to make sure that each was correctly identified and recorded in one of the five crop categories (vegetables, legumes, cereals, forages and cash crops). the study also involved asking participating farmers to share seed samples of particular landraces to form the nucleus of the ex situ collection. seed samples from 74 landraces, both field crops and vegetables, were collected from farmers across the four districts and planted for multiplication purposes in a safe location in sarmada, nw syria, near the syrian-turkish bab alhawa border crossing, using typical local cultural practices. at maturity, seeds were collected, dried and disinfected using thiram fungicide. each sample was then placed in a labelled paper bag on which the variety, original location, source of the variety, name of the farmer who had donated the sample (with their permission), sample size, collection date and storage date were recorded, prior to being stored in airtight plastic barrels with dry silica gel. at this stage, this modest seed-multiplication activity aims only to increase their quantity as part of this ex situ collection. additional funds will be required to expand seed-multiplication activities in order to support their distribution for use by local farmers. results the 75 interviewees and 25 fg participants took part willingly in the study, providing the information being sought by the study team. they had good knowledge of the crop landraces that had been widely cultivated in their local area over the previous 20 years, and which were still in cultivation in 2019. more than half of the 73 landraces recorded in the study as previously widely cultivated were of vegetable crops (54%) – the remaining comprising legumes (19%), cereals (18%), forages (7%), and cash crops (2%) (table 1). in terms of actual numbers (table 1), the study revealed that amongst the ten wheat landraces that were once widely cultivated, only five were still in use, and of the five species of legumes being grown in the study area, the number of landraces that were still in use had fallen from 23 to 11. the eight vegetable crop species had once included 27 landraces, whereas only 14 were still in widespread cultivation in 2019. of the four sunflower cash-crop landraces known to the farmers, only one was no longer grown, whereas the majority of forage landraces (six out of nine) genetic resources (2022), 3 (5), 51–58 conservation of crop landraces in syria 54 figure 1. study area. the inset map shows the research area in green, relative to the whole of syria. source: humanitarian data exchange https://data.humdata.org/dataset/syrian-arab-republic-administrative-boundaries-populated-places table 1. status of the cultivation of landraces by crop category across the study area (nw syria) in 2019. pwc, previously widely cultivated, i.e. common and widely available in the communities in terms of yield and seed during the period 1999–2019; cwc, currently widely cultivated, i.e. previously widely cultivated, and still common and widely available. category common name scientific name pwc cwc cereals wheat triticum aestivum 10 5 legumes broad bean vicia faba 4 3 chickpea cicer arietinum 6 2 common bean phaseolus vulgaris 5 3 cowpea vigna unguiculata 3 1 lentil lens culinaris 5 2 vegetables armenian cucumber cucumis melo flexuosus 5 1 eggplant solanum melongena 5 2 pepper capsicum annuum 4 3 tomato solanum lycopersicum 4 1 watermelon citrullus lanatus 3 2 zucchini cucurbita pepo 1 1 okra abelmoschus esculentus 3 2 squash cucurbita pepo 2 2 forages barley hordeum vulgare 4 1 corn zea mays 5 2 cash crops sunflower helianthus annuus 4 3 totals 73 36 had been abandoned. notably, none of the landraces that were previously widely cultivated are now only rarely cultivated or found only in restricted or very local cultivation. furthermore, no new landraces were recorded and none of the landraces currently widely cultivated were previously only cultivated on a small scale. figure 2 shows the decline in cultivation of the landraces that were once widely grown by crop category, which all the interviewees agreed had been well adapted to the climatic conditions in their area. understanding why particular landraces were still in cultivation or not was an important part of this study. interviewees and fg participants were asked to select one or more reasons from a list of possible causes 55 al darvish et al genetic resources (2022), 3 (5), 51–58 figure 2. percentage reduction in the number of landraces cultivated in the study area before and after 2019 by crop category. developed by the study team for each crop category (figure 3). participants gave good taste, pest resistance and drought tolerance as the most important reasons for the continued use of crop landraces. other factors, such as yield, historical interest and storage properties were deemed to be of less importance. when offered a second set of reasons as to why they might continue cultivating these landraces, the most significant were local sale (cited by 41% of participants) and seed production (34%). use for personal consumption (18%) and national sale (7%) were seen as less important. figure 4 shows that when 67 participating farmers were asked to consider why they no longer cultivated a particular landrace, the main factors given were lack of seeds (50%), competition from new hybrids (24%) and competition from more productive commercial varieties introduced from other parts of syria (18%). factors such as neglect by the competent authorities, disinterest amongst the next generation of farmers, lack of market demand and the absence of government control, were seen as less important. when the farmers were asked about seed exchange, 90% confirmed the exchange of small quantities with other growers, with the remaining 10% open, in principle, to the practice of regular seed exchange. this was figure 3. reasons for the continued cultivation of crop landraces in nw syria (based on the responses of the 75 interviewees and participants in the 25 fgs). figure 4. reasons for farmers no longer cultivating crop landraces (based on the responses of 67 farmers). reflected in their responses to questions on measures that might be taken to conserve landraces (figure 5). all expressed an interest in conserving landraces, with the exchange of seeds with other farmers (61%) considered the most important protection measure, although conservation in genebanks was also seen to be important (22%). although farmers did not consider the general conservation of crop landraces to be their responsibility, nevertheless, all those interviewed expressed a willingness to share their knowledge to support conservation of landraces through the establishment of a farmers’ association. discussion prior to this initiative, the most recent available information on the status of crop landraces in syria dated back to 2016 and focused on wheat. however, our 2019 study of vegetable, legume, cereal, forage, and cash-crop landraces reveals that about half of the landraces in cultivation in nw syria 20 years ago are no longer cultivated. although the range of crop species remains comparable, farmers no longer maintain or use all the landrace varieties that were cultivated in the past, indicating a significant loss of locally adapted crop diversity. figure 5. the importance given by the survey participants to different landrace conservation measures (based on the responses of the 75 interviewees and participants in the 25 fgs). genetic resources (2022), 3 (5), 51–58 conservation of crop landraces in syria 56 although the study did not allow us to determine confidently whether this erosion of local genetic resources had increased since the start of the syrian crisis in 2011, observations and knowledge gained throughout this period by the authors point strongly to an overall decline in biodiversity in nw syria (both on-farm and in the wild), particularly due to social pressures such as migration and a dependency on collection of local natural resources. although a number of the farmers who participated in the study did consider that an absence of government control and neglect by competent local authorities was a factor in the decline of their crop landraces, the ongoing crisis is clearly an underlying factor, pointing to the need for urgent interventions to protect these landraces from further genetic erosion. the reasons given for continuing to grow particular landraces were a mix of end-use and practical cultivation considerations. for example, flavour and texture were highlighted as important qualities, along with pest resistance and drought tolerance. the value of crop landraces for pest and disease resistance, as well as for drought tolerance, has been widely reported (almekinders et al, 1994; fao, 1995; veteläinen et al, 2008; dwivedi et al, 2016; fao, 2019a) interestingly, yield was ranked lower than good taste, pest resistance and drought tolerance as a reason for continuing to cultivate crop landraces in this study, and equally with tradition and historical considerations. this indicates that while yield is often considered the most important aspect of crop production, landraces continue to be cultivated for other qualities that are not available in high-yielding varieties. the importance of local sales as a reason may also speak to the value of taste or tradition of the crop in the local area. one evident barrier to the continued cultivation of landraces is a lack of seeds (noted by 50% of the participating farmers), pointing to a need for landrace seed regeneration, which could be organized through the establishment of a farmers’ cooperative. however, some landraces clearly yield sufficient seed quantities, as seed sales were given by 34% of the farmers as a reason for continuing to grow them. notably, the availability of seeds of competing hybrid varieties, regardless of their suitability for cultivation in the local environment, was given by 24% of the farmers as a strong reason for their cultivation over and above landraces. the quantity of good-quality landrace seeds may also account for the fact that many landraces are abandoned, rather than continuing to grow them only on a very restricted scale. ninety percent of the participating farmers noted the occasional exchange of landrace seeds with other growers – an action reliant on local trust-based seed supply networks. this indicates continued interest in the cultivation of landraces and the need for a formal seedmultiplication and quality-control system to maintain their production following the cessation of the activities of the gosm in nw syria since the start of the crisis. the effectiveness of the efforts of the sig to replace that function in nw syria has been limited and has not involved the seed multiplication of crop landraces. seed-exchange activities and local seed sales of landraces are a positive sign, although clearly insufficient to sustain landraces, given the study’s finding that half of those in cultivation 20 years ago in the study area are no longer being grown by the farmers involved in the study, or based on the knowledge of other interviewed participants (agricultural researchers and engineers). these two ongoing activities may be the sole reason for the continued existence of the landraces that remain in cultivation, which might otherwise have become extinct. the extent of the remaining diversity could be determined through morphological and molecular genetic analyses. although the farmers involved in the study did not see themselves as the custodians of landraces, or believe that working together in farmers’ associations would help, in the current crisis, they are nevertheless by default fulfilling this custodial role. the commonly held view was that this was the role of an external body, as had been the case prior to the crisis – a view illustrated by their willingness to support the practical measures initiated by the study team in the form of seed collection for ex situ storage and subsequent multiplication. additional funds will be required to expand current landrace seed-multiplication activities and allow for their distribution amongst local farmers. the results of this research point to the urgent need for complementary in situ (on-farm) and ex situ conservation to protect landraces that may otherwise be lost (maxted et al, 1997; camacho-villa et al, 2005). continuation of this work to create a local seedbank, and to support the regeneration and distribution of landrace seeds to farmers, could revitalize the cultivation and spread of these landraces in this war-torn region. the provision of seed samples to existing genebanks would provide additional protection against deleterious natural and human-induced events. the conservation of locally adapted crop landraces is an essential component of sustainable agricultural development for future food, nutrition and livelihood security for future generations in syria and worldwide. acknowledgements this research was supported by cara (the council for atrisk academics), united kingdom. the syrian research team wish to extend their thanks to peter hoebe, richard ellis, ian dawson and meriel g. jones for their assistance in developing an early draft of the manuscript. supplemental data interview questionnaire author contributions munzer al darvish coordinated the research and contributed to the research design and manuscript; anas al kaddour, akram bourgol and yasser ramazan contributed to the research design and manuscript, and 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(blackwell, chicester), 163-180. https://doi.org/10.1073/pnas.1421533112 https://doi.org/10.1073/pnas.1421533112 https://www.fao.org/3/am489e/am489e.pdf http://dx.doi.org/10.1017/s1742170513000240 http://dx.doi.org/10.1017/s1742170513000240 https://doi.org/10.1111/rsp3.12222 https://www.cbd.int/doc/world/sy/sy-nr-04-en.pdf https://www.cbd.int/doc/world/sy/sy-nr-04-en.pdf https://doi.org/10.3389/fpls.2019.00126 https://doi.org/10.3389/fpls.2019.00126 https://doi.org/10.1017/s1479262109990062 https://doi.org/10.1017/s1479262109990062 https://hdl.handle.net/10568/106154 introduction materials and methods results discussion acknowledgements supplemental data author contributions conflict of interest statement original article genetic resources (2022), 3 (5), 59–67 doi: 10.46265/genresj.kkxv5870 https://www.genresj.org issn: 2708-3764 optimum contribution selection (ocs) analyses prompted successful conservation actions for faroese horse population anne kettunen *,a,b, signa kallsoy joensen c and peer berg a,d a the nordic genetic resource center (nordgen), c/o norwegian university of biosciences, p.o. box 5003, nmbu, no-1432 °as, norway b nofima as, p.o. box 210, no-1431 °as, norway c felagik føroysk ross, á signabø 64, 416 signabøur, faroe islands d norwegian university of life sciences, p.o. box 5003, no-1432 °as, norway abstract: the faroese horse, an endangered indigenous horse breed, is a part of the cultural and societal heritage of the faroe islands. population history describes a severe bottleneck, prompting for quantification of the genetic diversity (level of inbreeding, probability of gene origin, effective population size) and assessment of sustainable conservation potential (optimum contribution selection, ocs) of the faroese horse population. the pedigree completeness (pci) of the faroese horse is adequate for a realistic estimation of the level of inbreeding (pci5 = 0.96). in concordance with the known population history, the average inbreeding is exceptionally high; in the last cohort, it was equal to 26.8%. an estimate of the effective population size, based on individual increase in inbreeding and coancestry, accounting for the whole population history, was eight. ocs offers a tool to understand and control the increase in the average relationships in the population. within a fixed number of matings, the repetitive use of stallions resulted in the lowest level of average relationships. successful follow-up of mating schemes planned together with a holistic assessment of the suitability of an individual as a breeding candidate, will minimize the increase in inbreeding in future generations and maximize the possibility to increase the census size of the faroese horse population. keywords: conservation, native breed, horse, effective population size, optimum contribution selection citation: kettunen, a., joensen, s. k., berg, p. (2022). optimum contribution selection (ocs) analyses prompted successful conservation actions for faroese horse population. genetic resources 3 (5), 59–67. doi: 10.46265/genresj.kkxv5870. © copyright 2022 the authors. this is an open access article distributed under the terms of the creative commons attribution license (cc by 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. introduction the faroese horse (føroyska rossik in faroese) represents an important part of the agricultural history and cultural integrity of the faroe islands. according to the domestic animal diversity information system (dad-is) of the food and agriculture organization of the un (fao), the breed status is defined as critical and at risk. the history of the faroese horse is not well documented and there is ambiguous evi∗corresponding author: anne kettunen (mailto:anne.kettunen@nofima.no) dence of the origin of the faroese horse. most likely the faroese horse descended from scandinavian and/or celtic horse breeds brought to the faroe islands by settlers in 500–800 ce (bjørk, 1984; joensen, 2019). in written sources, the faroese horse and its use are first described in the 1600s (bjørk, 1984; joensen, 2019). despite their small size, the faroese horses were traditionally used for carrying heavy loads, only seldom used for riding, and never as draught horses as there were no suitable roads for that (bjørk, 1984). horses were only brought to the villages when they were needed as a working force to carry manure to the fields, peat to the houses or during the harvest of hay and grain (bjørk, received: 05.01.2022 accepted: 14.03.2022 published online: 12.05.2022 https://www.genresj.org https://www.dx.doi.org/10.46265/genresj.kkxv5870 https://www.genresj.org https://www.dx.doi.org/10.46265/genresj.kkxv5870 mailto:mailto:anne.kettunen@nofima.no 60 kettunen et al genetic resources (2022), 3 (5), 59–67 1984). outside of these periods of gruelling work, horses were kept free ranging without supplemental feed all year round. consequently, the faroese horse developed into a small (115–125cm at withers), strong, resilient horse well adapted to the demanding climate, terrain and vegetation of the faroe islands. at its highest, the population size of faroese horses has been approximately 800 individuals (bjørk, 1984). in the late 19th century, a shift of labour from agriculture to fisheries and the mechanization of agriculture decreased the importance of horses as working power for agriculture. at the same time, the faroese horse became a valuable export commodity; a large number of faroese horses were exported to the british isles as pit ponies for the coal mining industry (bjørk, 1984; joensen, 2019). the need for pit ponies was increased when the use of women and children as workforce in the mines was made illegal. the reduced population size and decreased interest in pure faroese horses set off demand for the importation of foreign breeds to the faroe islands. consequently, norwegian fjord horses and icelandic horses were imported and interbred with faroese horses. crossbreeding increased the height at withers (125–132cm) and improved the working ability of the horse, but had a deteriorating effect on the overwintering ability (bjørk, 1984). although the documentation of the population management is deficient, heavy restrictions on stallion use during the 1930s when foreign breeds were strongly favoured, are described in literature (bjørk, 1984). by the 1960s, the faroese horse population was close to extinction with only five individuals alive. currently, a small population of faroese horses is maintained on the faroe islands by hobby breeders. today, this unique breed is used for recreational purposes and tourism. the current population stems from a few horses born between the 1940s and 1960s. in 1978, the organization felagik føroysk ross was established to bring together people dedicated to conserving this native breed. the genetic variation in the faroese horse population using 12 microsatellite markers was earlier assessed by mikko et al (2004). as expected, the population had low genetic variation but did not show any apparent signs of inbreeding. in recent years, the interest in the sustainable conservation of faroese horses has greatly increased. given the traditional knowledge of the population history, and due to the limited success in increasing the population size, there is a need for a knowledge-based management plan for this unique horse breed. genetic diversity describes the range of different traits inherited in a population. genetic diversity is a prerequisite for selective breeding and critical for a population’s adaptation potential in changing environments. endangered local breeds typically have small census sizes and low genetic diversity. consequently, minimization of the rate of inbreeding is imperative for the sustainable conservation of these breeds. optimum contribution selection (ocs) is a method that enables maximization of the selected parents’ genetic level and simultaneously constrains their average coancestry at a desired level (meuwissen, 1997; henryon et al, 2015). ocs is used in many conservation programmes, and for example, suggested by nielsen and kargo (2020) as part of the conservation strategy to save the endangered jutland horse. our hypothesis was that the faroese horse expresses extremely low genetic diversity and high levels of inbreeding, and that further rapid increase in inbreeding can be circumvented by using ocs. to test this hypothesis, we conducted a pedigree analysis of the faroese horse population to assess the level of genetic diversity. effective population size (ne), the key parameter for describing genetic diversity in animal populations, was estimated with several methods. additionally, we used ocs to investigate the possibilities for sustainable management of this horse breed. based on these results, management recommendations were drafted. materials and methods data pedigree data, comprising horses born between 1917 and 2016, were obtained from the felagik føroysk ross. out of the 178 individuals, 87 were males and 91 females. in the 1960s the population of faroese horses experienced a severe bottleneck with only five individuals alive (figure 1). these were one stallion born in 1962 and four mares born in 1946, 1952, 1956 and 1958. one of the mares is the dam of two of the other dams, both being a result of a sire–daughter mating. one of the mares alive in the 1960s is unrelated to the other four individuals. the number of horses born per year is presented in figure 2. a very low number of foals were born yearly until the recent years when invigorated national efforts for the sustainable conservation of the faroese horse have resulted in increased population size (figure 2). between 2000 and 2016, a total of 92 foals were born, out of which 66 were alive at the time of the study (december 2016). it is worth mentioning that additional 25 foals have been born between 2017 and august 2019. statistical methods population analyses pedigree completeness, level of inbreeding and additive genetic relationships were calculated using eva 2.1 (berg et al, 2006; henryon et al, 2015). two birth years were combined into biannual cohorts from the year 1991 and onwards due to the low number of foals born per year. estimates of effective population size and parameters derived from the probabilities of gene origin were obtained by endog v.4.8 (gutiérrez and goyache, 2005). more specifically, different methods were used to derive 4f and thereafter effective population was comgenetic resources (2022), 3 (5), 59–67 optimum contribution selection for faroese horse conservation 61 figure 1. pedigree for animals in the bottleneck. stallions are marked as squares and mares as circles. symbols with green filling are the five individuals alive in the 1960s. puted as ne = 1 24f , using the same estimators of rate of inbreeding as in a previous study on norwegian lundehund (kettunen et al, 2017). in short, three regression coefficients of the individual inbreeding coefficients on the number (method identification in parentheses) of i) complete generations (ia), ii) maximum number of generations (ib) and ii) equivalent generations (ic) traced back to founders for each individual, were computed to give information on the lower, upper and ’real’ limits of ne = 1 2b . further, two regression methods were used to approximate4f ; first, the regression coefficient of the individual inbreeding coefficients on birth years (ii), and second the regression coefficient of the inbreeding coefficients regressed on the number of equivalent generations (iv). the average generation interval (gutiérrez et al, 2003) was used to define the increase in inbreeding between two generations. in two other methods values of log(1− fi) were regressed either on birth years (iiia) or complete generation equivalents (iiib) (pérez-enciso, 1995). additionally, individual increases in inbreeding (v) (gutiérrez et al, 2008, 2009) and increase in coancestry (vi) for all pairs of individuals in the reference population (cervantes et al, 2011) were used for estimation of ne and the standard error of the estimated ne. additional details of the methods used are presented in kettunen et al (2017) and references therein. parameters derived from the probabilities of gene origin, that is the effective number of founders (fe), ancestors (fa), non-founders (fne) and the founder genome equivalent (fge) were used to describe the genetic variability in the faroese horse population. the definition and calculation of these parameters can be found in literature (lacy, 1989, 1995; boichard et al, 1997; caballero and toro, 2000). these parameters were used to calculate the degree of genetic diversity relative to the base accounting for loss of diversity due to unequal founder contribution, bottlenecks and genetic drift (kettunen et al, 2017). parameters fe , fa and fne were obtained from endog whereas fge was calculated as 1 2 − α , where − α was the average coancestry of all living individuals (caballero and toro, 2000). optimum contribution selection eva 2.1 software was used for the ocs analyses (berg et al, 2006; henryon et al, 2015). horses born between 1995 and 2014 were considered breeding candidates. individuals with known health or reproductive problems were excluded as breeding candidates prior to ocs analyses. this resulted in a total of 24 males and 28 females being available for breeding. genetic contributions were optimized for 6 to 12 matings, constraining the number of matings allowed for each stallion from 1 to 4. genetic contributions were optimized using default algorithm parameters (supplemental data) with full penalty on average relationships; this equals zero weight on genetic merit with all focus on minimization of the average relationships. results and discussion pedigree completeness and inbreeding pedigree completeness (maccluer et al, 1983) was computed three (pci3), five ( pci5) a nd s even (pci7) generations back (figure 3). for the foals born in 2016, pci3, pci5 and pci7 were 1.0, 0.96 and 0.83, respectively. pci is essentially a measure of the proportion of known ancestral contributions that could contribute to inbreeding. consequently, low population level pci will result in underestimation of the level of inbreeding and overestimation of the level of genetic diversity, as unknown relationships are treated as unrelatedness. giontella et al (2019) reported pci of 90% and 70% for the third and fifth parental generation of the indigenous maremmano horse breed, respectively. pcis for three different reference populations of hokkaido native horses were reported in onogi et al (2017). estimates for the last reference population (2006–2015) were identical to those for faroese horses. 62 kettunen et al genetic resources (2022), 3 (5), 59–67 figure 2. number of individuals per birth year between 1917–2016. the pedigree completeness of the tiny pedigree of the faroese horse is adequate for a realistic estimation of the level of inbreeding. the average co-ancestry and inbreeding in the last cohort in this study (foals born in 2016) were 32.7% and 26.8%, respectively. as the number of breeding animals was very limited, the use of very old individuals for breeding was common. for individuals born in 1980 and onwards the average age of dams was 11.7 years figure 3. pedigree completeness three (pci3), five (pci5) and seven (pci7) generations back over birth years. and sires 9.8 years, resulting in an average generation interval of 10.7 years. a total of 22 matings in the whole pedigree were between half-sibs (12.4%), 3 between full-sibs (1.7%) and 15 (8.4%) between parent and offspring. the average inbreeding over birth years fluctuated, and inbreeding in some birth years was governed by one or very few individuals (figure 4). the individual inbreeding coefficients in the total pedigree (excluding founders and half-founders) varied between 0.8 and 41.3%. sire–daughter mating between the same two individuals resulted in highly inbred individuals born in 1956 and 1958. there was a rapid increase in the level of inbreeding during the 1970s. this was a consequence of the bottleneck in the 1960s, when only five individuals were alive. from 1980 onwards, the increase in inbreeding was moderate (figure 4), with exception of the year 1990 (2004), where only one (two) foal(s) was born with a very high inbreeding coefficient. the overall average inbreeding was 21.9%, with 89.9% inbred animals. a total of 48.2% individuals have inbreeding coefficient corresponding to half-sib to full-sib mating, and 11.3% of the horses had inbreeding coefficients above 30%. average inbreeding of the faroese horse is much higher than that reported for different horse breeds in literature; average inbreeding in native horse populations is typically below 10% (gandini et al, 1992; sairanen et al, 2009; duru, 2017; onogi et al, 2017; giontella et al, 2019; mancin et al, genetic resources (2022), 3 (5), 59–67 63 2020; perdomo-gonzález et al, 2020; poyato-bonilla et al, 2020). slightly higher pedigree-based inbreeding coefficients have been reported for old kladruber horse, warmblood trotter and gotland russ (vostrá-vydrová et al, 2016; kvist et al, 2019). similarly to the faroese horse population, extremely high inbreeding (32.5%) has been reported in a small population of the endangered sorraia horse (lúıs et al, 2007). average coancestry among parents of a cohort expresses the expected level of inbreeding of the progeny that results from random mating. the realized inbreeding of the progeny in faroese horses is slightly lower than the expected inbreeding. this indicates that, despite the high relatedness between available breeding candidates, breeders have mated individuals less related than expected with random mating (figure 4). this said, the same phenomenon could arise if matings resulting in highly inbred progeny are partly unsuccessful, e.g. due to lethal allele combinations, and consequently fewer offspring would be born from these matings. although no apparent signs of inbreeding in the faroese horse were found in mikko et al (2004), it is expected that matings resulting in extremely high inbreeding are not always successful. adverse effects of inbreeding on reproduction have been documented in literature. results from sairanen et al (2009) confirmed that intense inbreeding as well as the age of both mare and stallion, have an adverse effect on foaling rate. reproductive problems, such as retained placenta and reduced sperm motility have been reported in friesian horses and shetland ponies with inbreeding above 7% and 2%, respectively (onogi et al (2017) and references therein). although the population size of faroese horses has increased during the past three decades, some mares had repeated fertility problems (joensen, 2019). these mares repeatedly experienced miscarriages, and repeatedly remained empty despite successful mating. a recent study on the faroese horse concluded that 30% of the mares are infected by equine endometritis, yet infection status was not fully coherent with the fertility status of the mares (joensen, 2019). the magnitude of inbreeding depression on the faroese horse reproduction has not been documented. that said, given the generally high level of relatedness of the population, mating of highly related individuals should be avoided. genetic contributions of founders and ancestors the base population, defined as the individuals with one or more unknown parents, consisted of ten individuals. the actual base population, where an animal with one unknown parent was defined as a half-founder, consisted of eight animals. the contribution of the ten founder individuals to the last recorded cohort of 2016 varied between 3.0–12.3%. the highest genetic contribution of an ancestor to the most recent cohort was 48.5% from the only living stallion in the 1960s bottleneck (figure 1). the second-largest genetic contribution of 37.1% was from its son born in 1972. only one figure 4. average observed and expected inbreeding and deviation from random mating (devf) between birth years 1980 and 2016. of the mares alive during the 1960s was unrelated, and contributed 24.6% to the 2016 cohort. the three remaining mares alive during the bottleneck were sired by one sire only; its genetic contribution to the latest cohort was 22.3%. the genetic contributions of the dam and its two daughters were 15.2%, 16.3% and 7.0%, respectively. only two ancestral individuals accounted for 50% of the variation in the population with known parents. the genetic contribution of the ancestors sums up to more than 100% as the genetic contributions in this extremely small pedigree are highly dependent. genetic diversity and effective population size effective number of founders (fe), ancestors (fa), nonfounders (fne) and genomic equivalents (fge) were 9, 3, 1.7 and 1.4, respectively, with reference population of all animals with both parents known. parameter fe is defined a s t he n umber o f e qually contributing founders that would result in the genetic diversity of the population under study. it only accounts for the loss in genetic variability due to unequal contribution of founders (lacy, 1989), thus it is not very useful for assessing genetic diversity. parameter fa is defined as the minimum number of ancestors explaining the complete genetic diversity of the population, and accounts for potential bottlenecks in the pedigree (boichard et al, 1997). the amount of genetic drift since the foundation of the population is reflected by fne. finally, fge represents the cumulative loss of genetic diversity since the base population and directly relates to ne (lacy, 1989, 1995; caballero and toro, 2000). we used probability of gene origin parameters to derive genetic diversity measures to estimate the degree of genetic diversity relative to the base accounting for loss of diversity due to unequal founder contribution, bottlenecks and genetic drift. the total loss of genetic diversity relative to the base population was 35.8% optimum contribution selection for faroese horse conservation 64 kettunen et al genetic resources (2022), 3 (5), 59–67 (gd = 1 − 1 2fge = 0.642) (lacy, 1995; caballero and toro, 2000). proportionally, 84.5% of this loss could be allocated to bottlenecks and random genetic drift (1 − fge fe ). consequently, 5.6% of the genetic diversity relative to the base (gd∗ = 1 − 1 2fe = 0.944) was lost due to unequal founder contribution (15.5% of the total diversity loss) (lacy, 1995). in literature, estimates of parameters of gene origin are frequently used to describe the genetic diversity of horse populations (duru, 2017; onogi et al, 2017; giontella et al, 2019; mancin et al, 2020; perdomogonzález et al, 2020). ratios of the parameters have been used to interpret the loss of genetic diversity (onogi et al, 2017; perdomo-gonzález et al, 2020), but ignoring the definitions of the quantification of the proportion lost/retained relative to the base (gd, gd*, gd*-gd) in a quantitative manner (lacy, 1995). this leads to ambiguous interpretations of the parameter ratios as indicators of the origin of the loss of the genetic variation between studies. following lacy (1995), the complete loss of genetic variation relative to the base for pura raza español (perdomo-gonzález et al, 2020), turkish arab horse (duru, 2017), the italian maremmano horse (giontella et al, 2019), the hokkaido native horse (onogi et al, 2017) and italian heavy draught horse (mancin et al, 2020) is small compared to the faroese horse: 5.7%, 5.2%, 3.6%, 2.7% and 0.7%, respectively. a slightly higher degree of loss in genetic diversity was estimated for the endangered old kladruber horse: 10.5–17%, depending on the reference population (vostrá-vydrová et al, 2016). the genetic diversity of the faroese horse based on the pedigree analysis is extremely low. regression coefficients of the individual inbreeding coefficients on the number of i) complete generations (ia), ii) maximum number of generations (ib) and ii) equivalent generations (ic) traced back to founders for each individual were computed. method ia and ic give information on the lower and upper limits of ne, whereas method ib should indicate the ’real’ ne (gutiérrez and goyache, 2005). the estimates of ne from the regression-based methods ranged from 8.54 to 24.39 (table 1). methods v and vi (table 1), based on individual increase in inbreeding and coancestry, account for the whole population history estimated ne to be 8. estimates of ne for local breeds such as turkish arab, italian maremmano, hungarian hucul and italian heavy draught horse estimated with methods v and vi (table 1 ) have been considerably larger than those for the faroese horse: 42–97 (duru, 2017; somogyvári et al, 2018; giontella et al, 2019; mancin et al, 2020). despite the slight decrease in the rate of inbreeding in recent years, all the estimates of ne for the faroese horse are critically low, and far from the recommended ne of 50 to 100 to ensure the sustainable management of this endangered horse breed. optimum contribution selection to test whether optimum contribution selection is successful in alleviating problems with inbreeding, we assessed breeding scenarios of 6 to 12 matings, where males were mated with a maximum of 1 to 4 females, relative to the average relationship (figure 5) and selected individuals (figure 6). within a fixed number of matings the repetitive use of stallions resulted in the lowest level of average relationships, although the differences between scenarios were very small and at maximum approximately 0.4% unit (figure 5, maxmate = 1, 6 vs 12 matings). in an extremely small and inbred population, as is the case of the faroese horse, an increase in the number of selected individuals inevitably results in the selection of more related individuals. a minimum of four and a maximum of seven stallions were selected for breeding over all the scenarios allowing repetitive use of stallions. a subset of the same three stallions was selected in all scenarios, and in total only seven out of the 24 available stallions were ever selected across all scenarios (figure 6). the additive genetic relationship of each of the selected males with all candidate males (0.60–0.62) and females (0.53–0.59) was equal to or lower than the average amongst male candidates (0.62) or between male and female candidates (0.59). additionally, the additive genetic relationship of the selected males with the individuals born during the last two years (2015–2016) was lower than the average of all candidate males: 0.57 vs. 0.60. similarly, the additive genetic relationship of the selected females with all candidate females (0.60) and males (0.57) was lower than that amongst female candidates (0.62) or between female and male candidates (0.59). the additive genetic relationship of the selected females with individuals in the last two figure 5. average relationship of selected individuals in different breeding scenarios. number of matings per stallion was constrained to 1–4 (maxmate) and a total of 6–12 matings were optimized. the points represent the lowest possible average relationship for a given number of matings and constraints of stallion use. genetic resources (2022), 3 (5), 59–67 65 cohorts was lower than that of all female candidates: 0.58 vs. 0.60. the potential of ocs for optimization of genetic gain in performance and increase in relatedness in e.g. the franches-montagnes horse, menorca horse and norwegian and north-swedish trotter has been assessed by hasler et al (2011), olsen et al (2013) and solé et al (2013). similar to the faroese horse, full weight on average relationships and minimization of inbreeding when optimizing matings for the endangered jutland horse were used in nielsen and kargo (2020); authors reported that preselection of sires and ocs successfully lowered the average inbreeding compared to random mating. sustainable breeding of an extremely small and a priori highly inbred population is of great importance. successful follow-up of mating schemes planned with ocs helps to avoid a rapid increase in inbreeding in future generations, as well as minimizes inbreeding of single matings. therefore, whether an individual is eligible as a breeding candidate should be evaluated table 1. estimates of effective population size (ne) calculated with different methods. for methods v and vi, standard error in parentheses. *, reference population of individuals with both parents known (n = 168); **, n = 163. full description of methods ii, iiib, v and vi are found in gutiérrez et al (2003), pérez-enciso (1995), gutiérrez et al (2008), gutiérrez et al (2009) and cervantes et al (2011), respectively. method n e whole pedigree ia: regression on complete generations 8.54 ib: regression on maximum generations 24.39 ic: regression on equivalent generations 12.69 ii: regression on birth date 11.04 iiia: log regression on birth date 11.57 restricted pedigree* iiib: log regression on equivalent generations 15.37 iv**: regression on equivalent generations 14.16 v: individual increase in inbreeding 7.99 (1.35) vi: individual increase in co-ancestry 7.88 (0.43) figure 6. breeding candidates with their relationships to all candidate males (n = 24) and females (n = 28) (each candidate having two indicators, one for same sex and one for opposite sex). individuals selected in any of the scenarios are marked with circles and individuals selected for all scenarios with 6–12 requested matings and repetitive use of males (2–4) with bold circles. optimum contribution selection for faroese horse conservation 66 kettunen et al genetic resources (2022), 3 (5), 59–67 thoroughly relative to the health of the individual and possible previous experiences regarding fertility and mothering characters. this is to avoid that the planned mating will not be realized or does not result in viable progeny. the felagik føroysk ross has adopted the use of a database, førøya fongur, to collect all information (e.g. pedigree, health, mating, progeny) on the faroese horse population. information collected in the database on reproduction and health will assist in the recognition of possible inbreeding depression, but this process is still in its infancy. the government of the faroe islands provides support (currently managed by the agricultural agency of the faroe islands) to horse owners if their horses are registered in the database and are producing offspring. unfortunately, the breeder can receive support even if the planned mating would not be appropriate relative to the resulting inbreeding of the offspring. overall, the maximization of reproductive success is of high priority to be able to expand the census size. conclusions and recommendations results from this study are in accordance with the documented population history of the faroese horse; the population has extremely high average relatedness and inbreeding, and low effective population size. consequently, the census size should be expanded as it sets the limit of constraining the future rate of inbreeding. it is recommended to optimize the contributions of parents to the next cohort to minimize increases in average coancestry of the population. due to the expected inbreeding depression, the fitness-related traits should be recorded in the database and carefully monitored. to increase the interest in the breed, actions to improve the economic value of the faroese horse, e.g. through export and increased use in tourism should be promoted. molecular genetic characterization is recommended to be able to assess the additive genetic relationships between individuals accurately and to heighten the potential of ocs in conserving the faroese horse. acknowledgements we thank the felagik føroysk ross for providing the data. supplemental data algorithm parameters used for eva analyses author contributions signa kallsoy joensen compiled and quality checked the pedigree data. anne kettunen analyzed the data, interpreted the results and drafted the manuscript. peer berg wrote eva. all authors read, revised and approved the final manuscript. data availability statement anonymized pedigree data is available upon request from the felagik føroysk ross. conflict of interest the authors declare no conflicts of interest. references berg, p., nielsen, j., and sørensen, m. k. 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(2020). ecpgr recommended simple sequence repeat loci for analyses of european plum (prunus domestica) collections. genetic resources 1 (1), 40-48. doi: 10.46265/genresj.2020.1.40-48. © copyright 2020 the authors. this is an open access article distributed under the terms of the creative commons attribution license (cc by 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. introduction simple sequence repeats (ssr), also known as microsatellites, were introduced in plant research almost three decades ago (akkaya et al, 1992). due to their abundance, reproducibility, and polymorphism, ssrs have proven highly useful for confirming identity of accessions in plant collections and in quantifying their relatedness. in clonally propagated crops, which include most of the fruit and berry crops, each cultivar originates from a single recombination event and all cultivated plants of a particular cultivar would therefore be expected to have the same ssr profile (nybom and weising, 2010). sports, e.g. mutants that differ in fruit ∗corresponding author: hilde nybom (hilde.nybom@slu.se) colour, usually differ so little from the original cultivar that they cannot be distinguished with ssrs. the ability to correctly identify plant material from different cultivars has economic importance (e.g. regarding infringement on plant variety ownership) as well as forms a basis for management of plant collections and their utilization in plant breeding and research. compared to some of the more recently developed approaches like single nucleotide polymorphisms (snp) and next generation sequencing (ngs), ssrs are comparatively easy to apply and interpret, and can be very cost-effective for developing smaller datasets. this means that specific questions asked by growers, plant nursery owners, amateur pomologists and genebank curators can be solved by analysing just a few plant samples and comparing their ssr profiles with received: 2020 04 24 accepted: 2020 06 12 published online: 2020 08 31 https://www.genresj.org https://www.dx.doi.org/10.46265/genresj.2020.1.40-48 https://www.genresj.org https://www.dx.doi.org/10.46265/genresj.2020.1.40-48 mailto:hilde.nybom@slu.se genetic resources (2020), 1 (1) 40–48 ssr loci for plums 41 previously obtained ssr profiles for other samples in a large database. the possibility of adding new ssr-based data to an already existing database, even when developed in a different laboratory, is thus regarded as a major asset. however, this option is dependent on the application of the same set of ssr markers and suitable standardisation procedures including the appointment of closely defined control or reference genotypes. in crops like apple (malus x domestica), where the standardization of loci and procedures has taken very long to achieve, use of separate sets of ssr loci has resulted in many datasets that cannot be compared across different research groups (sehic et al, 2013). this situation may have been improved recently since several apple studies are now based on the same set of loci as used in the international research project fruitbreedomics (urrestarazu et al, 2016). the european cooperative programme for plant genetic resources (ecpgr; www.ecpgr.cgiar.org) aims to ensure long-term conservation of important germplasm in europe as well as facilitate increased utilization of this germplasm, e.g. in plant breeding. an important task is to select unique and well-documented accessions with valuable traits, of european origin or important to europe, for the establishment of a decentralized european collection under the rules of aegis (a european genebank integrated system; www.ecpgr .cgiar.org/aegis). a second task is to assess variability among the accessions and investigate possible subgroupings due to, e.g. geographic differentiation. in fruit tree crops like pears (pyrus communis) and sweet cherries (prunus avium), sets of recommended ssr loci have been appointed by ecpgr and published (clarke and tobutt, 2009; evans et al, 2009). since then, numerous research groups have used these ssr loci for analyses of genetic identity and relatedness in these crops, and, in the case of cherry, also for sour cherries (prunus cerasus). until recently, relatively few ssr-based studies have been undertaken on genetic diversity in european plums, a major reason being that this is a hexaploid (x = 7; 2n = 6x = 42) crop and therefore less amenable to molecular marker-based analyses. european plums are usually treated as prunus domestica, although small-fruited primitive cultivars and landraces are sometimes referred to a separate mostly wild species prunus insititia or to a subspecies p. domestica subsp. insititia. p. domestica is generally thought to result from hybridization between the diploid cherry plum (prunus cerasifera), the tetraploid sloe (prunus spinosa) and potentially an additional species (reales et al, 2010). results and discussion selected ssr loci most of the hitherto published studies on ssr diversity in european plums are based on differing sets of loci (horvath et al, 2011; xuan et al, 2011; öz et al, 2013; gharbi et al, 2014; halapija kazija et al, 2014; makovics-zsohár et al, 2017; merkouropoulos et al, 2017; pop et al, 2018; abdallah et al, 2019; manco et al, 2019; urrestarazu et al, 2018). in order to facilitate harmonization between future studies, we propose a standard set of nine ssr loci, approved by the ecpgr prunus working group, for european plum (table 1). these loci have already been used for investigations of genetic diversity, first in plum cultivars and landraces from norway and sweden (sehic et al, 2015) and subsequently in two ecpgr-funded projects with accessions sampled in a total of 14 european countries: ‘prundoc’ (sehic et al, 2019) and ‘prunus alignment’ (gaši et al, 2020); reporting data from both ecpgr studies). seven of the nine chosen ssr loci were developed from genomic dna of peach (prunus persica): bppct007, bppct014, bppct034, bppct039 and bppct040 (dirlewanger et al, 2002), and udp96-005 and udp98-407 (cipriani et al, 1999). one locus, paca33, is an expressed sequence tag-ssr (est-ssr) from apricot (prunus dulcis)(decroocq et al, 2003), while cpsct026 originates from genomic dna of japanese plum (prunus salicina)(mnejja et al, 2004). in addition to studies based on eight or nine of these loci (sehic et al, 2015; gaši et al, 2020), five loci, bppct034, bppct039, bpct040, paca33 and udp96005, have also been used by halapija kazija et al (2013, 2014) for screening of plum accessions from croatia and neighbouring countries. marker reliability is critical for producing cultivar profiles to be used in shared databases. hexaploid plum accessions are considerably more difficult to genotype compared to diploid genotypes since each locus may have up to six alleles. overlooking a true but faint band is entirely plausible, as well as is mistakenly scoring an artefactual band as an allele (gaši et al, 2020). although 15 loci remained as serious candidates among a wider set of ssr loci screened initially, six of these had to be discarded due to unreliable amplification (paca18, paca49, pacb22, pacb26, pacb35, pacc13) (decroocq et al, 2003), thus leaving the nine chosen loci. very high reliability was shown recently when pollinizer success could be determined using seven of the chosen ssr loci for analysis of plum embryos harvested after open pollination (meland et al, 2020). the need for very accurate ssr allele scoring is objectively higher in trials that use the obtained dna profile database for paternity analyses compared to standard diversity studies. detected polymorphisms were very high for the nine chosen ssr loci when a joint biostatistical analysis was performed for plum accessions previously genotyped in the above-mentioned studies (sehic et al, 2015; gaši et al, 2020). although the study by gaši et al (2020) was based on only eight of the nine recommended ssrs, all plum accessions from that study had also been genotyped for the remaining microsatellite locus (bppct039). number of alleles ranged from 18 to 48, and nei’s gene diversity ranged from 0.88 to 0.93 (table 2). http://www.ecpgr.cgiar.org/aegis https://www.ecpgr.cgiar.org 42 nybom et al genetic resources (2020), 1 (1) 40–48 table 1. ssr loci, dna sequences, references and annealing temperature of the nine primer pairs selected by the ecpgr prunus working group for assessment of plum accessions. locus dna sequence reference annealing temp. ◦c cpsct026 3’-tctcacacgctttcgtcaac-5’ 3’-aaaaagccaaaaggggttgt-5’ mnejja et al (2004) 46 bppct034 3’-ctacctgaaataagcagagcc at-5’ 3’-caatggagaatggggtgc-5’ dirlewanger et al (2002) 56 udp96-005 3’-gtaacgctcgctaccacaaa-5’ 3’-cctgcatatcaccacccag-5’ cipriani et al (1999) 56 bppct014 3’-ttgtctgcctctcatcttaacc-5’ 3’-catcgcagagaactgagagc-5’ dirlewanger et al (2002) 58 bppct039 3’-attacgtaccctaaagcttctgc-5’ 3’-gatgtcatgaagattggagagg-5’ dirlewanger et al (2002) 58 bppct040 3’-atgaggacgtgtctgaatgg-5’ 3’-agccaaacccctcttatacg-5’ dirlewanger et al (2002) 58 udp98-407 3’-agcggcaggctaaatatcaa-5’ 3’-aatcgccgatcaaagcaac-5’ cipriani et al (1999) 58 paca33 3’-tcagtctcatcctgcatacg-5’ 3’-catgtggctcaaggatcaaa-5’ decroocq et al (2003) 58 bppct007 3’-tcattgctcgtcatcagc-5’ 3’-cagatttctgaagttagcggta-5’ dirlewanger et al (2002) 60 laboratory procedures all amplifications were performed according to dirlewanger et al (2002) with the minor changes of an increase to 1 u taq polymerase (thermo fischer scientific, surrey, uk) and the introduction of four different annealing temperatures (table 1). diluted pcr products were mixed with hi-di formamide (applied biosystems, beverly, ma, usa) and an in-house prepared size standard, after which the amplified fragments were separated on an abi 3130xl genetic analyser (applied biosystems). since errors due to competitive amplification are more difficult to spot in samples of hexaploid organisms compared to diploid ones, we recommend that all amplifications are performed in simplex. whether to pool the amplification products from two (or three) loci together before allele sizing in, e.g., an automated gene sequencer, is less critical and therefore a matter of what is regarded as most convenient in each laboratory. reference genotypes a set of seven reference accessions has been appointed: the large-fruited canadian eggplum ‘valor’ (cross between ‘imperial epineuse and ‘grand duke’), the small-fruited german eggplums ‘hanita’ (‘president’ x ‘auerbacher’) and ‘topfirst’ (‘čačanska najbolja’ x ‘ruth gerstetter’), the french greengage ‘reine claude violette’ (offspring of ‘reine claude verte’), the largefruited american prune ‘stanley’ (‘d’ente double’ x ‘grand duke’), the french mirabelle ‘mirabelle de nancy’, and the east european small-fruited prune ‘bistrica’. allele sizes of these references (supplementary table 1) can be used as a basis for determination of the size adjustment needed to render data from other laboratories comparable. leaves of these genotypes can table 2. allele size range, number of alleles and gene diversity (nei, 1978) for 9 ssr loci, calculated among 175 plum accessions investigated by gaši et al (2020) and by sehic et al (2015), as well as among 7 reference cultivars. locus code size range (bp) no. alleles gene diversity size range (bp) no. alleles gene diversity plum accessions (n=175) reference cv. (n=7) udp 98-407 156/231 29 0.8825 164/203 10 0.8995 pac a 33 169/254 37 0.9275 169/252 15 0.8907 cpsct 026 165/216 22 0.9118 165/208 13 0.8989 bppct 040 113/154 18 0.8811 120/146 8 0.8640 bppct 007 121/163 19 0.9121 123/147 10 0.8872 bppct 014 186/294 48 0.9319 186/258 16 0.9298 bppct 034 213/277 25 0.9117 215/259 12 0.9013 udp 96-005 92/169 34 0.9218 100/165 11 0.8462 bppct039 113/187 33 0.9255 126/179 17 0.9339 mean 29.4 0.9118 12.4 0.8946 genetic resources (2020), 1 (1) 40–48 ssr loci for plums 43 be obtained from the institute of pomology (croatian centre for agriculture, food, and rural affairs), located in donja zelina, croatia. alternatively, replicate samples could be used from trees already present in many plum collections following an initial dna-based confirmation of their identity against the original reference genotypes. evaluation of band profiles in previous ecpgr-recommended sets of ssr markers (clarke and tobutt, 2009; evans et al, 2009), 16 loci were selected for both pear and sweet cherries, although only six loci were defined as first priority in cherries followed by three loci as 2nd priority, three loci as 3rd priority and four loci as 4th priority. in these diploid crops, the maximum number of alleles scored would thus reach 32 although the number is usually lower due to the presence of two copies of the same allele in several loci. since all european plums are hexaploid, up to six different alleles can be expected in each ssr locus, amounting to a maximum of 54 alleles for nine loci. however, the total number of alleles in a plum sample analysed with the proposed nine loci has only reached 35 on average (sehic et al, 2015). the discrepancy here is also probably due, at least in part, to multiple copies of the same allele in some of the locus/genotype combinations. in a study of 78 presumably hexaploid plum genotypes screened with all nine loci, 59 accessions revealed six alleles in one to three loci, while the remaining 19 accessions revealed a maximum of five different alleles in any locus (sehic et al, 2015). in another study of 110 accessions analysed with 8 of the proposed ssr loci, 85 accessions revealed six alleles in at least one locus, while 23 accessions revealed five alleles and two accessions revealed a maximum of only four alleles (gaši et al, 2020). counting the total number of bands for each genotype is thus recommended, since an unusually low number of alleles can be an indicator of poor amplification. analyses of genetic diversity multilocus ssr profiles are generally scored as ‘allelic phenotypes’ based on the presence of alleles but not their frequencies. since the likelihood of actually overlooking alleles is much higher in hexaploid samples compared to samples of lower ploidy, the threshold for determination of whether two (or more) samples are identical, has to be relatively low. in a study of european plums, all pairwise comparisons with the fraction of shared bands, sxy [= 2nxy/(nx + ny)], reaching 0.88 or higher, were interpreted as resulting from the same recombination event and thus being genetically identical except for possible minor somatic mutations (gaši et al, 2020). availability of biostatistical software which can be used on genotyping data of allopolyploid accessions, such as the hexaploid plum, is significantly restricted compared to software solutions for diploid accessions. however, some programs provide options to overcome the challenges of allele dosage ambiguity. population genetics software spagedi 1.3 (hardy and vekemans, 2002) and polysat (clark and jasieniuk, 2011), an r package for polyploid microsatellite analysis, enable the replacement of “missing alleles” in loci where fewer than six different alleles (de facto maximum in hexaploid plum) are scored, with the average allele size. this enables the calculation of allele frequency, as well as gene diversity (nei, 1978) and f statistics (weir and cockerham, 1984). additionally, population structure can be investigated using the bayesian model-based cluster procedure within structure version 2.2.3 (pritchard et al, 2000). genetic differentiation among groups of genotypes (based on various criteria such as geographical origin, morphological or taxonomic traits, breeding status, or obtained as reconstructed panmictic populations in structure), can be examined using the genotype/genodive package (meirmans and tienderen, 2004) which enables analyses of molecular variance (amova) (excoffier et al, 1992) among polyploids, as well as among a mix of genotypes with varying levels of ploidy. although the visualization of relationships among polyploid genotypes is easily accomplished through hierarchical clustering, such as upgma dendrograms using a matrix with pairwise comparisons based on the jaccard similarity coefficient, the use of factorial correspondence analysis (fca) on ssr data of diploid crops has become increasingly common. this multivariate analysis can be conducted on a matrix of binary microsatellite allele presence/absence data using the “dudi.coa” routine in r 2.15.2 (r core team, 2012) as suggested by muller and mccusker (2009). construction of the mentioned matrix is quite simple and appropriate for polyploid genotypes. a graphical display of the fca results can then be achieved with the rgl package version 0.93.945 (adler and murdoch, 2013) in the same statistical software. compiled dataset and genetic structure a joint dataset covering 165 accessions (subsequent to removing the duplicates between studies) was compiled from the two datasets (sehic et al, 2015; gaši et al, 2020). the 8 loci from the original study by gaši et al (2020) were complemented by the genotyping of bppct039, resulting in 9 loci scored for all accessions. most of these accessions had never been genotyped before and they were chosen so as to represent both the major coverage of cultivars in plum-growing countries in europe as well as the whole range of material in germplasm collections, from local and landrace accessions to commercial cultivars produced in modern breeding programs. this dataset is available in supplementary table 1 and can be regarded as a starting point for a shared international dataset to be used by different research groups. genetic structure among accessions in this compiled dataset was investigated with a bayesian modelbased cluster procedure using structure version 44 nybom et al genetic resources (2020), 1 (1) 40–48 figure 1. plot of deltak values from the bayesian genetic structure analyses of 165 plum accessions. 2.2.3 (pritchard et al, 2000). for individuals with fewer than six allelic variants per locus, absent alleles were treated as missing data. k (unknown) reconstructed panmictic populations (rpps ) were computed on individuals, testing k (log-likelihood) = 1–10 for all samples, assuming that the sampled accessions were from unknown origin. ten independent runs were conducted for each k. a burn-in period of 200,000 and 500,000 iterations was applied. structure harvester version 0.6.1 (earl and holdt, 2011), which implements the evanno method (evanno et al, 2005), was used to estimate k values for the analysed data (figure 1). k = 2, 4 and 9 were used to assign individuals to specific clusters. all input files were compiled using madc version 1.2 (grahić and grahić, unpublished data). genetic structure analyses for k = 2, accessions were divided into two approximately equal rpps (mostly red or mostly green) and a large number of admixed genotypes (figure 2). among the green-coloured samples (rpp2:1) were p. insititia cultivar no. 81 ‘kozlienka’ from slovakia, the feral p. insititia accession no. 82 ‘krikon’ from sweden, the hungarian plums no. 106 ‘potyó fehér’ and no. 107 ‘potyó szilva’, and the central–eastern european prunes also known as zwetschen (e.g., no. 108 ‘požegača’). redcoloured samples (rpp2:2) instead included the french prunes of ‘d’agen’ type (no. 36 and 37), most of the greengages (no. 118–121, 123 and 124) and largefruited cultivars grown across europe as dessert plums (e.g. no. 153 ‘victoria’). for k = 4, the previous rpp2:1 (≈ p. insititia) was split into one large (green, rpp4:1) and one smaller (red, rpp4:2) rpp (figure 3). rpp4:1 contained the above-mentioned ‘kozlienka’, ‘krikon’, ‘potyó fehér’, ‘potyó szilva’ and ‘požegača’, as well as small-fruited plums of the damson or bullace type like the italian ‘ramassin’ (no. 115–117). rpp4:2 instead comprised several norwegian landrace plums but also e.g. ‘spilling’ (no. 135) collected in denmark but most likely of german origin. the previous rpp2:2 (p. domestica s.s.) was split into one larger rpp (yellow, rpp4:3) with ‘victoria’ and many other large-fruited dessert plums, and one smaller (blue rpp4:4) with greengages like ‘reine claude bålnäs’ (no. 118) and ‘reine claude grande verte’ (no. 124) as well as the french prunes. for k = 9, most samples showed an admixed genotype (figure 4). the previous rpp4:1 was divided into three rpps with the largest (dusty pink, rpp9:1) containing small-fruited accessions like ‘cariadoggia’ (no. 33) and ‘muninca’ (no. 101) from italy, ’karsavas’ (no. 73) from latvia and ‘moravka’ (no. 98) from serbia. the zwetschen (e.g. ‘požegača’) were found in the second (orange, rpp9:2), while two putatively diploid samples (no. 2 and no. 51) and the greek ‘asvestochoriou’ (no. 10) made up the third (red, rpp9:3). the previous rpp4:2 was split into two rpps containing mainly norwegian landraces (green, rpp9:4, and brown, rpp9:5, respectively). the previous rpp4:3 was divided mainly into two rpps with the german ‘gräfin cosel’ (no. 49) and ‘ruth gerstetter’ (no. 127) in rpp9:6 (dark blue) and some other large-fruited cultivars in rpp9:7 (pale blue). several well-known cultivars like ‘victoria’ were denoted as an admixture of these two rpps. the previous rpp4:4 corresponded relatively closely to rpp9:8 (yellow) and contained mainly greengages. the mirabelles (no. 95–97, 161 and possibly also no. 52) formed a rpp of their own (purple, rpp9:9) in spite of having admixed genotypes at lower k-values. conclusions in this contribution we present, for the first time, a set of nine ssr loci recommended by the ecpgr prunus working group for use in genotyping of european plums, and for analyses of genetic variation and structure. a set of seven reference cultivars is proposed. a compiled dataset with allelic information for 165 accessions is presented as a resource to allow comparison of further datasets. genotyping of these 165 accessions showed that all loci produce highly polymorphic genetic profiles, while analysis of genetic structure revealed a major dichotomy between p. insititia-related accessions and cultivars belonging to p. domestica sensu stricto, as well as differentiation among minor subgroups defined by pomological traits and geographical origin. by adding genetic profiles for new samples into this dataset, researchers can easily check whether they are synonymous with any of the present 165 accessions and also check for parent-offspring relations. in addition, performance of genetic structure analyses with all available samples is likely to provide valuable information about pomological grouping of the new samples in genetic collections as well as in plant breeding programs. acknowledgements our sincere thanks to jasna sehic, who has performed a major part of the laboratory work and allele sizing genetic resources (2020), 1 (1) 40–48 ssr loci for plums 45 figure 2. bar plot of the results from a bayesian genetic structure analysis with k = 2, green rpp2:1, red rpp2:2. for accession names, see supplementary table 1. figure 3. bar plot of the results from a bayesian genetic structure analysis with k = 4, green rpp4:1, red rpp4:2, yellow rpp4:3, blue rpp4:4. for accession names, see supplementary table 1. 46 nybom et al genetic resources (2020), 1 (1) 40–48 figure 4. bar plot of the results from a bayesian genetic structure analysis with k = 9, dusty pink rpp9:1, orange rpp9:2, red rpp9:3, green rpp9:4, brown rpp9:5, dark blue rpp9:6, pale blue rpp9:7, yellow rpp9:8, purple rpp9:9. for accession names, see supplementary table 1. for the appointed ssr loci. we wish to acknowledge the ecpgr for providing both the suitable international framework of collaboration and the necessary funds to bring this work to completion. supplemental data supplementary table 1. ssr profiles for 165 european plum accessions. sample numbers are the same as used in the bayesian genetic structure analyses (figures 2, 3 and 4), while study = 1 refers to accessions analysed in gaši et al (2020) and study = 2 refers to accessions analysed in sehic et al (2015). reference accessions (ref) are given at the end of the table. profiles for each ssr are indicated as allele sizes in base pairs. author contributions hn managed projects where the proposed set of ssr loci were used, wrote a major part of the manuscript. dg is the chair of 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(2020). genetic resources – a new attempt at serving the community. genetic resources 1 (1), 1-3. doi: 10.46265/genresj.2020.1.1-3. © copyright 2020 the authors. this is an open access article distributed under the terms of the creative commons attribution license (cc by 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. history between 1970 and 2009, fao and ipgri/bioversity international were jointly producing the plant genetic resources newsletter (pgrn)1, a peer reviewed journal focusing on “genetic resources of useful plants, resulting from new work, historical study, review and criticism in genetic diversity, ethnobotanical and ecogeographical surveying, herbarium studies, collecting, characterization and evaluation, documentation, conservation, and genebank practice”. paper issues were originally distributed free of charge and eventually the publication was converted into an online open access publication. this magazine covered a specific niche of interest for the plant genetic resources community, publishing information that normally would not be suitable for existing scientific journals. it was open to contributions from all regions and offered a free of charge publication service. ∗corresponding author: l maggioni (l.maggioni@cgiar.org) 1 plant genetic resources newsletter archive: https://www.bioversityin ternational.org/e-library/library-services/plant-genetic-resources-new sletter/ the fao journal animal genetic resources2, supporting the implementation of the global plan of action for animal genetic resources (gpa-angr, (fao, 2007), had a similar history and was discontinued in 2016. over a 30-year period, more than 600 papers were published and available in 3 languages (english, french and spanish). the papers published between 2010 and 2016 are freely available online. a majority of the articles were focused on the ruminant species and about 60% of them were related to the characterization and monitoring of animal genetic resources. the discontinuation of pgrn and animal genetic resources was determined by strategy changes and redefinition of priorities of the international organizations that were managing these journals. in the field of plant genetic resources, at least two peer-reviewed magazines (genetic resources and crop evolution and plant genetic resources: characterization and utilization) were already in operation at the time of pgrn and continue today to offer the opportunity to 2 animal genetic resources archive: https://www.cambridge.org/core /journals/animal-genetic-resources-resources-genetiques-animales-re cursos-geneticos-animales received: 17 08 2020 accepted: 17 08 2020 published online: 31 08 2020 https://www.genresj.org https://www.dx.doi.org/10.46265/genresj.2020.1.1-3 https://www.genresj.org https://www.dx.doi.org/10.46265/genresj.2020.1.1-3 mailto:l.maggioni@cgiar.org http://www.bioversityinternational.org/e-library/library-services/plant-genetic-resources-newsletter/ https://www.cambridge.org/core/journals/animal-genetic-resources-resources-genetiques-animales-recursos-geneticos-animales 2 maggioni et al genetic resources (2020), 1 (1) 1–3 publish scientific contributions. however, the possibility was lost to formally publish ‘grey literature’ type of contributions, i.e. methods, strategies, guidelines, reports, case studies, etc., with the support of an international institution as a publisher, offering free of charge services. in fact, since the termination of pgrn, the secretariat of the european cooperative programme for plant genetic resources (ecpgr) received various inputs from its members, inquiring about the possibility to resurrect the journal and soliciting action. as for the field of angr, several journals are offering the possibility of publishing work related to animal genetic resources, provided that their scientific interest and scientific quality is in scope with the magazines’ objectives, however none of them are specifically dedicated to angr. in europe, an alternative was sometimes found through oral or poster presentations during scientific conferences in europe, such as the european federation of animal science (eaap) conference. the annual conference of the eaap organizes a specific session devoted to angr every year, coordinated by erfp, fao and eaap. a new attempt the opportunity to attempt the re-establishment of a journal that would take on the heritage of pgrn and animal genetic resources in a new context was provided by the decision of the three european networks on plant (ecpgr), animal (european regional focal point for animal genetic resources, erfp) and forest (european forest genetic resources programme, euforgen) genetic resources to join forces within the horizon 2020 project ‘genres bridge joining forces for genres and biodiversity management’3, which was approved for funding by the european commission (ec) for the period 2019-2021. as part of this project a task dedicated to the creation of the journal genetic resources was included, thus securing funding for the journal’s initial establishment and the production of the first three issues. it is the intention of the three networks to develop a sustainability plan for the continuation of the journal after the end of the project, depending on its success and the feedback received from users, thereby exploring the possibility in the future to share costs and responsibilities as part of their own budgets, as well as seeking support from other possible sponsors. prior to the launch of the journal, a survey was carried out of selected audiences about the main gaps that a journal on genetic resources would be expected to fill (maggioni, 2019). the survey gave an indication of an existing interest for a new journal on genetic resources (91% of respondents). although agricultural plants (80%), including wild relatives (59%) raised the highest interest, the forestry (24%) and farm animals (9%) domains also registered a significant interest, considering that only a limited number of stakeholders 3 www.genresbridge.eu from these domains were included among the survey recipients. the journal was started under the management of the ecpgr secretariat and is published by bioversity international on behalf of the ecpgr. the editorial board includes members of the plant and animal genetic resources community with expertise in different sectors of relevance. the journal’s focus is on publishing original research as well as methods, strategies, guidelines, case studies or reviews on topics of interest on the present and future use of genetic resources, thus serving a variety of stakeholders across sectors. genetic resources uses the open-source web publishing platform open journal systems (ojs4), and a webbased production software (www.typeset.io), which guarantee that running costs of the publication are limited, allowing the journal to provide real open access publications, which are free to publish and free to read. the journal is a member of crossref5, a not-for-profit cooperative effort among publishers to enable persistent cross-publisher citation linking in online academic journals. it also adheres to publication ethics as published by the committee on publication ethics (cope)6. the active support of voluntary reviewers recruited worldwide and particularly within the european networks ensures timely processing of manuscript submissions. the european networks also offer hubs of expertise to support the editorial and language review of articles. based in europe and mainly supported by the european networks, genetic resources is open to worldwide contributions, offering a public free of charge service also to other regions. this new journal can thus be seen as a contribution of the european region to the implementation of the fao global plans of action on genetic resources (fao, 2011, 2014, 2007), particularly in terms of promoting access to communication, data and information exchange. the importance of genetic resources, the challenges ahead (primarily habitat destruction and climate change) and the existing gaps of knowledge call for intensified actions and global collaboration, as illustrated in the keynote review by the commission for genetic resources for food and agriculture (cgrfa) in this first issue (pilling et al, 2020). this journal has the aspiration to contribute to encouraging a fertile framework of collaboration and exchange of information. it is hoped that the opportunity to use the publication services of genetic resources, starting with this issue, will be increasingly used and appreciated by contributors and readers. their feedback will be taken into account and their judgement will eventually determine whether this enterprise deserves to grow and to continue into the future. 4 https://pkp.sfu.ca/ojs/ 5 www.crossref.org 6 https://publicationethics.org/ http://www.genresbridge.eu http://www.typeset.io https://pkp.sfu.ca/ojs/ http://www.crossref.org https://publicationethics.org/ genetic resources (2020), 1 (1) 1–3 new journal for genres community 3 acknowledgements the authors acknowledge funding received from the european union’s horizon 2020 research and innovation programme under grant agreement no 817580. references fao (2007). the global plan of action for animal genetic resources and the interlaken declaration. url: http://www.fao.org/3/a-a1404e.pdf. fao (2011). second global plan of action for plant genetic resources for food and agriculture. url: http://www.fao.org/3/i2624e/i2624e00.pdf. fao (2014). global plan of action for the conservation, sustainable use and development of forest genetic resources. url: http://www.fao.org/3/a-i3849e.pdf. maggioni, l. (2019). survey on gaps to be filled by a new journal. url: https://www.genresj.org/index. php/grj/genresbridge survey. pilling, d., belanger, j., diulgheroff, s., koskela, j., leroy, g., mair, g., and hoffmann, i. (2020). global status of genetic resources for food and agriculture: challenges and research needs. genetic resources 1(1), 4–16. doi: 10.46265/genresj.2020.1.4-16 http://www.fao.org/3/a-a1404e.pdf http://www.fao.org/3/i2624e/i2624e00.pdf http://www.fao.org/3/a-i384 9e.pdf https://www.genresj.org/index.php/grj/genresbridge_survey https://www.genresj.org/index.php/grj/genresbridge_survey http://doi.org/10.46265/genresj.2020.1.4-16 history a new attempt original article genetic resources (2020), 1 (2), 12–22 doi: 10.46265/genresj.shbd3744 https://www.genresj.org issn: 2708-3764 phenotypic characterization of sheep populations in tahtay maichew district, northern ethiopia abebe hailu a, amine mustefa *,a, tesfalem aseged a, abraham assefa a, seble sinkie a and semere tsewene b a ethiopian biodiversity institute, p.o.box 30726, addis ababa, ethiopia b ethiopian biodiversity institute, mekelle biodiversity center, p.o.box 30726, mekelle, ethiopia abstract: eighteen quantitative measurements and fourteen qualitative characteristics taken from 306 adult sheep (57 rams and 249 ewes) were used to phenotypically characterize sheep populations of tahtay maichew district, ethiopia. most traits showed significant variation by agro-ecological zone, sex and age groups with higher values generally recorded for rams as compared to ewes. middle age group animals displayed highest values for several traits, reflecting the optimal production age. agro-ecological zone affected ewes more than rams. the highland sheep had shortest height at withers, widest shoulder points and longest hair, indicative of adaptation to their environment. qualitative characteristics of the studied sheep populations such as tail shape, plain coat color pattern, unpigmented skin, hairy fiber and the absence of horn, toggle, ruff and beard suggest that they constitute a previously undescribed sheep breed. tan coat color differentiated high and midland sheep from lowland sheep where white and brown colors were dominant. canon bone length, height at withers and tail length were the three most important morphometric variables used in discriminating the sheep populations. on average 66% of the animals could be classified into their respective agro-ecological zone. our data suggest that highland sheep populations are distantly related to lowland sheep, while midland sheep are more closely related to lowland sheep. it can be concluded that breeding programs specific to each agro-ecological zone need to be designed for sustainable utilization and conservation of the studied sheep populations. furthermore, molecular based studies might allow further characterization of ethiopian sheep breeds. keywords: indigenous breed, sheep genetic resources, morphological characterization, qualitative traits, tigray region, ethiopia citation: hailu, a., mustefa, a., aseged, t., assefa, a., sinkie, s., tsewene, s. (2020). phenotypic characterization of sheep populations in tahtay maichew district, northern ethiopia. genetic resources 1 (2), 12–22. doi: 10.46265/genresj.shbd3744. © copyright 2020 the authors. this is an open access article distributed under the terms of the creative commons attribution license (cc by 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. introduction the ethiopian livestock sector, which is mainly dominated by indigenous animal genetic resources, contributes significantly to the economy and food security of the country (central statistical agency, 2018), providing livelihood for 37-87% of the country’s population (central statistical agency, 2005). this sector contributes 15 — 17% and 35 — 49% of the total and agricultural gross domestic product, respectively (michael et al, 2016). within the livestock sector, small rumi∗corresponding author: amine mustefa (aminemustefa32@gmail.com) nants, especially sheep, provide a sustainable option for smallholder low input-output production systems. indigenous sheep genetic resources play a major role in developing countries like ethiopia, as are better adapted to environments which are harsh, marginal and degraded, have low body weight and excellent grazing skills (misra and singh, 2002; degen, 2007). the indigenous sheep genetic resources account for 99.81% of the total sheep population in ethiopia (central statistical agency, 2018). conducting phenotypic characterization is a prerequisite for sustainable utilization, conservation and improvement of a breed through designing appropriate sheep breeding programs (fao, 2012). this will furreceived: 04.06.2020 accepted: 20.10.2020 published online: 21.12.2020 https://www.genresj.org https://www.dx.doi.org/10.46265/genresj.shbd3744 https://www.genresj.org https://www.dx.doi.org/10.46265/genresj.shbd3744 mailto:aminemustefa32@gmail.com 13 hailu et al genetic resources (2020), 1 (2), 12–22 ther maximize sustainable food security while minimizing pressure on the environment. ethiopia, one of the major gateways for domestic sheep to africa (devendra and mcleroy, 1982), is believed to have the largest livestock population in africa with 31.3 million sheep (central statistical agency, 2018) categorized into 14 traditionally recognized and phenotypically distinct sheep populations (9 breeds within 6 breed groups) (gizaw et al, 2008; ebi, 2016). the 9 sheep breeds of ethiopia are simien, short fat tailed, washera, gumz, horro, arsi, bonga, afar and black head somali (bhs) (gizaw et al, 2008). in literature, sheep populations of the current study area were generally classified as sekota traditional population and further categorized under the short fat tailed breed (gizaw et al, 2008). however, samples were not taken from nearby areas of the current study areas. sekota sheep populations were characterized as short fat tail turned-up at end and fused with main part. the population is medium-sized, predominantly brown or white coat color, few blacks with brown belly. the white animals have finer hair or wooly udder-coat, semi-pendulous or rudimentary ears in wag himra and tigray while predominantly rudimentary in tekeze valley. sekota sheep population were reared by agew, tigray and amhara communities (gizaw et al, 2008). however, due to the country’s high ecological and production system variations, some of the breeds were re-characterized in more recent studies, including simien sheep (melaku et al, 2019), short fat tailed sheep (hayelom et al, 2014; bimerow et al, 2011; getachew et al, 2009), washera sheep (mengistie et al, 2010), arsi sheep (worku, 2018); afar sheep (getachew et al, 2009). in addition to these studies, some work was done in the tigray region on abergelle sheep (tajebe et al, 2011) and tigray highland sheep (gebreyowhens and tesfay, 2016). despite the efforts made to characterize the ethiopian indigenous sheep genetic resources as mentioned above, they have not yet been exhaustive in covering all regions of the country in general and the tigray region in particular. they also focused on only a few specifically well-known sheep populations. as a consequence, a high sheep diversity remains unstudied, along with the associated diversified ecology, production systems and ethnic groups. therefore, there is an urgent need for continued characterization and identification to understand the relationships within and among breeds. thus, the present study was initiated to cover these gaps and phenotypically describe the indigenous sheep populations of tahtay maichew district, central tigray zone, ethiopia. materials and methods study areas the study was carried out in the tahtay maichew district, which is located in the central zone of tigray national regional state (figure 1). the district covers a total area of 18,618 km2 with estimated livestock table 1. climatic factors and sheep population size of the threeagro-ecological zones in the tahtay maichew district of tigray region, ethiopia. variables highland midland lowland altitude in meters > 2500 1500 – 2500 < 1500 temperature in ◦c (mean) 9.9 19.9 30.3 annual rainfall in mm (mean) 600 – 700 500 – 600 400 – 500 sheep population size 11,816 8,903 4,476 population size of 247,907, consisting 75,707 cattle, 55,517 goats, 110 mules, 6,716 donkeys, 25,195 sheep and 84,102 poultry (atsbeha et al, 2015). the studied areas were categorized as highland, midland and lowland based on the climatic factors in table 1. site selection and data collection available background information on the existence of unstudied sheep populations adapted to different agro-ecological zones was captured through short pilot survey and focus group discussions with livestock experts and keepers. two sites (kebeles) were sampled randomly from each agro-ecological zone (see table 1). quantitative and qualitative data were recorded from a total of 306 adult sheep (57 rams and 249 ewes) based on data collection procedures outlined in fao guidelines (fao, 2012). studied animals were carefully handled by trained personnel. quantitative measurements were taken early in the morning of the day before feeding and watering when the animals were calm and standing in an upright position on flat ground. eighteen quantitative measurements were collected: body length (cm), body weight (kg), heart girth (cm), height at withers (cm), chest depth (cm), shoulder point width (cm), subs height (cm) rump length (cm), rump width (cm), tail length (cm), tail width (cm), head length (cm), head width (cm), shin circumference (cm), horn length (cm), hair length (cm), canon bone length (cm), ear length (cm), testis circumference (cm). fourteen qualitative characteristics were also collected: coat color pattern, coat color, skin color, fiber type, ear orientation, head profile, back profile, rump profile, tail type, tail shape and presence of toggle, horn, beard and ruff were recorded. statistical analysis data were entered and managed using microsoft excel© worksheet. detection of outliers and testing the normality of the quantitative measurements data was performed using the univariate procedure of statistical analysis software (sas) 9.0 (sas institute, 2002). analysis of data on quantitative measurements and qualitative characteristics was carried out using the general linear model (glm) procedure and the frequency (freq) procedure of sas 9.0 software, genetic resources (2020), 1 (2), 12–22 characterization of sheep in northern ethiopia 14 figure 1. map of the studied areas. respectively. linear measurements least square means (lsm) were separated using the adjusted tukey-kramer test (sas institute, 2002). quantitative and qualitative data were analyzed using the following model:yijk = µ+ai+bj +ck+eijk where yijk is an observation, µ is the overall mean, ai is the fixed effect of environment, bj is the fixed effect of the sex, ck is the fixed effect of age group and eijk is the random error attributed to the nth observation. environment, sex and age group were fitted as class variables throughout the analysis, while sex effect was removed from the class variables when the analysis was done separately for each sex. multivariate analysis was performed on quantitative measurements. stepwise discriminant function analysis (stepdisc) with forward selection procedure was used to find out the quantitative variables that better discriminate populations from different environment. percentage assignment of observations to environment and probabilities of misclassifications were evaluated by discriminant function analysis (discrim). canonical discriminant function analysis (candisc) was also performed to find out linear combination of quantitative variables that provide maximal separations between environments. the scored canonical variables were used to plot pairs of canonical variables to get visual interpretation of environmental differences. pairwise squared mahalanobis distances between environments were computed as:d2 (i|j) = (xi − xj) ′ cov−1 (xi − xj). where d2 (i|j) is the distances between environments zones i and j, cov−1 is the inverse of the covariance matrix of measured variables, xi and xj are the means of variables in the ith and jth populations. results quantitative measurements level of significance (p-values) outputs of the class variables for both the overall analysis and separately for each sex are presented in table 2. overall, most of the studied traits were significantly affected by agroecological zone, age and sexual differences. effect of agro-ecological zone on some quantitative traits (heart girth, height at withers, shoulder point width, rump length, tail length, head length, hair length, canon bone length, and ear length) was more significant on ewes than rams. the overall mean with the respective standard error and deviation, and the effect of agro-ecological zone, sex and age on the quantitative measurements are presented in tables 3, 4 and 5. the highland sheep population had the shortest height at withers, widest shoulder points and longest hair, while midland sheep population 15 hailu et al genetic resources (2020), 1 (2), 12–22 table 2. level of significance for the overall analysis and separately for both sexes traits overall rams ewes agro-eco zone age sex agro-eco zone age agro-eco zone age bl (cm) 0.2688 <0.0001 0.3394 0.3930 0.0028 0.3873 0.0004 bw (kg) 0.5309 <0.0001 0.9515 0.3384 0.0061 0.6135 <0.0001 hg (cm) 0.0330 <0.0001 0.6983 0.0772 0.0002 0.0399 <0.0001 haw (cm) 0.0005 0.0250 0.0617 0.0562 0.0009 0.0075 0.5451 cd (cm) 0.0486 0.0013 0.0267 0.1216 0.0120 0.2068 0.0397 spw (cm) 0.0012 <0.0001 0.0017 0.8511 0.0008 <0.0001 0.0078 sh (cm) 0.5553 0.5924 0.2194 0.1241 0.9660 0.8881 0.7606 rl (cm) <0.0001 0.2364 0.2377 0.8307 0.8157 <0.0001 0.1604 rw (cm) 0.7008 0.0028 0.4727 0.2884 0.0001 0.3402 0.0504 tl (cm) 0.0003 0.9584 0.0040 0.1598 0.2868 0.0007 0.2635 tw (cm) 0.3219 0.3538 <0.0001 0.5269 0.0064 0.2726 0.8968 hl (cm) <0.0001 0.0002 0.0410 0.2124 0.0213 <0.0001 0.0062 hw (cm) 0.0283 0.0092 <0.0001 0.2161 0.9169 0.0597 0.0122 sc (cm) 0.4564 0.0003 <0.0001 0.5994 0.0001 0.6909 0.2794 hrl (cm) <0.0001 0.0076 0.0007 0.1643 0.3147 <0.0001 0.0123 cbl (cm) <0.0001 0.0377 0.2666 0.6187 0.0349 <0.0001 0.1417 el (cm) <0.0001 0.0365 0.6856 0.5261 0.4147 0.0002 0.0153 tc (cm) 0.0253 0.0114 bl = body length, bw = body weight, hg = heart girth, haw = height at withers, cd = chest depth, spw = shoulder point width, sh = subs height, rl =rump length, rw = rump width, tl = tail length, tw = tail width, hl =head length, hw = head width, sc = shin circumference, hrl = hair length, cbl = canon bone length, el = ear length, tc = testis circumference. possessed the shortest tail and ear. almost half of the measured traits were affected by sex of the animals showing higher values for males. most of the overall agro-ecological zone differences were due to the differences within the ewes. however, testis circumference, the only trait among the rams which is affected by agro-ecological zone, increases significantly as we shift from highland to lowland. the majority of the quantitative measurements were significantly affected by the age of the animals (figures 2, 3 and 4). accordingly, values of some traits (body weight, chest depth, shoulder point width, rump width, testis circumference, body length, heart girth, and height at withers) gradually increased towards the optimum age of three years and then decreased towards the oldest age (5 years; figures 2 and 3). however, this was not true in some traits (head length, head width, shin circumference, hair length, canon bone length, and ear length; figure 4). on the other hand, age did not affect subs height, rump length, rump width, tail length, and tail width. qualitative characteristics the outputs of the chi-squared tests, if the qualitative characteristics of the sheep populations from the three agro-ecological zones differ, are presented in table 5. accordingly, ear orientation, back profile, head profile, rump profile and coat color of the three agro-ecological zones were significantly different (table 5). on the other hand, the coat color pattern, fiber type, skin color, tail type, tail shape and presence of toggle, horn, beard figure 2. effect of age on body weight(bw), chest depth (cd), shoulder point width (spw), rump width (rw), and testis circumference (tc). figure 3. effect of age on body length (bl), heart girth (hg), and height at withers (haw) genetic resources (2020), 1 (2), 12–22 characterization of sheep in northern ethiopia 16 table 3. overall mean (x), standard error (se), standard deviation (sd) and pairwise mean comparison (least square means and standard errors) for the effect of agro-ecological zone and sex. means within a column bearing different superscripts are significantly different; a is given to the highest value. traits overall agro-ecological zone sex x±se sd highland midland lowland rams ewes n 306 102 102 102 57 249 bl (cm) 55.2±0.2 4.0 54.7±0.5 55.4±0.4 55.5±0.4 55.6±0.6 54.9±0.3 bw (kg) 23.5±0.2 3.4 22.9±0.4 23.4±0.3 23.3±0.4 23.2±0.5 23.2±0.2 hg (cm) 71.0±0.3 4.9 71.8±0.5a 70.1±0.5b 70.7±0.5ab 71.0±0.7 70.7±0.3 haw (cm) 63.9±0.2 3.2 63.2±0.4b 64.5±0.3a 64.9±0.4a 64.8±0.5 63.7±0.2 cd (cm) 29.8±0.2 3.1 30.0±0.4 30.0±0.3 30.8±0.3 30.9±0.5 29.6±0.2 spw (cm) 18.7±0.1 2.3 19.7±0.3a 18.6±0.3b 19.0±0.3b 19.8±0.3 18.4±0.2 sh (cm) 32.9±0.1 2.5 32.8±0.3 33.1±0.3 33.1±0.3 33.3±0.4 32.7±0.2 rl (cm) 14.9±0.1 2.2 15.6±0.3a 15.2±0.2a 14.1±0.2b 15.2±0.3 14.7±0.2 rw (cm) 21.3±0.1 2.3 21.2±0.3 21.4±0.2 21.2±0.3 21.1±0.4 21.4±0.2 tl (cm) 16.8±0.2 3.1 17.5±0.4a 16.3±0.3b 18.0±0.3a 18.1±0.5 16.4±0.2 tw (cm) 16.8±0.2 3.0 18.0±0.3 17.5±0.3 17.5±0.3 19.0±0.5 16.3±0.2 hl (cm) 13.9±0.1 1.5 14.4±0.2a 14.0±0.2a 13.4±0.2b 14.2±0.2 13.6±0.1 hw (cm) 10.2±0.1 1.3 10.4±0.1b 10.8±0.1a 10.4±0.1b 11.1±0.2 9.9±0.1 sc (cm) 6.8±0.1 0.8 7.0±0.1 7.1±0.1 6.9±0.1 7.3±0.1 6.6±0.1 hrl (cm) 4.5±0.1 1.3 5.2±0.1a 4.6±0.1b 4.5±0.1b 5.2±0.2 4.4±0.1 cbl (cm) 12.3±0.1 1.2 12.7±0.1a 12.5±0.1a 11.8±0.1b 12.5±0.2 12.2±0.1 el (cm) 7.6±0.2 3.0 7.8±0.4a 6.4±0.3b 8.4±0.4a 7.7±0.5 7.4±0.2 n = number of observations, bl = body length, bw = body weight, hg = heart girth, haw = height at withers, cd = chest depth, spw = shoulder point width, sh = subs height, rl =rump length, rw = rump width, tl = tail length, tw = tail width, hl =head length, hw = head width, sc = shin circumference, hrl = hair length, cbl = canon bone length, el = ear length. table 4. pairwise mean comparison (least square means and standard errors) for the effect of agro-ecological zonewithin each sex. means within a column bearing different superscripts are significantly different; a is given to the highest value. traits rams ewes highland midland lowland highland midland lowland n 14 26 17 88 76 85 bl (cm) 56.3±1.3 56.1±0.9 57.6±1.0 54.7±0.5 55.5±0.5 55.3±0.5 bw (kg) 23.2±1.1 23.7±0.7 24.8±0.8 23.3±0.4 23.7±0.4 23.3±0.4 hg (cm) 73.2±1.5 70.6±1.0 73.3±1.2 72.2±0.6a 70.6±0.6b 70.6±0.6b haw (cm) 65.0±1.1 65.7±0.7 67.6±0.8 63.1±0.4b 64.4±0.4a 64.4±0.4a cd (cm) 30.2±1.2 30.8±0.8 32.5±0.9 29.7±0.3 29.4±0.4 30.2±0.4 spw (cm) 20.0±0.9 19.9±0.6 20.3±0.7 19.6±0.2a 18.1±0.3b 18.5±0.2b sh (cm) 32.0±1.0 34.0±0.6 33.3±0.7 32.7±0.3 32.7±0.3 32.8±0.3 rl (cm) 15.3±0.8 14.8±0.5 14.9±0.6 15.5±0.3a 15.2±0.3a 13.7±0.3b rw (cm) 22.6±0.8 21.4±0.5 22.1±0.6 21.6±0.3 21.9±0.3 21.4±0.3 tl (cm) 19.2±1.2 17.2±0.8 18.7±0.9 16.9±0.3a 15.7±0.4b 17.4±0.3a tw (cm) 19.9±1.1 18.7±0.7 19.3±0.8 16.9±0.3 16.4±0.4 16.3±0.3 hl (cm) 15.2±0.6 14.6±0.4 14.1±0.4 14.2±0.2a 13.8±0.2a 13.2±0.2b hw (cm) 10.4±0.6 11.4±0.4 11.2±0.5 9.8±0.1 10.2±0.1 9.7±0.1 sc (cm) 7.4±0.3 7.7±0.2 7.5±0.3 6.7±0.1 6.7±0.1 6.6±0.1 hrl (cm) 4.9±0.5 5.4±0.3 4.5±0.4 5.0±0.1a 4.1±0.2b 4.2±0.1b cbl (cm) 12.6±0.4 12.9±0.2 12.6±0.3 12.8±0.1a 12.4±0.1a 11.7±0.1b el (cm) 7.6±1.1 7.2±0.7 8.3±0.8 7.5±0.4a 6.1±0.4b 8.2±0.4a tc (cm) 23.2±1.1b 24.0±0.7ab 26.1±0.8a n = number of observations, bl = body length, bw = body weight, hg = heart girth, haw = height at withers, cd = chest depth, spw = shoulder point width, sh = subs height, rl = rump length, rw = rump width, tl = tail length, tw = tail width, hl = head length, hw = head width, sc = shin circumference, hrl = hair length, cbl = canon bone length, el = ear length, tc = testis circumference. 17 hailu et al genetic resources (2020), 1 (2), 12–22 figure 4. effect of age on head length(hl), head width (hw), shin circumference (sc), hair length (hrl), canon bone length (cbl), and ear length (el) and ruff were not significantly different among the studied agro-ecological zones. accordingly, the sheep populations can be characterized as hair type sheep with plain coat color pattern. additionally, all of the studied sheep populations possess cylindrical thin tail with turned up at end, and straight head profile. the results also revealed that almost none of the sheep sampled have pigmented skin, horns, toggle, ruff and beard. tan coat color was dominantly observed in the high and midland agro-ecological zones while white and brown colors were dominant in the lowland sheep (figures 5, 6 and 7). multivariate analysis for discrimination of sheep populations according to stepwise discriminant function analysis, canon bone length, height at withers and tail length were the three most important morphometric variables used in discriminating the sheep populations from different agro-ecological zones (table 6). chest depth, subs height, body length, and tail width were found not to be useful variables due to their lowest discriminatory power (table 6). the probabilities of all main multivariate tests over the canonical structures were significant (table 7). figure 5. coat color of sheep populations from different agro-ecological zones; significant (p<0.0001) effect of agroecological zones was observed over the coat color of the studied sheep populations. table 5. percentages and their respective chi-squared probabilities of some qualitative characteristics of the sheep populations from different agro-ecological zones. χ2values in bold are statistically significant. qualitative traits agro-ecological zones highland midland lowland χ2 coat color pattern 0.1445 plain 77.5 85.3 82.4 patchy 17.6 12.7 17.6 spotted 4.9 2.0 nr fiber type 0.8211 hairy 85.3 86.3 88.2 wooly 14.7 13.7 11.8 ear orientation 0.0127 erect 1.0 1.0 4.9 semipendulous 48.0 34.3 37.2 pendulous 25.5 16.7 21.6 carried horizontal 25.5 48.0 36.3 head profile 0.0100 straight 71.6 65.7 85.3 concave 12.7 11.8 8.8 convex 15.7 22.5 5.9 back profile 0.0002 straight 70.5 40.2 62.8 slopes up towards the rump 25.5 56.9 34.3 slopes down from withers 2.0 2.9 2.9 curved 2.0 0 0 rump profile <0.0001 flat 27.5 59.8 50.0 sloping 72.5 40.2 48.0 roofy 0 0 2.0 toggle 0.0987 present 3.9 11.8 10.8 absent 96.1 88.2 89.2 canonical correlation coefficients of the quantitative variables and class means outputs from the two canonical structures are shown in table 8. the first canonical structure (can 1) explains the majority (69%) of the variability with eigenvalue of 0.48. the first canonical correlation (57%) was the greatest multiple correlation with the classes that was achieved by using the linear combination of the quantitative variables. the results revealed that can 1 separates the sheep populations (class means) from different agro-ecological zones. results of a discriminant function analysis (table 9) shows the classification of data into a known agroecological zone. accordingly, an average of 66% of the sampled animals were classified into their respective genetic resources (2020), 1 (2), 12–22 characterization of sheep in northern ethiopia 18 table 6. summary of the stepwise discriminant function analysis; ascending order of traits used in discriminating the sheep populations from different agro-ecological zones. step variables entered partial r-squared f value pr > f wilk’s lambda pr < lambda 1 canon bone length 0.1237 21.39 <0.0001 0.8763 <0.0001 2 height at withers 0.0564 9.02 0.0002 0.8269 <0.0001 3 tail length 0.0557 8.87 0.0002 0.7809 <0.0001 4 rump length 0.0554 8.79 0.0002 0.7376 <0.0001 5 head width 0.0529 8.36 0.0003 0.6986 <0.0001 6 hair length 0.0460 7.18 0.0009 0.6664 <0.0001 7 head length 0.0525 8.23 0.0003 0.6315 <0.0001 8 shoulder point width 0.0308 4.70 0.0098 0.6120 <0.0001 9 shin circumference 0.0258 3.91 0.0210 0.5962 <0.0001 10 heart girth 0.0172 2.58 0.0777 0.5860 <0.0001 11 body weight 0.0268 4.03 0.0188 0.5703 <0.0001 12 rump width 0.0147 2.18 0.1154 0.5619 <0.0001 chest depth 0.0073 1.07 0.3432 subs height 0.0013 0.18 0.8314 body length 0.0010 0.15 0.8595 tail width 0.0008 0.12 0.8876 agro-ecological zone. the overall error rate was 34%, while higher error rates were obtained from the classification of midland sheep populations. pairwise squared distances between agro-ecological zones are shown in table 10. all distances were significant. highland sheep populations are distantly related to the lowland sheep. on the other hand, midland sheep relates more towards lowland sheep than highland sheep. table 7. multivariate statistics and f approximates statistic value f value num df den df pr > f wilks’ lambda 0.5559 6.14 32 576 <0.0001 pillai’s trace 0.5015 6.04 32 578 <0.0001 hotellinglawley trace 0.6957 6.24 32 510.82 <0.0001 roy’s greatest root 0.4811 8.69 16 289 <0.0001 table 8. eigen values, canonical correlations and class means can 1 can 2 multivariate statistics canonical correlation 0.5699 0.4203 eigenvalue 0.4811 0.2146 proportion 0.6916 0.3084 class (agro-ecological zones) highland 0.9139 -0.2290 lowland -0.7539 -0.4141 midland -0.1600 0.6430 discussion along with the most observable qualitative traits, quantitative measurements produce reliable information in characterization and differentiation of sheep populations. in our current study, more traits showed significant differences among ewes than among rams in different agro-ecological zones. this might be due to either the larger sample size taken for ewe populations or due to the similarity of rams over the studied agro-ecological zones, which could be attributed to common markets table 9. number and percent of observations classified into agro-ecological zones. from highland lowland midland total highland 70 (68.6) 20 (19.6) 12 (11.8) 102 (100) lowland 12 (11.8) 71 (69.6) 19 (18.6) 102 (100) midland 19 (18.6) 23 (22.6) 60 (58.8) 102 (100) total 101 (33.0) 114 (37.3) 91 (29.7) 306 (100) error rate 0.31 0.30 0.41 0.34 priors 0.33 0.33 0.33 table 10. squared mahalanobis distance between agroecological zones; output of the multivariate analysis calculated using the quantitative measurements from highland lowland midland highland 0 lowland 2.82*** 0 midland 1.91*** 1.47*** 0 *** shows the significance of the distance calculations at p<0.0001. 19 hailu et al genetic resources (2020), 1 (2), 12–22 figure 6. lowland sheep flock in tahtay maichew district. where farmers select and purchase rams for sire purposes. most of the studied traits were affected by agroecological zone differences, which might be due to the differences in physiological adaptation mechanism of sheep types to different environments, management, availability of different feed and nutrition and/or the variations being caused by genetic factors. for example, the majority of lowland sheep possess higher values for height at withers, testis circumference and short hair which might help them to adapt to a hot environment. these results are in line with the results of getachew et al (2009) and gizaw et al (2008) for menz and afar sheep who reported that such measurements of the lowland afar sheep were higher than the highland menz sheep populations. most of the body measurements were higher for rams than ewes, which might be attributed to enhanced muscle mass and skeletal development in males due to testosterone hormone secretions (baneh and hafezian, 2009). these results follow rensch’s rule where the males of a particular species are usually heavier than the females (rensch, 1950). size differences may be ascribed to the differences in the endocrine system of the two sexes; estrogen hormone was shown to have a limited effect on growth in females (baneh and hafezian, 2009). these results are in agreement with mustefa et al (2019) and getachew et al (2009) who reported that males were higher than the females in most growth traits in goats and sheep respectively. in agreement with the results of the current study, most scholars report differences in traits between the sexes with rams being dominant over ewes (rensch, 1950; baneh and hafezian, 2009; mustefa et al, 2019; getachew et al, 2009). however, differences due to sex was not observed in tigray highland sheep populations (hayelom et al, 2014). in contrast to this, hayelom et al (2014) report the dominance of ewes over the rams on most of the linear body measurements of elle sheep populations. in a different study, no significant differences were observed among body weight of simien sheep ewes and rams (melaku et al, 2019). the overall mean body weight (23.50 kg) presented in the current study was higher than those reported by tajebe et al (2011) for abergelle sheep (21.25 kg), and by gebreyowhens and tesfay (2016) for tigray highland sheep (22.10 kg), while the values were lower than those reported by hayelom et al (2014) for tigray highland sheep (27.52 kg), and melaku et al (2019) for simien sheep (24.90 kg) which show their difference from the sheep populations of the neighboring areas. although the body weight of rams and ewes presented in this study were higher than those reported for genetic resources (2020), 1 (2), 12–22 characterization of sheep in northern ethiopia 20 figure 7. highland sheep flock in tahtay maichew district. abergelle and tigray highland sheep (tajebe et al, 2011; gebreyowhens and tesfay, 2016), other traits such as body length, height at withers, heart girth and tail length were comparable between these sheep populations. on the other hand, higher values for all morphological measurements were reported by edea et al (2010) for the country’s most known sheep breeds (bonga and horro sheep). the results also revealed that linear body measurements among the studied sheep population differ with age. three-year-old sheep showed the highest values for most of the measurements, reflecting the optimum growth age. these results are in contrast with results of getachew et al (2009) for menz and afar sheep and melaku et al (2019) for simien sheep who reported that the body weight of the sheep continued to increase with age. in addition to the quantitative measurements, the qualitative characteristics of a population also allow to easily differentiate genetic resources. among the obvious qualitative characteristics which differentiate the current sheep populations from the previously characterized sheep populations are the complete absence of beard, horn, ruff, toggle and pigmented skin. similarly, variations in coat color were also observed among the different agro-ecological zones. accordingly, the majority of the highland and midland sheep populations from the current study possess tan coat color which makes them unique among the other tigray highland sheep populations. sheep populations sampled from the lowland area display dominantly white and light colors which is in agreement with the report of getachew et al (2009) for the lowland afar sheep. the majority of the sheep populations from the current study possessed hairy fiber type coats, which was in contrast to the results of hayelom et al (2014), who report course wool for tigray highland sheep. however, huge variations were not observed among the other qualitative characteristics of the sheep types from the current study and earlier studies of gebreyowhens and tesfay (2016) and hayelom et al (2014) for tigray highland sheep. discriminant function analysis allowed the classification of an average 66% of the studied animals into their respective environments zone. lowest classification of individuals into their respective agro-ecological zone was observed in midland sheep populations, indicating a lack of uniqueness within them. all of the pairwise comparisons between populations from different agro-ecological zones were found to be highly significant with the largest difference observed between the highland and lowland sheep populations. these results reflect the large altitudinal differences between the two agro-ecological zones. the shortest distance calculated 21 hailu et al genetic resources (2020), 1 (2), 12–22 between the lowland and midland sheep populations show their relative similarities as compared with the highland sheep. these differences among different agroecological zones show the presence of potential genetic resource variations which can be useful for maintaining diversity and further selection-based genetic improvement programs. in conclusion, using a combination of quantitative and qualitative characteristics we were able to discriminate the sheep populations from three agro-ecological zones in the tahtay maichew district, tigray region of ethiopia and to group them into two distinct populations (the highland and the lowland sheep). sheep populations from the midland agro-ecological zone were considered to be part of the lowland group. therefore, it is better to consider the highland and lowland sheep as different traditional populations until molecular characterization results provide further evidence for population differentiation. additionally, the molecular characterization studies will show the within population genetic diversity and level of inbreeding which can be used for selecting appropriate genetic improvement plans (selection or crossbreeding). according to the reports of gizaw et al (2008), the sheep genetic resources of most parts of the tigray region were generally referred as sekota sheep population under the short fat tailed breed. however, results from our study indicate that there are several sheep populations that cannot be categorized under the sekota traditional sheep population. therefore, it is advisable to include these genetic resources for further molecular studies to understand the genetic diversity within and among populations. acknowledgements the authors are highly indebted to the ethiopian biodiversity institute (ebi) for covering all the budget needs of the work. our special appreciation also goes to the smallholder farmers /breeders for providing their animal to this work for free. we also take this opportunity to thank the animal science experts and development agents in the district for their endless help during the data collection. a special word also goes to our friend and work partner mr. tadesse hunduma for mapping the study area. author contributions all authors contributed to the study conception and design. material preparation, and data collection were performed by ah, ta, ss and st. data analysis and writing the first draft of the manuscript was performed by am and ah. aa commented on previous versions of the manuscript. all authors read and approved the final manuscript. conflict of interest statement the authors declare no conflicts of interest. references atsbeha, g., demissew, s., woldu, z., and edwards, s. 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(2018). morphometrical characterization and traditional breeding objectives of native sheep reared in east shoa and west arsi zone of oromia regional state. m.sc. thesis, hawassa university, hawassa, ethiopia. https://dx.doi.org/10.1017/s1014233900002704 10.1017/s1014233900002704 https://www.iiste.org/journals/index.php/jbah/article/view/15182 https://www.iiste.org/journals/index.php/jbah/article/view/15182 http://www.ijagbio.com/volume-8-no-4-2019/ http://www.ijagbio.com/volume-8-no-4-2019/ http://www.lrrd.org/lrrd22/9/taye22169.htm http://www.lrrd.org/lrrd22/9/taye22169.htm https://iiste.org/journals/index.php/jnsr/article/view/32220/33107 https://iiste.org/journals/index.php/jnsr/article/view/32220/33107 https://doi.org/10.1016/s0921-4488(02)00187-6 https://doi.org/10.1016/s0921-4488(02)00187-6 http://www.lrrd.org/lrrd31/6/amine31089.html http://www.lrrd.org/lrrd31/6/amine31089.html https://www.sas.com/en_us/software/stat.html https://hdl.handle.net/10568/70955 introduction materials and methods study areas site selection and data collection statistical analysis results quantitative measurements qualitative characteristics multivariate analysis for discrimination of sheep populations discussion author contributions conflict of interest statement original article genetic resources (2021), 2 (4), 44–54 doi: 10.46265/genresj.ugud8568 https://www.genresj.org issn: 2708-3764 teak genetic diversity in ghana shows a narrow base for further breeding and a need for improved international collaboration for provenance exchange tieme h v wanders *,a, james n ofori b, alexander amoako c, maarten postuma b, cornelis a m wagemakerd, elmar m veenendaal b and philippine vergeer b,d a form international, hattem, the netherlands b plant ecology and nature conservation group, wageningen university, wageningen, the netherlands c form ghana, sunyani, ghana d department of experimental plant ecology, radboud university, nijmegen, the netherlands abstract: we evaluated the genetic diversity of teak (tectona grandis l.) provenances at a newly established provenance trial with 52 provenances collected from africa, south america and asia in tain ii forest reserve in central ghana. this provenance trial was established to widen the genetic basis for teak establishment in west africa. using genotyping by sequencing (gbs) we analysed the genetic diversity of these provenances. results of the study revealed that, although acquired from a wide geographical range, most teak provenances in the trial belong to only two distinct groups that are closely related. the implication of this finding is that, for breeding, a wider range of provenances is needed from the original teak distribution areas, and more specifically from southern india. we conclude that urgent protection of older existing sources of genetic variation in teak, as well as an improvement of international collaboration under the nagoya protocol with countries with native teak populations, is necessary. keywords: teak, tectona grandis l, provenance trial, ghana, gbs citation: wanders, t. h. v., ofori, j. n., amoako, a., postuma, m., wagemaker, c. a. m., veenendaal, e. m., vergeer, p. (2021). teak genetic diversity in ghana shows a narrow base for further breeding and a need for improved international collaboration for provenance exchange. genetic resources 2 (4), 44–54. doi: 10.46265/genresj.ugud8568. © copyright 2021 the authors. this is an open access article distributed under the terms of the creative commons attribution license (cc by 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. introduction teak is a high-quality timber species of great importance in plantation establishment throughout the tropics. teak (tectona grandis l.) is naturally distributed in myanmar, india, laos and thailand (white, 1991) but can now be found in about 36 countries in tropical asia, south america and africa (koskela et al, 2014). a record area of about 5.7 million ha of teak has been planted (bhat and hwan, 2004; nair, 2007), underlining the economic importance of teak for tropical forestry. teak has reached ∗corresponding author: tieme h v wanders (t.wanders@forminternational.nl) such a large plantation area because it is easy to cultivate in a wide climatic amplitude (orwa et al, 2009), grows fast and produces valuable timber. growth rates of 15 to 20 m3/ha/year are not uncommon on good sites. (ugalde-arias, 2013; jerez and coutinho, 2017) and the value of the timber can reach up to 800 usd/m3 c/f (itto, 2020). except for the establishment of provenance trials in the 1970s, little effort seems to have gone into the improvement of genetic diversity in the countries where teak is widely planted. in fact, the trend seems to point to a narrowing of the genetic basis due to the wide use of the so called “solomon island clones”, which indeed provide superior growth and received: 22.01.2021 accepted: 06.10.2021 published online: 10.11.2021 https://www.genresj.org https://www.dx.doi.org/10.46265/genresj.ugud8568 https://www.genresj.org https://www.dx.doi.org/10.46265/genresj.ugud8568 mailto:t.wanders@forminternational.nl genetic resources (2021), 2 (4), 44–54 analysis of the genetics and origin of teak in ghana 45 shape of the trees under many circumstances in various countries (chaix et al, 2011; goh and monteuuis, 2012; ugalde-arias, 2013). although such clones contribute to the present success and financial attractiveness of teak planting, tree planters should heed their genetic diversity. to help mitigate against the effects of climate change, the appearance of new diseases, and to allow for improvement of other qualities such as growth speed and heartwood formation, genetic diversity is key and should receive more attention (graudal and moestrup, 2017). it is important to initiate and support selection and testing of superior individuals in local breeding programmes because many traits such as bole straightness, proportion of heartwood and fine branching, which are important for commercial production of teak, have a genetic background (kjær et al, 1996, 1999; fofana et al, 2008), but the phenotypic manifestation of traits is not the same in each locality. in ghana, teak is the prime plantation species with well over 150,000 hectares planted since 2002 (fsdfc ghana, 2017). at present, for the development of teak plantations in ghana, a very limited number of seed sources are available (wanders, 2014; fsd-fc ghana, 2017). most of these seed sources are ‘unproven’, which means that the stands have been identified as seed stands, but progeny trials are not available to support the selection of these stands for this purpose. the material from these stands is now systematically evaluated at the tain ii forest reserve provenance trial at form ghana ltd. the stands that are currently used as seed sources were mostly planted in the 1980s and 1990s, many of them with material from kihuhwi in tanzania (fsdfc ghana, 2017). other sources of planting material are what remains of an international teak provenance trialing effort containing 13 provenances from india, laos, indonesia and ghana planted between 1972 and 1975 by the danish development cooperation danida and the forest research institute of ghana forig (keiding et al, 1986). a clonal seed orchard has been developed based on material from this trial by forig at jimira in ghana. from these sources, plantation developers presently obtain seeds for plantation establishment. some also resort to importation of seed from other countries. recognising the need for a wider genetic pool to source seeds, form ghana ltd started a new provenance trial in 2015 in which, over several years, 52 accessions of teak coming from ghana, tanzania, côte d’ivoire, malaysia, brazil, costa rica, honduras, and indirectly from india and indonesia, were planted (wanders, 2020). while these provenances represent a global distribution of teak, there is great uncertainty on their genetic kinship. in this study, we investigated the genetic diversity of teak provenances presently grown for testing in ghana. we evaluated how closely related the provenances are and whether the aim of a wider genetic base for the teak industry in west africa can be achieved under the present conditions and with the material currently accessible. materials and methods trial location the trial is located in block a42 in the tain ii forest reserve (figure 1). the coordinates of the location are 7◦37’53.78”n and 2◦38’26.31”w. the layout of the trial is a block design with blocks of 49 trees per provenance. for most provenances there is at least one replicate, but some have several replicates. planting started in 2015 and new material has been added annually, while also adding already present material to make comparisons within and between the years possible. in 2020 the trial covered 12 hectares. genetic sampling and sample library preparation leaf samples (one sample per provenance) were collected at form ghana’s tain ii provenance trial and at form ghana’s nursery in may 2019. leaf samples were immediately dried with silica gel and stored for further processing. a total of 41 trees of 37 different accessions were sampled. genomic dna extraction was done using a nucleospin 96 plant ii kit from bioké, following the manufacturer’s instructions. genetic variation was measured using genotyping by sequencing (gbs) (elshire et al, 2011). first, 88 to 278 ng of genomic dna (gdna) of each of 41 samples was digested by two restriction enzymes (asei and nsii) after which, two indexed adapters were ligated to the dna fragments. the main change in the adapter design was the incorporation of three random unique molecule identifier (umi) nucleotides per adapter for the identification of pcr duplicates within each amplified gbs library. after ligation, individual samples were cleaned by two subsequent nucleo-mag (macherey-nagel, germany) cleanup steps of 1x and 0.8x beads. a small volume test pcr (15 cycles) was performed using kapa hifi hotstart readymix (roche diagnostics, switzerland). the resulting product was diluted 10,000 x prior to qpcr quantification (kapa library quantification kit for hts, roche diagnostics, switzerland). the result of the qpcr was subsequently used to equimolarly pool the original cleaned digestion/ligation products. this pooled product was concentrated using a column-based nucleomag pcr clean-up (macherey-nagel, germany) and nick repaired using dna polymerase i (50 µl reaction). the nick repaired product was amplified in five reactions of 10 µl each and cleaned by two subsequent nucleomag (macherey-nagel, germany) clean-up steps using 1x and 0.8x beads, respectively. the average library size was 1,177 bp. the final gbs library was quantified by qpcr, pooled with other libraries and spiked with 10% phix prior to sequencing. this increases the dna complexity of the library in order to improve the hiseq colour matrix estimation for which the first 11 sequencing cycles are 46 wanders et al genetic resources (2021), 2 (4), 44–54 figure 1. map showing the location of the provenance trial in ghana, and the various planting years sampled. used overlapping with our index region. sequencing was performed by novogene (hong kong) on an illumina hiseq x-ten sequencer, producing 2x150bp paired-end (pe) sequencing reads. in total 0.4 sequence lane was devoted to the 41 teak gbs libraries, providing a total of 232,995,422 raw reads. data analysis demultiplexing, de novo reference construction, mapping and snp calling of the dna sequences were conducted using stacks version 2.4 (catchen et al, 2013). pcr duplicates were removed using clone filter based on the umi nucleotides, followed by demultiplexing using process radtags. to identify snps from the reads we used the “denovo map.pl” script using -m 3 -m 5 -n 5 based on exploratory runs using a range of values (m 2-6, -m 3-7 and n=m) to maximise the quality of snps for this dataset (paris et al, 2017). after mapping, data was filtered using vcf tools (danecek et al, 2011). the applied filter first removed all loci which were not present in more than 50% of individuals, had a genotype quality below 30 or had a mean depth lower than six. after this, individuals with more than 80% missing data were removed. all snps, which were not present in all individuals and had an individual sample depth less than 10, were removed. four duplicate datasets were removed from the analysis, resulting in a total of 37 samples. we used structure (pritchard et al, 2000) on 1000 randomly selected snps to assess patterns of genetic structure in the samples, with a number of assumed populations (k) of 1-7, with 10 replicates per k. we used 1,000,000 burn-in and 500,000 reps. afterwards, the output data was analysed using structure selector (li and liu, 2018). clustering was done using the adgenet package (jombart and ahmed, 2011) in r version 3.5.3 (r core team, 2019). genetic distance was calculated using the r-package adegenet, using dist(method=”euclidian”). principal component analysis was done with the function dudi.pca from the rpackage ade4. all scripts used in this analysis are available at https://github.com/maartenpostuma/teak-anal ysis. demultiplexed reads are available under bioproject prjna756980 at ncbi (https://www.ncbi.nlm.nih.gov /bioproject/prjna756980). https://www.ncbi.nlm.nih.gov/bioproject/prjna756980 https://www.ncbi.nlm.nih.gov/bioproject/prjna756980 https://github.com/maartenpostuma/teak-analysis https://github.com/maartenpostuma/teak-analysis genetic resources (2021), 2 (4), 44–54 analysis of the genetics and origin of teak in ghana 47 table 1. analysed provenances, id and their origin in this study. sample no. id / provenance country country expected origin literature describing the source 1 brazil 700 brazil imported clones through proteca solomon islands (goh and monteuuis, 2009; monteuuis and goh, 2015)2 brazil 701 brazil imported clones through proteca solomon islands 3 bouaké (64) côte d’ivoire bouaké côte d’ivoire (wanders (2014) 4 bamoro côte d’ivoire bamoro seed stand sodefor côte d’ivoire (dupuy and verhaegen, 1993; kadio, 2006; kokou, 2010) 5 bangsri pati ghana danida provenance trial in nsoatre indonesia (keiding et al, 1986; kjær et al, 1995) 6 brazil t brazil mato grosso brazil 8 catie costa rica 278 peñas blancas costa rica 9 dunkwa ghana oppon mansi forest reserve tanzania (wanders, 2014) 10 brazil f brazil mato grosso brazil 11 goaso ghana bia tano forest reserve tanzania (wanders, 2014) 12 honduras honduras la cumbre seed stand honduras 13 jimira ghana jimira forest reserve mixed from nsoatre pt in ghana (wanders, 2014) 14 kihuhwi p68 tanzania provenance trial in longuza tanzania (madoffe and maghembe, 1988) 15 kihuwi tanzania kihuhwi forest reserve tanzania 16 kiroka tanzania kiroka seed stand tanzania 17 kvtc tanzania imported from kvtc seed stand tanzania 18 la téné côte d’ivoire la téné seed stand, sodefor côte d’ivoire (but based on pt, many provenances possible) (dupuy and verhaegen, 1993; kadio, 2006; kokou, 2010) 19 longuza tanzania imported from sfi tanzania (madoffe and maghembe, 1988) 20 luasong malaysia luasong, tawau, sabah malaysia goh and monteuuis (2009); monteuuis and goh (2015) 21 miro 1 costa rica catie 194/16625 costa rica 22 miro 2 costa rica catie 212/09b11 costa rica 23 miro 3 costa rica catie 221/45c12 costa rica 24 mtibwa p61 tanzania provenance trial in longuza tanzania (madoffe and maghembe, 1988) 25 mtibwa tanzania mtibwa seed stand tanzania 26 nigeria p67 tanzania provenance trial in longuza tanzania continued on next page 48 wanders et al genetic resources (2021), 2 (4), 44–54 table 1 continued sample no. id / provenance country country expected origin literature describing the source 27 nilambur ghana danida provenance trial in nsoatre kerala, india (keiding et al, 1986; kjær et al, 1995) 28 north india north india north india north india 29 oumé côte d’ivoire oumé côte d’ivoire 30 pampawie ghana pampawie forest reserve ghana (wanders, 2014) 31 perlis malaysia perlis seed stand, taliwas, lahad datu, sabah malaysia (goh and monteuuis, 2009; monteuuis and goh, 2015) 32 sangoué côte d’ivoire sangoué seed stand, sodefor côte d’ivoire (dupuy and verhaegen, 1993; kadio, 2006; kokou, 2010) 33 t1 ghana danida provenance trial in nsoatre not traceable (likely nilambur) (keiding et al, 1986; kjær et al, 1995) 34 t4 ghana danida provenance trial in nsoatre not traceable (likely bangsri pati) (keiding et al, 1986; kjær et al, 1995) 35 taliwas malaysia taliwas, lahad datu, sabah malaysia (goh and monteuuis, 2009; chaix et al, 2011; monteuuis and goh, 2015) 36 temandsang ghana danida provenance trial in nsoatre indonesia (keiding et al, 1986; kjær et al, 1995) 37 topslip ghana provenance trial in longuza cöımbatore, india (madoffe and maghembe, 1988) 38 worawora ghana wora wora forest reserve ghana (wanders, 2014) genetic resources (2021), 2 (4), 44–54 analysis of the genetics and origin of teak in ghana 49 results a total of 23,182 snps were obtained after filtering, which ensured the accuracy and reliability of subsequent genetic diversity and population structure analyses. the optimal value of k was determined by evanno’s delta k method (evanno et al, 2005). two clearly defined main clusters (k=2; figure 2) and a maximum of 4 clusters (k=4; figure 2) were revealed. the most likely number of clusters was k=2. these two main clusters are at a great genetic distance from each other and are clustered primarily according to geographical region: one cluster consisted of teak stands originating from asia and the pacific and the other cluster originating from south america and africa. in addition, k=3 and k=4 showed some variation within the two main clusters. when k=3, accessions originating from côte d’ivoire and malaysia for example could be distinguished and at k=4 accessions from the solomon islands separated from the other accessions from the asia-pacific cluster. the list of provenances, id and their collected and expected origin (as inferred from the genetic analysis) is shown in table 1. ‘origin of collection’ in this table means how form ghana obtained the material and the ‘expected origin’ in the table refers to the origin to which the material can be traced in the literature. further analysis of the two main clusters clearly showed more genetic similarities within the south america–africa cluster indicating less genetic variability as compared to the asia–pacific cluster, which had less genetic similarities indicating more variation in genotype especially in the asian cluster. this was also illustrated by a principal component analysis based on genetic distance (figure 3) and the number of polymorphic sites (snps) within the two clusters. in the asia–pacific cluster, 94% of snps were polymorhpic compared to 74% of polymorphic snps in the south america–africa cluster, even though the latter had more individuals. in addition, only 1364 private alleles were found in the south america–africa cluster as compared to 6201 private alleles in the asia–pacific cluster. mean euclidean-based genetic distance was calculated as 87.25 ± 19.35 within the south america–africa cluster (green + pink), 117.19 ± 43.9 within the asia–pacific cluster (yellow + red), and 161.35 ± 32.35 between these two main clusters (figure 2). these data showed substiantally higher levels of genetic variation in the asia–pacific cluster and suggest low levels of genetic variation in the south america–africa cluster. discussion the results in figure 2 show two main clusters of genetic variation for the 37 teak provenances sampled in the tain ii forest reserve. the first cluster mainly consists of teak stands from africa and south america and the second of provenances from asia and the pacific. data on the number of polymorphic sites in the two clusters and genetic distance within and between the clusters indicate less genetic variation between provenances in the africa-south america cluster and a high genetic variation between provenances within the asia-pacific cluster and especially in the asia cluster. grouping the material shows that material from indonesia and africa is closely related which confirms the conclusions of verhaegen et al (2010) that teak from ghana and indonesia could be originating from laos while teak from other african places can be traced back to north india (fofana et al, 2008). together, they form a group that is different from the thai and south india provenance groups. in this study we can now add the south american provenances to the latter group. the attribution of the indonesian provenances to laos was also found by hansen et al (2017), who unfortunately did not sample from ghana. the grouping of material from malaysia and india in one group can be explained by the collection of indian provenances in provenance trials in côte d’ivoire for the establishment of the malaysian plots (goh and monteuuis, 2009). the link between the material from the solomon islands and india should not be surprising as the solomon islands have no indigenous teak population and their population was built up from foreign material which mostly came from india (raomae, 2012). most teak provenances within the africa-south america cluster showed less genetic variation in this study which confirms that african teak provenances most likely originate from a limited range in north india and none of the african provenances are from south india (verhaegen et al, 2010). some uncertainty on the exact origin of provenances in our study remains. attribution to a certain origin as indicated by the structure analysis was based on the genetic relatedness of samples from single trees representing each provenance. based on this, provenances that were genetically more related were then assigned to the same cluster. however, some provenances originated from mixed clonal seed orchards (jimira, kiroka, sangoué and la téné) as presented in table 1. the seed obtained from such seed orchards is potentially more diverse and sampling may have covered only part of the locally present diversity. as a consequence, more sampling in the same population of seedlings from such orchards could potentially also identify genetic material from the other cluster. despite the uncertainty of the origin of some provenances, the results show that although imports were made from very different areas in the tropics, the achieved gain in genetic diversity is very limited and reflects that, over time, teak provenances from a limited number of sources have spread over a wide area (fofana et al, 2008). this also means that at present, new imports of teak seeds into e.g. ghana, mostly do not constitute a new genetic accession added to the gene pool. before going through the process of obtaining permits and importing seeds from a presumed new accession, it is important to compare its genetic makeup with the existing provenances. it is also important to further investigate the current collection of provenances 50 wanders et al genetic resources (2021), 2 (4), 44–54 figure 2. clustering of teak stands of different provenances. shown is a dendrogram based on genetic distance (right part) and the different clusters as identified by the structure analysis (green, cluster 1; yellow, cluster 2; pink, cluster 3 and red, cluster 4). the dendrogram was generated by hierarchical clustering (upgma) based on genetic dissimilarity. vertical lines in the dendrogram give the amount of genetic dissimilarity and represent genetic lineages. each row represents an individual tree per provenance, with the length of the different colour segments representing the proportion of a cluster in an individual’s genetic makeup. k = 2–4 indicates the number of genetic clusters that were revealed in this structure analysis from 2 to 4. the most likely number of clusters was k=2. so that the search for additional genetic material for teak provenance pools in west africa can be conducted with more focus. our findings emphasise the need for acquiring teak provenances from areas of its original distribution that are high in genetic diversity and are not in the present provenance trial, one such area being south-west india (hansen et al, 2017) and the semi-moist east coast of india (hansen et al, 2015). the analysis of vaishnav and ansari (2018) indicates that genetic resources in india may be a source for screening resilient superior provenances for improvement strategies for sustainable production of quality timber on a large scale. various examples exist for the benefit of matching specific provenances to specific local conditions. indigenous teak populations from annamalai hills in the indian states of kerala and tamil nadu contain well performing provenances for tanzania (madoffe and maghembe, 1988; pedersen et al, 2007), while a nilambur provenance from india and a savannahket provenance from laos have been assessed as very suitable for ghana (adu-bredu et al, 2019). currently it is difficult to obtain accessions from some of the countries containing the high diversity areas, as they have banned the export of seeds and sometimes also of clones of their genetic heritage. genetic resources (2021), 2 (4), 44–54 analysis of the genetics and origin of teak in ghana 51 figure 3. principal components analysis on genetic distance of the different teak accessions. the different accessions are indicated by numbers, the two main clusters as identified by structure analysis by different colours. it is, for instance, impossible to import seeds from india (government of india , 2002). this makes it all the more urgent to get a full view of the genetic make-up of trees planted in old (pre-nagoya protocol) provenance trials such as the series of international provenance trials planted in the 1970s (keiding et al, 1986). more and more of these trials are lost to felling, e.g. recently longuza provenance trial in tanzania (wanders, personal observation) and to disaster as is the case of st. croix in puerto rico, which was part of the series of international provenance trials set up by danida and was destroyed by hurricanes (morgan, personal communication, 2016). the original series of international provenance trials by danida contained 75 provenances which were under test on over 50 locations, with 41 original teak provenances originating from the natural range of teak (keiding et al, 1986). these trials potentially remain a very important source of genetic variety for any breeding programme (koskela et al, 2014; adu-bredu et al, 2019) and their conservation should be a high priority. as the climate is changing and forestry is to adapt to the climate, becoming either wetter or drier, the need to access a wider range of genetic material may become more and more important in tropical forestry (koskela et al, 2014). at present, the nagoya protocol on access to genetic resources and benefit sharing (abs) (cbd, 2011) could govern the sharing of benefits resulting from exchanges of genetic material in a more structured and mutually beneficial manner. it is not yet clear if the signing of the nagoya protocol will make it possible to again obtain seeds from countries having interesting genetic resources, but prohibiting export of seeds and other propagation materials. documents that need to be elaborated per seedlot, such as the prior informed consent and benefit sharing agreement, create barriers that need urgent addressing at supranational level. koskela et al (2014) provide an insight into the amount of paperwork necessary in order to plant a provenance trial, which is another argument to carefully conserve pre-nagoya planted trials and exchange genetic material from these. the amount of work going into the drafting and signing of abs and mutually agreed terms (mat) may make it worthwhile to engage in only for commercially high returning crops. in international forestry, non-profit initiatives to exchange seeds exist. one of these, camcore (https:/ /camcore.cnr.ncsu.edu/) has done excellent work on the collection and distribution of seeds for broad testing of pinus and eucalyptus species. camcore has organised expeditions for the collection of seeds of species interesting for forestry and tree breeding and distributed these seeds to be planted in trials at member organisations and companies. camcore has recently also started work on teak (hodge et al, 2019). the cost of the membership fee for this organisation, however, is not accessible to all organisations involved in plantation development. more exchange would certainly improve the possibilities of increasing the gene pool for teak breeding. conclusion as our work has shown, it can be difficult to have access to diverse genetic materials. with the uncertainties about the long-term fitness of currently available genetic material under climate change and possible disease vulnerability, having access to genetic diversity is becoming increasingly important. because not all genetic diversity has a direct commercial interest, the creation and maintenance of a national gene bank or national collection (nccpg, 2007; fao, 2014) should be a national priority. conservation of teak genetics in thailand has been described by kaosa-ard et al (1998) and graudal et al (1999). in india genebanks have also been created such as the national teak germplasm bank in chandrapur (maharashtra) whose genetic diversity has been analysed (mahesh et al, 2016). lack of formal protection of tree genetic resources can cause genetic material to be lost unnoticed. through cooperation between the countries that took part in past international provenance trials on teak, each participant country could, through exchanges, build up a collection of most, if not all, accessions of teak originally distributed. this should be done in addition to addressing of international barriers for exchange of genetic material of teak from its original range mentioned earlier. a national teak genebank collection for ghana (and other african teak producing countries) would then become an excellent centre for the distribution and conservation of genetic material. the facility managing such a collection should become a member of camcore or a similar organisation to further facilitate exchange. acknowledgements we would like to thank form ghana ltd and, more specifically, mr willem fourie, for the possibility to sample their provenance trial and for the financial https://camcore.cnr.ncsu.edu/ https://camcore.cnr.ncsu.edu/ 52 wanders et al genetic resources (2021), 2 (4), 44–54 contribution to the analysis of the findings. this study also received co-funding from wageningen university. author contributions tieme wanders, philippine vergeer, james ofori and elmar veenendaal conceived and planned the experiments; james ofori, alexander amoako and tieme wanders collected data; philippine vergeer, niels wagemaker, maarten postuma, james ofori, tieme wanders and elmars veenendaal contributed to analysis and interpretation of the results; tieme wanders took the lead in writing the manuscript. all authors provided critical feedback and helped shape the research, analysis and manuscript. conflict of interest statement the authors declare no conflicts of interest. references 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(1991). teak: some aspects of research and development. rapa publication, bangkok, 70p. https://doi.org/10.1515/sg-2007-0022 https://doi.org/10.1515/sg-2007-0022 https://doi.org/10.1093/genetics/155.2.945 https://doi.org/10.1093/genetics/155.2.945 https://www.r-project.org/ http://www.fao.org/3/i3825e/i3825e60.pdf http://www.fao.org/3/i3825e/i3825e60.pdf https://doi.org/10.1007/s11105-018-1101-3 https://doi.org/10.1007/s11105-018-1101-3 https://doi.org/10.1007/s11295-010-0286-x https://doi.org/10.1007/s11295-010-0286-x introduction materials and methods trial location genetic sampling and sample library preparation data analysis results discussion conclusion author contributions conflict of interest statement supplemental data for nduche m., magos brehm j., abberton m., omosun g. and maxted n. (2021) west african crop wild relative checklist, prioritization and inventory. genetic resources 2(4) 55-65. doi: 10.46265/genresj.eifl1323 supplemental table s1 related crop and concept level of priority cwr for west africa s/n family genus taxon related crop common crop name concept type concept level 1 convolvulaceae ipomoea ipomoea acanthocarpa (choisy) hochst. ex schweinf. & asch. ipomoea batatas (l.) lam. sweet potato taxon group 4 2 convolvulaceae ipomoea ipomoea alba l. ipomoea batatas (l.) lam. sweet potato taxon group 4 3 convolvulaceae ipomoea ipomoea aquatica forssk. ipomoea batatas (l.) lam. sweet potato taxon group 4 4 convolvulaceae ipomoea ipomoea argentaurata hallier f. ipomoea batatas (l.) lam. sweet potato taxon group 4 5 convolvulaceae ipomoea ipomoea asarifolia (desr.) roem. & schult. ipomoea batatas (l.) lam. sweet potato taxon group 4 6 convolvulaceae ipomoea ipomoea barteri baker ipomoea batatas (l.) lam. sweet potato taxon group 4 7 convolvulaceae ipomoea ipomoea blepharophylla hallier f. ipomoea batatas (l.) lam. sweet potato taxon group 4 8 convolvulaceae ipomoea ipomoea chrysochaetia hallier f. ipomoea batatas (l.) lam. sweet potato taxon group 4 9 convolvulaceae ipomoea ipomoea coptica (l.) roth ex roem. & schult. ipomoea batatas (l.) lam. sweet potato taxon group 4 10 convolvulaceae ipomoea ipomoea intrapilosa rose ipomoea batatas (l.) lam. sweet potato taxon group 4 11 convolvulaceae ipomoea ipomoea ochracea (lindl.) sweet ipomoea batatas (l.) lam. sweet potato taxon group 4 12 convolvulaceae ipomoea ipomoea prismatosyphon welw. ipomoea batatas (l.) lam. sweet potato taxon group 4 13 convolvulaceae ipomoea ipomoea rubens choisy ipomoea batatas (l.) lam. sweet potato taxon group 4 14 dioscoreaceae dioscorea dioscorea abyssinica hochst. ex kunth dioscorea rotundata poir. white yam gene pool 2 15 dioscoreaceae dioscorea dioscorea baya de wild. dioscorea rotundata poir. white yam gene pool 3 16 dioscoreaceae dioscorea dioscorea burkilliana j.miège dioscorea rotundata poir. white yam gene pool 3 17 dioscoreaceae dioscorea dioscorea minutiflora engl. dioscorea rotundata poir. white yam gene pool 2 18 dioscoreaceae dioscorea dioscorea praehensilis benth. dioscorea rotundata poir. white yam gene pool 2 19 dioscoreaceae dioscorea dioscorea quartiniana a.rich. dioscorea rotundata poir. white yam taxon group 4 20 dioscoreaceae dioscorea dioscorea sagittifolia pax dioscorea rotundata poir. white yam taxon group 4 21 dioscoreaceae dioscorea dioscorea sagittifolia var. lecardii (de wild.) nkounkou dioscorea rotundata poir. white yam gene pool 2 22 dioscoreaceae dioscorea dioscorea sansibarensis pax dioscorea rotundata poir. white yam taxon group 4 23 dioscoreaceae dioscorea dioscorea schimperiana hochst. ex kunth dioscorea rotundata poir. white yam taxon group 4 24 dioscoreaceae dioscorea dioscorea smilacifolia de wild. & t.durand dioscorea rotundata poir. white yam gene pool 2 25 dioscoreaceae dioscorea dioscorea togoensis r.knuth dioscorea rotundata poir. white yam gene pool 2 26 euphorbiaceae manihot manihot esculenta subsp. peruviana crantz manihot esculenta crantz cassava gene pool 3 27 malvaceae gossypium gossypium anomalum wawra gossypium hirsutum linn. upland cotton gene pool 3 28 malvaceae gossypium gossypium barbadense linn. gossypium hirsutum linn. upland cotton gene pool 1b 29 malvaceae gossypium gossypium herbaceum var. acerifolium (guill. & perr.) a. chev. gossypium hirsutum linn. upland cotton gene pool 3 30 papilionaceae phaseolus phaseolus lunatus linn. phaseolus vulgaris linn. common bean taxon group 4 31 papilionaceae phaseolus phaseolus vulgaris subsp. abarigineus l. phaseolus vulgaris linn. common bean gene pool 1b 32 papilionaceae vigna vigna ambacensis welw. ex bak. vigna unguiculata (linn.) walp. cowpea taxon group 4 33 papilionaceae vigna vigna desmodioides wilczek vigna unguiculata (linn.) walp. cowpea taxon group 4 34 papilionaceae vigna vigna filicaulis hepper vigna unguiculata (linn.) walp. cowpea taxon group 4 35 papilionaceae vigna vigna gracilis (guill. & perr.) hook. f. vigna unguiculata (linn.) walp. cowpea taxon group 4 https://doi.org/10.46265/genresj.eifl1323 s/n family genus taxon related crop common crop name concept type concept level 36 papilionaceae vigna vigna luteola (jacq.) benth. vigna unguiculata (linn.) walp. cowpea taxon group 4 37 papilionaceae vigna vigna macrorrhyncha (harms) milne-redhead vigna unguiculata (linn.) walp. cowpea taxon group 4 38 papilionaceae vigna vigna marina (burm.) merill vigna unguiculata (linn.) walp. cowpea taxon group 4 39 papilionaceae vigna vigna multinervis hutch. & dalz. vigna unguiculata (linn.) walp. cowpea taxon group 4 40 papilionaceae vigna vigna nigritia hook. f. vigna unguiculata (linn.) walp. cowpea taxon group 4 41 papilionaceae vigna vigna oblongifolia a. rich. vigna unguiculata (linn.) walp. cowpea taxon group 4 42 papilionaceae vigna vigna racemosa (g. don) hutch. & dalz. vigna unguiculata (linn.) walp. cowpea taxon group 4 43 papilionaceae vigna vigna reticulata hook. f. vigna unguiculata (linn.) walp. cowpea taxon group 4 44 poaceae echinochloa echinochloa colonum (l.) link echinochloa esculenta (a. braun) h. scholz barnyard millet taxon group 4 45 poaceae echinochloa echinochloa crus-galli (l.) p.beauv. echinochloa esculenta (a. braun) h. scholz barnyard millet gene pool 1b 46 poaceae echinochloa echinochloa crus-pavonis (kunth) schult echinochloa esculenta (a. braun) h. scholz barnyard millet taxon group 4 47 poaceae echinochloa echinochloa frumentacea link echinochloa esculenta (a. braun) h. scholz barnyard millet gene pool 2 48 poaceae echinochloa echinochloa pyramidalis (lam.) hitchc. & chase echinochloa esculenta (a. braun) h. scholz barnyard millet taxon group 4 49 poaceae eleusine eleusine africana kenn. -o'byrne eleusine coracana (l.) gaertn. finger millet gene pool 3 50 poaceae eleusine eleusine indica (l.) gaertn. eleusine coracana (l.) gaertn. finger millet gene pool 2 51 poaceae eragrostis eragrostis japonica (thunb.) trin. eragrostis tef (zuccagni) trotter teff taxon group 4 52 poaceae eragrostis eragrostis prolifera (sw.) steud. eragrostis tef (zuccagni) trotter teff taxon group 4 53 poaceae eragrostis eragrostis unioloides (retz.) nees ex steud. eragrostis tef (zuccagni) trotter teff taxon group 4 54 poaceae hordeum hordeum bulbosum l. hordeum vulgare l. barley gene pool 2 55 poaceae oryza oryza barthii a.chev. oryza sativa l. rice gene pool 1b 56 poaceae oryza oryza brachyantha a.chev. & roehr. oryza sativa l. rice gene pool 3 57 poaceae oryza oryza eichingeri peter oryza sativa l. rice gene pool 2 58 poaceae oryza oryza glaberrima steud. oryza sativa l. rice gene pool 1b 59 poaceae oryza oryza longistaminata a.chev. & roehr. oryza sativa l. rice gene pool 1b 60 poaceae oryza oryza punctata kotschy ex steud. oryza sativa l. rice gene pool 2 61 poaceae panicum panicum comorense mez panicum miliaceum l. proso millet taxon group 4 62 poaceae panicum panicum repens l. panicum miliaceum l. proso millet gene pool 3 63 poaceae saccharum saccharum spontaneum subsp. spontaneum l. saccharum officinarum l. sugarcane gene pool 3 64 poaceae saccharum saccharum spontaneum subsp. aegyptiacum (willd.) hack. saccharum officinarum l. sugarcane gene pool 2 65 poaceae sorghum sorghum purpureosericeum (hochst. ex a.rich.) schweinf. & asch sorghum bicolor (l.) moench sorghum gene pool 3 66 poaceae sorghum sorghum virgatum (hack.) stapf sorghum bicolor (l.) moench sorghum taxon group 4 67 sterculiaceae cola cola acuminata (p. beauv.) schott & endl. cola nitida (vent.) schott & endl. kola nut gene pool 1b 68 sterculiaceae cola cola altissima engl. cola nitida (vent.) schott & endl. kola nut taxon group 4 69 sterculiaceae cola cola angustifolia k. schum. cola nitida (vent.) schott & endl. kola nut taxon group 4 70 sterculiaceae cola cola argentea mast. cola nitida (vent.) schott & endl. kola nut taxon group 4 71 sterculiaceae cola cola attiensis aubrév. & pellegr. cola nitida (vent.) schott & endl. kola nut taxon group 4 72 arecaceae phoenix phoenix reclinata jacq. phoenix dactylifera l. date palm gene pool 1b 73 dioscoreaceae dioscorea dioscorea alata l. dioscorea rotundata poir. white yam gene pool 2 74 dioscoreaceae dioscorea dioscorea bulbifera l. dioscorea rotundata poir. white yam gene pool 2 75 dioscoreaceae dioscorea dioscorea cayenensis lam. dioscorea rotundata poir. white yam gene pool 1b s/n family genus taxon related crop common crop name concept type concept level 76 euphorbiaceae manihot manihot carthagenensis (jacq.) müll.arg. manihot esculenta crantz cassava gene pool 2 77 euphorbiaceae manihot manihot carthagenensis subsp. glaziovii (müll.arg.) allem manihot esculenta crantz cassava gene pool 2 78 euphorbiaceae manihot manihot dichotoma ule manihot esculenta crantz cassava gene pool 2 79 euphorbiaceae manihot manihot esculenta subsp. flabellifolia crantz manihot esculenta crantz cassava gene pool 1b 80 papilionaceae phaseolus phaseolus vulgaris var aborigines l. phaseolus vulgaris linn. common bean gene pool 1b 81 papilionaceae vigna vigna unguiculata subsp. aduensis (l.) walp. vigna unguiculata (linn.) walp. cowpea gene pool 2 82 papilionaceae vigna vigna unguiculata subsp. alba (l.) walp. vigna unguiculata (linn.) walp. cowpea gene pool 2 83 papilionaceae vigna vigna unguiculata subsp. baoulensis (l.) walp. vigna unguiculata (linn.) walp. cowpea gene pool 2 84 papilionaceae vigna vigna unguiculata subsp. burundiensis (l.) walp. vigna unguiculata (linn.) walp. cowpea gene pool 2 85 papilionaceae vigna vigna unguiculata subsp. dekindtiana (l.) walp. vigna unguiculata (linn.) walp. cowpea gene pool 1b 86 papilionaceae vigna vigna unguiculata subsp. letouzeyi(l.) walp. vigna unguiculata (linn.) walp. cowpea gene pool 2 87 papilionaceae vigna vigna unguiculata subsp. pawekiae (l.) walp. vigna unguiculata (linn.) walp. cowpea gene pool 2 88 papilionaceae vigna vigna unguiculata subsp. pubescens (l.) walp. vigna unguiculata (linn.) walp. cowpea gene pool 2 89 papilionaceae vigna vigna unguiculata subsp. stenophylla (l.) walp. vigna unguiculata (linn.) walp. cowpea gene pool 1b 90 papilionaceae vigna vigna unguiculata subsp. tenuis (l.) walp. vigna unguiculata (linn.) walp. cowpea gene pool 1b 91 papilionaceae vigna vigna unguiculata subsp. unguiculata var. spontanea (l.) walp. vigna unguiculata (linn.) walp. cowpea gene pool 1b 92 poaceae digitaria digitaria barbinodis henrard digitaria exilis (kippist) stapf fonio gene pool 3 93 poaceae digitaria digitaria ciliaris (retz.) koeler digitaria exilis (kippist) stapf fonio gene pool 2 94 poaceae digitaria digitaria fuscescens (j.presl) henrard digitaria exilis (kippist) stapf fonio gene pool 2 95 poaceae digitaria digitaria iburua stapf digitaria exilis (kippist) stapf fonio gene pool 2 96 poaceae eleusine eleusine coracana subsp. coracana (l.) gaertn. eleusine coracana (l.) gaertn. finger millet gene pool 1b 97 poaceae eragrostis eragrostis pilosa (l.) p.beauv. eragrostis tef (zuccagni) trotter teff gene pool 1b 98 poaceae hordeum hordeum vulgare subsp. spontaneum l. hordeum vulgare l. barley gene pool 1b 99 poaceae sorghum sorghum bicolor subsp. verticilliflorum (l.) moench sorghum bicolor (l.) moench sorghum gene pool 1b 100 poaceae sorghum sorghum drummondii (nees ex steud.) millsp. & chase sorghum bicolor (l.) moench sorghum gene pool 2 101 poaceae triticum triticum turgidum l. triticum aestivum l. wheat gene pool 1b 102 poaceae triticum triticum turgidum subsp. durum (desf.) husn. triticum aestivum l. wheat gene pool 1b supplemental table s2 endemicity and international union for conservation of nature and natural resources (iucn) category of priority cwr s/n taxon common crop name distribution distribution status assessment level iucn category 1 ipomoea acanthocarpa (choisy) hochst. ex schweinf. & asch. sweet potato niger, nigeria, senegal regional endemic global least concern 2 ipomoea alba l. sweet potato ghana, guinea, côte d'ivoire, liberia, nigeria, sierra leone, togo regional endemic global least concern 3 ipomoea aquatica forssk. sweet potato gambia, ghana, guinea, côte d'ivoire, liberia, mali, mauritania, nigeria, senegal, sierra leone, togo regional endemic global least concern 4 ipomoea argentaurata hallier f. sweet potato ghana, guinea, côte d'ivoire, nigeria, sierra leone, togo regional endemic global least concern 5 ipomoea asarifolia (desr.) roem. & schult. sweet potato gambia, ghana, guinea, côte d'ivoire, mali, mauritania, niger, nigeria, senegal regional endemic global least concern 6 ipomoea barteri baker sweet potato guinea, côte d'ivoire, mali, nigeria, senegal, sierra leone regional endemic global least concern 7 ipomoea blepharophylla hallier f. sweet potato benin, ghana, guinea, côte d'ivoire, mali, nigeria, senegal regional endemic global least concern 8 ipomoea chrysochaetia hallier f. sweet potato guinea, côte d'ivoire, nigeria, senegal, sierra leone regional endemic global least concern 9 ipomoea coptica (l.) roth ex roem. & schult. sweet potato ghana, guinea, mali, mauritania, nigeria, niger, senegal, togo regional endemic global least concern 10 ipomoea intrapilosa rose swee tpotato nigeria national endemic global least concern 11 ipomoea ochracea (lindl.) sweet sweet potato ghana, guinea-bissau, guinea, côte d'ivoire, mauritania, nigeria, senegal, sierra leone, togo regional endemic global least concern 12 ipomoea prismatosyphon welw. sweet potato nigeria national endemic global least concern 13 ipomoea rubens choisy sweet potato benin, ghana, guinea, côte d'ivoire, mauritania, niger, nigeria, senegal regional endemic global least concern 14 dioscorea abyssinica hochst. ex kunth white yam benin, burkina faso, ghana, côte d'ivoire, liberia, mali, nigeria, senegal regional endemic global least concern 15 dioscorea baya de wild. white yam côte d'ivoire, liberia regional endemic global least concern 16 dioscorea burkilliana j.miège white yam benin, côte d'ivoire, liberia, sierra leone regional endemic global least concern 17 dioscorea minutiflora engl. white yam benin, burkina faso, guinea, côte d'ivoire, liberia, nigeria, senegal, sierra leone, togo regional endemic global least concern 18 dioscorea praehensilis benth. white yam benin, burkina faso, gambia, ghana, guinea, guinea-bissau, côte d'ivoire, liberia, nigeria, sierra leone, togo regional endemic global least concern 19 dioscorea quartiniana a.rich. white yam benin, burkina faso, gambia, ghana, guineabissau, côte d'ivoire, liberia, nigeria, senegal, sierra leone regional endemic global least concern 20 dioscorea sagittifolia pax white yam benin, burkina faso, gambia, guinea, guineabissau, côte d'ivoire, liberia, nigeria, senegal, sierra leone, togo regional endemic global least concern 21 dioscorea sagittifolia var. lecardii (de wild.) nkounkou white yam benin, gambia, guinea, guinea-bissau, côte d'ivoire, liberia, mali, senegal, sierra leone, togo regional endemic global least concern 22 dioscorea sansibarensis pax white yam benin, guinea, côte d'ivoire, nigeria, togo regional endemic global near threatened s/n taxon common crop name distribution distribution status assessment level iucn category 23 dioscorea schimperiana hochst. ex kunth white yam burkina faso, nigeria regional endemic global least concern 24 dioscorea smilacifolia de wild. & t.durand white yam benin, burkina faso, ghana, guinea, côte d'ivoire, liberia, nigeria, sierra leone, togo regional endemic global least concern 25 dioscorea togoensis r.knuth white yam benin, burkina faso, gambia, ghana, guinea, côte d'ivoire, liberia, nigeria, senegal, sierra leone, togo regional endemic global least concern 26 manihot esculenta subsp. peruviana crantz cassava unknown unknown global least concern 27 gossypium anomalum wawra upland cotton mali, niger regional endemic global near threatened 28 gossypium barbadense linn. upland cotton ghana, liberia, nigeria, senegal, sierra leone, togo regional endemic global least concern 29 gossypium herbaceum var. acerifolium (guill. & perr.) a. chev. upland cotton benin, senegal, nigeria regional endemic global data deficient 30 phaseolus lunatus linn. common bean benin, côte d'ivoire, liberia, nigeria, senegal, sierra leone, togo regional endemic global least concern 31 phaseolus vulgaris subsp. abarigineus l. common bean unknown unknown global least concern 32 vigna ambacensis welw. ex bak. cowpea nigeria national endemic global least concern 33 vigna desmodioides wilczek cowpea sierra leone, nigeria regional endemic global endangered 34 vigna filicaulis hepper cowpea ghana, côte d'ivoire regional endemic global least concern 35 vigna gracilis (guill. & perr.) hook. f. cowpea gambia, guinea-bissau, mali, liberia, nigeria, senegal, sierra leone regional endemic global least concern 36 vigna luteola (jacq.) benth. cowpea benin, ghana, liberia, senegal, sierra leone, togo regional endemic global least concern 37 vigna macrorrhyncha (harms) milne-redhead cowpea nigeria national endemic global least concern 38 vigna marina (burm.) merill cowpea liberia, nigeria regional endemic global least concern 39 vigna multinervis hutch. & dalz. cowpea nigeria, togo regional endemic global least concern 40 vigna nigritia hook. f. cowpea ghana, côte d'ivoire, nigeria, sierra leone regional endemic global least concern 41 vigna oblongifolia a. rich. cowpea unknown unknown global least concern 42 vigna racemosa (g. don) hutch. & dalz. cowpea ghana, gambia, guinea-bissau, mali, nigeria, senegal, togo regional endemic global least concern 43 vigna reticulata hook. f. cowpea ghana, nigeria, senegal, sierra leone, togo regional endemic global least concern 44 echinochloa colonum (l.) link barnyard millet benin, burkina faso, côte d'ivoire, gambia, ghana, guinea, guinea-bissau, mali, mauritania, liberia, niger, nigeria, senegal, sierra leone, togo regional endemic global least concern 45 echinochloa crus-galli (l.) p.beauv. barnyard millet burkina faso, côte d'ivoire, togo regional endemic global least concern 46 echinochloa crus-pavonis (kunth) schult barnyard millet benin, ghana, guinea, guinea-bissau, côte d'ivoire, liberia, niger, nigeria, sierra leone, togo regional endemic global least concern 47 echinochloa frumentacea link barnyard millet gambia, nigeria, togo regional endemic global least concern 48 echinochloa pyramidalis (lam.) hitchc. & chase barnyard millet benin, burkina faso, côte d'ivoire, gambia, ghana, guinea, guinea-bissau, mali, mauritania, niger, nigeria, senegal, sierra leone, togo regional endemic global least concern s/n taxon common crop name distribution distribution status assessment level iucn category 49 eleusine africana kenn. -o'byrne finger millet benin, burkina faso, gambia, ghana, guinea, guinea-bissau, mali, niger, nigeria, senegal, sierra leone, togo regional endemic global least concern 50 eleusine indica (l.) gaertn. finger millet benin, burkina faso, côte d'ivoire; gambia, ghana, guinea, guinea-bissau, liberia, mali, mauritania, niger, nigeria, senegal, sierra leone, togo. regional endemic global least concern 51 eragrostis japonica (thunb.) trin. teff benin, burkina faso, côte d'ivoire; gambia, ghana, guinea, guinea-bissau, mali, mauritania, niger, nigeria, senegal, sierra leone, togo. regional endemic global least concern 52 eragrostis prolifera (sw.) steud. teff benin, burkina faso, côte d'ivoire, gambia, ghana, mali, mauritania, niger, nigeria, senegal, togo. regional endemic global least concern 53 eragrostis unioloides (retz.) nees ex steud. teff guinea, liberia, sierra leone regional endemic global least concern 54 hordeum bulbosum l. barley mauritania national endemic global least concern 55 oryza barthii a.chev. rice benin, burkina faso, côte d'ivoire, gambia, ghana, guinea, guinea-bissau, liberia, mali, mauritania, niger, nigeria, senegal, sierra leone, togo. regional endemic global least concern 56 oryza brachyantha a.chev. & roehr. rice côte d'ivoire, guinea, guinea-bissau, mali, mauritania, niger, nigeria, senegal, sierra leone regional endemic global data deficient 57 oryza eichingeri peter rice côte d'ivoire national endemic global least concern 58 oryza glaberrima steud. rice benin, burkina faso, côte d'ivoire, gambia, guinea, guinea-bissau, mali, mauritania, niger, senegal, sierra leone, togo. regional endemic global least concern 59 oryza longistaminata a.chev. & roehr. rice benin, burkina faso, côte d'ivoire, gambia, ghana, guinea, guinea-bissau, liberia, mali, niger, nigeria, senegal, sierra leone, togo. regional endemic global least concern 60 oryza punctata kotschy ex steud. rice benin, ghana, côte d'ivoire, nigeria, togo regional endemic global least concern 61 panicum comorense mez proso millet ghana, nigeria, togo regional endemic global least concern 62 panicum repens l. proso millet benin, côte d'ivoire, gambia, ghana, guinea, guinea-bissau, liberia, mali, mauritania, niger, nigeria, senegal, sierra leone, togo regional endemic global least concern 63 saccharum spontaneum subsp. spontaneum l. sugarcane unknown unknown global least concern 64 saccharum spontaneum subsp. aegypt iacum (willd.) hack. sugarcane burkina faso, ghana, nigeria, niger, togo regional endemic global least concern 65 sorghum purpureosericeum (hochst. ex a.rich.) schweinf. & asch sorghum burkina faso, mali, nigeria regional endemic global least concern 66 sorghum virgatum (hack.) stapf sorghum mali, mauritania, niger, senegal regional endemic global least concern 67 cola acuminata (p. beauv.) schott & endl. kola nut benin, côte d'ivoire, liberia, nigeria, sierra leone, togo regional endemic global least concern 68 cola altissima engl. kola nut nigeria national endemic global least concern 69 cola angustifolia k. schum. kola nut sierra leone national endemic global least concern 70 cola argentea mast. kola nut nigeria national endemic global least concern 71 cola attiensis aubrév. & pellegr. kola nut côte d'ivoire national endemic global least concern s/n taxon common crop name distribution distribution status assessment level iucn category 72 phoenix reclinata jacq. date palm benin, burkina faso, gambia, ghana, guineabissau, guinea, côte d'ivoire, liberia, nigeria, senegal, sierra leone, togo regional endemic not applicable not evaluated 73 dioscorea alata l. white yam benin, burkina faso, guinea-bissau, mali, senegal, togo regional endemic not applicable not evaluated 74 dioscorea bulbifera l. white yam benin, burkina faso, ghana, guinea-bissau, guinea, côte d'ivoire, liberia, mali, nigeria, senegal, sierra leone, togo regional endemic not applicable not evaluated 75 dioscorea cayenensis lam. white yam benin, burkina faso, gambia, ghana, guineabissau, guinea, côte d'ivoire, liberia, mali, niger, nigeria, senegal, sierra leone, togo regional endemic not applicable not evaluated 76 manihot carthagenensis (jacq.) müll.arg. cassava benin, burkina faso, gambia, togo regional endemic not applicable not evaluated 77 manihot carthagenensis subsp. glaziovii (müll. arg.) allem cassava benin, burkina faso, gambia, togo regional endemic not applicable not evaluated 78 manihot dichotoma ule cassava unknown unknown not applicable not evaluated 79 manihot esculenta subsp. flabellifolia crantz cassava unknown unknown not applicable not evaluated 80 phaseolus vulgaris var aborigineusl. common bean unknown unknown not applicable not evaluated 81 vigna unguiculata subsp. aduensis(l.) walp. cowpea unknown unknown not applicable not evaluated 82 vigna unguiculata subsp. alba (l.) walp. cowpea unknown unknown not applicable not evaluated 83 vigna unguiculata subsp. baoulensis(l.) walp. cowpea benin, cote d'ivoire, ghana, liberia, nigeria, sierra leone, togo regional endemic not applicable not evaluated 84 vigna unguiculata subsp. burundiensis(l.) walp. cowpea unknown unknown not applicable not evaluated 85 vigna unguiculata subsp. dekindtiana(l.) walp. cowpea unknown unknown not applicable not evaluated 86 vigna unguiculata subsp. letouzeyi(l.) walp. cowpea unknown unknown not applicable not evaluated 87 vigna unguiculata subsp. pawekiae(l.) walp. cowpea unknown unknown not applicable not evaluated 88 vigna unguiculata subsp. pubescens(l.) walp. cowpea unknown unknown not applicable not evaluated 89 vigna unguiculata subsp. stenophylla (l.) walp. cowpea unknown unknown not applicable not evaluated 90 vigna unguiculata subsp. tenuis (l.) walp. cowpea unknown unknown not applicable not evaluated 91 vigna unguiculata subsp. unguiculata var. spontanea (l.) walp. cowpea unknown unknown not applicable not evaluated 92 digitaria barbinodis henrard fonio mali, nigeria regional endemic not applicable not evaluated 93 digitaria ciliaris (retz.) koeler fonio benin, burkina faso, gambia, ghana, côte d'ivoire, guinea, guinea-bissau, liberia, mali, mauritania, niger, nigeria, senegal, sierra leone regional endemic not applicable not evaluated 94 digitaria fuscescens (j.presl) henrard fonio ghana, côte d'ivoire, liberia, niger, nigeria regional endemic not applicable not evaluated s/n taxon common crop name distribution distribution status assessment level iucn category 95 digitaria iburua stapf fonio benin, côte d'ivoire, nigeria, niger, togo regional endemic not applicable not evaluated 96 eleusine coracana subsp. coracana (l.) gaertn. finger millet unknown unknown not applicable not evaluated 97 eragrostis pilosa (l.) p.beauv. teff benin, burkina faso, côte d'ivoire, gambia, ghana, guinea, guinea-bissau, liberia, mali, mauritania, niger, nigeria, senegal, sierra leone, togo regional endemic not applicable not evaluated 98 hordeum vulgare subsp. spontaneum l. barley unknown unknown not applicable not evaluated 99 sorghum bicolor subsp. verticilliflorum (l.) moench sorghum unknown unknown not applicable not evaluated 100 sorghum drummondii (nees ex steud.) millsp. & chase sorghum mali, niger regional endemic not applicable not evaluated 101 triticum turgidum l. wheat unknown unknown not applicable not evaluated 102 triticum turgidum subsp. durum (des f.) husn. wheat unknown unknown not applicable not evaluated supplemental table s3: gross production value of socioeconomically valuable crops in west africa s/n gross production (1000 int. $) socioeconomically valuable crop benin burkina faso cote d’ivoire gambia ghana guinea guinea bissau liberia mali mauritania niger nigeria senegal sierra leone togo total 1 date palm 2010 1204 no data no data no data no data no data no data no data 100 20704 15723 no data no data no data no data 2011 1228 no data no data no data no data no data no data no data 610 20764 16228 no data no data no data no data 2012 1264 no data no data no data no data no data no data no data 19 20853 16525 no data no data no data no data 2013 1274 no data no data no data no data no data no data no data 653 20938 15985 no data no data no data no data 2014 1299 no data no data no data no data no data no data no data 714 18333 14751 no data no data no data no data 2015 1326 no data no data no data no data no data no data no data 711 21279 17141 no data no data no data no data 2016 1344 no data no data no data no data no data no data no data 679 21755 18229 no data no data no data no data 2017 1368 no data no data no data no data no data no data no data 640 21728 18353 no data no data no data no data 2018 1390 no data no data no data no data no data no data no data 651 21592 18791 no data no data no data no data 2019 1413 no data no data no data no data no data no data no data 651 21314 19217 no data no data no data no data total 13110 5428 209260 170943 398741 2 sweet potato 2010 15969 19122 9722 no data 24801 39627 no data 4134 42312 551 9066 716562 14467 42614 982 2011 10130 28947 9329 no data 26868 42667 no data 4547 54763 559 12253 726809 8391 43467 980 2012 10790 19183 9621 no data 27901 43995 no data 4754 71267 496 16125 742472 6200 45640 4125 2013 13597 34629 9903 no data 27901 45370 no data 4795 84186 517 20210 759889 7647 46553 2343 2014 13535 7800 10198 no data 28576 47464 no data 4793 78986 1083 16801 771884 7834 41567 2376 2015 11451 9720 10520 no data 29270 49206 no data 4812 62796 1073 23573 797201 7234 49129 1716 2016 12017 14571 10830 no data 29815 51012 no data 4872 112691 1066 22512 810301 10813 30885 566 2017 23989 12142 11181 no data 30504 52884 no data 4933 210950 1011 24695 825792 14832 31660 1805 2018 13363 13338 11290 no data 31193 54825 no data 4994 104280 992 26859 841283 14881 32455 1767 2019 14121 13720 10795 no data 31882 58221 no data 5054 64582 1050 35790 856774 18476 36944 1792 total 138962 173172 103389 288711 485271 47688 886813 8398 207884 7848967 110775 400914 18452 10719396 3 white guinea yam 2010 694291 25832 1426774 no data 1577093 27170 no data 5160 23850 749 no data 9876712 no data no data 187987 2011 723620 26388 1463683 no data 1665722 20600 no data 5556 21746 753 no data 8767014 no data no data 192556 2012 743699 29990 1501475 no data 1756587 32184 no data 5689 21948 794 no data 8551300 no data no data 228715 2013 782860 24230 1516563 no data 1871871 34603 no data 5689 20948 794 no data 9424324 no data no data 174948 2014 852157 11630 1640081 no data 1883626 26455 no data 5697 24688 786 no data 11946718 7501 no data 208073 2015 701299 7610 1759532 no data 1930498 29825 no data 5604 21913 769 no data 12085986 9261 no data 206757 2016 804687 12611 1824357 no data 1968653 33328 no data 5574 31450 771 no data 13590177 18521 no data 215373 2017 858461 12366 1891299 no data 2078867 22623 no data 5580 20861 774 no data 14309918 19051 no data 218699 2018 779207 9501 1909146 no data 2079214 49726 no data 5587 24071 777 no data 13229566 18521 no data 227227 2019 817190 13270 1898909 no data 2192985 51812 no data 5594 16917 780 no data 13243583 20514 no data 231323 total 7757471 173428 15405045 19005116 328326 55730 228392 7747 115025298 93369 2091658 160171580 4 cassava 2010 500494 602 335145 1365 1961918 154325 9927 71625 5546 no data 16418 6179359 26331 472177 132027 2011 529692 610 342725 1436 2068960 161640 8764 71862 6835 no data 11089 6710670 22501 502732 145071 2012 529700 654 350477 1525 2113476 169188 9927 75515 8157 no data 15584 7402225 27527 520865 139456 2013 568063 632 353982 1671 2323071 177089 3347 74722 8977 no data 22679 6887410 21216 553590 131170 2014 590825 594 615900 1800 2585783 207265 6419 81811 7577 no data 19337 8183585 37375 589309 167527 2015 496965 627 739056 1670 2500725 218892 8021 86503 6152 no data 26821 8374603 63862 639070 150969 2016 565484 602 660748 1741 2585783 233482 7415 101351 17269 no data 21293 8653931 77579 596947 149275 2017 575242 593 779735 1799 2761650 254495 7907 78162 14739 no data 46440 8000565 102395 620181 151339 2018 554954 589 813637 1856 3028569 275369 7858 84084 20052 no data 54105 8106198 148732 643415 158282 2019 565846 588 761029 1914 3261266 311703 8146 81100 10215 no data 74628 8599855 149728 666649 162409 total 5477265 6091 5752434 16777 25191201 2163448 77731 806735 105519 308394 77098401 677246 5804935 1487525 124973702 s/n gross production (1000 int. $) socioeconomically valuable crop benin burkina faso cote d’ivoire gambia ghana guinea guinea bissau liberia mali mauritania niger nigeria senegal sierra leone togo total 5 cotton 2010 123385 477132 157376 523 10811 33333 5045 no data 219442 no data 3153 542735 23464 no data 38738 2011 238898 397346 234508 541 15315 36036 5135 no data 401178 no data 9009 484384 22853 no data 75322 2012 216240 546836 317236 495 15315 37838 4504 no data 408846 no data 8108 259097 29052 no data 72725 2013 276895 690285 365063 495 12613 37838 4504 no data 396418 no data 8559 242980 25962 no data 70135 2014 343837 806285 369367 495 12613 37838 4504 no data 494318 no data 9009 261406 23932 no data 95495 2015 242532 692725 405403 482 13340 38014 4911 no data 462670 no data 5946 250019 27928 no data 73073 2016 406493 707008 279277 373 12869 39593 5060 no data 583111 no data 6036 251188 21621 no data 101801 2017 538723 760659 295494 378 11779 39159 5020 no data 656398 no data 5991 261681 18018 no data 105549 2018 682878 434387 328985 400 11558 39292 5059 no data 591494 no data 6757 235908 13622 no data 123662 2019 713386 652457 320710 393 11196 39424 5098 no data 640294 no data 7553 210002 14875 no data 105025 total 3783267 6165120 3073419 4575 127409 378365 48840 4854169 70121 2999400 221327 861525 22587537 6 common bean 2010 81836 no data 25232 no data 143364 no data no data no data 105547 8095 9680 no data no data no data 58075 2011 65170 no data 25620 no data 146333 no data no data no data 339149 8488 13873 no data no data no data 58285 2012 72150 no data 26484 no data 170132 no data no data no data 100949 8810 14483 no data no data no data 101100 2013 78356 no data 27334 no data 152753 no data no data no data 80760 9138 13720 no data no data no data 79991 2014 73018 no data 28339 no data 153325 no data no data no data 113790 9795 13422 no data no data no data 127704 2015 75541 no data 29672 no data 153325 no data no data no data 142452 9571 14653 no data no data no data 135234 2016 77612 no data 30268 no data 109165 no data no data no data 137263 10007 14657 no data no data no data 126646 2017 81180 no data 31159 no data 129954 no data no data no data 114336 10264 15067 no data no data no data 151959 2018 107091 no data 32051 no data 134666 no data no data no data 114336 10713 15477 no data no data no data 158204 2019 96172 no data 32942 no data 145084 no data no data no data 121817 10879 15887 no data no data no data 154529 total 808126 289101 1438101 1370399 95760 140919 1151727 5294133 7 cowpea 2010 no data 209167 no data no data no data no data 183 no data 43078 2619 592540 1125240 16329 no data no data 2011 no data 147331 no data no data 79068 no data 183 no data 44553 2601 524212 549165 9391 no data no data 2012 no data 199950 no data no data 74583 no data 200 no data 47586 2611 446161 1719137 18379 no data no data 2013 no data 200378 no data no data 66948 no data 200 no data 56216 2616 597924 1546936 13593 no data no data 2014 no data 187992 no data no data 67235 no data 205 no data 49872 2630 532233 714213 21410 no data no data 2015 no data 190857 no data no data 67924 no data 210 no data 50111 2631 558817 770438 27686 no data no data 2016 no data 185172 no data no data 68946 no data 213 no data 53034 2636 663835 1252929 33382 no data no data 2017 no data 185688 no data no data 70646 no data 217 no data 49183 2641 654164 1294444 36301 no data no data 2018 no data 228230 no data no data 71943 no data 222 no data 86170 2646 793999 1169254 51033 no data no data 2019 no data 217967 no data no data 67728 no data 227 no data 71971 2650 797342 1194764 61515 no data no data total 1952732 635021 2060 551774 26281 6161227 11336520 289019 20954634 8 fonio 2010 612 13749 13123 no data no data 288626 393 no data 39297 no data 4104 59372 1031 no data no data 2011 561 10886 13002 no data no data 307043 394 no data 38302 no data 3724 61515 1302 no data no data 2012 791 15509 13299 no data no data 322365 435 no data 13053 no data 4454 61173 1124 no data no data 2013 847 14930 12620 no data no data 338453 542 no data 22931 no data 5308 61456 1117 no data no data 2014 1227 6428 14098 no data no data 355341 631 no data 27990 no data 2794 61762 1636 no data no data 2015 1158 9828 14489 no data no data 373072 480 no data 15235 no data 4359 62363 2423 no data no data 2016 999 8210 14864 no data no data 373072 471 no data 12567 no data 4589 62063 1739 no data no data 2017 1047 7558 15315 no data no data 366583 465 no data 34675 no data 4660 62224 2896 no data no data 2018 4780 8461 15496 no data no data 358417 459 no data 20921 no data 4805 62385 2944 no data no data 2019 2857 7686 15446 no data no data 398051 453 no data 30433 no data 4539 62548 3867 no data no data total 14879 103245 141752 3481023 4723 255404 43336 616861 20079 4681302 s/n gross production (1000 int. $) socioeconomically valuable crop benin burkina faso cote d’ivoire gambia ghana guinea guinea bissau liberia mali mauritania niger nigeria senegal sierra leone togo total 9 finger millet 2010 8080 344479 14653 47421 65707 70234 4503 no data 412135 1004 1151628 1551628 244067 9834 15384 2011 7409 248702 14653 26179 55194 48168 4193 no data 438783 184 828541 381534 144274 12004 15113 2012 8877 323616 14800 34838 53923 63284 5088 no data 504254 970 1159019 384334 198848 11696 8314 2013 7051 323675 14933 28149 46554 64265 5419 no data 556431 1186 876876 272956 154659 11809 19339 2014 7103 291855 15688 23052 46515 66288 3001 no data 514679 547 996846 419735 122737 12004 5264 2015 6494 283946 16565 22033 47226 67398 4201 no data 559470 837 1021772 445759 225035 13204 11603 2016 7557 271609 17496 19528 47720 71476 4201 no data 542142 944 1166198 465923 148050 11404 7154 2017 7417 248550 18486 15605 49061 72538 4855 no data 447939 983 1137374 450145 262730 11704 7816 2018 7845 356838 19506 11404 54487 64445 5402 no data 552274 974 1157275 635904 269359 11404 7827 2019 7502 291146 19506 10503 57018 66988 6002 no data 563739 966 981451 600193 242191 11404 8044 total 75335 2984416 166286 238712 523405 655084 46865 5091846 8595 1047698 0 5608111 2011950 116467 105858 28109910 10 barley 2010 no data no data no data no data no data no data no data no data no data 260 no data no data no data no data no data 2011 no data no data no data no data no data no data no data no data no data 262 no data no data no data no data no data 2012 no data no data no data no data no data no data no data no data no data 265 no data no data no data no data no data 2013 no data no data no data no data no data no data no data no data no data 268 no data no data no data no data no data 2014 no data no data no data no data no data no data no data no data no data 279 no data no data no data no data no data 2015 no data no data no data no data no data no data no data no data no data 273 no data no data no data no data no data 2016 no data no data no data no data no data no data no data no data no data 279 no data no data no data no data no data 2017 no data no data no data no data no data no data no data no data no data 285 no data no data no data no data no data 2018 no data no data no data no data no data no data no data no data no data 306 no data no data no data no data no data 2019 no data no data no data no data no data no data no data no data no data 307 no data no data no data no data no data total 2784 2784 11 rice 2010 48874 105846 471690 39064 192251 631081 81827 115792 506886 52578 40210 1749066 236223 401500 43060 2011 85889 94195 341410 19998 181446 701110 68520 113801 681037 61797 28991 1803853 158705 441650 43891 2012 84723 124904 610813 21203 188157 750400 77629 116148 812026 72042 27879 2124653 183665 446373 62938 2013 141798 119426 756389 27259 222723 803006 82014 105589 773406 79210 34065 1886257 170566 491011 101842 2014 91567 135897 803069 18253 236206 770608 52012 104024 847381 114669 40648 2347524 218616 470856 57851 2015 79899 127152 841973 27375 250868 800662 66482 115757 911604 87209 35521 2446620 354445 340893 55122 2016 110058 150441 803648 19162 268930 835419 72739 131079 1087527 80951 40825 2958070 369952 342305 53618 2017 108998 127319 829067 11732 282383 859534 64526 96788 1058843 117712 44047 3060557 395477 350823 54953 2018 179623 137028 784877 10168 300889 915004 68828 100894 1238724 126315 39735 3286157 471859 359700 56896 2019 158774 147248 736775 8604 361739 1016454 73130 66482 1249990 149780 47617 3298672 451971 370524 57508 total 1090203 1269456 6979711 202818 2485592 8083278 707707 1066354 9167424 942263 379538 24961429 3011479 4015635 587679 64950566 12 sugarcane 2010 538 20422 80813 no data 6508 12702 283 11894 16114 no data 9985 38147 38152 3411 no data 2011 312 20647 87098 no data 6508 12702 283 11894 15934 no data 7930 33924 39498 3456 no data 2012 450 21769 83788 no data 6643 13241 285 11894 15879 no data 8734 48872 40396 3456 no data 2013 1012 21545 88374 no data 6733 13465 285 11894 15067 no data 8734 57095 39947 3456 no data 2014 916 21458 88138 no data 6688 13464 289 11928 15735 no data 10186 63445 42845 3494 no data 2015 597 21634 89859 no data 6715 13696 297 12099 16353 no data 14854 65081 44705 3544 no data 2016 539 21707 82228 no data 6835 13715 301 12127 16294 no data 9697 66751 48395 3502 no data 2017 449 21814 81960 no data 6836 13807 305 12169 16410 no data 11349 67008 48395 3522 no data 2018 449 21921 88014 no data 6866 13907 309 12212 16504 no data 11584 64348 48395 3542 no data 2019 449 22028 88349 no data 6897 14003 313 12254 16609 no data 14393 65354 49090 3561 no data total 5711 214945 858621 67229 134702 2950 120365 160899 107446 570025 439818 31533 2714244 s/n gross production (1000 int. $) socioeconomically valuable crop benin burkina faso cote d’ivoire gambia ghana guinea guinea bissau liberia mali mauritania niger nigeria senegal sierra leone togo total 13 sorghum 2010 36514 432339 10362 8472 70475 9725 3822 no data 271727 25422 282800 1551241 35322 5571 53151 2011 28938 327051 10093 4465 62360 6641 4127 no data 258727 5348 167338 1236077 18870 6026 52845 2012 31236 417910 10609 5028 60821 8726 5115 no data 287127 27394 298844 1268001 29885 6517 54502 2013 19133 408495 10964 6602 55771 8861 5837 no data 318991 19762 286799 1151384 19992 6517 61980 2014 21777 370947 11154 4407 56263 8255 3041 no data 276292 12425 309768 1495266 22228 6517 66816 2015 28169 311866 11969 5739 57056 7921 3476 no data 331811 16213 416721 1521709 40948 8319 58840 2016 28167 361439 12838 5975 49877 6256 3693 no data 302782 17378 392811 1641415 27483 10644 59255 2017 30063 296716 13772 6099 49963 6387 4345 no data 309198 10644 422544 1507367 46811 12382 59992 2018 69335 419220 15206 6236 68696 13158 4562 no data 319262 18899 456227 1477172 64184 10644 60225 2019 43446 406611 15206 6517 74945 13552 4779 no data 328260 14772 412009 1447845 58689 10862 61386 total 336778 3752594 122173 59540 606227 89482 42797 3004177 168257 3445861 14297477 364412 83999 588992 26962766 14 wheat 2010 no data no data no data no data no data no data no data no data 2380 571 1223 26156 no data no data no data 2011 no data no data no data no data no data no data no data no data 8015 684 306 39077 no data no data no data 2012 no data no data no data no data no data no data no data no data 9490 1443 737 23683 no data no data no data 2013 no data no data no data no data no data no data no data no data 6496 1583 1342 18946 no data no data no data 2014 no data no data no data no data no data no data no data no data 10816 1672 427 21623 no data no data no data 2015 no data no data no data no data no data no data no data no data 8468 1670 1916 14210 no data no data no data 2016 no data no data no data no data no data no data no data no data 9506 1828 1950 14210 no data no data no data 2017 no data no data no data no data no data no data no data no data 6635 1983 1422 15868 no data no data no data 2018 no data no data no data no data no data no data no data no data 6912 1962 1138 14210 no data no data no data 2019 no data no data no data no data no data no data no data no data 1948 2046 1165 14210 no data no data no data total 70666 15442 11626 202193 299927 15 kola nut 2010 436 no data 48935 no data 14554 no data no data no data no data no data no data 105853 no data 5281 no data 2011 438 no data 48710 no data 17527 no data no data no data no data no data no data 100777 no data 5510 no data 2012 438 no data 45383 no data 17635 no data no data no data no data no data no data 98587 no data 5454 no data 2013 438 no data 42473 no data 16842 no data no data no data no data no data no data 96396 no data 5584 no data 2014 440 no data 41386 no data 16607 no data no data no data no data no data no data 122456 no data 5854 no data 2015 444 no data 46174 no data 17595 no data no data no data no data no data no data 129266 no data 6055 no data 2016 447 no data 44842 no data 17713 no data no data no data no data no data no data 114347 no data 6044 no data 2017 448 no data 44438 no data 17968 no data no data no data no data no data no data 115695 no data 6161 no data 2018 451 no data 44121 no data 18222 no data no data no data no data no data no data 116522 no data 6288 no data 2019 453 no data 43864 no data 18478 no data no data no data no data no data no data 117672 no data 6407 no data total 4433 450326 173141 1117571 58638 1804109 original article genetic resources (2021), 2 (3), 1–10 doi: 10.46265/genresj.dmat2233 https://www.genresj.org issn: 2708-3764 collecting and regenerating populations of the allium ampeloprasum complex from greece chris kik *,a, liesbeth de groot a, gerlof bottema b, marc op ’t hof c, niels de visserd, peter willems e, toon van doormalen f, stelios samaras g, elias polemish and dimitris tzanoudakis i a centre for genetic resources, wageningen university & research, wageningen, 6700 aa, the netherlands b enza zaden research and development b.v, haling 1/e, 1602 cb, enkhuizen, the netherlands c hazera seeds b.v, schansereind 27, 4921 pm, made, the netherlands d rijk zwaan zaadteelt en zaadhandel b.v, burgemeester crezéelaan 40, 2678 kx, de lier, the netherlands e bejo zaden b.v, trambaan 1a, 1749 cz, warmenhuizen, the netherlands f nunhems netherlands b.v, napoleonsweg 152, 6083 ab, nunhem, the netherlands g hellenic agricultural organization-demeter, directorate general of agricultural research, institute of plant breeding and phytogenetic resources, thermi-thessaloniki, 57001, greece h laboratory of general and agricultural microbiology, agricultural university of athens, athens, 11855, greece i section of plant biology, department of biology, university of patras, patras, 26500, greece abstract: collecting expeditions are of prime importance to acquire genetically unique material, as for many crops and their wild relatives, large gaps are present in collections worldwide. this is also true for the three species of the allium ampeloprasum complex, native to greece, which are considered as the crop wild relatives of cultivated leek (allium porrum). therefore, a collecting expedition was carried out in greece in 2009. a total of 62 populations of a. ampeloprasum, 20 populations of a. bourgeaui, 19 populations of a. commutatum and three mixed species populations were sampled. the sampled populations were mostly small (less than 50 plants), but sometimes large populations (more than 10,000 plants) were encountered, especially for a. commutatum. two different reproduction systems were observed in a. ampeloprasum, which is probably due to ploidy level differences. the sexual type was predominantly found along cultivated fields, whereas the asexual type occurred in abandoned fields together with sarcopoterium spinosum (l). spach and cistus spp. regeneration protocols were developed for these species as the phenology of cultivated leek is different from its wild relatives. regenerating a. ampeloprasum was more difficult compared to the other two species. ten years after the collecting mission only one-third of the collected material has been regenerated. this is partly due to the characteristics of the material and partly because the dutch collectors and the greek competent national authorities on access and benefit sharing were not able to conclude a specific arrangement which also involved the commercial use of the material. keywords: collecting expedition, regeneration, mode of reproduction, access and benefit sharing, leek, crop wild relatives citation: kik, c., de groot, l., bottema, g., op ’t hof, m., de visser, n., willems, p., van doormalen, t., samaras, s., polemis, e., tzanoudakis, d. (2021). collecting and regenerating populations of the allium ampeloprasum complex from greece. genetic resources 2 (3), 1–10. doi: 10.46265/genresj.dmat2233. © copyright 2021 the authors. this is an open access article distributed under the terms of the creative commons attribution license (cc by 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. introduction crop wild relatives (cwr) are nowadays considered important gene reservoirs for the genetic improvement ∗corresponding author: chris kik (chris.kik@wur.nl) of their related crops. however, the occurrence of cwr in genebanks worldwide is often poor and the wild relatives of cultivated leek (allium porrum l.), namely allium ampeloprasum l., a. bourgeaui rech.f. and a. commutatum guss., are no exception to this (keller and kik, 2018). the breeding of new leek cultivars takes received: 05.03.2020 accepted: 18.01.2021 published online: 24.03.2021 https://www.genresj.org https://www.dx.doi.org/10.46265/genresj.dmat2233 https://www.genresj.org https://www.dx.doi.org/10.46265/genresj.dmat2233 mailto:chris.kik@wur.nl 2 kik et al genetic resources (2021), 2 (3), 1–10 many years as the crop is a cross-fertilizing (segmental) autotetraploid (2n=4x=32). major challenges in leek breeding are the identification and subsequent introgression of disease (e.g phytophthora porri) and pest resistances (e.g. thrips tabaci) as these organisms cause major yield reductions when a crop is infected. until present no adequate resistances have been found to many diseases and pests in cultivated leek germplasm. the three cwr of leek form the allium ampeloprasum complex (von bothmer, 1970) which can be crossed successfully with their cultivated crop species leek (kik et al, 1997; kik, 2002a). genecological aspects of the three cross-fertilizing wild species have been treated by von bothmer (1970, 1974), taxonomic issues by de wilde-duyfjes (1976), mathew (1996) and hirschegger et al (2010). breeding aspects of leek have been treated by de clercq and bockstaele (2002). the geographic distribution of the three cwr species of leek is quite different (hirschegger et al, 2010). a. bourgeaui is predominantly found on the greek aegean islands, but populations of the species have also been recorded from the aegean coasts of both east continental greece and west asia minor. a. commutatum has a broader distribution range compared to a. bourgeaui, as it occurs also along the adriatic coast and on the mediterranean islands and islets west of the italian peninsula (sicily, sardinia and corsica). a. ampeloprasum has a very broad distribution range and can be found in the entire mediterranean region and as far as afghanistan in the east. the ecological preferences of the three species differ to a large extent. a. ampeloprasum is a ruderal species which occurs in disturbed places such as cultivated and abandoned fields; population sizes range from small to large. a. bourgeaui is a chasmophyte and can be found on limestone cliffs and inland rocky slopes and occurs mostly in small populations. a. commutatum occurs in coastal habitats and can be found on islets and shores and is able to dominate the area (von bothmer, 1974). the first collecting expeditions focusing on the three species of the a. ampeloprasum complex took place between 1964-1969 (von bothmer, 1970) as part of a larger floristic and biogeographical project (19571969) aimed at studying the effects of genetic and reproductive drift in small populations in the central aegean archipelago (runemark, 1969, 1970). in case of the allium study, a total of 66 a. ampeloprasum, 56 a. bourgeaui and 64 a. commutatum populations were sampled (von bothmer, 1974). in 1982 q.p. van der meer (ivt, wageningen, the netherlands) received from r. von bothmer (university of lund, sweden) 21, 16 and 39 populations of a. ampeloprasum, a. bourgeaui and a. commutatum respectively from this expedition. after 20 years only ten, one and 20 populations of a. ampeloprasum, a. bourgeaui and a. commutatum respectively were still present in this working collection (kik, 2002b). in the global genesys database (https://www.genesys-pgr.org/) no allium material collected between 1964 and 1969 in greece is included and only a few populations of a. bourgeaui and a. commutatum are present in this database. given the low numbers of leek cwr accessions in public genebanks and considering the near absence of in situ management of the three allium species, a new collecting expedition was clearly needed. the aim of the present paper is to report on a collecting expedition in greece which took place in 2009, describing the ecologies of the three allium species, recording the geographic locations of the populations sampled, reporting on the challenges in collecting, regeneration and utilization, and providing two newly developed regeneration protocols for the three wild relatives of leek. material and methods germplasm acquisition a field collecting form, based on the modified fao multicrop passport descriptor list (alercia et al, 2015), was used to document the passport data of the populations sampled (for collecting details: https://www.wur.nl/en /research-results/statutory-research-tasks/centre-forgenetic-resources-the-netherlands-1/genebank/specia l-collections.htm). all sampled material received a collecting number, in this case tkxxx (i.e. tzanoudakis kik followed by a number). latitude, longitude and altitude were determined via gps (garmin, etrex series venture hc) with an inaccuracy of 1-5 meters, using wgs84 as map datum and ddd.dddd◦ as position format. digital pictures were taken of all collecting sites (kik, 2009). considering the peculiarities of the geography of greece, the organisation of the mission (continental and insular areas to be visited, period and duration of the expedition) was mainly based on the information from the rich allium collection (mainly herbarium specimens) deposited at the botanical museum of the university of patras (upa, patras, greece) and on the field experience of the greek participants, especially of the senior author (d.t.) who is an expert on the taxonomy and geographical distribution of the genus allium in greece. the collecting mission took place from july 2 to august 21 2009. collecting took place in the coastal area of the peloponnese on the mainland and on the islands karpathos, crete, andros, kythera and lesbos (figure 1). the three allium species could easily be recognized in the surrounding vegetation because of the height of their flower stalks (ca. 1 m). as a rule of thumb, at least three individual plants needed to be seen at a first glance at a specific location to start an area survey. the area explored by foot per population varied from ca. 0.1 to 2 ha. the inflorescences of all three species were collected in linen bags (20 x 35 cm). for each population collected, a label with the collecting number was put inside the bag and a label attached to the rope which tied up a bag. whenever possible, around 20 inflorescences https://www.genesys-pgr.org/ https://www.wur.nl/en/research-results/statutory-research-tasks/centre-for-genetic-resources-the-netherlands-1/genebank/special-collections.htm genetic resources (2021), 2 (3), 1–10 ex situ management of leek crop wild relatives 3 figure 1. overview of the collecting sites of the leek cwr collecting mission 2009. the blue, yellow and red dots indicate allium ampeloprasum, a. commutatum and a. bourgeaui, respectively. for collecting site details: https://www.wur.nl/en/research-result s/statutory-research-tasks/centre-for-genetic-resources-the-netherlands-1/genebank/special-collections.htm per population were randomly collected and bulked in a linen bag. in almost all cases, the seed was not visible from the outside. therefore, the quality of the seed was inspected by opening the green fruit capsule (ovary) manually and by checking if the seed was black or white. if fruit capsules in a number of inflorescences contained black (mature) seed, a seed sample was taken. the populations collected at the southeastern coast of crete always had black seeds as in this part of greece summer and high temperatures arrive early. together with the inflorescence, 15-20 cm of the flower stalk was also collected to provide the maturing seeds with nutrients. if the seeds were still translucent/white, 510 bulblets (small bulbs inside the foliage leaves of the storage bulbs; not to be confused with bulbils which are present in the inflorescence) were collected per plant. in a few cases bulbs were also collected. upon arrival at cgn the material was dried outdoors in open plastic cages (day/night temperatures: +16/10°c; relative humidity: ~75%) in linen bags for two weeks. then the material was transferred for three months to a conditioned storage room with a temperature of 15°c and 15% relative humidity. the seed cleaning also took place in this room. subsequently, the dried seeds were placed in vacuum-sealed, threelayered aluminum foil bags and stored at -18°c, awaiting future regeneration. the bulblets and bulbs sampled during the expedition were planted in mid-november in a conditioned greenhouse. ex situ regeneration of the collected seed material protocols had to be developed for the regeneration of allium ampeloprasum, a. bourgeaui and a. commutatum as the life cycle of cultivated leek is quite different from its wild relatives. firstly, a protocol (regeneration protocol i) for regeneration only under the climatic conditions of the netherlands was developed. however, as regeneration in the netherlands took three years and plants were lost during the regeneration process, an alternative protocol (regeneration protocol ii) was developed which involved a two stage regeneration process: the first stage took place under the climatic conditions of the netherlands (wageningen) and the second step under the climatic conditions of south-east spain (cartagena). this protocol took two years and fewer plants were lost. regeneration protocol i 1. sowing during the last week of august to first week of september of 300 seeds in sowing trays with normal potting soil in a heated glasshouse (day/night temperatures: +15/12°c). 2. after the first seedlings have appeared in the heated glasshouse, the sowing trays should be placed for 3-4 days at 4°c to enhance germination; subsequently, the sowing trays are transferred back to the heated glasshouse for 2-3 weeks and the entire procedure of placing the sowing trays at 4°c should be repeated if not enough seeds have germinated. this procedure may be repeated 34 times. in the meantime, emerging seedlings are planted in 24 cm diameter pots (3 plants per pot) and placed in a heated greenhouse; care should be taken that plants do not receive too much water. 3. during next may the watering is gradually decreased to a minimum and in the summer months no watering takes place. watering will start again in september. 4. every year in august the bulbs are uprooted and bulblets are removed from the main bulb and bulbs replanted again at the end of august or beginning of september. https://www.wur.nl/en/research-results/statutory-research-tasks/centre-for-genetic-resources-the-netherlands-1/genebank/special-collections.htm 4 kik et al genetic resources (2021), 2 (3), 1–10 5. when the main bulb has a minimum diameter of 4-5 cm flowering is possible. it usually takes about 2.5 years after sowing until the bulb has reached this size. 6. when the plants start flowering in april/may, flowering plants are isolated in cages together with plants from the same population. regeneration takes place with a minimum of 12 plants per population, using blow flies for pollination. bulbs that did not produce a flowering stalk can be maintained in the same pot in the greenhouse for the next season. 7. in august, when the seeds have a black colour, the seed stalks are placed upside down in linen bags for drying during 2 weeks, subsequently the seeds are threshed and placed in a paper bag in a drying room (15°c and 15% rh). regeneration protocol ii 1. sowing during the last week of august to first week of september of 300 seeds in sowing trays with normal potting soil in a heated glasshouse (day/night temperatures: +15/12°c). 2. after the first seedlings have appeared in the heated glasshouse, the sowing trays should be placed for 3-4 days at 4°c to enhance germination; subsequently, the sowing trays are transferred back to the heated glasshouse for 2-3 weeks and the entire procedure of placing the sowing trays at 4°c should be repeated if not enough seeds have germinated. this procedure may be repeated 34 times. in the meantime, emerging seedlings are planted in 24 cm diameter pots (3 plants per pot) and placed in a heated greenhouse; care should be taken that plants do not receive too much water. 3. during may the watering is gradually decreased to a minimum and in the summer months no watering takes place. 4. the bulbs (diameter ca. 1 cm) are harvested in july-august and sent to cartagena (spain), where they are planted in the field by the end of august or beginning of september. individual plants are planted 30 cm apart from each other (within and between rows) to allow sufficient bulb development. 5. when the main bulb has a minimum diameter of 4-5 cm flowering is possible. in this environment/these conditions the bulb usually reaches this size 1.5 years after sowing. 6. when the plants start flowering in april/may, flowering plants are isolated by placing cages around plants from the same population. regeneration takes place with a minimum of 12 plants per population. blow flies are used for pollination. 7. in august, when the seeds have a black colour, the seed stalks are placed upside down in linen bags for drying during one week, subsequently the seeds are threshed and sent to the netherlands for further processing. figure 2. allium ampeloprasum l. (tk072; mylopotamos, kythera) in its natural habitat. specific collecting mission arrangements an agreement on the basis of which the collecting mission could take place, the so-called mutually agreed terms (mat), was negotiated between the greek ministry of rural development and food, the university of patras and cgn for the collecting mission. an authorization (prior informed consent; pic) to collect the three leek cwrs in greece was issued by the greek competent national authorities on access and benefit sharing (cna-abs) and countersigned by cgn, as a basis for collecting and subsequent distribution of the material. in this arrangement it was agreed that the material collected would be regenerated by cgn and a fair share of each successfully regenerated population will be sent to the greek genebank. another agreement was concluded between cgn and a number of breeding companies. in this agreement it was stipulated that breeding companies would co-finance the expedition and help to regenerate the material sampled. furthermore, an embargo period of five years after the successful regeneration of an accession was negotiated before the material would become available in public databases. results ecogeographic description of species of the allium ampeloprasum complex allium ampeloprasum allium ampeloprasum was mainly found adjacent to cultivated fields or abandoned agricultural fields (figure 2). contrary to its name (‘ampelos’ in greek means ‘vineyard’ and ‘prason’ means ‘leek’) the species was only observed growing between grapevines on a few occasions. the populations sampled often consisted of 115 plants and were found in altitudes ranging from 5 to 567 meters above sea level (masl). two types of a. ampeloprasum plants were observed (von bothmer, 1974): a sexual type (type genetic resources (2021), 2 (3), 1–10 ex situ management of leek crop wild relatives 5 i) with a small inflorescence (ca. 10 cm in diameter), small bulblets (bulbs in between foliage leaves which embrace the large renewal bulb; < 7 mm length) and with seeds; and an asexual type (type ii) with a large inflorescence (ca. 20 cm in diameter), larger and more flattened bulblets, and with shrivelled, nonviable seeds (figure 3). allium bourgeaui the populations sampled consisted of 1-50 plants collected at altitudes ranging from 2 to 522 masl. according to mathew (1996) a. bourgeaui comprises three subspecies, subsp. bourgeaui rech.f., subsp. cycladicum bothmer and subsp. creticum bothmer. the three subspecies are distinguished from each other on the basis of the colour and shape of papillae of the perianth. as collecting took place when the flowering period was over and seeds had set, it was not possible to recognize the three subspecies on the basis of these traits. identification of the three subspecies is nevertheless possible as the three subspecies have a distinct and scarcely overlapping distribution pattern (mathew, 1996). more precisely, ssp. creticum can only be found in crete (n=5 populations sampled), ssp. bourgeaui only on the east aegean islands of rhodos, karpathos and kasos (n=6; figure 4) and ssp. cycladicum on cyclades, ikaria and the eastern part of the greek mainland (n=9). the habitats in which these subspecies were found ranged from rocky gorges and steep hillsides to more accessible locations like field borders and hillsides along roads. figure 3. the two allium ampeloprasum types differ in their mode of reproduction. inflorescences and bulblets of both types are shown. on the left the asexual type (type ii) and on the right the sexual type (type i). figure 4. allium bourgeaui ssp.bourgeaui rech.f. (tk012; achata beach, karpathos) in its natural habitat. allium commutatum the population size of this species varied considerably between locations: from 1-15 plants (diakofti, kythera) to over 100,000 plants (methoni, monemvasia, kythera chora castles; figure 5), but it mostly occured in relatively large (>50 plants) populations. when it occured in large populations, it formed mats, where almost no other plant species could be found. these large populations were also found on islets near the coast. during the expedition the species was collected on islets in front of the coast (andros), but also on coastal shores (xerokambos, crete) and close to old castles (e.g. monemvasia, peloponnese). one population was found on a steep rocky inland hill on the western peloponnese (lake/lagoon of kaiafa; ca. 1 km from the actual shoreline). however, this can be considered a relict population as in the past, due to the fluctuation of the sea level, the present location of the population was actually a coastal area. only a number of islets in front of the southwestern coast of andros were visited during this mission due to the relative inaccessibility of these locations. inhabitants of the islands have knowledge of the presence of allium species on these islets as these islets are often called ‘prasoudha’ or ‘prasonisi’ (islet of prason). mixed allium species locations on three occasions we observed that next to a. commutatum or a. bourgeaui also a. ampeloprasum was present (table 1). the a. commutatum / a. ampeloprasum combination was found on a sandy beach plain (kythera). the a. bourgeaui / a. ampeloprasum combination was found on a clearly disturbed rocky slope along a road (karpathos) and on a (probably disturbed) rocky slope along a road (peloponnese). the mixed populations were located at altitudes between 2 and 177 masl. plants with intermediate morphological characters suggesting hybridization between the two species have not been observed in the localities concerned. 6 kik et al genetic resources (2021), 2 (3), 1–10 figure 5. allium commutatum guss (tk076; methoni castle, peloponnese) in its natural habitat. collecting: number of populations collected in total, around 4,000 km were travelled in greece by car, boat and plane and 104 populations were collected: 62 single species populations of a. ampeloprasum, 20 of a. bourgeaui, 19 of a. commutatum and three mixed populations, which consisted of two allium species (table 1). collecting: number of seeds, bulblets and bulbs sampled from 65 single species populations enough black mature seed (criterium: > 0.5 g = ca. 200 seeds) were harvested to be sure that ca. 80 plants per population could be obtained for regeneration. for 36 single species populations this was not the case as the number of mature seeds obtained was below the threshold. for the three mixed populations enough seed could be collected for each population (figure 6). a. ampeloprasum clearly differed from the other two species as 42% (26 out of 62) of the populations did not yield enough seeds, compared to 25% and 26% for a. bourgeaui and a. commutatum respectively. possible reasons for this could be a) a lower number of plants made up the population; b) immature seeds were collected; and c) presence of plants with different reproduction systems occurring in some populations of a. ampeloprasum. in nine a. ampeloprasum populations plants of the two reproduction types (see above) were observed and care was therefore taken to select plants with smaller inflorescences (i.e. sexual plants) and with black matured seed present within green fruit capsules. in three a. ampeloprasum and three a. bourgeaui populations, only a few plants could be harvested, thus seed sampling could only be taken from fewer than five plants, thus yielding only low seed amounts. from two a. bourgeaui populations vegetative material (bulblets/bulbs) had been collected, which allowed for the multiplication of plants of these populations. when collecting immature seeds, which occurred in four populations of a. ampeloprasum, two of a. bourgeaui and five of a. commutatum, translucent white seeds present within the green fruit capsules were collected, assuming that these seeds could still mature on the stalk. therefore, inflorescences with ca. 20 cm flower stalk were collected. however, this precaution proved to be not enough to obtain mature black seeds for these 11 populations. however, as a precautionary measure vegetative material was also sampled for these 11 populations and thus these samples resulted in viable accessions. regeneration initially, a protocol (material and methods: regeneration protocol i) was developed for regeneration in the netherlands. however, the next generation could only be harvested three years after sowing the originally collected seeds. therefore, a second protocol (material and figure 6. the quantity of seeds and the number of bulblets and bulbs sampled in the various leek cwr populations. when a category on the x-axis is presented as an interval the lower value is excluded from the interval and the higher value is included. genetic resources (2021), 2 (3), 1–10 ex situ management of leek crop wild relatives 7 table 1. the number of collected leek cwr populations per location visited in greece; mixed species populations: populations in which two species are present. location a. ampeloprasum a. bourgeaui a. commutatum mixed species populations total a. ampeloprasum/ a. commutatum a. ampeloprasum/ a. bourgeaui karpathos 9 7 1 17 crete 25 5 3 33 andros 1 4 6 11 peleponnese 4 4 6 1 15 kythera 10 4 1 15 lesbos 13 13 total 62 20 19 1 2 104 methods: regeneration protocol ii) was developed in which the first stages of the regeneration took place the netherlands and the later stages in cartagena (spain). this procedure shortened the production of the next generation of seeds by one year. as the collecting took place in 2009, the regeneration of the material started in 2010. in 2013 the first eight populations were successfully regenerated and, subsequently these populations were included as accessions in the dutch genebank (cgn) and placed in the public domain (https://www.wur.nl/en/research-results/sta tutory-research-tasks/centre-for-genetic-resources-thenetherlands-1/genebank/special-collections.htm). this was only in 2019, due to an embargo period of five years after the successful regeneration of a population, which had been agreed upon by the breeding companies co-financing the mission and cgn on the material and associated information (table 2). in the coming years all accessions which were successfully regenerated will be placed in the public domain. the percentage of populations that have been successfully regenerated until present is 39% (42/107). the mean germination percentages of the populations regenerated until present are 78%, 65% and 89% for a. ampeloprasum, a. bourgeaui and a. commutatum, respectively. eight populations (seven a. ampeloprasum and one a. bourgeaui) have been donated to the czech genebank in olomouc as no seeds could be produced from these populations, but enough bulbs/bulblets were present for vegetatively maintaining these populations. during regeneration, up to now, ten populations of a. ampeloprasum (15% losses; 10/65), three of a. bourgeaui (14% losses; 3/22) and two of a. commutatum (10% losses; 2/20) were lost due to no germination or decay of bulbs. discussion ecogeography of the species from the allium ampeloprasum complex during the collecting mission more a. ampeloprasum populations were collected than a. bourgeaui and a. commutatum populations. this is probably due to the habitat preferences of the three species: a. ampeloprasum has a broad ecological amplitude, whereas the other two species have a narrow ecological amplitude. in terms of generalist and specialist species (fried et al, 2010), a. ampeloprasum can be considered as a generalist and a. bourgeaui and a. commutatum as specialists. greece can be considered as an important biodiversity centre for allium subgenus allium which is represented in greece by more than 100 species (tzanoudakis, 2001; dimopoulos et al, 2013). in situ management of these species is currently not taking place in greece, but in case of a. bourgeaui and a. commutatum this should be considered due to their specific environmental requirements. the amount of seeds that could be collected from natural populations of the three species varied to a large extent among these species. it was observed that in many populations of a. ampeloprasum (42%) only a few seeds could be harvested. the reasons for this are unclear, but it is possible that this is due the presence of a polyploid series a. ampeloprasum, which is much larger compared to the polyploid series in a. commutatum and a. bourgeaui, and which might affect seed production (von bothmer, 1970, 1974). in this context, the presence of plants with different modes of reproduction (sexual and asexual) in a. ampeloprasum in cultivated and abandoned fields, especially observed in kythera, was an interesting phenomenon, probably reflecting different selective forces acting on the species. these forces are probably related to human activities, changes in agricultural methods and land use in general. the sexual type was observed on its own in cultivated fields only, probably due to its generative capacity which allowed the species to co-evolve with the cultivated crops (potatoes, tomatoes, cucumbers, onions). ploughing in these cultivated fields occurs in early spring and irrigation takes place by running water from springs or by underground well-water. the asexual type was mainly collected from places more dry and remote from villages on larger pieces of land formerly cultivated (ploughed during winter, not irrigated) with crops like cereals (triticum, avena, hordeum) or legumes (vicia, lathyrus). after abandoning these fields, species like sarcopoterium, cistus and others perennial species dominate. https://www.wur.nl/en/research-results/statutory-research-tasks/centre-for-genetic-resources-the-netherlands-1/genebank/special-collections.htm 8 kik et al genetic resources (2021), 2 (3), 1–10 preliminary flow cytometry of a few sexual and asexual individuals showed that the asexual type had higher 2c peaks compared to the sexual type, indicating a higher ploidy level than the tetraploid level (kik, unpubl. results). most probably, the sexual type is a (segmental) autotetraploid with 2n=4x=32 and not a diploid (2n=2x=16; guenaoui et al (2013), whereas the asexual type has an increased ploidy level (2n>32), which might impair sexual reproduction and leads to strong(er) vegetative growth and thus stronger competitive strength. no cultivation of leek was observed during the collecting mission. however, instead of cultivated leek, wild leek species, i.e. only allium ampeloprasum and a. commutatum, as a. bourgeaui is difficult to collect, is used as a condiment in the traditional greek kitchen (salads, soups, pies etc; stavridakis (2006). the absence of leek cultivation implies that it is highly likely that no hybridisation and subsequent introgression of genes from the cultivated species into the three wild relatives of leek has taken place. consequently, no replacement of wild genes has taken place in the three species, which increases their value as a gene reservoir for research and breeding. collecting and regenerating the species from the allium ampeloprasum complex collecting the theoretical framework concerning the sampling of populations is well developed for collecting a natural population, using probability-based sampling approaches (volk et al, 2007). however, in collecting expeditions one deals with more than one population, raising the question of how to balance the number of plants to be collected in populations versus the number of populations to be sampled in a given area to adequately acquire a significant percentage of the genetic variation present in these populations. until present only a few studies have addressed sampling in more than one population and simulation approaches were used to study the effects of various factors on the collecting of genetic variation present in these populations. in this context hoban and scharlbaum (2014) argued that it was not realistic to focus on obtaining 95% of the variation present in the populations sampled. a more practical approach would be to collect around 75% of the variation present in these populations. this would require sampling seeds from 20-30 plants per population in case of a diploid species and even less in case of a tetraploid species (bray, 1983). therefore, in the case of the three species of the allium ampeloprasum complex, the aim was to collect seeds from 20-25 plants per population. another issue which needs to be taken into account is how to collect in populations where a strong vegetative propagation can be assumed, for example in and around monemvasia, methoni and kythera chora castles, where large populations (> 100,000 plants) of a. commutatum are present. to sample these populations appropriately, knowledge about the clonal structure of the populations is essential. however, in case of a. commutatum no literature on this issue has been found. the way this problem has been approached in the current collecting expedition was to collect seeds from plants that were meters apart from each other, hoping that they were genetically different from each other. regeneration bulk sampling took place within populations instead of sampling seeds from individual plants and keeping their progenies apart. this was done for logistic reasons, as it proved unfeasible to isolate between 10 and 15 biparental crosses per population rather than using one isolation cage for all the plants of a population for each of the 104 collected populations. bulk sampling and subsequent bulk regeneration per population involves a risk as genetic variation in the subsequent generations could be lost due to random sampling effects (gale and table 2. overview of the regeneration process of leek cwr. 1) year when material will be publicly available; 2) number of populations; 3) 107 = 104 (populations) + 3 (mixed species populations that were subdivided during the regeneration process, based on their species identity and thus resulting in three extra populations for regeneration). year1 a. ampeloprasum a. bourgeaui a. commutatum total succesfully regenerated, already available 2019 22 3 3 8 2020 1 4 6 11 succesfully regenerated, available in 2021 2 3 1 6 2022 0 0 5 5 2023 2 2 1 5 2024 3 1 0 4 2025 1 1 1 3 to be regenerated 37 4 1 42 lost 10 3 2 15 donated to czech genebank 7 1 0 8 total 65 22 20 1073 genetic resources (2021), 2 (3), 1–10 ex situ management of leek crop wild relatives 9 lawrence, 1984; cross and wallace, 1994). however, the decline in genetic variation in tetraploid species is less compared to diploid species (bray, 1983). furthermore, the loss of genetic variation after a number of generations also depends upon the frequency of genes in the collected samples and the ratio of effective population size versus total population size (ne/n) in case of cross-fertilizing species, such as the three species of the allium ampeloprasum complex. however, very important in maintaining the genetic variation in a sample is to keep the number of regenerations as low as possible and to use as many plants as possible per population. therefore, good storage conditions are essential in this context (van treuren et al, 2013). regenerating the cwr of leek proved to be more difficult than expected, especially as it was thought that the regeneration protocol used for cultivated leek could be applied for its cwr species. however, due to differences in flowering phenologies this was not the case, i.e. the cwrs of leek cannot be regenerated in the same way as leek. leek is sown in march and grows until autumn in the netherlands, whereas the cwrs grow during the winter. leek does not grow in winter, but resumes growth in spring and flowers/sets seed in junejuly. the cwrs of leek, on the other hand, are sown in august/september and grow from autumn until spring. at the end of spring the foliage dies back when the bulb has been formed. after the summer (august/september) the bulb sprouts and the growth resumes. when the bulb is large enough, flowering and subsequent seed setting takes place next may/june. in regeneration protocol i, the regeneration takes place in the netherlands and takes three years, whereas in regeneration protocol ii, the regeneration takes place in the netherlands and in spain and takes two years. the difference between both protocols is attributable to the better growing conditions in spain compared to the netherlands, which favour bulb growth from the end of august to may. a relatively large number of seeds had to be sown as germination percentages varied between 55-83%, depending upon the species. a. ampeloprasum was the species where most losses occurred, so for this species it may be suggested to sow twice as many seeds (n= 600). access and benefit-sharing issues access and benefit sharing (abs) is an important aspect in the two international agreements that regulate the exchange of plant genetic resources (pgr), namely the convention on biodiversity (cbd) and the international treaty on plant genetic resources for food and agriculture (it-pgrfa). in this context, national focal points (nfps) and competent national authorities for abs (cna-abs) have been appointed in the countries that ratified one or both agreements; greece and the netherlands ratified both the two agreements. pic and mat were concluded before the collecting mission took place, and in the mat an article was present referring to the bonn guidelines. in this context, a problem was encountered which appeared after the collecting mission when the dutch collectors and the greek cna-abs were not able to conclude a specific arrangement in which it was stipulated that the material collected could also be used for commercial purposes. the consequence was that breeding companies, who were involved in this project, preferred to focus their contributions primarily on material that it could certainly be used in their own breeding programmes. this meant that only a small number of greek populations could be regenerated annually as the dutch national genebank cgn did not have sufficient capacity to carry out a large number of regenerations per year, which resulted in a low speed of the regeneration for these three species. another consequence of not being able to conclude a specific arrangement was that the utilization of the material collected is currently limited to research purposes. therefore, collecting missions should ensure the appropriateness of the terms and conditions of abs in all its aspects well before initiating expeditions. acknowledgements the dutch ministry of agriculture, nature and food quality is gratefully acknowledged for funding this research which was carried out in the framework of the programme genetic resources (wot-03). in addition, we would like to thank bejo zaden b.v., enza zaden research and development b.v., nunhems netherlands b.v., rijk zwaan zaadteelt en zaadhandel b.v., hazera seeds b.v. and syngenta seeds b.v. for supporting the collecting and regeneration of the material collected. furthermore, the manuscript was clearly improved by the contributions of two anonymous reviewers. author contributions all authors have contributed substantially to the study design, execution, data analysis and interpretation, drafting and revision of the submitted manuscript. conflict of interest statement the authors declare that they have no conflict of interest, that the work submitted is their own, that copyright has not been breached in seeking its publication, and that the work submitted has not previously been published and is not being considered for publication elsewhere. references alercia, a., diulgheroff, s., and mackay, m. 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(1974). studies in the aegian flora xxi. biosystematic studies in the allium ampeloprasum complex. opera botanica 34, 1–104. https://doi.org/10.1007/bf01254001 https://doi.org/10.1007/bf01254001 https://doi.org/10.1186/1472-6785-10-20 https://doi.org/10.1186/1472-6785-10-20 https://doi.org/10.1007/s10722-012-9819-5 https://doi.org/10.1016/j.ympev.2009.08.030 https://doi.org/10.1016/j.ympev.2009.08.030 https://doi.org/10.1016/j.biocon.2014.06.014 https://doi.org/10.1007/s001220050438 https://doi.org/10.1007/s001220050438 https://doi.org/10.2307/1217954 https://doi.org/10.1007/s10722-012-9929-0 https://doi.org/10.1007/s10722-012-9929-0 https://doi.org/10.1007/bf01254001de https://doi.org/10.1186/1472-6785-10-20gale https://doi.org/10.1007/s10722-012-9819-5hirschegger https://doi.org/10.1016/j.ympev.2009.08.030hoban https://doi.org/10.1016/j.biocon.2014.06.014keller https://doi.org/10.1007/s001220050438mathew https://doi.org/10.2307/1217954stavridakis https://doi.org/10.1007/s10722-012-9929-0volk introduction material and methods germplasm acquisition ex situ regeneration of the collected seed material regeneration protocol i regeneration protocol ii specific collecting mission arrangements results ecogeographic description of species of the allium ampeloprasum complex allium ampeloprasum allium bourgeaui allium commutatum mixed allium species locations collecting: number of populations collected collecting: number of seeds, bulblets and bulbs sampled regeneration discussion ecogeography of the species from the allium ampeloprasum complex collecting and regenerating the species from the allium ampeloprasum complex collecting regeneration access and benefit-sharing issues author contributions conflict of interest statement 1 supplemental data for koodalugodaarachchi, v., kekulandara, d. s., gimhani, d. r. (2022). genetic diversity of oryza sativa ‘dahanala’ traditional red rice and molecular markers associated with trichome density on adaxial surfaces. genetic resources 3 (5), 10–23. doi: 10.46265/genresj.aveo9374 supplemental data 1: description of seed morphological characters – modified based on ipgri rice descriptors (ipgri, 2007) 1. seed shape (7.5.22) time: at maturity, shape of the grains is classified as: 1) round 2) semi-round 3) half-spindle shaped 4) spindle-shaped 5) very spindle-shaped 2. lemma and palea colour are classified into 19 classes (7.5.5) (modified): 0) straw 1) gold and/or gold furrows on straw background 2) brown spots on straw 3) brown furrows on straw 4) brown 5) reddish to light purple 6) purple spots on straw background 7) purple furrows on straw background 8) purple 9) black 10) white 11) brownish black 12) yellowish gray 13) yellowish brown 14) brown furrows on gold background 15) reddish brown spots on gold background 16) black spots on straw background 17) brownish black furrows on gold background 18) brownish black furrows on straw background 19) brown furrows on yellowish background 3. seed coat (bran) colour (7.5.23) (modified): time: at maturity, brown rice (dehulled grains) is classified into: 0) off white 1) white 2) light brown 3) speckled brown 4) brown 5) red 6) variable purple 7) purple 8) off white with black apiculus 9) off white with brown apiculus https://10.0.180.185/genresj.aveo9374 2 10) brown seed coat with white strips 11) red seed coat with white strips 12) off white seed coat with white strip with black apiculus 13) off white seed coat with white strips 14) brown seed coat with black apiculus 15) light brown seed coat with white strip 16) brown seed coat with white strip and black apiculus 4. lemma and palea pubescence (7.5.4): time: at maturity, pubescence of the hull is classified as: 1) glabrous 2) hairs on lemma keel 3) hairs on upper portion 4) short hairs 5) long hairs (velvety) 5. grain length (7. 5. 15): time: at maturity, grain length is measured as the distance from the base of the lowermost sterile lemma to the tip (apiculus) of the fertile lemma or palea, whichever is longer. in the case of awned varieties, the grain is measured to a point comparable to the tip of the apiculus 6. grain width (7. 5. 16): time: at maturity, width of grain is measured in millimeters as the distance across the fertile lemma and the palea at the widest point 3 supplemental figure 1: additional ssr marker profiles amplified from dna bulks (rm201, rm220, rm241, rm259, rm270). the arrowhead indicates corresponding scored alleles. b1–b8, dna bulks; b9, bg360; l25, l100, l200, size markers; bp, basepairs. supplemental figure 2: pcr profile or heterogenic marker rm515 of dna bulks. 4 supplemental figure 3: pcr profiles of expanded dna bulks for ssr markers (rm84, rm207, rm224, rm480, rm518, rm536) amplification of bulked dna samples and expanded bulks with a) rm480 (product size: 199–221bp), b) rm84 (product size: 118–124bp), c) rm224 (product size: 124–142bp), d) rm207 (product size: 110–132bp), e) rm536 (product size: 220–230bp), f) rm518 (product size: 158–180bp) (resulted with monomorphic alleles) (product size: 132– 154bp). the arrowhead indicates corresponding scored alleles. b1–b8, dna bulks; b9, bg360; l25, l100, l200, size markers; bp, basepairs. 8, 15, 3, 11, 6, 21, 7, 22, 28, 20, 32, 33, 19, 4, 5, 17, 31, 27, 23, 13, 16, 26, 30, 29, 9, 24, 12, 18, 14, 9, 24, 12, 18, 14 individual accessions included in bulks, numbered as in table 1. b) 5 supplemental figure 4: pcr profiles of expanded dna bulks for ssr markers (rm217, rm237, rm418, rm440, rm515, rm571) amplification of bulked dna samples and expanded bulks with a) rm237 (product size: 124–138bp), b) rm571 (product size: 182–192bp, c) rm418 (product size: 245–290bp), d) rm440 (product size: 161–217bp), e) rm515 (product size: 211–219bp), f) rm217 (product size: 114–144bp). the arrowhead indicates corresponding scored alleles. b1– b8, dna bulks; b9, bg360; l25, l100, l200, size markers; bp, basepairs. 8, 15, 3, 11, 6, 21, 7, 22, 28, 20, 32, 33, 19, 4, 5, 17, 31, 27, 23, 13, 16, 26, 30, 29, 9, 24, 12, 18, 14, 9, 24, 12, 18, 14 individual accessions included in bulks, numbered as in table 1. 6 supplemental figure 5: map position of markers rm277 and rm279 on rice chromosomes 12 and 2. (adapted from temnykh et al., 2000) 7 references international plant genetic resources institute, and west africa rice development association. descriptors for wild and cultivated rice (oryza spp.). biodiversity international, 2007. https://www.bioversityinternational.org/fileadmin/user_upload/online_library/ publications/pdfs/232.pdf temnykh, s., park, w.d., ayres, n., cartinhour, s., hauck, n., lipovich, l., cho y.g., ishii, t., mccouch, s,r. (2000). mapping and genome organization of microsatellite sequences in rice (oryza sativa l.). theoretical applied genetics 100, 697–712. https://www.bioversityinternational.org/fileadmin/user_upload/online_library/publications/pdfs/232.pdf https://www.bioversityinternational.org/fileadmin/user_upload/online_library/publications/pdfs/232.pdf original article genetic resources (2020), 1 (2), 1–11 doi: 10.46265/genresj.bjcv8100 https://www.genresj.org issn: 2708-3764 assessment of wild tomato accessions for fruit yield, physicochemical and nutritional properties under a rain forest agro-ecology dorcas o. ibitoye a, adesike o. kolawole *,b and roseline t. feyisola c a genetic resources unit, national horticultural research institute, pmb 5432, ibadan, nigeria b department of crop production and soil science, ladoke akintola university of technology, pmb 4000, ogbomoso, nigeria c department of plant science, olabisi onabanjo university, ago-iwoye, nigeria abstract: tomato (solanum lycopersicum l.) is a broadly consumed fruit vegetable globally. it is one of the research mandate vegetable of the national horticultural research institute (nihort), ibadan, nigeria. the institute’s genebank contains diverse collections of tomato accessions and wild relatives, without utilization information for the african continent. with the decline in diversity and potential of cultivars, a robust tomato breeding pipeline with broad genetic base that eliminates redundancy in the development of lines with desired horticultural traits is paramount. this study evaluated the mean performance and variations of thirteen wild tomato accessions obtained from the c.m. rick tomato genetic resource center, university of california, davis, usa, evaluated for agronomic, nutritional and physicochemical traits under a rain forest agro-ecology zone in nigeria. the accessions were planted and grown in three replications with randomized complete block design. agronomic traits, physicochemical and nutritional parameters were measured and analyzed. there was significant (p < 0.001) variation among accessions for all traits measured. accession la0130 was separated from others by cluster analysis and was outstanding for its unique attributes which include: fruit yield parameters, total soluble solids, titratable acidity and lycopene content. the principal component analysis suggests fruit yield related traits, titratable acidity and lycopene contributed most to the variation among the 13 accessions. the results obtained can be used to breed materials adapted to a rain forest agro-ecology. these wild tomato accessions have genes with desirable agronomic, nutritional and physicochemical traits that could be introgressed into breeding lines to improve commercial tomato varieties. keywords: agronomic traits, breeding programme, fruit quality, variation, wild relatives citation: ibitoye, d. o., kolawole, a. o., feyisola, r. t. (2020). assessment of wild tomato accessions for fruit yield, physicochemical and nutritional properties under a rain forest agro-ecology. genetic resources 1 (2), 1–11. doi: 10.46265/genresj.bjcv8100. © copyright 2020 the authors. this is an open access article distributed under the terms of the creative commons attribution license (cc by 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. introduction tomato (solanum lycopersicum l. formerly lycopersicon esculentum mill.) is one of the most famous and broadly consumed vegetable crops throughout the world (nowicki et al, 2013; ajayi and hassan, 2019). nigeria was ranked the largest producer of tomato in west africa and the 16th largest producer in the world with 4.2 million metric tonnes fao (2016). ∗corresponding author: adesike o. kolawole (aokolawole@lautech.edu.ng) these data suggest prospects for nigeria tomato breeding programmes to enhance production efficiency by improving the quantity and quality of tomato fruit. however, extensive breeding efforts and selection over the years have modified tomato (blanca et al, 2015). the decline in diversity and potential of cultivated germplasm has been reported (jatoi et al, 2008; chen et al, 2009). to enlarge the gene pool of cultivars, breeders now focus on introgression of desirable genes from wild relatives (singh, 2006) . wild tomato species have a rich reservoir of useful genetic traits needed to improve cultivated tomareceived: 05.05.2020 accepted: 09.09.2020 published online: 21.12.2020 https://www.genresj.org https://www.dx.doi.org/10.46265/genresj.bjcv8100 https://www.genresj.org https://www.dx.doi.org/10.46265/genresj.bjcv8100 mailto:aokolawole@lautech.edu.ng 2 ibitoye et al genetic resources (2020), 1 (2), 1–11 toes and serve as sources of genetic variability (hanson et al, 2007). miller and tanksley (1990) reported that the genomes of cultivated tomato contain 5 % of the genetic variation of their wild relatives. these wild tomato species are native to western south america and distributed from central ecuador, through peru to northern chile, and in the galápagos islands (darwin et al, 2003). there are 16 wild species of tomato, namely solanum habrochaites, s. pennellii, s. pimpinellifolium, s. cheesmaniae, s. galapagense, s. peruvianum, s. corneliomulleri, s. chilense, s. chmielewskii, s. arcanum, s. neorickii, s. huaylasense, s. lycopersicoides, s. ochranthum, s. jugandifolium, and s. sitiens (rick and fobes, 1975; peralta et al, 2008; zuriaga et al, 2009). the closest wild ancestor to cultivated tomato is s. pimpinellifolium l. found in the centre of origin of tomato from the northern part of chile to colombia. most of these wild relatives are vulnerable to extinction because of their small population sizes (bai and lindhout, 2007; onyia et al, 2019). therefore, wild tomato accessions stored in genebanks need to be evaluated in time and space in order to identify significant traits and valuable potential. documented information about the performance of wild tomato accessions will aid future use in tomato breeding programmes. currently, the breeding programme at the national horticultural research institute (nihort), ibadan, nigeria has made diverse collections of landraces, cultivars and wild tomato accessions. only the landraces and cultivars have been extensively studied for their agronomic performance and resistance to abiotic and biotic stresses (olaniyi et al, 2010; nnabude et al, 2015). however, the wild accessions obtained from the c.m. rick tomato genetic resource centre, university of california, davis, usa have not been studied for the identification of useful agronomical traits, nutritional and physicochemical parameters in nigeria. in addition to fruit yield and improved agronomical traits, the fruit quality and nutritional parameters are essential breeding objectives from a consumer’s point of view and even in the processing industries (bauchet and causse, 2012; bergougnoux, 2014). fruit quality and physicochemical parameters are cultivar dependent (riahi et al, 2009; ilahy et al, 2011; domı́nguez et al, 2012). fertilization, cultural practices and postharvest storage could influence fruit quality and physicochemical parameters (rosales et al, 2011; beckles, 2012). production of quality tomato fruits depends on the climatic conditions, sunlight availability, good agronomic practices and genetic variability among cultivars (causse et al, 2003; peixoto et al, 2018). in order to identify, select and develop novel tomato lines with desired horticultural traits for a nigerian breeding programme, it is crucial to evaluate tomato accessions which are wild to the nigerian germplasm. these could then be subjected to diverse breeding methodologies and agronomic practices (chitarra and chitarra, 2005). knowledge of desirable traits from the evaluated tomato accessions will help to identify those that could be used as parents in a tomato breeding programme, hence, promoting improved nutrition and increased production (causse et al, 2003; álvaro toledo and burlingame, 2006). this study evaluated mean performance and variation among wild tomato accessions based on agronomic, nutritional and physicochemical traits under rainforest agro-ecology in nigeria. materials and methods germplasm the accessions evaluated in this study are from the c.m. rick tomato genetic resource centre (tgrc), university of california, davis, usa (table 1). they are part of the tomato wild relatives core collection of the nihort germplasm bank without previous utilization information that could be included in breeding programmes. nursery and field operations seeds were sown into perforated nursery trays filled with sterilized soil and grown for three weeks in a greenhouse at nihort, ibadan, oyo state (rain forest zone; 3◦ 56’e, 7◦ 33’ n; 168 meters above sea level). the perforated nursery trays were kept moist by regular watering on daily basis at sunrise and sunset with tap water using a watering can. seedlings were transplanted to the field in paired rows in plots that were 2 m long with spacing of 0.5 m between rows and 0.5 m between plants within a row. spacing between plots was 1 m. seedlings were arranged in a randomized complete block design with three replications. n-p-k (15-15-15) fertilizer was applied at the rate of 120 kg/ha three weeks after transplanting. the plants were trellised to prevent lodging and loss of fruits due to diseases and pests. manual weeding was carried out at two-week intervals. to protect the leaves from defoliating pests, plants were sprayed with the pyrethroid insecticide cymbush containing cypermethrin at 2, 6, and 9 weeks after transplanting at the rate of 450 ml of active ingredients per 100 liters of water per hectare using a knapsack sprayer. no disease infestation was observed during the experiment and data were collected on five randomly selected plants per plot. sample preparation uniformly ripe, healthy fruit at the red-ripe stage were harvested (hanson et al, 2004). a total of 1015 representative fruit were collected from pre-tagged plants (from the first 3 clusters) to minimise intra-plant variability (borja et al, 1998). tomato samples (100 g) were homogenized in 50 ml of water in a water bath at 4◦c and low light (to reduce antioxidant loss) for physicochemical analysis. all analyses were done in triplicate for each sample at the product development laboratory of nihort. genetic resources (2020), 1 (2), 1–11 performance of wild tomato accessions in nigeria 3 table 1. description of the 13 species of wild tomato accessions used for the experiment no. accession id species origin (area of collection) 1 la0103 solanum peruvianum (l.) miller cajamarquilla lima, peru 2 la0130 solanum chilense dunal moquegua, peru 3 la0411 solanum pimpinellifolium (l.) miller. pichilingue, los rios, ecuador 4 la1028 solanum chmielewskii (c.m. rick et al.) casinchichua, apurimac, peru 5 la1041 solanum cheesmanii l. riley santa cruz, el cascajo, galapagos islands, ecuador 6 la1136 solanum cheesmanii l. riley gardner st, floreana islet, galapagos islands, ecuador 7 la1208 solanum esculentum var. cerasiforme dunal sierra nevada, colombia 8 la1272 solanum pennellii (correll) d’arcy pesquera, lima, peru 9 la1293 solanum peruvianum f. glandulosum (c.f. mull) matucana, lima, peru 10 la2641 solanum parviflorum (c.m. rick et al.) apurimac, peru 11 la4113 solanum sitiens i.m. johnst. estación ceres, antofagasta chile 12 la4115 solanum sitiens i.m. johnst. quebrada desde cerro, oeste de paqui, antofagasta, chile 13 la4138 solanum pimpinellifolium (l.) miller el corregidor, la molina, lima, peru physicochemical and nutritional analyses total soluble solid (◦brix) (g 100 g−1) of the juice was measured using the eclipse hand-held refractometer [pn# 45-01 (0-15 ◦brix)] and the ph of the fruit juice was measured using a benchtop ph meter (sper scientific benchtop) with the ph meter calibrated with standard buffers ph 4 or 9. for determination of titratable acidity (g 100 g−1) and vitamin c content (mg 100 g−1), 10 ml of juice from 10 fruits was diluted in 100 ml of distilled water and titrated with naoh (0.1 n) to ph 8.2. for vitamin c, the solution was titrated with iodine (0.1 n) until a colour change was observed (international plant genetic resources institute, 1996) . to determine lycopene content (mg 100 g−1), 5 ml of acetone-n-hexane mixture in the ratio 4:6 was added to 0.8 g of tomato pulp for each sample and mixed well. the mix was centrifuged at 5000 rpm for 5 min at 4◦c; the supernatant was extracted and absorbance measured with a spectrophotometer (model 6400, jenway) at 503 nm using the acetone-n-hexane mix as blank (rosales et al, 2006). lycopene content was calculated using an extinction coefficient (e%) of 3150. agronomic data collection data were collected on the following traits: number of leaves at maturity (nlm), plant height at maturity (ph), number of clusters per plant (ncp), number of fruits per cluster (nfc), fruit weight (fw), fruit length (fl), fruit circumference (fc), number of fruits per plant (nfp) and fruit size index (fsi). fruit yield of tomato was adjusted to t ha−1using the following formula: fruit yield (t ha−1) = fruit yield per plot (kg) x 10,000 / plot area (m2) x 1,000. statistical analysis the data was subjected to analysis of variance (anova) using proc glm in sas (sas institute, 2010) . means were separated using fisher’s least significant difference (lsd) test (p < 0.05). a rank summation index (rsi) (mulumba and mock, 1978) was constructed to create the aggregate trait by ranking accessions with regard to high fruit weight, fruit yield, improved agronomics, nutritional and physicochemical traits. ranks were summed for each accession to select the top five. pearson’s correlation analysis was done to determine associations among all traits measured with sas. hierarchical cluster analysis was performed using sas proc cluster based on centroid distance and a dendrogram constructed by proc tree in sas to identify divergent groups. to identify patterns of morphological variation, principal component analysis (pca) was conducted. those pcs with eigen values >1 were selected (jeffers, 1967). the pca analysis reduces dimensions of a multivariate data to a few principal axes, generates an eigen vector for each axis and produces component scores for characters (sneath and sokal, 1973). results the anova produced significant mean squares for all agronomic, nutritional and physicochemical traits of the tomato accessions indicating genetic variations for all measured traits (table 2). the coefficient of variation (cv) used to measure the precision of the experiment indicated the data was reliable (table 2). phenotypic variation in the biological growth stages of tomato accessions revealed that la4113 was tallest and la2641 shortest (table 3). the most fruits per cluster were observed for accession la0103 and least for la1041. the most fruits per plant were observed for accession la0411, the least for la4138. for fruit yield related 4 ibitoye et al genetic resources (2020), 1 (2), 1–11 table 2. mean squares from analysis of variance of agronomic, nutritional andphysicochemical traits of wild tomato accessions source df nlm ph (cm) ncp nfc fw (g) fruit yield (t/ha) fl (cm) fc (cm replication 2 752.03 65.89 4.58 31.57∗∗ 57571.79∗∗ 1.44∗∗ 0.19∗ 0.15 accessions 12 3254.65∗∗∗ 148.54∗∗∗ 31.89∗∗ 28.52∗∗∗ 24458.12∗ 0.61∗ 0.37∗∗∗ 0.65∗∗∗ error 24 408.16 37.18 10.53 4.80 11010.68 0.28 0.05 0.11 cv 17.77 13.67 33.11 29.22 68.32 68.32 13.20 19.79 source df nfp fsi vit c (mg/100 g) tss (obrix) (g/100 g) ta (g/100g) fruit juice ph lycopene (mg/100 g) replication 2 1381.87 0.02 2.3 0.0004 0.0003 0.003 0.51 accessions 12 4605.24∗∗∗ 0.08∗∗∗ 147.40∗∗∗ 0.51∗∗∗ 0.17∗∗∗ 0.08∗∗∗ 106.12∗∗∗ error 24 841.79 0.02 2.54 0.03 0.002 0.002 0.31 cv 42.25 12.37 4.94 4.14 4.74 0.95 2.38 *, **, *** significant at 0.05, 0.01 and 0.001 probability levels, respectively. cv = coefficient of variation, nlm = number of leaves at maturity, ph = plant height at maturity, ncp = number of cluster per plant, nfc = number of fruits per cluster, fw = fruit weight, fl = fruit length, fc = fruit circumference, nfp = number of fruits per plant, fsi = fruit size index, vit c = vitamin c, tss = total soluble solids, ta = titratable acidity. traits, tomato accession la0130 had the heaviest fruit and most fruit yield, la1293 had the lowest fruit weight and least fruit yield. accession la0411 had the highest concentration of vitamin c. la1028 had the highest levels of total soluble solids while accessions la4113, la4138, la1041 had the lowest levels. accessions la1208 and la4133 had the lowest titratable acidity, la2641, la1293 and la0130 had the lowest fruit juice ph. lycopene content was highest in tomato accession la0130 and lowest in la1208 (table 3). based on a rank summation index (rsi) of 13 accessions, la0130 was identified as best performing among all tested accessions, with the best fruit yield performance, desirable agronomic, nutritional and physicochemical traits. tomato accession la0130 was characterized by moderate plant height, highest number of fruits per cluster, fruit weight and fruit yield (table 3). accession la0130 also had the highest titratable acidity and lycopene content, but moderate fruit juice ph (table 4). pearson’s correlation coefficient was calculated to determine associations among traits and showed variation for some trait combinations (table 5). fruit yield was significantly positively correlated with fruit weight coupled with a significant negative correlation with number of leaves at maturity. fruit size index was significantly negatively correlated with fruit circumference and significantly positively correlated with vitamin c content. total soluble solid was significantly positively correlated with number of leaves at maturity and vitamin c content. titratable acidity was significantly positively correlated with number of fruit per plants and total soluble solid. fruit juice ph was significantly negatively correlated with number of fruit per plant, total soluble solid and titratable acidity. lycopene content was positively significantly correlated with vitamin c content and titratable acidity. all agronomic traits, nutritional and physicochemical parameters measured which showed significant variations were adopted to construct a hierarchical cluster based on the centroid distances among the 13 tomato accessions as in figure 1. cluster analysis differentiated the accessions into 4 distinct groups, where la0130 differed from the other three groups. cluster i consisted of five accessions, cluster ii had four accessions, and clusters iii and cluster iv had three and one accessions, respectively indicating variation among the accessions. additionally, contribution of each measured trait to the total variation within the accession was further determined through principal component analysis (pca) based on correlation matrix of the variables. the scree plot of the pca indicated six eigenvalues corresponding to the entire percent variance with eigenvalues >1. pca1 accounted for about 22 % of variation, pca2 for 19 %, pca3 for 15 %, pca4 for 11 %, pca5 for 10 % and pca6 for 6 % (table 6). the first principal component axis (pca1) was mainly loaded positively by fruit yield, fruit yield related traits and titratable acidity. in pca2 traits which had positive contribution were number of leaves at maturity, plant height at maturity, titratable acidity and lycopene. fruit length, fruit circumference, total soluble solids and lycopene had positive contributions in pca3. in pca4 plant height at maturity, number of clusters per plant, fruit weight, fruit yield and fruit juice ph had positive contributions were. in pca5 number of clusters per plant, number of fruits per cluster, fruit length, fruit circumference and total soluble solids had positive contributions. in pca6 traits which had positive contributions were fruit length, fruit size index and vitamin c content. discussion significant phenotypic variations among the accessions for all agronomic, nutritional and physicochemical traits validate availability of genetic diversity in the collection from the c.m. rick tomato genetics genetic resources (2020), 1 (2), 1–11 performance of wild tomato accessions in nigeria 5 table 3. mean ranking of agronomic traits of wild tomato accessions evaluated under the rainforest agro-ecology zone in nigeria s/n accession ph (cm) nfc nfp fw (g) fruit yield (t/ha) nlm ncp fl (cm) fc (cm) fsi rsi 1 la0130 44.14 11.72 95.42 380.00 1.90 99.67 9.72 2.30 2.05 1.15 30 2 la0411 52.97 8.64 143.78 113.33 0.57 137.67 9.78 1.22 1.17 1.05 51 3 la1136 45.22 8.50 79.33 236.67 1.18 109.67 8.44 1.33 1.20 1.13 63 4 la2641 29.64 10.94 106.72 140.00 0.70 96.00 7.28 1.67 1.40 1.19 65 5 la0103 48.39 10.20 86.23 143.33 0.72 108.00 18.39 1.85 1.90 0.98 67 6 la1272 35.96 9.39 94.50 156.67 0.78 107.00 7.73 1.60 1.90 0.86 69 7 la1208 39.74 4.28 28.95 220.00 1.10 49.00 8.55 1.50 2.27 0.67 71 8 la1028 46.29 5.67 28.83 93.33 0.47 171.67 8.89 2.35 2.79 0.84 79 9 la1293 50.92 6.34 95.72 40.00 0.20 145.00 10.11 1.38 1.45 0.99 80 10 la4115 44.96 3.95 27.94 200.00 1.00 138.33 9.39 1.58 1.67 0.95 83 11 la4138 40.85 3.67 20.72 90.00 0.45 138.33 7.16 1.78 1.37 1.31 83 12 la1041 44.91 3.33 30.50 130.00 0.65 68.00 7.17 1.71 1.69 1.02 84 13 la4113 56.00 10.89 53.98 53.33 0.27 110.00 14.78 1.32 1.40 0.95 85 minimum 17.92 2.67 11.25 10.00 0.05 34.00 5.83 1.20 1.00 0.66 maximum 62.08 15.83 205.83 500.00 2.50 176.00 29.17 2.41 2.90 1.46 mean of top 5 44.07 10.00 102.30 202.67 1.01 110.20 10.72 1.67 1.54 1.10 grand mean 44.61 7.50 68.66 153.59 0.77 113.72 9.80 1.66 1.71 1.01 sel. differential (%) -1.22 33.32 48.98 31.95 31.95 -3.09 9.41 0.78 -9.83 9.40 lsd (5 %) 10.28 3.69 48.89 176.83 0.88 34.05 5.47 0.37 0.57 0.21 ph = plant height at maturity, nfc = number of fruits per cluster, nfp = number of fruits per plant, fw = fruit weight, nlm = number of leaves at maturity ncp = number of cluster per plant, fl = fruit length, fc = fruit circumference, fsi = fruit size index. rsi = rank summation index. sel. differential = selection differential is estimated as a proportion (%) of mean of all accessions table 4. ranking of the mean performance of nutritional and physicochemical parameters of wild tomato accessions evaluated under the rainforest agro-ecology zone in nigeria s/n accession vit c (mg/100 g) tss (obrix) ta (g/100 g) fruit juice ph lycopene (mg/100 g) rsi 1 la0130 42.46 4.50 1.38 4.85 32.54 30 2 la0411 46.28 4.50 1.26 4.9 31.02 51 3 la1136 29.21 3.75 0.76 5.3 24.42 63 4 la2641 32.07 3.88 0.98 4.8 15.11 65 5 la0103 33.63 3.75 0.74 5.15 21.57 67 6 la1272 21.02 3.75 0.95 5.00 16.13 69 7 la1208 26.01 4.00 0.63 5.30 13.43 71 8 la1028 34.56 4.85 0.82 5.05 22.16 79 9 la1293 25.53 4.50 1.31 4.85 26.16 80 10 la4115 25.38 3.75 1.02 5.10 27.65 83 11 la4138 40.21 3.50 0.70 5.05 26.51 83 12 la1041 31.38 3.50 0.85 5.30 31.24 84 13 la4113 30.64 3.50 0.69 5.15 18.63 85 minimum 20.90 3.25 0.6 4.80 12.64 maximum 46.29 5.20 1.39 5.30 32.81 mean of top 5 36.73 4.08 1.02 5.00 24.93 grand mean 32.18 3.98 0.93 5.06 23.58 sel. differential (%) 14.13 2.42 10.01 -1.22 5.73 lsd (5 %) 2.95 0.31 0.08 0.09 1.04 vit c = vitamin c, tss = total soluble solids, ta = titratable acidity. rsi = rank summation index. sel. differential = selection differential is estimated as a proportion (%) of mean of all accessions 6 ibitoye et al genetic resources (2020), 1 (2), 1–11 table 5. pearson’s correlation coefficient (r) of agronomic, nutritional and physicochemical traits of wild tomato accessions evaluated under a rainforest agro-ecology in nigeria nlm fw (g) fruit yield (t/ha) fc (cm) nfp fsi vit c (mg/100 g) tss (obrix) ta (g/100 g) ph fw -0.32∗ fruit yield -0.32∗ 1.00∗∗∗ fc 0.03 0.01 0.01 nfp -0.05 0.18 0.18 -0.34∗ fsi 0.18 0.05 0.05 -0.60∗∗∗ 0.18 vit c 0.19 0.08 0.08 -0.07 0.22 0.40∗ tss 0.37∗ 0.08 0.08 0.30 0.30 -0.10 0.38∗ ta 0.24 0.15 0.15 -0.08 0.50∗∗∗ 0.18 0.32 0.66∗∗∗ ph -0.35∗ 0.04 0.04 0.03 -0.49∗∗∗ -0.21 -0.31 -0.51∗∗∗ -0.76∗∗∗ lycopene 0.27 0.15 0.15 -0.14 0.10 0.35∗ 0.58∗∗∗ 0.32∗ 0.57∗∗∗ -0.14 *, *** significant at 0.05 and 0.001 probability levels, respectively. nlm = number of leaves at maturity, fw = fruit weight, fc = fruit circumference, nfp = number of fruits per plant, fsi = fruit size index, vit c = vitamin c, tss = total soluble solids, ta = titratable acidity, ph = fruit juice ph. table 6. eigenvalue, proportion of variability and estimated traits of wild tomato accessions contributing to first six principal components traits pc1 pc2 pc3 pc4 pc5 pc6 nlm -0.08 0.31 0.20 -0.04 0.00 -0.07 ph (cm) -0.16 0.34 0.06 0.32 -0.07 -0.20 ncp -0.08 0.18 -0.12 0.48 0.21 0.13 nfc 0.29 0.07 -0.28 0.12 0.24 0.15 fw (g) 0.37 -0.26 0.06 0.27 -0.06 -0.08 fruit yield (t/ha) 0.37 -0.2 0.06 0.27 -0.06 -0.08 fl (cm) 0.03 -0.05 0.41 0.07 0.30 0.51 fc (cm) -0.09 -0.13 0.32 0.09 0.54 0.09 nfp 0.32 0.22 -0.21 -0.05 0.06 -0.08 fsi 0.14 0.11 0.03 -0.10 -0.48 0.53 vit c (mg/100 g) 0.16 0.25 0.22 0.16 -0.16 0.33 tss (obrix) 0.19 0.21 0.31 -0.11 0.24 -0.29 ta (g/100 g) 0.30 0.26 0.20 -0.18 0.02 -0.24 fruit juice ph -0.24 -0.27 -0.10 0.29 -0.17 -0.08 lycopene (mg/100 g) 0.13 0.19 0.34 0.12 -0.34 -0.11 eigenvalue 4.15 3.68 2.82 2.12 1.98 1.08 proportion (%) 22 19 15 11 10 6 cumulative (%) 21 41 56 67 78 83 nlm = number of leaves at maturity, ph = plant height at maturity, ncp = number of cluster per plant, nfc = number of fruits per cluster, fw = fruit weight, fl = fruit length, fc = fruit circumference, nfp = number of fruits per plant, fsi = fruit size index, vit c = vitamin c, tss = total soluble solids, ta = titratable acidity. genetic resources (2020), 1 (2), 1–11 performance of wild tomato accessions in nigeria 7 figure 1. dendrogram of 13 wild tomato accessions based onagronomic traits, nutritional and physicochemical parameters generated by centroid hierarchical cluster analysis resource center (chetelat, 2004, 2006). previous research reported significant variations for agronomic traits for cultivated tomato varieties grown in various environments in africa (chernet and zibelo, 2014; shiberu, 2016; regassa et al, 2016). this study indicates a wealth of genetic variability for fruit quality traits of wild tomato accessions. accessions la0411 and la2641 had the highest number of fruit per plant which could be ascribed to genetic variation in flower abortion (kanneh et al, 2017). numbers of fruit per plant from this study were higher than the values reported by ceballosaguirre and vallejo-cabrera (2012), but similar with the report of agong et al (2001). the mean performances for fruits per cluster and fruit weight in our study were higher than the results presented by ceballosaguirre and vallejo-cabrera (2012) who worked on wild tomato accessions from the tomato genetics resources center (tgrc), university of californiadavis. disparities in the results from this study may be due to difference in the accessions evaluated, number of days before transplanting, agronomic practices used, and the environment. important quality traits that determine flavor, shelf life and market-related attributes of tomato are total soluble solids, fruit juice ph, titratable acidity, lycopene and vitamin c content. the quality of tomato fruit for industrial processing and paste production depends on a high value of total soluble solids. total soluble solids recorded in this present study ranged from 3 to 5 ºbrix which is comparable to the minimum value of total soluble solids (4.5 ºbrix) reported by campos et al (2006) but considered low for industrial tomatoes. previous studies have reported a range from 4 to 6 ºbrix for total soluble solids of tomato fruits (alcántar et al, 1999; cramer et al, 2001; pascale et al, 2001). high total soluble solid increases tomato paste efficiency and must be between 5.0 and 6.5 % in industrial tomatoes (teka, 2013). the range of 4.80 – 5.30 for tomato fruit juice ph reported in this study is considerably high. tomato fruit juice ph values can vary from 4.25 to 4.78 and fruits with high ph values may not be recommended for fresh tomato consumption or industrial processing (paulson and stevens, 1974; anthon et al, 2011; rajae et al, 2018). a ph below 4.50 is desirable because it reduces proliferation of microorganisms and indicates quality (mohammed et al, 1999; tigist et al, 2013). however, the ph of ripe tomatoes may exceed 4.50 because a higher ph value is associated with flavor (stevens, 1972). titratable acidity in this study was higher than previously reported (george et al, 2004; tigist et al, 2013; rajae et al, 2018). tomatoes are considered the main source of lycopene compounds and a major source of carotenoids in the human diet (willcox et al, 2003). lycopene imparts the red color to tomato and affects quality. the range for lycopene content reported in the literature is between 0.58-6.50 mg 100 g−1 (rickman et al, 2007; saha et al, 2010), which is lower than reported in this study. the vitamin c concentrations reported in this study for all accessions were higher than reported by aoun et al (2013), but consistent with the range reported by franke et al (2004) and saha et al (2010). our results show that wild tomato accessions contain significant antioxidants and may be useful for nutritional improvement in tomato breeding programmes (tigchelaar, 1986) . all fruit quality and nutritional traits measured in this study reveal the value of the wild tomato accession as a source of useful alleles and their utilization as interesting donor parents in cultivar development. selection of the top outstanding five accessions with rsi may be useful as donor parent through intra and interspecific hybridization (ghani et al, 2020) and may result in a significant increase in tomato fruit weight and fruit yield. this gain in fruit weight and yield could also be associated with improvement in number of fruits per cluster, number of fruits per plant, titratable acidity, lycopene content and fruit juice ph. to improve breeding efficiency and selection indices in crop improvement, knowledge about correlation among traits is essential (nzuve et al, 2014). results from pearson’s correlation coefficients indicate that as tomato fruit yield increases, number of leaves at maturity decreases significantly. selection based on fruit weight and reduction in number of leaves at maturity could lead to tomato fruit yield improvement. there were significant negative correlations between fruit juice ph and titratable acidity. this implies that increased fruit juice ph was accompanied by a decrease in titratable acidity and acid concentrations and is associated with maturity (teka, 2013). significant positive correlations between total soluble solids and titratable acidity in this study corroborate findings of aoun et al (2013), and also indicated that plants with high sugar content have more free organic acids than plants with low sugar content (saliba-colombani et al, 2001; georgelis, 2002; getinet et al, 2008). with positive correlations, genes controlling these traits could be linked to, or be under control of, pleiotropic effects (boćanski et al, 2009). positive and/or negative desirable relationships among 8 ibitoye et al genetic resources (2020), 1 (2), 1–11 some agronomic, nutritional and physicochemical traits indicate that desirable genes in these wild accessions could be exploited in further breeding activities for cultivar improvement (sujiprihati et al, 2003). furthermore, the wild tomato accessions were arranged in 4 clusters; with cluster iv appearing as the most phenotypically diverse. the best performing accession la0130 in cluster iv has the highest number of fruits per cluster; highest fruit weight, fruit yield, vitamin c concentration, lycopene content, and moderate total soluble solids, titratable acidity and fruit juice ph. this accession might harbor novel traits that are lacking in cultivated tomato and may be used as potential parent in tomato breeding to develop high yielding cultivars with desirable nutritional and physicochemical traits. the eigenvalue from pca indicates importance of each principal component axis and its contribution to variability in traits of the tomato accessions. fruit size index and vitamin c concentration play a role in explaining the variation but are less important than the first four factors. conclusion this study identifies variability among the 13 wild tomato accessions evaluated. accession la0130 was outstanding for its unique attributes which included high number of fruits per cluster, fruit weight, fruit yield, total soluble solids, titratable acidity and lycopene content amongst others. thus, this wild tomato accession may be considered promising to broaden the genetic variability for tomato improvement programmes. consequently, this accession may be incorporated into the tomato breeding programme in the national institutes and could be used in hybridization for developing lines with desirable horticultural traits. documentation of the agronomic, nutritional and physicochemical performance of the evaluated wild tomato accessions is informative for their utilization in breeding programmes. these results are useful for breeders working on the development and improvement of tomato, as desirable traits from these wild tomatoes can be transferred into the commercial tomato varieties suitable for the growth conditions in the rainforest agro-ecology zone of nigeria and to boost production and diversity. conflicts of interest: the authors declare no conflict of interest. acknowledgements the authors appreciate mrs. bola bello for providing technical support during planting, field management, data collection and tomato harvesting. author 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(2009). classification and phylogenetic relationships in solanum section lycopersicon based on aflp and two nuclear gene sequences. genetic resources and crop evolution 56(5), 663–678. url: https://dx.doi.org/ 10.1007/s10722-008-9392-0. doi: 10.1007/s10722008-9392-0 https://dx.doi.org/10.1002/jsfa.3720 https://dx.doi.org/10.1002/jsfa.3720 https://dx.doi.org/10.2307/2484764 https://dx.doi.org/10.1002/jsfa.2825 https://dx.doi.org/10.1002/jsfa.2825 https://dx.doi.org/10.1002/jsfa.4166 https://dx.doi.org/10.1002/jsfa.2546 https://dx.doi.org/10.1002/jsfa.2546 https://dx.doi.org/10.1002/jsfa.3802 https://dx.doi.org/10.1007/s001220051643 https://support.sas.com/documentation/93/ https://support.sas.com/documentation/93/ https://dx.doi.org/10.1080/10408690390826437 https://dx.doi.org/10.1080/10408690390826437 https://dx.doi.org/10.1007/s10722-008-9392-0 https://dx.doi.org/10.1007/s10722-008-9392-0 https://doi.org/10.3923/ajps.2003.51.57 https://doi.org/10.1007/s13197-011-0378-0 https://doi.org/10.1007/s13197-011-0378-0 https://doi.org/10.20431/2454-6224.0203001 introduction materials and methods germplasm nursery and field operations sample preparation physicochemical and nutritional analyses agronomic data collection statistical analysis results discussion conclusion conflicts of interest: author contributions original article genetic resources (2020), 1 (2), 29–41 doi: 10.46265/genresj.bnhb8715 https://www.genresj.org issn: 2708-3764 robust microsatellite markers for hybrid analysis between domesticated pigs and wild boar donovan anderson*,a, yuki negishi a, rio toma a, junco nagata b, hidetoshi tamate c and shingo kaneko a,d a symbiotic systems science and technology, fukushima university, fukushima city, fukushima, japan b forestry and forest products research institute, ibaraki, tsukuba, japan c department of biology, yamagata university, yamagata city, yamagata, japan d institute of environmental radioactivity, fukushima university, fukushima city, fukushima, japan abstract: hybridization between wild boar (sus scrofa) and their domestic relative, pigs, is a global issue and gene flow between these populations has been known to negatively impact biodiversity with increased aggression, litter sizes, and growth. however, establishing a cost-effective analysis for long-term monitoring of possible gene flow of wild pigs into wild boar populations is challenging due to common alleles at multiple loci and often it is difficult to distinguish boar specific lineages. therefore, there is a need to select loci with lineage specific alleles for hybrid detection. to determine these loci, we calculated allele frequencies and polymorphism measurements from successfully amplified microsatellite loci with dna extracted from domestic pigs and wild boar populations from the period prior to, and after, the evacuations and disasters in fukushima, japan, in 2011, which resulted in an uncontrolled release of domestic pigs. thirty-two microsatellite loci showed pig putative alleles suggesting these selected loci can be useful genetic markers. seventeen loci successfully distinguished pig and wild boar hybridization in fukushima populations. identified loci from this study provide a cost-efficient tool for genetic analysis and will provide a wealth of information on how an uncontrolled release of domestic livestock from natural or anthropogenic disasters may impact their wild relatives. keywords: microsatellite, hybridization, alleles, pigs, polymorphism citation: anderson, d., negishi, y., toma, r., nagata, j., tamate, h., kaneko, s. (2020). robust microsatellite markers for hybrid analysis between domesticated pigs and wild boar. genetic resources 1 (2), 29–41. doi: 10.46265/genresj.bnhb8715. © copyright 2020 the authors. this is an open access article distributed under the terms of the creative commons attribution license (cc by 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. introduction hybridization between wild species and their domesticated relatives has been detected in multiple environments across the globe (pierpaoli et al, 2003; godinho et al, 2011; goedbloed et al, 2013a). invasive species and hybridized individuals compete with native populations, and cause negative impacts to biodiversity (rhymer and simberloff, 1996; randi, 2008; harrison and larson, 2014). invasive pigs are known to successfully disperse in wild environments and cause considerable impact on the gene pool of native wild boar populations (vernesi et al, 2003; koutsogiannouli et al, 2010; goedbloed et al, 2013b). multiple countries have implemented management programs to reduce wild boar population expansion (waithman et al, 1999; scandura et al, 2008; saito et al, 2011), but hybrid individuals may have increased litter sizes, aggression, and growth rates (goedbloed et al, 2013b; dzialuk et al, 2018). areas of suspected hybridization between invasive pigs and wild boar populations should be continuously monitored to understand the extent of introgression of pig genes in the wild boar gene pool. microsatellite marker analysis is a well-established monitoring tool to evaluate possible introgression of invasive species and hybridization detection (nijman et al, 2003; randi, 2008; uemura et al, 2018). the selection of reliable microsatellite markers by optimizing amplification protocols prior to monitoring a target population is of great importance because it has consequences for subsequent genotyping (hoffman and amos, 2005; kolodziej et al, 2012). however, received: 22.06.2020 accepted: 30.11.2020 published online: 29.12.2020 ∗corresponding author: donovan anderson (s1871003@ipc.fukushima-u.ac.jp) https://www.genresj.org https://www.dx.doi.org/10.46265/genresj.bnhb8715 https://www.genresj.org https://www.dx.doi.org/10.46265/genresj.bnhb8715 mailto:s1871003@ipc.fukushima-u.ac.jp 30 anderson et al genetic resources (2020), 1 (2), 29–41 genotyping hybrid individuals (e.g. crossed pig and boar) can be challenging due to common or shared alleles at multiple loci (larson et al, 2005; grossi et al, 2006; choi et al, 2014) and it is necessary to select suitable loci with lineage specific alleles for hybridization detection. validating appropriate microsatellite markers with possible hybridized wild boar and the pigs involved in the hybridization will provide the necessary genetic composition data to develop a cost-efficient monitoring tool to evaluate the introgression of pig genes to the wild boar gene pool. such cost-efficient analyses have provided monitoring opportunities to estimate abundancy of hybrids (qi et al, 2010; matsumoto et al, 2019), population characteristics (goedbloed et al, 2013a; sharma et al, 2013) and local genetic structures (tadano et al, 2016; touma et al, 2020) in animals. genetic diversity and ancestry of wild boar have been well studied, including areas of south east asia, and information from hybridization occurrences with domestic pigs is of increasing interest due to possible genetic alterations (choi et al, 2014; todesco et al, 2016). wild boar populations inhabiting fukushima prefecture, in japan, are suggested to be threatened by hybridization following the uncontrolled release of domesticated pigs after mandated evacuations due to the fukushima nuclear disasters in 2011 (okuda et al, 2018; anderson et al, 2019). additionally, hybridization in this area has not altered the morphological characteristics of wild boar (anderson et al, 2019) and possible hybrids can only be detected using dna. thus, estimating appropriate genotypes of wild boar from the period prior to 2011, after 2011, and from domestic pigs in this area, with microsatellite markers will provide an important source of information for better understanding hybridization effects with native species following such events. adequate selection of microsatellite markers from this area will establish a cost-efficient tool to easily distinguish if a wild boar population has been impacted by hybridization. in this study, we selected robust microsatellite markers used in european and asian pig studies (rohrer et al, 1994; krause et al, 2002; karlskov-mortensen et al, 2007; fao, 2011) that could differentiate wild boar or pig alleles. our goal for this study was two-fold: first, we aimed to select useful microsatellite markers for hybrid analysis between domesticated pig and wild boar populations; and second, to use these loci to perform a preliminary check of the introgression of pig alleles into wild boar populations in fukushima prefecture following the disasters in 2011. materials and methods analysed samples and dna extraction thirty-one muscle tissue samples were collected from three populations (hereafter referred to as pop1, pop2 and pop3) and were selected based on mitochondrial dna (mtdna) haplotype and year sampled. sample haplotype and date were prioritized for optimal determination of reliable microsatellite screening of hybridization between wild boar and domesticated pigs after the fukushima disasters in 2011. pop1 samples were from 10 unrelated domestic pigs (sus scrofa domesticus) that were collected from a local pig slaughterhouse or local markets within fukushima prefecture in 2016-2017. pop2 samples were from 13 wild boar (sus scrofa) muscle samples that were collected in 20032004, prior to the evacuations and fukushima disasters, from a wild boar population in northern ibaraki prefecture, south of fukushima prefecture. the mtdna analysis has shown that this population is the same haplotype (d42172) and has extremely high genetic similarity to the wild boar population in eastern fukushima prefecture (nagata et al, 2006). pop3 samples were collected in 2015-2016, after the fukushima disasters, from eight suggested hybridized wild boar that had a typical mtdna haplotype of pig (suggested pig ancestor in maternal lineage; mk801664, see anderson et al (2019)). all animals were legally culled by licensed hunters, and this entire study was approved by fukushima university’s institutional animal care and use committee. all experiments were performed in accordance with relevant guidelines and regulations. all samples were stored individually at −20 ◦c in 99.5% ethanol until extraction. total genomic dna was extracted from muscle tissue using the gentra puregene tissue kit (qiagen), according to manufacturer’s instructions. microsatellite loci genotyping a total of 52 unlinked microsatellite loci were selected from previously developed phage libraries (rohrer et al, 1994; krause et al, 2002; karlskovmortensen et al, 2007) and recommended microsatellite markers from the food and agriculture organisation of the united nations database (fao, 2011) and screened for amplification success on all 31 samples. pcr amplification was performed in 5 µl reactions using the qiagen multiplex pcr kit (qiagen) and a protocol for fluorescent dye-label (blacket et al, 2012). each sample reaction contained 10 to 20 ng of genomic template dna, 2.5 µl of multiplex pcr master mix, 0.1 µm of forward primer, 0.2 µm of reverse primer, and 0.1 µm of fluorescently labeled primer. amplification conditions consisted of 95 ◦c for 15 minutes followed by 33 cycles of denaturation at 94 ◦c for 30 seconds, annealing at 57 ◦c for 1.5 minutes, and extension at 72 ◦c for 1 minute and an extension at 60 ◦c for 30 minutes. all thermal cycling conditions used in t100 thermal cycler (bio-rad laboratories, inc., hercules, ca, usa). product sizes were determined using an abi prism 3130 genetic analyzer and genemapper software (applied biosystems, foster city, ca, usa). characterization of microsatellite markers successful markers were identified after our initial screening by clear peak patterns following amplifications. number of alleles (na), observed heterozygosity genetic resources (2020), 1 (2), 29–41 markers for pig and wild boar hybridization 31 table 1. characteristics of 32 microsatellite markers selected. ta = annealing temperature locus primer sequence (5’ → 3’) forward/reverse repeat motif range of alleles (bp) ta (◦c) fluorescent label genebank accession no. sw632 tgggttgaaagatttcccaa (ac)21 115-138 55 fam af225099 ggagtcagtactttggcttga s0090 ccaagactgccttgtaggtgaata (ac)24 227-253 55 fam m95002 gctatcaagtattgtaccattagg sw24 ctttgggtggagtgtgtgc (gt)13 99-135 55 vic af235245 atccaaatgctgcaagcg swr1941 agaaagcaatttgatttgcataatc (tg)20 215-255 55 vic af253904 acaaggacctactgtatagcacagg sw857 tgagaggtcagttacagaagacc (ca)22 165-187 55 ned af225105 gatcctcctccaaatcccat s0228 ggcataggctggcagcaaca (ac)17 93-112 55 pet l29195 agcccacctcatcttatctacact sw2008 caggccagagtagcgtgc (gt)25 148-170 55 fam af253773 cagtcctcccaaaaataacatg sw240 agaaattagtgcctcaaattgg (tg)17 164-186 55 vic af235246 aaaccattaagtccctagcaaa s0097 gacctatctaatgtcattatagt (ac)28 135-155 55 ned m95020 ttcctcctagagttgacaaactt umnp147 gccttcgttacatggcattc (gt)23 151-167 58 pet af511119 tctctgtgaggtcatggtgg umnp239 cttacaaaaccaccaccatcg (ac)18 96-112 60 fam af511146 tcaatatcaacattgcgtgttg umnp296 cagggaactctcttcaatatcc (tg)13 151-181 58 ned af511184 acatttgatttccaaagttgtg umnp298 gctataagaaccgcctcattg (gt)22 157-169 58 ned af511185 tgtgtgctgctgaagcatg umnp351 tcagtgtcacccctcatcac (ac)15 143-169 58 fam af511222 tctccttgaccttctaagcacc umnp358 aagtcatttcacacctctgtgc (ca)22 160-176 58 vic af511230 cgttgcagttactattccaagc umnp362 gatgtgtagctgatttgcaatg (ac)21 125-135 60 pet af511231 gacaagaatctgaaaaggagcg umnp381 ccgattagacccctagtctgg (ac)22 169-185 60 ned af511244 continued on next page 32 anderson et al genetic resources (2020), 1 (2), 29–41 table 1 continued locus primer sequence (5’ → 3’) forward/reverse repeat motif range of alleles (bp) ta (◦c) fluorescent label genebank accession no. cagattagcgttccctgtttg umnp405 cagagttcacctctccctttac (ac)21 148-162 62 vic af511255 tccttgctgagtcccagg umnp442 atccaagctgctgaagttgg (tg)12 122-124 60 ned af511283 aaacatttccacaagaaaatgg umnp453 tcattctctatctcaagatgcatg (ac)17 122-140 58 pet af511291 ctgaggtacctttgcctagagg umnp480 agtgatttctgcccaggatg (tg)21 143-155 58 vic af511308 cctaggaatttccctctgcc umnp485 cctcaggctcagctctgc (tg)17 187-213 57 pet af511313 gttgtccgtgagtccctagc umnp489 aagcaccataggagaagactgg (ac)12 115-141 60 pet af511317 ctcggaagcaagtaagtggg umnp494 ctgcctgattggcacattag (ac)23 114-142 60 fam af511320 ggtaatgggaaagcctagcc umnp500 tgaggctatcacctgcagtg (ag)24 229-251 60 fam af511324 gactgaacccttaacagatggg umnp502 tggcaaacgttgctttagg (gt)22 164-172 60 vic af511325 tagggaaatatctgaaatctaaaatg umnp509 aaactacatccattctcttggg (gt)21 138-164 60 fam af511328 gttgtgccagttacacttctgc umnp511 gatcactgtgtgagtgcatgc (gt)14 107-117 60 vic af511329 aacagagttccattttgcgg umnp539 caacgttgctgtggctgtag (ca)32 171-181 60 ned af511346 ttctggtttatggttcccatg umnp548 tccaagttagactgcctgcc (ca)14 172-180 60 ned af511353 actgctgcttatttctcaaggg umnp610 ctttggctcaatctcattcatg (ac)33 168-178 60 vic af511389 tgggcttttgaaaatttaaatg umnp640 tatgccatgtgcgtggtc (ac)13 123-145 60 fam af511399 acaaactgcaccacagaatagc genetic resources (2020), 1 (2), 29–41 markers for pig and wild boar hybridization 33 (ho), and expected heterozygosity (he) were calculated using genaiex version 6.5 (peakall and smouse, 2012) for successfully amplified loci. calculation of inbreeding coefficients (fis) and test of deviation from hardy–weinberg equilibrium (hwe) for polymorphic loci were tested using fstat version 2.9.3 (goudet, 1995). allele frequency in each locus for pop1, pop2, and pop3 was calculated using genalex version 6.41 (peakall and smouse, 2006). genetic differentiation among pop1, pop2, and pop3 (i.e. differentiation among pig, wild boar, and hybrids) was evaluated using amova, calculating pairwise codominant genotypic distances (smouse and peakall, 1999), and performing principal coordinates analysis (pcoa) using genalex version 6.41 (peakall and smouse, 2006). results of the initial 52 microsatellite loci selected, 32 loci were successfully amplified with all wild boar and pig samples. marker information is provided in table 1. twenty loci were eliminated based on low amplification success or unclear peak patterns in wild boar dna samples collected from pop2 and pop3. polymorphism measurements for the 32 amplified microsatellite loci in pop1, pop2, and pop3 are summarized in table 2. for pop1, ho and he per locus ranged from 0.10 to 1.00 (mean, 0.64) and from 0.10 to 0.82 (mean, 0.65), respectively. the range of fis was -0.46 to 0.47 (mean, 0). for pop2, ho and he per locus ranged from 0.00 to 0.77 (mean, 0.36) and from 0.00 to 0.73 (mean, 0.39), respectively. the range of fis was -0.28 to 0.85 (mean, 0.07). for pop3, the ho and he per locus ranged from 0.00 to 0.88 (mean, 0.45) and from 0.00 to 0.76 (mean, 0.44), respectively. the range of fis was -0.62 to 1.00 (mean, -0.01). all 32 loci showed no evidence of significant deviation from hwe (p > 0.05). in total, 231 putative alleles were identified that ranged from 1 to 8 per locus (mean, 4), as outlined in table 2 (no. alleles). the mean number of alleles was 5.1, 3.2, and 3.3 for pop1, pop2, and pop3, respectively. of the 231 alleles, 52 (23%) were putative to the wild boar populations and 95 (41%) were putative to domestic pigs (table 3). additionally, 68 (30%) alleles were shared by pigs and one of the wild boar populations or by all three populations. among the 68 shared alleles, 21 were shared by pigs and the wild boar population from the period after the fukushima disasters in 2011 (bolded alleles in table 4), indicating introgression of pig genes into the wild boar gene pool. the allele frequencies of amplified microsatellite markers, including those that distinguished these shared alleles between pop1 and pop3, are provided in table 4. amova suggested strong genetic differentiation between the three populations (fst = 0.318, p < 0.001). genetic differentiation is also well-supported by clear divisions among the three populations with pcoa (figure 1). pop1 is uniquely distinguished along the first axis and pop2/pop3 are distinguished along the second axis. furthermore, codominant genotypic distances describe 35.40% and 7.4% of the variation with the first and second axes, respectively. taken together, our data strongly indicate genetic differentiation of pigs, wild boar from the period prior to, and after, the disasters in fukushima in 2011. discussion all 32 loci selected from previous studies (rohrer et al, 1994; krause et al, 2002; karlskov-mortensen et al, 2007; fao, 2011) showed pig putative alleles suggesting they can be useful for wild boar and pig hybrid analysis in fukushima and elsewhere. the presence of pig-specific alleles at certain loci depends on the genetic composition of the target population and the pig population involved in the hybridization. in this study, pig samples were from slaughterhouses and farms nearby the evacuated area to improve the likelihood of detecting newly introgressive pig alleles in the hybridized wild boar. one of the pigs sampled had the mtdna haplotype that was the same as hybrid wild boar in fukushima (anderson et al, 2019), and the sampled pig population in this study had high genetic variation (mean na = 5.1). therefore, we were confident in the representation of the pig genetic composition involved in the hybridization for this study and were able to distinguish an appropriate set of markers for hybrid analysis. our selective use of markers with low frequencies of common alleles in source pig individuals and target wild boar populations is highly suggested for cost-effective analysis. the highest number of alleles was observed in the pig population, which was expected because of human mediated translocations of domestic populations with high genetic diversity (scandura et al, 2008; yang et al, 2017). additionally, if the 95 pig putative alleles were excluded, then 27% of the detected alleles were shared by all three populations in this study (table 3). the high percentage of shared alleles between pig and wild boar verifies the challenge of identifying appropriate markers for hybridization analysis. seventeen of the 32 microsatellite loci distinguished hybridization between pigs and wild boar in this study and these can be used as robust markers, specifically for wild boar populations in fukushima. these seventeen markers detected at least one of the 21 alleles that were only shared between pop1 and pop3 (bolded alleles in table 4). alleles only shared by pop1 and pop3, and not detected in pop2, would suggest that the alleles were introgressive through mixing of pigs and wild boar during the period after the 2011 evacuations and fukushima disasters. the higher percent of total shared alleles between pop1 and pop3 (9%), compared to pop1 and pop2 (4%), indicates that there is likely more genetic mixing between pop1 and pop3 (table 3), which would also support the hypothesis of hybridization occurring after disasters in 2011. 34 anderson et al genetic resources (2020), 1 (2), 29–41 table 2. polymorphism measurements of microsatellite loci of each sampled population. n = no. samples, na = no. alleles, ho = observed heterozygosity, he = heterozygosity, fis = breeding coefficient locus pop1 (n = 10) pop2 (n = 13) pop3 (n = 8) na ho he fis na ho he fis na ho he fis sw632 5 0.80 0.79 -0.01 2 0.38 0.50 0.23 3 0.50 0.40 -0.25 s0090 6 0.90 0.77 -0.17 2 0.08 0.07 -0.04 3 0.43 0.36 -0.20 sw24 6 0.56 0.77 0.27 4 0.31 0.39 0.21 6 0.88 0.76 -0.15 swr1941 5 0.60 0.72 0.16 2 0.15 0.26 0.41 2 0.14 0.13 -0.08 sw857 6 1.00 0.75 -0.33 3 0.31 0.27 -0.14 3 0.38 0.32 -0.17 s0228 5 0.40 0.59 0.32 5 0.77 0.69 -0.11 4 0.38 0.66 0.44 sw2008 5 0.90 0.70 -0.29 4 0.31 0.38 0.19 3 0.63 0.48 -0.31 sw240 6 0.70 0.77 0.08 2 0.31 0.43 0.28 3 0.50 0.55 0.10 s0097 6 0.60 0.79 0.24 4 0.38 0.48 0.19 4 0.63 0.63 0.01 umnp147 7 0.80 0.81 0.01 4 0.08 0.51 0.85 3 0.13 0.23 0.45 umnp239 4 0.50 0.62 0.19 4 0.62 0.56 -0.10 4 0.75 0.68 -0.10 umnp296 7 0.80 0.81 0.01 3 0.69 0.61 -0.14 5 0.75 0.66 -0.14 umnp298 6 0.80 0.76 -0.05 3 0.23 0.21 -0.10 2 0.13 0.49 0.75 umnp351 5 0.40 0.76 0.47 5 0.62 0.49 -0.26 3 0.50 0.57 0.12 umnp358 7 0.60 0.81 0.25 4 0.31 0.33 0.08 2 0.50 0.38 -0.33 umnp362 2 0.30 0.38 0.20 1 0.00 0.00 n/a 1 0.00 0.00 n/a umnp381 2 0.10 0.10 -0.05 5 0.62 0.73 0.15 3 0.13 0.23 0.45 umnp405 3 0.40 0.34 -0.19 1 0.00 0.00 n/a 2 0.13 0.12 -0.07 umnp442 3 0.30 0.27 -0.13 1 0.00 0.00 n/a 2 0.00 0.22 1.00 umnp453 6 0.80 0.73 -0.10 2 0.08 0.07 -0.04 1 0.00 0.00 n/a umnp480 5 0.80 0.60 -0.34 2 0.31 0.36 0.13 2 0.38 0.30 -0.23 umnp485 6 0.80 0.61 -0.32 3 0.38 0.52 0.26 3 0.88 0.54 -0.62 umnp489 4 0.56 0.52 -0.07 3 0.54 0.48 -0.13 5 0.86 0.65 -0.31 umnp494 4 0.50 0.59 0.15 4 0.62 0.48 -0.28 4 0.75 0.67 -0.12 umnp500 5 0.70 0.76 0.07 4 0.46 0.63 0.27 6 0.88 0.64 -0.37 umnp502 4 0.80 0.67 -0.20 5 0.31 0.34 0.09 5 0.50 0.50 0.00 umnp509 5 0.70 0.67 -0.05 4 0.38 0.49 0.21 5 0.63 0.50 -0.25 umnp511 4 0.90 0.62 -0.46 4 0.77 0.67 -0.16 4 0.38 0.62 0.39 umnp539 8 0.70 0.82 0.14 3 0.46 0.41 -0.12 4 0.63 0.55 -0.13 umnp548 3 0.50 0.51 0.01 2 0.15 0.36 0.57 2 0.13 0.12 -0.07 umnp610 6 0.60 0.72 0.17 3 0.15 0.14 -0.06 4 0.38 0.41 0.09 umnp640 7 0.70 0.70 -0.01 3 0.69 0.54 -0.27 4 0.75 0.65 -0.16 table 3. number of putative and shared alleles by population with putative allele origin. percentage indicates proportion of alleles related to total alleles detected in this study. population(s) putative allele origin number of alleles (% of total alleles) pop1 (pig) pig 95 (41%) pop2 (wild boar) wild boar 18 (8%) pop3 (hybrid boar) pig and/or wild boar 16 (7%) shared pop1 and pop2 pig and/or wild boar 10 (4%) shared pop1 and pop3 pig 21 (9%) shared pop2 and pop3 wild boar 34 (15%) shared pop1, pop2, and pop3 pig and/or wild boar 37 (16%) total alleles 231 genetic resources (2020), 1 (2), 29–41 markers for pig and wild boar hybridization 35 table 4. allele frequencies for selected microsatellite markers. pop1 n=10; pop2 n=13; pop3 n=8. allelep indicates putative pig alleles. alleleb indicates putative wild boar alleles. bolded alleles indicate pig alleles putatively introgressed into the wild boar population. locus allele frequency locus allele frequency pop1 pop2 pop3 pop1 pop2 pop3 sw2008 102b 0 0.15 0.69 umnp362 124p 0.25 0 0 106b 0 0.77 0.19 126p 0.75 0 0 108 0.05 0.04 0 132b 0 1 1 110p 0.25 0 0 umnp381 165p 0.05 0 0.06 112 0.45 0.04 0 167b 0 0.35 0 114p 0.1 0 0.13 173 0.95 0.27 0.88 116p 0.15 0 0 175b 0 0.08 0.06 sw24 121 0 0 0.06 179b 0 0.27 0 123p 0.28 0 0 181b 0 0.04 0 125 0.06 0.77 0.13 umnp405 140 0 0 0.06 127b 0 0.08 0.06 144 0.15 1 0.94 129 0.33 0.08 0.38 148p 0.05 0 0 131 0.17 0.08 0.25 156p 0.8 0 0 133p 0.11 0 0 umnp442 118 0 0 0.13 135p 0.06 0 0 120 0.1 1 0.88 139 0 0 0.13 122p 0.85 0 0 sw240 107p 0.1 0 0 124p 0.05 0 0 111p 0.4 0 0 umnp453 122 0.05 0.96 1 113p 0.15 0 0 130p 0.05 0 0 119b 0 0.69 0.5 132p 0.1 0 0 121p 0.1 0 0 134p 0.1 0 0 123p 0.1 0 0 136 0.3 0.04 0 125 0.15 0.31 0.44 138p 0.4 0 0 127 0 0 0.06 umnp480 136p 0.15 0 0 sw632 160 0 0.5 0.75 138b 0 0.77 0.81 172p 0.25 0 0 144p 0.6 0 0 174 0.2 0.5 0.19 146 0.1 0.23 0.19 180p 0.25 0 0 148p 0.1 0 0 182p 0.15 0 0.06 152p 0.05 0 0 184p 0.15 0 0 umnp485 185b 0 0.58 0.38 sw857 156p 0.05 0 0 193b 0 0.38 0.56 164p 0.15 0 0 195b 0 0.04 0 166p 0.35 0 0 203p 0.1 0 0 168 0.05 0.12 0.06 207p 0.6 0 0 170 0.3 0.85 0.81 209p 0.1 0 0 172 0.1 0.04 0.13 211p 0.05 0 0.06 219p 0.05 0 0 225p 0.1 0 0 continued on next page 36 anderson et al genetic resources (2020), 1 (2), 29–41 table 4 continued locus allele frequency locus allele frequency pop1 pop2 pop3 pop1 pop2 pop3 swr1941 224b 0 0.85 0.93 umnp489 116p 0.11 0 0.07 228 0.3 0.15 0.07 126p 0.67 0 0.07 232p 0.4 0 0 128 0.11 0.65 0.5 234p 0.15 0 0 130 0.11 0.04 0 236p 0.1 0 0 134 0 0 0.07 238p 0.05 0 0 140b 0 0.31 0.29 s0090 252p 0.05 0 0 umnp494 108p 0.5 0 0 254 0.35 0.96 0.79 114 0 0 0.06 256 0.15 0.04 0.07 126b 0 0.15 0.31 258p 0.2 0 0.14 128b 0 0.12 0.19 260p 0.2 0 0 130 0.4 0.69 0.44 262p 0.05 0 0 132p 0.05 0 0 s0097 230p 0.1 0 0 134p 0.05 0 0 232p 0.2 0 0 141b 0 0.04 0 238 0 0 0.13 umnp500 219p 0.3 0 0 240b 0 0.08 0 221p 0.15 0 0 244b 0 0.04 0 223 0.05 0.12 0.06 250p 0.3 0 0 225p 0.3 0 0.13 252b 0 0.69 0.5 227b 0 0.54 0.56 254p 0.1 0 0 229 0 0 0.13 256p 0.25 0 0.06 237b 0 0.12 0.06 258p 0.05 0 0 239b 0 0.23 0.06 260b 0 0.19 0.31 245p 0.2 0 0 s0228 239p 0.6 0 0 umnp502 156 0.2 0.81 0.69 241p 0.05 0 0 158 0.05 0.04 0.06 243b 0 0.42 0 160 0.45 0.04 0.06 245 0.05 0.04 0 162p 0.3 0 0 247b 0 0.23 0.19 164 0 0 0.13 251 0 0 0.19 166b 0 0.08 0.06 255b 0 0.27 0.13 168b 0 0.04 0 257 0.2 0.04 0.5 umnp509 133 0 0 0.06 259p 0.1 0 0 141b 0 0.08 0.13 umnp147 141p 0.3 0 0 143b 0 0.12 0.69 147p 0.1 0 0 145b 0 0.69 0.06 148p 0.05 0 0 147p 0.05 0 0 149p 0.2 0 0 151 0.4 0.12 0 153b 0 0.04 0.06 153p 0.4 0 0.06 157p 0.2 0 0 155p 0.1 0 0 159 0.05 0.23 0.88 157p 0.05 0 0 163 0 0 0.06 165b 0 0.65 0 167 0.1 0.08 0 continued on next page genetic resources (2020), 1 (2), 29–41 markers for pig and wild boar hybridization 37 table 4 continued locus allele frequency locus allele frequency pop1 pop2 pop3 pop1 pop2 pop3 umnp239 90b 0 0.54 0.31 umnp511 98 0 0 0.13 92p 0.35 0 0 102b 0 0.46 0.56 94p 0.5 0 0.13 106p 0.35 0 0.13 96p 0.1 0 0 108 0.1 0.12 0.19 98p 0.05 0 0.13 110 0.5 0.31 0 104b 0 0.38 0.44 112 0.05 0.12 0 106b 0 0.04 0 umnp539 159p 0.05 0 0 108b 0 0.04 0 161p 0.05 0 0 umnp296 147b 0 0.15 0.06 165p 0.25 0 0 149p 0.1 0 0.06 167 0.05 0.23 0.19 151p 0.15 0 0 169p 0.2 0 0 155p 0.2 0 0.06 171 0.05 0.04 0 159p 0.3 0 0 173 0.25 0.73 0.63 161p 0.05 0 0 175 0 0 0.13 167p 0.15 0 0 177p 0.1 0 0.06 171 0.05 0.35 0.44 umnp548 168p 0.65 0 0.06 177b 0 0.5 0.38 170 0.25 0.23 0 umnp298 153 0.25 0.88 0.56 176b 0 0.77 0.94 155 0.2 0.08 0.44 178p 0.1 0 0 167 0.35 0.04 0 umnp610 162p 0.45 0 0.06 169p 0.05 0 0 164 0.2 0.92 0.75 185p 0.1 0 0 166b 0 0.04 0 191p 0.05 0 0 170b 0 0.04 0.06 umnp351 130p 0.1 0 0 174p 0.15 0 0.13 136p 0.3 0 0 178p 0.05 0 0 140 0.3 0.15 0.56 180p 0.05 0 0 142 0.2 0.08 0 186p 0.1 0 0 144p 0.1 0 0.13 umnp640 113p 0.05 0 0 156b 0 0.04 0 117 0.45 0.23 0.38 162b 0 0.69 0.31 119 0 0 0.06 166b 0 0.04 0 121p 0.3 0 0 umnp358 154 0.05 0.81 0.75 127 0.05 0.62 0.44 158 0.25 0.08 0.25 129p 0.05 0 0.13 160p 0.2 0 0 135p 0.05 0 0 164p 0.05 0 0 137p 0.05 0 0 166b 0 0.08 0 139b 0 0.15 0 168b 0 0.04 0 169p 0.05 0 0 170p 0.15 0 0 172p 0.25 0 0 38 anderson et al genetic resources (2020), 1 (2), 29–41 the loci identified in this study (table 4) provide a unique tool to contribute to determining a timeline of hybridization for these populations. similar frequencies of pig alleles in other wild boar populations may suggest early stages of hybridization, as our data indicates recent occurrence of hybridization in fukushima prefecture, following the release of domestic pigs into the wild boar populations in 2011 (okuda et al, 2018). additionally, the identified loci can contribute to determining if the introgressive alleles are being retained or lost due to natural causes (e.g. backcross) in the hybridized wild boar population using introgressive allele frequencies over time. studies have been published to determine wild boar and pig hybridization hotspots, recent occurrences, and genetic impacts using variable genetic markers, such as mtdna sequence (ishiguro et al, 2002; mccann et al, 2014), rad-seq analysis (goddard and hayes, 2007; iacolina et al, 2018) or microsatellite markers (murakami et al, 2014). however, next generation sequencing (ngs), such as rad-seq, have disadvantages including that a large amount of high quality dna is required, and the amount of data to be analyzed becomes demanding. therefore, general genetic markers, such as microsatellite markers, are still useful for analysis of degraded dna extracted from feces in the field and old specimens of bones (kierepka et al, 2016). selected robust markers from our study will show their advantages in future hybrid analysis and are cost-effective for immediate or continuous monitoring for small sample sizes or dna analysis of degraded samples. also, comparing ngs and microsatellite marker data from a common population in future studies will not only give more indepth information about that target population, but will more clearly show the advantages and disadvantages of each marker. data availability statement the data that support the findings of this study are available from the corresponding author upon reasonable request. acknowledgements thanks to dr hiroko ishiniwa and dr kei okuda for support with sampling. additionally, we are thankful to all prefectural hunters for their support in obtaining samples. funding for this study was partially provided by the nippon life insurance foundation. author contributions da and sk contributed to the writing, drafting, and execution of the manuscript. da, yn and sk, contributed to data analysis and interpertation. all authors contributed to study design, revision, and approval of the submitted manuscript. all authors declare that the submitted work is their own and that copyright has not been breached in seeking its publication. additionally, the submitted work has not been previously published and is not being considered elsewhere. conflict of interest statement the authors declare no conflict of interest. references anderson, d., toma, r., negishi, y., okuda, k., ishiniwa, h., hinton, t. g., nanba, k., tamate, h. b., and kaneko, s. 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(2017). genome-wide snp data unveils the globalization of domesticated pigs. genetics selection evolution 49(1), 71–71. doi: https://dx.doi.org/10. 1186/s12711-017-0345-y https://dx.doi.org/10.1046/j.1365-294x.2003.01763.x https://dx.doi.org/10.1046/j.1365-294x.2003.01763.x https://dx.doi.org/10.2307/3802513 https://dx.doi.org/10.1186/s12711-017-0345-y https://dx.doi.org/10.1186/s12711-017-0345-y introduction materials and methods analysed samples and dna extraction microsatellite loci genotyping characterization of microsatellite markers results discussion data availability statement author contributions conflict of interest statement original article genetic resources (2022), 3 (6), 1–14 doi: 10.46265/genresj.uyml5006 https://www.genresj.org issn: 2708-3764 genetic differentiation between coexisting wild and domestic reindeer (rangifer tarandus l. 1758) in northern eurasia gulnara r svishcheva a,b, olga v babayan c, taras p sipkod, sergey n kashtanov a, marina v kholodovad and yurii a stolpovsky a a vavilov institute of general genetics, russian academy of sciences, 119333, moscow, russia b institute of cytology and genetics, siberian branch of the russian academy of sciences, 630090, novosibirsk, russia c skolkovo innovation centre, gordiz ltd, 121205, moscow, russia d a.n. severtsov institute of ecology and evolution, russian academy of sciences, 1190713, moscow, russia abstract: rangifer tarandus l. 1758 is one of the few modern hoofed species in which domestic and wild forms coexist in the same territory. the genetic differentiation of domestic and wild reindeer in northern eurasia was examined using microsatellite data. a total of 780 animals were studied at 16 microsatellite loci. samples of wild reindeer were taken from seven populations inhabiting different natural areas, and samples of domestic animals were selected from the evenki, evens, chukchi and nenets breeds, including two ecotypes, tofalar and todzha reindeer. the levels of genetic diversity and variation in wild reindeer were higher than in domestic ones. bayesian clustering analysis allowed us to distinguish domesticated reindeer populations by the degree of taming, but failed to detect differences in genetic structure between wild reindeer populations. these differences were found using the pairwise fst values. overall, the microsatellite analysis revealed a significant genetic differentiation between domestic and wild forms and the structuring of populations within each form, which may be important for the development of strategies for animal conservation. keywords: domestic and wild forms of reindeer, geographic population, microsatellites, phylogenetic tree, biodiversity citation: svishcheva, g. r., babayan, o. v., sipko, t. p., kashtanov, s. n., kholodova, m. v., stolpovsky, y. a. (2022). genetic differentiation between coexisting wild and domestic reindeer (rangifer tarandus l. 1758) in northern eurasia. genetic resources 3 (6), 1–14. doi: 10.46265/genresj.uyml5006. © copyright 2022 the authors. this is an open access article distributed under the terms of the creative commons attribution license (cc by 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. introduction reindeer (rangifer tarandus l. 1758) is a widespread circumpolar species on the planet. the geographic distribution of reindeer mainly covers the northern part of the arctic region, including arctic and subarctic regions of eurasia and north america (williams and heard, 1986). although the range of reindeer is very large, the species has been classified as vulnerable by the international union for conservation of nature (iucn) because of a 40% decline over the last decades (gunn, 2016). the decline in population size is the result of the intensification of industrial development in the arctic and climate warming (yannic et al, 2014). for the indigenous arctic ethnic groups living in northern eurasia, reindeer are of great economic, social, cultural and ecological importance, since they are a source of meat, hide and milk, as well as a means for transportation. in the historical process, the employment of reindeer has been of crucial importance in the colonization of the northernmost parts of eurasia. currently, reindeer continue to play a central role in the cultures of the indigenous nenets, chukchi, evenki, sami, evens peoples and other peoples of northern eurasia (helskog and indrelid, 2011; bjørklund, 2013). reindeer husbandry is undoubtedly a historical branch of animal keeping in the northern regions of russia. unlike other arctic countries, reindeer husbandry in russia is much differentiated. representatives of 18 peoples are engaged in the industry, thereby preserving their national traditions, and 16 of them are included in the received: 01.12.2021 accepted: 15.04.2022 published online: 08.07.2022 https://www.genresj.org https://www.dx.doi.org/10.46265/genresj.uyml5006 https://www.genresj.org https://www.dx.doi.org/10.46265/genresj.uyml5006 2 svishcheva et al genetic resources (2022), 3 (6), 1–14 official list of indigenous small-numbered people of the north (jernsletten and klokov, 2002). by now, four native (nenets, evens, evenki and chukchi) breeds of reindeer have been officially registered in russia. the nenets breed is widespread in the taymyr district of the krasnoyarsk territory, as well as in the yamal-nenets, nenets and khantymansi autonomous districts, the komi republic and the murmansk region. currently, this reindeer breed is the most numerous (over 880,000 heads) and prosperous. the chukchi breed inhabits the territory of the chukotka and kamchatka peninsulas and the northeast of yakutia. the evenki breed is bred in the taiga zone of siberia and the far east, from the yenisei river region to the shores of the sea of okhotsk and sakhalin island. the evens breed habitat is the mountain-taiga regions of yakutia and kamchatka, as well as the region of magadan (by its type, this breed occupies an intermediate position between the chukchi and evenki breeds). all breeds differ in productivity and conformation, as well as in adaptation to specific natural and climatic conditions. based on our own observations and fao data (zabrodin and borozdin, 1989), we summarized the descriptive characteristics of the four breeds mentioned above (table 1 ). the breed-forming processes in reindeer husbandry have much in common with the breed genesis of other species of domesticated aboriginal animals, such as horses, sheep, yaks, camels, etc. the reindeer breed specialty is based on the folk traditional breeding system and the complete lack of commercial incentives, features that later also appear in stud breeding in europe and north america (kharzinova et al, 2015). all the reindeer breeds are aboriginal and the result of folk selection by various northern people (zabrodin and borozdin, 1989; kharzinova et al, 2015). within the breeds, there are some ecotypes that are characterized by their own morphological features, but they are taxonomically indistinguishable (davydov et al, 2007). in particular, within the evenki breed, two independent ecotypes, tofalar and todzha reindeer, have been identified (kharzinova et al, 2015). the tofalar reindeer, bred by the small indigenous ethnic group, tofalars, are the largest of the domesticated reindeer. currently, tofalar reindeer husbandry numbers less than 100 heads, compared with several thousand in the 1990s, and the population of the todzha reindeer, bred by todzha-tuvans, is less than 1,000 animals. the tofalar and todzha herds, which define the southern borders of reindeer husbandry, are the most isolated from other reindeer herds bred by the indigenous peoples. the reindeer husbandry of the tofalars and that of the todzha-tuvans can be combined into one sayan group of reindeer husbandry, based on their common characteristics. in russia, along with domesticated reindeer, there are many wild herds, whose ranges cover almost the entire tundra, forest-tundra and taiga zones, including mountainous areas. depending on the habitat of wild reindeer, several ecological forms (macroecotypes) are distinguished, namely island (inhabiting arctic deserts), tundra (migrating to the forest-tundra for the winter), taiga and mountain-taiga (carrying out vertical migrations) (davydov et al, 2007). the most significant differences in size are observed between the tundra and taiga forms of reindeer. as a rule, forest reindeer are more long-legged animals, with elongated body. however, the intraspecific taxonomy of rangifer tarandus l. is still controversial and needs to be specified using molecular genetic markers. on the territory of eurasia, several subspecies of wild reindeer are distinguished (danilkin, 1999). the svalbard reindeer (r. tarandus platyrhynchus) is a non-migratory tundra subspecies inhabiting the higharctic archipelago of svalbard. the novaya zemlya reindeer (r. tarandus pearsoni) is an island subspecies living only on the northern island of the novaya zemlya archipelago. the european reindeer (r. tarandus tarandus) is a subspecies that lives in the european part of eurasia in the tundra and northern taiga regions up to the ural mountains. the siberian reindeer (r. tarandus sibiricus) is a subspecies that lives in tundra and partly forest-tundra zones of siberia (from the ural mountains to the lena river and lake baikal). finally, the okhotsk reindeer (r. tarandus phylarchus) is a subspecies inhabiting the coast of the sea of okhotsk, the amur river basin, the northern part of sikhote-alin, the kamchatka peninsula and sakhalin island (from the lena river and lake baikal to the sea of okhotsk). these geographic subspecies differ in the body type, increasing in size with the transition of the habitat from west to east, but genetic and phenotypic variabilities and differences in subspecies have been insufficiently studied. it is worth noting that the bulk of the wild reindeer populations (~85%) is concentrated in the taymyr peninsula, northern yakutia, and the central part of chukotka (kolpaschikov et al, 2015; kharzinova et al, 2018). the taymyr herd of wild reindeer is the largest and most monitored wild reindeer population in eurasia, inhabiting a vast area in the north of central siberia (petrov et al, 2012; kolpaschikov et al, 2015). three large, relatively isolated herds of wild reindeer are found in the continental tundra of yakutia, namely yana-indigirka, sundrun (indigirkakolyma) and lena-olenek (bulun) (safronov, 1996). in addition, there are sedentary taiga reindeer populations, distributed primarily in the mountains of the south and west yakutia. in recent years, a difficult demographic situation has developed with the populations of wild reindeer on the kola peninsula (baranova et al, 2016). the main direct cause of the decline in these populations is poaching, while forest fires and deforestation, leading to the depletion of food resources, are considered indirect causes. wild reindeer of the murmansk region of the russian federation (the kola peninsula) are endangered and listed in the latest red data book of murmansk region (konstantinova et al, 2014). on the contrary, the number of wild reindeer in the magadan genetic resources (2022), 3 (6), 1–14 genetic differentiation of reindeer populations 3 region is growing thanks to constant monitoring and restrictions on poaching. wild and domesticated reindeer have distinct craniological parameters and differ genetically according to results obtained by various methods of biochemical genetics (kharzinova et al, 2017). dna markers are the most popular tool for studying genetic diversity of reindeer populations. the analysis of mitochondrial dna polymorphism is widely used in phylogenetic studies (flagstad and røed, 2003; davydov et al, 2007; wang et al, 2019; røed et al, 2020). microsatellite markers are widespread for studying the genetic structure and allelic pools of individuals, as well as for their identification a nd d ifferentiation ( røed and midthjell, 1998; jepsen et al, 2002; mcloughlin et al, 2004; mcdevitt et al, 2009; kharzinova et al, 2016; zhai et al, 2017; stolpovsky et al, 2020). with the development of high-throughput snp genotyping platforms, snp markers are getting more popular in animal agriculture. although snp platforms are becoming available for genomic research in reindeer, they are not cost-effective for genotyping several hundred animals. this study aimed to identify genetic diversity among poorly examined wild reindeer populations and genetic differences between wild and coexisting domesticated reindeer living in different eco-climatic zones using microsatellite data. materials and methods animal sample information initially, 790 animals were collected, which were selected from all breeds registered in russia (nenets, evens, evenki and chukchi), including two ecotypes (tofalar and todzha reindeer), as well as from seven wild populations of magadan, amur, taymyr, yakutia, tura, murmansk and chukotka regions (figure 1 and supplemental table s1). for the study of domesticated reindeer, we selected clinically healthy animals according to phenotypic traits corresponding to their breeds. the age of the selected animals varied from 3 to 7 years. special attention was paid to calving females, since only females with a good conformation, regularly bearing healthy calves and capable of raising strong offspring, should be selected for further reproduction. for females, the presence of a calf was determined by the condition of the udder and by interviewing reindeer herders. males were selected in august-september, and females were selected in october-november, i.e. during periods of their maximum fatness. for the study of wild reindeer, we used the biological material from animals that were obtained from hunters. as biological material, pieces of the cartilaginous part of the horn or auricle were taken, which were placed in test tubes and fixed with ethanol (96%). a total of 13 population samples were tested. it is worth noting that the sample of nenets reindeer breed combined three subsamples taken from different breeding regions, since no significant genetic differences have been found between these subsamples (stolpovsky et al, 2020). dna extraction and microsatellite genotyping animals were genotyped using 16 polymorphic microsatellite markers (bms1788, bms745, c143, c217, c276, c32, fcb193, nvhrt16, oheq, rt1, rt24, rt30, rt6, rt7, rt9 and t40). the description of microsatellites is given in supplemental table s2. dna was extracted from velvet antlers, muscles, skin, and ear notches using the cordis sprint pcr compatible reagent kit (ooo gordiz moscow, russia, https://gordiz.ru/en/products/animal-kits/cordis-rangifer/) according to the protocol of the manufacturer. pcr was performed using applied biosystems thermal cyclers under the conditions recommended by the manufacturer of the used reagent kit. separation of pcr products was carried out by capillary electrophoresis using an abi 3130 automatic genetic analyzer (applied biosystems). data analysis and determination of allele variants in the specimens for each locus were performed using the genemapper id-x 1.4 software (applied biosystems). the genotyping quality was controlled using a standard specimen supplied as part of the reagent kit. for each animal, the genotypes of microsatellites were obtained as the allelic lengths in base pairs. the used multiplex short tandem repeat (str) panel consisted of ten dinucleotide loci (rt6, bms1788, rt30, rt1, rt9, rt7, rt24, fcb193, bms745 and nvhrt16) and six tetranucleotide str markers (oheq, c217, c32, 40, c276 and c143). of these, ten loci have been described for caribou: nvhrt16, bms745, fcb193, oheq, bms1788, rt6, rt24, rt30, rt9, rt1 and rt7 (yannic et al, 2014), and other six loci have been described for north american subspecies of red deer: c32, c143, c276, t40 and c217 (jones et al, 2002) (meredith et al, 2005) . this panel has recently been tested on reindeer (stolpovsky et al, 2020; dodokhov et al, 2021). statistical analysis the minimum number of markers needed to discriminate between animals was estimated using the genotype curve() function with 50,000 replicates from the poppr r package (kamvar et al, 2014). in each population sample, the individuals with duplicated genotypes were removed using the clonecorrect() function from the same package. linkage disequilibrium between loci was assessed using the pair.ia() function with 50,000 replicates from the same package. testing the departure from hardy–weinberg equilibrium (hwe) was performed using the hw.test function from the pegas r pack-age (paradis, 2010). here we applied two tests: the classical c2-test based on the expected genotype fre-quencies calculated from the allelic frequencies, and an exact test based on monte carlo permutations of alleles (with 50,000 replicates). to control the false discovery rate, the p-values of both hwe tests were https://gordiz.ru/en/products/animal-kits/cordis-rangifer/ 4 svishcheva et al genetic resources (2022), 3 (6), 1–14 table 1. comparative characteristics of four domestic breeds of reindeer in russia. characteristics were summarized from our observations and fao data (zabrodin and borozdin, 1989). breed predominant pelage colour animal physique live weight of males before rut (♂) and females (♀) [kg] slaughter yield of live weight [%] chukchi dark brown short height, squat, wide and at the same time elongated body, short limbs, well-developed skeleton and musculature. ♂: 125-130 ♀: 93-96 ~51-55 nenets brown and dark brown medium height, rather long and wide body, well-developed skeleton, relatively wide head. ♂: 130-135 ♀: 90-95 ~50 evenki light brown and grey of various shades tall height, rather long body, deep chest, well-developed skeleton and musculature ♂:140-175 ♀: 108-120 ~49 evens light brown and grey colour of various shades tall height, long body, a narrow deep chest, narrow long pelvis, well-developed skeleton, narrow long head. ♂: 130-140 ♀:91-103 ~49-51 figure 1. geographical positions of the studied reindeer samples. the population samples are numbered as follows. breeds: 1. nenets; 2. chukchi; 3. evens; 4. evenki; 5. todzha; 6. tofalar. wild reindeer herds: 7. taymyr; 8. tura; 9. murmansk; 10. amur; 11. magadan; 12. yakutia; 13. chukotka. the map was downloaded from https://www.google.com/maps. genetic resources (2022), 3 (6), 1–14 genetic differentiation of reindeer populations 5 adjusted using the p.adjust r function with the option method=‘fdr’ (benjamini and hochberg, 1995). the main population genetic statistics for each reindeer sample were computed in the adegenet r package (jombart, 2008). allele frequencies, allelic richness and the number of private alleles were assessed by the popgenreport r package. polymorphism information content was estimated using the polysat r package (clark and jasieniuk, 2011). for each ‘locus-population’ pair, fixation index and the observed and expected heterozygosity were estimated using the divbasic() function from the the diversity r package (keenan et al, 2013). nei’s pairwise fst and corresponding p-values were obtained by the pairwise.fst() function from the hierfstat r package (goudet, 2005) and visualized by the corrplot r package (wei et al, 2017). fit, fst and fis for each locus were calculated using the fst() function from the pegas r package. the bayesian clustering approach implemented in the structure v.2.3.4 programme (pritchard et al, 2000) was used to study population differentiation. the optimal number of clusters was determined by the method proposed by (evanno et al, 2005) and implemented in the structure harvester and clumpak programs (earl, 2012; kopelman et al, 2015). the dendrogram based on nei’s genetic distances was constructed by the neighbour-joining algorithm implemented in the poppr r package, with bootstrap support from 5,000 replicates. isolation by distance was assessed by regressing pairwise genetic distance against the natural logarithm of geographical distance (km) with the mantel test (mantel, 1967) with 10,000 permutations using the ade4 r package (dray and dufour, 2007). slatkin’s linearized fst was adopted as the measure of genetic distance (rousset, 1997). the geographic centre of allelic richness among the studied populations was calculated as the weighted midpoint from a series of latitude/longitude coordinates by a method that converts longitude/latitude coordinates (in radians) to a 3d cartesian coordinate system (x, y, z): x = cos(lat) cos(lon), y = cos(lat) sin(lon) and z = sin(lat). this method then calculates the weighted means of these coordinates ( − x, − y, − z) and converts them back to longitude/latitude coordinates using the formulas: lon = atan2 ( − y, − x ) and lat = atan2 ( − z, √ − x 2 + − y 2 ) , where atan2 is a function defined as the angle in the euclidean plane. results data quality checks initially, 790 individuals were genotyped at 16 loci. to ensure that we had enough information to accurately identify multilocus genotypes, we estimated the minimum number of loci needed to discriminate between animals to be 15 markers and determined the presence of clones. according to the results, four individuals genotyped by less than 15 microsatellites and six individuals with duplicated genotypes were removed from further analyses. linkage disequilibrium between loci was assessed using the standardized index of association, rd (agapow and burt, 2001), which corrects for sample size; rd between loci was not high and varied from -0.019 to 0.072. a significant deviation from hwe was detected by both hwe tests considered here only in the sample of wild yakut reindeer at the c276 locus (supplemental tables s3–s5). since deviation from hwe is most often associated with genotyping errors, we had to exclude c276 from the data for the wild yakut sample. thus, the final dataset included 780 individuals. genetic variability for the 16 microsatellites analyzed, a total of 236 alleles were found, with 3 (c143 and c217) to 30 (oheq) alleles per marker. the mean number of alleles per marker across all samples was 14.8. the average percentage of the total number of alleles observed per marker varied from 40.94% (domestic tofalar reindeer) to 81.8% (wild yakut reindeer) (table 2). detailed results for each marker and reindeer population studied are available in supplemental table s6. in all studied samples of reindeer, we found alleles with high frequency (af) (af > 0.7). in particular, c217.215, t40.302, c32.330 and c143.180 had the highest frequencies. the polymorphism information content (pic) values were calculated for each combination ‘sample-locus’ (supplemental table s7). the highest polymorphism levels were found for pairs ‘w.yakut–oheq’ (pic = 0.920), ‘w.taym–bms1788’ (pic = 0.915) and ‘w.yakut–bms1788’ (pic = 0.911). the mean pic value over loci and samples appeared to be rather high, 0.71 ± 0.05. out of 236 alleles in 780 reindeer genotyped, 16 alleles were private alleles with allelic counts ≥ 2 and af > 0.008 (table 3). the largest number of private alleles per marker (3) was detected for bms1788 and oheq, and the largest numbers of private alleles adjusted for sample size were in the wild chukchi herd and todzha ecotype (24.4% and 14.4%, respectively of their total numbers). the presence of private alleles with frequencies above 0.01 in the wild herds suggests that each of these herds most likely has a unique genepool (svishcheva et al, 2020). in total, private alleles were found in four wild reindeer herds and two domesticated ones (table 3). in each sample of animals, we evaluated genetic variability in terms of the number of alleles (a), allelic richness (ar), observed (ho) and expected (he) heterozygosity, and fixation index (fis) (table 2 and supplemental table s6). ar ranged from 3.29 (tofalar herd) to 5.39 (wild yakut herd) with a mean of 4.60 ± 0.17. overall, we found a significant correlation between ar and he (r2. 0.96, p-value = 2.92 × 10−9). the highest ho value (0.77) corresponded to the wild taymyr herd and the lowest (0.57) to the tofalar herd. for each sample, we computed fis as (he-ho)/he. for evens and chukchi breeds the fis values were equal to zero, while for other samples, we observed a slight 6 svishcheva et al genetic resources (2022), 3 (6), 1–14 table 2. population parameters for the domesticated and wild reindeer samples studied. size: population sample size; n: the average number of animals genotyped per marker; a: the number of alleles per sample; %: the percentage of total alleles observed across population samples per population sample per locus; ar: the mean allelic richness across markers; ho and he: observed and expected heterozygosity, respectively; fis: fixation index; ic(se): mean (standard error) of inbreeding coefficients estimated for all animals. herds of wild reindeer from taimyr, tura, murmansk, amur, magadan, yakutia and chukotka are designated as ’w.taym’, ’w.tura’, ’w.murm’, ’w.amur’, ’w.magad’, ’w.yakut’ and ’w.chuk’, respectively. domestic form size n a % ar ho he fis ic (se) nenets 224 223.88 132 60.94 4.31 0.64 0.66 3.03e-02 0.167(0.017) todzha 42 42 89 46.66 3.77 0.64 0.62 -3.23e-02 0.171(0.043) tofalar 47 46.94 77 40.94 3.31 0.57 0.56 -1.79e-02 0.182(0.039) evens 44 43.56 130 60.98 4.78 0.73 0.73 0.00e+00 0.152(0.040) evenki 50 50 113 52.96 4.27 0.67 0.68 1.47e-02 0.165(0.041) chukchi 118 118 131 58.87 4.43 0.7 0.7 0.00e+00 0.156(0.023) wild form w.taym 21 21 142 65.88 5.34 0.77 0.76 -1.32e-02 0.130(0.047) w.tura 12 11.81 115 54.12 5.01 0.73 0.75 2.67e-02 0.138(0.048) w.murm 29 28.94 124 57.44 4.83 0.7 0.71 1.41e-02 0.161(0.053) w.amur 20 20 111 52.76 4.53 0.71 0.7 -1.43e-02 0.154(0.058) w.magad 6 6 89 43.31 4.44 0.71 0.68 -4.41e-02 0.134(0.075) w.yakut 126 118.12 193 81.8 5.27 0.74 0.77 3.90e-02 0.151(0.024) w.chuk 41 41 163 73.89 5.38 0.74 0.76 2.63e-02 0.154(0.043) table 3. private alleles by locus and sample sample locus allele size [bp] allelic frequency allelic count w.yakut rt6 168 0.036 9 w.yakut oheq 302 0.024 6 w.yakut rt24 260 0.02 5 w.yakut rt24 262 0.02 5 w.yakut bms1788 162 0.008 2 w.yakut c32 314 0.008 2 w.taym t40 335 0.045 2 w.murm oheq 268 0.052 3 w.chuk rt7 262 0.122 10 w.chuk fcb193 124 0.049 4 w.chuk bms1788 176 0.024 2 w.chuk fcb193 120 0.024 2 todzha bms745 131 0.071 6 todzha oheq 322 0.024 2 chukchi rt6 192 0.042 10 chukchi bms1788 166 0.008 2 deficiency a nd e xcess o f m icrosatellite heterozygotes. the mean fis value in the pooled sample was 0.0023 ± 0.0071. pairwise fst values to assess the levels of genetic differentiation between the populations over all microsatellites, nei’s pairwise fst values were computed (figure 2, supplemental table s8). according to a 5% significance level, the wild reindeer samples, except for ‘w.yakut’ and ‘w.chuk’, were genetically differentiated. the wild reindeer from chukotka differed only from the amur wild population sample, and the wild reindeer from yakutia were genetically similar to all sampled wild reindeer populations. when comparing only domesticated reindeer, we found stronger genetic differentiation relative to each other (all p-values < 0.03) than in group of wild reindeer. when comparing wild and domestic forms, we found significant differences (p-values < 0.05) for most (38 out of 42) pairs of populations, except for the pairs ‘w.magad’–’nenets’, ‘w.magad’–’chukchi’, ‘w.yakut’–’evens’ and ‘w.yakut’–’evenki’. the tofalar and todzha samples were the most distant from all other breeds and from each other (all p-values < 0.03). wright’s f statistics for each locus for 16 loci, the overall inbreeding coefficient (fit) varied from 0.0411 to 0.2296 (table 4) with a mean of genetic resources (2022), 3 (6), 1–14 genetic differentiation of reindeer populations 7 figure 2. a visualization of pairwise fst values. colour intensity and size of circles are proportional to the pairwise fst values between two population samples. crosses indicate that no significant differences were found between two samples (p-value threshold = 0.05). the two black triangles separate domestic and wild reindeer forms. 0.0077. the highest fit value reflecting a heterozygosity deficiency corresponded to marker c143, likely due to the small number of alleles at this locus (cornuet and luikart, 1996). the inbreeding coefficient, fis, ranged from -0.0388 to 0.0977 with a mean of 0.0188, and the fixation index, fst, ranged from 0.0486 to 0.1462 with a mean of 0.0808. based on fst, all loci (especially c143, rt30 and rt6) contributed to the differentiation between the herds. bayesian clustering analysis to infer the population structure, bayesian clustering was carried out using the markov chain monte carlo methods. we performed 50 runs for each number of clusters from 1 to 13. we selected an admixture model with correlated allele frequencies. to find the optimal number of clusters, we performed simulations of 1,000,000 iterations with a burn-in period of 100,000 generations. for each animal, the genetic origin was assessed as the proportion of genetic membership to each cluster. based on the statistic deltak with values of 216.98, 2.58, 520.04, 2.97, 0.14 and 0.72 for k = 2-6 and k13, respectively, we found the optimal number of clusters, (k = 4, deltak = 520.04) (figure 3). the first cluster consists only of the nenets breed (red bars), the second one includes tofalar and todzha ecotypes (green bars), the third one (purple bars) comprises the remaining three breeds (evens, evenki and chukchi), while the fourth cluster combines all wild reindeer. at k = 13, when the maximum proportion of a sample’s membership in any of these clusters (pm) is above 75%, we acknowledge ‘pure ancestry’ (svishcheva et al, 2020). pure ancestry was thus detected at four population samples: tofalar (92.5%), wild amur (87%), todzha (82%) and wild murmansk reindeer (79%). other samples were found to have mixed ancestry. the 8 svishcheva et al genetic resources (2022), 3 (6), 1–14 table 4. wright’s f statistics for each locus. fit: the inbreeding coefficient of an individual relative to the entire population; fst: the inbreeding coefficient of the subpopulation relative to the entire population expected under random mating; fis: the inbreeding coefficient of an individual relative to its own subpopulation. locus number of alleles fit fst fis bms1788 28 0.109 0.078 0.034 bms745 13 0.071 0.049 0.023 c143 3 0.23 0.146 0.098 c217 3 0.041 0.077 -0.039 c276 8 0.085 0.066 0.021 c32 9 0.118 0.085 0.037 fcb193 15 0.099 0.081 0.019 nvhrt16 13 0.047 0.052 -0.005 oheq 30 0.078 0.077 0.001 rt1 17 0.072 0.074 -0.002 rt24 17 0.117 0.084 0.037 rt30 15 0.163 0.131 0.037 rt6 17 0.087 0.091 -0.004 rt7 14 0.055 0.074 -0.02 rt9 14 0.081 0.08 0.001 t40 20 0.11 0.049 0.065 wild reindeer sample from tura showed the lowest pm values (< 20%). phylogenetic analysis we reconstructed an unrooted phylogenetic tree by the neighbour-joining algorithm using nei’s genetic distances (figure 4). the domestic reindeer were grouped into two clusters with 100% bootstrap support (bs). the cluster highlighted in green contained only main breeds (bs = 100%), while the cluster highlighted in red included two ecotypes (bs = 100%). among the wild populations, we did not identify any group (all bs < 45%). isolation by distance analysis and centre of allelic richness we performed an isolation-by-distance (ibd) analysis based on the mantel test. the original value of the correlation between genetic distances, measured as pairwise fst/(1− fst), and geographic (euclid) distances is represented by the black dot (figure 5a), and the histogram describes the permuted values in the absence of spatial structure. since the original value is inside the reference distribution, the spatial structure is nonsignificant (p-value = 0.135, r2= 0.03). for the studied population samples, we theoretically determined the geographic region/centre of allelic diversity using their geographic coordinates as well as allelic richness and indices defining natural (climatic) zones as weights (see supplemental table s1). we estimated that this region is located in the north-western part of yakutia (along the coast of the laptev sea), which is the core area of seasonal ranges and migration routes traditionally used by reindeer. we detected a significant negative correlation (cor = -0.57, p = 4.1×10−2) between the level of allelic richness in the population sample and the geographic distance from the sample to the theoretically calculated geographic centre (latitude ≈72◦, longitude ≈125◦). figure 5b demonstrates the distribution density of population samples plotted using a bivariate kernel density estimate. discussion in recent years, a difficult d emographic s ituation has developed in wild reindeer herds of some geographic regions of eurasia due to the industrial development of the arctic and adjacent territories, uncontrolled hunting, loss of grazing land and climatic changes (kirpotin et al, 2021). a severe economic and cultural disruption has been observed in some herds of domesticated reindeer, such as tofalar and todzha ecotypes. these reindeer are bred by small indigenous groups and mark the southern limits of reindeer husbandry. although the reindeer is an extremely important species for the indigenous arctic ethnic groups living in the arctic and subarctic regions of eurasia, information on the population structure of this species is limited. it is clear that genetic studies provide knowledge that can be useful in the conservation of genetic variation by identifying the intraspecific genetic structure and also in the management of wild hunting and poaching. the first s tep t o t his i s t o i dentify the population structure of this arctic species. we studied the genetic structure of rangifer tarandus l. from the eurasian part of the range. our study included 13 population samples of animals, covering most of the range, six of which were domesticated to varying degrees, and the remaining seven samples belong to the wild form of reindeer from tundra, taiga and mountainous nature zones. the studied samples from wild reindeer populations relate to the different subspecies according to (danilkin, 1999). the murmansk reindeer selected from the western part of the range, namely from the kola peninsula, belongs to a subspecies r. tarandus tarandus; the taymyr and tura reindeer selected from the central part of the range, namely from the taymyr peninsula and central siberian plateau, belong to a subspecies r. tarandus sibiricus; the amur, magadan, yakutia and chukotka reindeer samples selected from the eastern part of the range belong to a subspecies r. tarandus phylarchus. all studied wild populations of reindeer differ in morphological features. our population structure analysis used a newly developed panel of 16 polymorphic microsatellites with high polymorphism information content. when analyzing allele content, we detected private alleles specific for four wild reindeer herds (‘w.yakut’, ‘w.taym’, ‘w.murm’ and ‘w.chuk’) and for two domesticated ones (‘todzha’ and ‘chukchi’), which suggests that each of these herds probably has formed a unique genepool under different (selection, geographical or feed) conditions. besides, for the todzha ecotype and genetic resources (2022), 3 (6), 1–14 genetic differentiation of reindeer populations 9 figure 3. results of structure analysis based on microsatellite genotypes. colour indicates the proportion of membership of each animal to k assumed clusters. figure 4. phylogenetic tree constructed by the neighbour-joining algorithm. numbers at the branch nodes indicate the confidence values for each branch obtained using the bootstrap procedure. the red and green rectangles show clusters with bs > 95%. 10 svishcheva et al genetic resources (2022), 3 (6), 1–14 figure 5. a) histogram of simulated values. results of mantel test of correlations between genetic differentiation (slatkin’s linearized fst) and geographic distance (spatial euclidean). the black dot represents the original value of the correlation between genetic distances, measured as pairwise fst/(1− fst), and geographic (euclid) distances. b) distribution density of population samples plotted using a bivariate kernel density estimate. the linear regression trend is shown with a dashed line; colours represent degrees of density (blue: low; yellow: medium; red: high density). the wild murmansk reindeer, we found a high degree of ‘pure ancestry’, since the estimated maximum proportion of animal membership in these herds was 82.0% and 79%, respectively. along with these two herds, we identified t wo m ore g enetically h omogeneous herds: the tofalar ecotype (92.5%) and the wild amur (87%) reindeer. for the todzha and tofalar samples, this is explained by a high degree of taming of reindeer due to taiga-specific (spatially limited) breeding and keeping conditions and, of course, indigenous traditions (klokov, 2020). for the wild population samples from the amur and murmansk regions, ‘pure ancestry’ can be explained by their geographical remoteness from the crossings of major migration routes. by analyzing the heterozygosity and allelic richness, we suggest a comparatively low genetic variability in the tofalar and todzha ecotypes, whereas a high genetic variability was observed in the wild herds from the taymyr, chukotka and yakut regions. this is due to the large population sizes and long-distance migration capability of the wild reindeer herds compared to domestic tofalar and todzha reindeer. it is interesting that according to the fis index (table 2), we observed no noticeable inbreeding in the studied populations. however, analysis of the distribution of inbreeding coefficients calculated f or each animal, based on maximum likelihood method, showed comparably high inbreeding in the isolated samples of todzha and tofalar reindeer (supplemental table s9). results of bayesian clustering analysis showed that all wild reindeer herds were clustered together. however, the samples of domesticated animals were distributed among the three clusters according to the degree of their domestication. as expected, the todzha and tofalar herds having the deepest level of taming, without seasonal migrations, formed a separate cluster, while the main breeds living in conditions of large herd formation, including long seasonal migrations (stolpovsky et al, 2020), formed a cluster including only the nenets breed and a cluster including the remaining breeds. the special genetic status of the domesticated nenets reindeer is associated with the peculiarities of their historical formation (dotsev et al, 2017) (kharzinova et al, 2015). however, the phylogenetic analysis did not provide a high bootstrap support for joint clustering of wild herds and did not distinguish the nenets breed from the rest, but confirmed the joint clustering of two ecotypes of reindeer, as well as the joint grouping of reindeer from three breeds (evenki, evens and chukchi), adding to them the nenets breed. the analysis of paired fst confirmed the population genetic differentiation between domestic and wild forms of the eurasian reindeer, as well as the genetic structuring within each form (figure 3). when comparing these two forms, we found significant differences only for 4 out of 42 pairs of populations. we suppose that for pairs ‘w.magad’–‘nenets’, ‘w.magad’–‘chukchi’ this can be attributed to the small size of the magadan sample (n = 6) versus the large sample sizes of the chukchi (n = 120) and nenets breeds (n = 228). the genetic isolation of tame reindeer from wild ones living in the same geographic region is also worth mentioning. in particular, for the ‘w.amur’–‘evenki’ pair from genetic resources (2022), 3 (6), 1–14 genetic differentiation of reindeer populations 11 the stanovoy range region and the ‘w.chuk’–‘chukchi’ pair from the chukotka mountains region we did not see the effect from an exchange of some individuals. the comparison of only the five population samples of the wild herds (‘w.taym’, ‘w.tura’, ‘w.murm’, ‘w.amur’ and ‘w.magad’) showed that they are genetically differentiated from each other, but to a lesser extent than breed samples. the ‘w.yakut’ sample has no genetic differences from all others, and ‘w.chuk’ differs only from ‘w.murm’ and ‘w.amur’, which have characteristic feeding and behaviour patterns (baskin, 1986). the wild reindeer from yakutia and chukotka belong to very large herds (with a high level of genetic diversity) that during seasonal migrations cover vast territories of the tundra and taiga. probably, the exchange of genetic material between the wild reindeer herds takes place on overlapping migration routes. the population from yakutia is especially distinguished in the group of samples of wild reindeer, which occupies the central part of the range relative to the studied populations, and has the maximum level of genetic diversity. we showed that the number of common alleles in ‘w. yakut’ is about 82% of the total allelic diversity of the studied population samples, which significantly exceeds these indicators for the rest of the regional reindeer samples (table 3). according to the results of our study, yakutia is the centre of the allelic diversity of the genepool of the rangifer tarandus l. species in the eurasian part of the range. when comparing only the domestic herds, we found a higher level of genetic differentiation than among wild reindeer. this fact is consistent with an ecologically determined selection process that led to the emergence of divergent breeds in different geographic regions due to their different adaptability. in particular, the isolated samples of the todzha and tofalar herds showed the highest level of differentiation from all the breed samples (fst = 0.0190.091, p-values < 0.05), while a low but significant difference was observed between the evens and chukchi breed samples (fst = 0.014, p-value < 0.05), which can be explained by the fact that the breeds have been created on the basis of the same constantly migrating natural populations of the eastern part of the reindeer range. although the ibd analysis did not reveal a spatial structure of the population samples, which may be associated with long-term seasonal migrations of animals, we theoretically determined the geographic region of allelic diversity for the studied group of population samples. as expected, this region is located in the north-western part of yakutia: the coast of the laptev sea, where the major seasonal ranges and migration routes pass. the yakutsk, taymyr and chukotka wild populations were the geographically closest to the centre of allelic diversity. among the factors that limit species distributions and effect on genetic structure, undoubtedly the last glacial maximum (lgm) has been the major force shaping reindeer geographic ranges in northern eurasia. as it has been shown (grosswald, 1999), in this part of the continent, exactly in the area of the taymyr peninsula, the lgm boundary has been discovered, extending to the northeast. however, the laptev sea region, which is part of west beringia, has not been glaciated since at least the tazov glacial (wetterich et al, 2011). therefore, an eastern part of the species range has been significantly preserved. the species rangifer tarandus l. has lived here both during the ice age and in the subsequent period of natural disasters caused by the melting of glaciers. the pleistocene glaciation undoubtedly has influenced the genogeography of the species. this confirms the maximum genotypic diversity of reindeer in the eastern part of the range. from this region, resettlement has likely occurred throughout eurasia range at the end of the lgm period. longterm migrations have been inevitably accompanied by a loss of genetic diversity, a fact also known from studies of other species inhabiting this territory, for example, sable (kashtanov et al, 2015). conclusion overall, statistical analysis of microsatellite data revealed a significant genetic differentiation between domestic and wild reindeer and confirmed population structures within each form. our results highlight the importance of collecting microsatellite data from wild and domesticated reindeer in providing new insights into the genetic diversity and population structure of reindeer, which can help design strategies for genetic conservation and improvement as well as support resource use. author contributions grs: development of statistical methods, statistical analysis, interpretation of results, development of a concept, draft of the work, writing of the manuscript, final approval of the manuscript; snk: interpretation of results, writing of the manuscript, final approval of the manuscript; ovb: microsatellite analysis, final approval of the manuscript; tps: collecting of biological material, discussion of the results, final approval of the manuscript; mts: dna extracting; mvk: collecting of biological material, discussion of the results, final approval of the manuscript; yas: development of a concept, draft of the work, collecting of biological material, interpretation of results, writing of the manuscript, final approval of the manuscript. funding this research was supported by rsf grant no. 22-1600062. conflict of interest statement the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. 12 svishcheva et al genetic resources (2022), 3 (6), 1–14 ethics statement all experiments were approved by the local ethics committee of the vavilov institute of general genetics of the russian academy of sciences, russia. the animal use procedures were carried out in accordance with the national veterinary guidelines. data accessibility microsatellite genotyping data was deposited in zenodo: 10.5281/zenodo.6376769. supplemental data supplemental table s1. characteristics and geographic locations of the analyzed population samples of reindeer supplemental table s2. description of microsatellite markers supplemental table s3. p-values of the hardy–weinberg equilibrium test with the frd-based correction for each combination of sample and locus supplemental table s4. p-values of the hardy–weinberg equilibrium test for each locus supplemental table s5. the proportions of loci that are out of hwe for each population supplemental table s6. population parameters calculated for 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(2022). morphological characterization of 23 malus domestica borkh cultivars from central spain. genetic resources 3 (6), 22–37. doi: 10.46265/genresj.hjif8839. © copyright 2022 the authors. this is an open access article distributed under the terms of the creative commons attribution license (cc by 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. introduction the apple tree (malus domestica borkh.) is the most important temperate fruit tree crop, with more than 126 million tonnes harvested worldwide in 2020 (fao, 2022). such production is in line with the second report on the state of the world’s plant genetic resources for food and agriculture (fao, 2010), which reported malus l. genetic resources to be among the largest ex situ collections. morphological characterization of malus has been essential for an adequate description of germplasm collections, for breeding programmes (božović et al, 2015) and taxonomic studies (höfer et al, 2014; wagner et al, 2014). currently, although the information provided by genetic markers (such as microsatellites) is preferred against phenotyping due to their stability and economy (reddy et al, 2002; ban et al, 2014), the study of agricultural germplasm by morphological traits is still ∗corresponding author: alberto arnal (alberto.arnal@madrid.org) relevant and useful in diversity analysis (božović et al, 2015; király et al, 2015; kumar et al, 2018). as a result, apple morphological descriptions were conducted in bosnia and herzegovina (gaši et al, 2011), canada (watts et al, 2021), hungary (király et al, 2015), india (dolker et al, 2021), iran (farrokhi et al, 2013), italy (martinelli et al, 2008), the kashmir valley (dar et al, 2015), macedonia (kiprijanovski et al, 2020), montenegro (božović et al, 2015), serbia (mratinić et al, 2012) and turkey (karatas, 2022), reporting high morphological diversity. the most common morphological descriptors used in those works belong to international guidelines such as ibpgr (1982) and upov (2005) and they focus on fruit characteristics because sensorial characteristics and consumer demand focus on fruits (pereira-lorenzo et al, 2018). in spain, several studies also reported great phenotypic apple diversity (royo and itoiz, 2004; ramoscabrer et al, 2007; santesteban et al, 2009; pérezromero et al, 2015), but they did not include old cultivars from some central regions. this lack of informareceived: 20.12.2021 accepted: 24.05.2022 published online: 31.08.2022 https://www.genresj.org https://www.dx.doi.org/10.46265/genresj.hjif8839 https://www.genresj.org https://www.dx.doi.org/10.46265/genresj.hjif8839 mailto:alberto.arnal@madrid.org genetic resources (2022), 3 (6), 22–37 morphological characterization of apple cultivars from central spain 23 tion should be filled, as some of these undescribed old apple cultivars are valued for their sensorial qualities, others are suspected to be exclusive to central spain, and the abandonment of rural landscapes threatens their survival (aceituno-mata, 2010; comunidad de madrid, 2018; arnal et al, 2020). this study aims to quantify the morphological diversity of old apple cultivars growing in rural areas of central spain, compare it with a previous genetic study with simple sequence repeats (arnal et al, 2020) and see whether the old traditional cultivars were distinguishable by morphological descriptors. this work also provides the foundations for further agronomic and sensorial studies to complete the spanish apple morphological description and provide valuable information that will increase the knowledge of apple genetic resources. material and methods plant material a collection of 67 individuals from 41 accessions belonging to 23 old spanish apple cultivars was evaluated to assess their morphological diversity (table 1). the collection is located in arganda del rey (madrid) and belongs to the instituto madrileño de investigación y desarrollo rural, agrario y alimentario imidra (figure 1). for each accession, in 2009 two scions were grafted onto seedlings of m. domestica in a frame of 5m, being the aisles oriented in the sw-ne direction. two individuals of nine main reference varieties and sports (grafted in rootstocks from the same nursery) curated in the same orchard and environmental conditions were included as controls: ‘fuji aztec’, ‘fuji kiku 8’, ‘gala buckeye’, ‘gala schniga’, ‘golden delicious’, ‘golden reinders’, ‘granny smith’, ‘reineta blanca’ and ‘verde doncella’ (table 2). those references were selected because they are widespread in spain (iglesias et al, 2009). the annual maintenance of the collection was conducted as follows: goblet pruning at the end of autumn, soil amendment at the beginning of winter and a preventive application of pesticides against aphids at the end of spring. trees were irrigated every two weeks from may to september to reduce water stress during summer. morphological descriptors a set of 67 morphological descriptors (25 quantitative and 42 qualitative, of which 3 were discrete, 23 nominal and 16 ordinal) were assessed on for 4 organs: 8 descriptors on winter 1-year-old wooden branches (or shoots), 15 on leaves, 16 on flowers and 28 on fruits (table 3). the descriptors were obtained from ibpgr (1982), upov (2005) and urbina and dalmases (2014) and new descriptors and further categories in some qualitative traits were also considered, such as watercore (arnal, 2021). ten (10) to 20 fruits, 20 leaves, 10 flowers and 20 shoots were collected from different orientations of the tree crown in two years (except flowers) from summer 2016 to autumn 2019 and stored at 4–7ºc until processing. in particular, leaves and shoots were collected in 2016 and 2017, and flowers and fruits through the four years of the study. continuous descriptors of shoots, flowers and fruits were measured manually using a jp selecta model 5900601 digital caliper with a precision of 0.01mm. leaf quantitative descriptors were captured with imagej (schneider et al, 2012), so leaves were previously scanned attached to a 2d-scale. finally, apple fruit weight (ten per individual) was registered with a sartorius cp 2202 s digital scale with a precision of 0.01g. data analysis phenotypic diversity. arithmetic means for 25 quantitative, medians for 16 discrete and ordinal, and modes for 26 nominal descriptors were calculated to obtain the central values by accession and cultivar. all qualitative descriptors were translated into a numerical value to meet computing requirements. student’s tand cohen’s d-tests were conducted to identify ifferences between types of cultivar descriptors (references and old apple cultivars). lastly, tukey’s hsd test was performed to detect different groups among old apple cultivars. the significance level (α) was set at 0.05. correlations. a correlation matrix was calculated to explore significant correlations between descriptors in old traditional cultivars. correlations between continuous descriptors were done with pearson, whereas the rest were computed with spearman. no correlations between nominal descriptors were performed. descriptors with no variance were removed at this stage. figure 1. collection sites of the old apple cultivars from central spain. the two upper ellipses indicate accessions from sierra norte de madrid and the lower one, from the tagus river basin. the yellow flag indicates the location of the imidra collection. mdt25 2015 and bdlje 2018, cc-by 4.0 ign.es. 24 arnal et al genetic resources (2022), 3 (6), 22–37 table 1. cultivar name, individual, accession andcollection site of the 23 old apple cultivars sampled in rural areas of central spain. old cultivar name individual/s accession collection site amarillo de el paular apra1/2 apra rascafŕıa camuesa cabu1/2 cabu bustarviejo capr1/2 capr prádena del rincón camueso tard́ıo ctap1/2 ctap puebla de la sierra camueso temprano ctep1 ctep puebla de la sierra de chapa chca1/2 chca canencia chva1 chva valdemanco esperiega espu1/2 espu puebla de la sierra agridulce mamo1/2 mamo montejo de la sierra hojancas mhho1/2 mhho horcajuelo de la sierra mhpr1/2 mhpr prádena del rincón del ortel orca1/2 orca canencia ormo3 ormo morata de tajuña pero de aragón paho1/2 paho horcajuelo de la sierra papr1/2 papr prádena del rincón papu1/2 papu puebla de la sierra pepita de melón peca1/2 peca canencia pehi1/2 pehi la hiruela peva2 peva valdemanco pero gordo pghi2 pghi la hiruela pero pardo pphi1 pphi la hiruela ppmo1/2 ppmo montejo de la sierra pppu1/2 pppu puebla de la sierra pero real prbu1/2 prbu bustarviejo prhi1/2 prhi la hiruela rabudas rahi1/2 rahi la hiruela reineta reho11 reho1 horcajuelo de la sierra reho21 reho2 remo1 remo montejo de la sierra repr1/2 repr prádena del rincón rojillo rjho1/2 rjho horcajuelo de la sierra rojo rjpr1/2 rjpr prádena del rincón rjpu1/2 rjpu puebla de la sierra de rosa rora1/2 rora rascafŕıa rojillo temprano rtev1 rtev valdemanco san felipe sfca1 sfca carabaña temprano tepi1 tepi pinilla del valle verde doncella vdca1/2 vdca canencia vdho1/2 vdho horcajuelo de la sierra vdti1 vdti tielmes genetic resources (2022), 3 (6), 22–37 morphological characterization of apple cultivars from central spain 25 table 2. reference cultivars curated in the imidra collection. reference cultivar name accession number origin fuji aztec 4 worldwide cultivar 7 fuji kiku 8 11 worldwide cultivar 14 gala buckeye 115 worldwide cultivar 117 gala schniga 109 worldwide cultivar 113 golden delicious 104 worldwide cultivar 108 golden reinders 101 worldwide cultivar 203 granny smith 206 worldwide cultivar 210 reineta blanca 211 worldwide cultivar 215 verde doncella 307 national cultivar 311 multivariate analysis. a principal coordinate analysis (pcoa) was performed to visualize the possible groups of cultivars and detect the descriptors that better describe the differences among individuals. in the cluster analysis, a distance matrix between accessions was calculated with nei’s distance (nei, 1973) and the dendrogram was plotted using the unweighted pair group method with arithmetic mean (upgma) hierarchical agglomerative method (sokal and michener, 1958). analysis computing. statistics were performed in r studio v.3.4.1 (r core team, 2017) and a set of packages: ‘effsize’ for cohen’s d-test torchiano (2018), ‘corrplot’ (wei and simko, 2017) for the correlation matrix and ‘agricolae’ (de mendiburu, 2019) for tukey hsd. multivariate analysis was computed with an adapted version of the ‘morphotools’ script (koutecký, 2015). results phenotypic diversity thirty-two morphological descriptors (48% of the total; 19 continuous and 13 non-continuous), showed significant differences between references and old apple cultivars (table 4). in general, old apple cultivars registered lower quantitative values than references, but level frequencies in many qualitative descriptors were less skewed. shoots, leaves and flowers from both types of cultivars were similar, as there were only 14 significant descriptors out of 39 (36%). some significantly different descriptors found in those organs were the width of the apical bud (shw; p = 6.63 × 10−14), the petal length (flpetl; p = 0.0012) and the petal width (flpetw; p = 0.023). in fruit, 18 out of 28 descriptors were significantly different (64%). the calyx opening diameter (frccd; p = 4.26 × 10−24), fruit peduncle length (frpedl; p = 5.57 × 10−37), and peduncle width (frwp; p = 7.37 × 10−18) stood out by their significance and effect size (cohen’s d). in fact, it was observed that the peduncles from old apple cultivars were around 1cm shorter than those from references (figure 2a). other important quantitative descriptors such as the peduncular cavity width (frscw; p = 0.0163), the calyx cavity width (frccw; p = 0.0163), fruit length (frl; p = 0.0026) and fruit weight (frm; p = 0.0275) were significant, b ut t he e ffect s ize ( cohen’s d ) was not large. alternatively, no significant differences existed in fruit width (frw; p = 0.2081). in the fruit qualitative descriptors, the depth of the calyx cavity (frccdep) was ’intermediate’ in old apple cultivars and ’strong’ in references, with significant differences (p = 0.006), describing more diversity in old apple cultivars, as it was relatively easy to find a pples with a ’weak’, ’intermediate’, ’strong’, or ’very strong’ calyx. regarding over colour (frupcol), it was found that old apple cultivars had significantly l ess o ver c olour than references (p = 2.69 × 10−06). nevertheless, among apples with cheeks, ’red’ and ’yellow’ were the most abundant colours. means, medians and modes were also computed by cultivar. as a result, means of quantitative descriptors from references were contained in the tukey’s hsd groups of old apple cultivars. the average fruit length (frl) of the apples was in the 43–63mm range and the fruit width (frw) was between 50 and 81.5mm. regarding fruit weight (frm), apples weighed 125g on average. their shape (frshp) was mostly conical (sum of ’conical globose’, ’conical oblong’ and ’conical truncated’), with a minority of ellipsoidal and flat globose shapes. the cultivar ‘agridulce’ showed significant larger sizes than the other old apple cultivars and even references, as its measurements belonged to the ’a’ group of tukey’s hsd in 17 out of the 25 quantitative descriptors (almost 50%), such as fruit length (frl), fruit weight (frm) and fruit width (frw). ‘hojancas’ and ‘pero gordo’ also tended to have larger organs. the rest of the cultivars presented intermediate size organs, except ‘esperiega’, and ‘san felipe’, which showed small organs. correlations there was significant c orrelation i n 4 4 o ut o f 67 descriptors studied, as the total average significant correlation was 0.43 (figure 3). shoot colour (shcol) was removed as no variance was detected. by organ, the means of correlation was 0.37 in the shoot, 0.54 in the leaf, 0.42 in the flower, and 0.44 in the fruit. correlations within each organ were mainly positive, such as leaf area 26 arnal et al genetic resources (2022), 3 (6), 22–37 table 3. morphological descriptors evaluated in 23 old apple cultivars curated at the imidra collection. type of descriptor (c, continuous;d, discrete; n, nominal; o, ordinal). the hash (#) indicates that the descriptor has been altered (by adding or removing some levels of the published descriptors). sources:1, ibpgr (1982); 2, upov (2005); 3, urbina and dalmases (2014); 4, this paper. organ descriptor name code type levels source shoot pubescence on the apical bud shbpub o 0, glabrous; 1, intermediate; 2, tomentose 3 shoot colour shcol 1, brown; 2, reddish brown, 3; green; 4, grey; 5, purple; 6, red; 7, brown reddish; 8, light brown 2 shoot diameter (mm) shdia c – 2 length of the apical bud (mm) shl c – 3 lenticels# shlent o 1, very few; 3, few; 5 frequent; 7, densely populated 2, 3 apical shoot shape# shshp 1, semispherical; 2, ovoid; 3 intermediate; 4, conical 3 pubescence on shoot shspub o 0, glabrous; right, 9, tomentose 2 width of the apical bud (mm) shw c – 4 leaf leaf area (cm2) learea c – 4 asymmetry of the leaf blade leasim 0, symmetric; 1, asymmetric 4 shape of the base of the leaf blade lebas 1, cuneate; 2, rounded cuneate; 3, rounded; 4, asymmetric; 5, cordate; 7, truncated 3 petiole colour lecol 1, purple; 2, green and purple; 3, green 2 leaf edge shape leedg 1, crenate; 2, bicrenate; 3, serrate-1; 4, serrate-2; 5, biserrate-2; 6, biserrate-1; 7, triserrate 2, 3 foliar blade folding lefold 1, folded; 2, turned; 3, convex; 4, ondulate; 5, flat 3 leaf blade length (cm) lel c – 2, 3 maximum width of the leaf blade (cm) lemwl c – 4 petiole length (cm) lepetl c – 3 pubescence on the reverse lepub o 0, not pubescent; 1, pubescent at the base of the midrib; 3, little pubescent; 5, pubescent; 7, very pubescent; 9, tomentose 2, 3 leaf blade shape leshp o 1, ovate; 2, elliptical; 3, obovate 4 leaf petiole stipules lesti 1, rudimentary; 3, short filiform; 5, long filiform; 7, narrow foliar; 9, wide foliar 3 leaf apex length (mm) letip c – 3 shape of the leaf apex letipshp 2, rounded; 3, acute; 5, mucronate; 7, acuminated; 9, cuspidate 4 leaf blade width (cm) lew c – 2, 3 continued on next page genetic resources (2022), 3 (6), 22–37 morphological characterization of apple cultivars from central spain 27 table 3 continued flower androecium length (mm) fland c – 4 dominance of the gynoecium over androecium fldom o 1, dominated; 2, balanced; 3, dominant 2 gynoecium length (mm) flgin c – 3 pedicel colour flpedcol o 1; purple; 2, purple and green; 3, green 4 flower pedicel length (mm) flpedl c – 4 petal colour flpetcol 0, white; 1, pink white; 2, purple white; 3, purple 3 petal length (mm) flpetl c – 2, 3 number of petals flpetn d integer counting 3 petal width (mm) flpetw c – 2, 3 pubescence on pedicel flpub o 1, glabrous; 2, slightly pubescent; 3, tomentose 4 relative position of the petals flrpp o 0, free; 1, tangent; 2, overlapped 2, 3 sepal length (mm) flsepl c – 4 flower shape flshp 1, flat turned; 2, turned cupuliform; 3, cupuliform; 4, slightly cupuliform; 5, flat 3 flower pedicel stipules flsti 0, no present; 1, present 4 type of petal fltyp 0, flat; 1, wavy; 2, concave; 3, convex 4 welding point of the stamens flweld o 1, welded at the base; 2 welded in pairs up to a certain height; 3 completely welded 3 fruit calyx opening diameter (mm) frccd c – 2, 3 depth of the calyx cavity# frccdep o 0, external; 1, very weak; 2, weak 3, intermediate; 4, strong; 5, very strong 2, 3 length of the calyx cavity (mm) frccl c – 2, 3 shape of the opening of the calyx cavity frccshp 0, without sepals; 1, convergent; 2, partially extended or extended; 3, erect 2, 3 calyx cavity width (mm) frccw c – 2, 3 over colour distribution# frdiscol o 0, uniform (no cheeks); 1, blurred; 2, blurred and stripped; 3, stripped 1, 3 opening of the calyx cavity# freye 0, closed; 1 open 3 flattening# frflat o 1, dominated; 2, balanced; 3 dominant 2, 3 surface colour frgrocol 1, green; 2, light green; 3, yellowish green; 4, light yellow and 5, yellow 1, 2, 3 opening of the locules frhea o 0, closed; 1, semi-open; 2 open 2, 3 fruit length (mm) frl c – 2 number of loculi frloc d integer counting 1 continued on next page 28 arnal et al genetic resources (2022), 3 (6), 22–37 table 3 continued fruit weight (g) frm c – 1, 3 pulp colour frmcol 0, white; 1, greenish white; 2, green; 3, yellowish green; 4, white-yellowish; 5, greenish yellow; 6, yellow 2, 3 fruit peduncle length (mm) frpedl c – 2, 3 russeting in the calyx cavity# frrcc 0, no russeting; 1, russeting 2, 3 russeting on fruit faces# frrf 0, no russeting; 1, russeting 2, 3 ribs frrib o from 1, absent; to 5, very prominent 2, 3 russeting in the peduncular cavity# frrs 0, no russeting; 1, russeting 2, 3 peduncular cavity length (mm) frscl c – 2, 3 peduncular cavity width (mm) frscw c – 2, 3 number of seeds frseed d integer counting 4 fruit shape frshp 1, globose 2, conical globose; 3, wide conical globose; 4, flat; 5, flat globose; 6, conical; 7, narrow conical; 8, conical truncate; 9 ellipsoidal; 10, conical ellipsoidal; 11, oblong; 12, conical oblong; 13, asymmetric 1, 2, 3 over colour frupcol 0, without over colour (no cheeks); 1, white; 2, yellowish; 3, yellow; 4, orange; 5, reddish-pink; 6, red; 7, purple; 9, brown 1, 2, 3 vitrification (or watercore) frvitr 0, absent; 1, present 4 fruit width (mm) frw c – 2 bloom of skin frwax o 1; weak; 2, moderate; 3, intense 2, 3 peduncle width (mm) frwp c – 2, 3 genetic resources (2022), 3 (6), 22–37 morphological characterization of apple cultivars from central spain 29 (learea) with leaf width (lew; 0.91), petal length (flpetl) with petal width (flpetw; 0.81) and fruit weight (frm) with fruit width (frw; 0.92) and fruit length (frl; 0.71). some significant c orrelations w ere n egative, such as the one found between the fruit peduncle length (frpedl) and peduncle width (frwp; -0.68, figure 2b), and the length of the apical bud (shl) with peduncle width (frwp; -0.63). correlations between two different organs were also positive, for example between the fruit peduncle length (frpedl) and flower pedicel length (flpedl; 0.86) and with length of the apical bud (shl; 0.72). we found some negative significant correlations, such as the ones that involved the flower pedicel length (flpedl) with peduncle width (frwp; -0.63) and with the length of the apical bud (shl; -0.55). multivariate analysis the two multivariate analyses showed that the morphological diversity of apple references was lower than the one from old cultivars. results from pcoa (table 5) showed that the first three pcos explained 31% of the variability assessed, with seven coordinates necessary to reach 50% variability. by coordinates, pco 1 represented 14% of the variance, pco 2 10%, and pco 3 7%. the most important correlations in pco 1 were negative: outstanding fruit weight (frm; -0.76), fruit length (frl; 0.75), leaf area (learea; -0.75), peduncular cavity width (frscw;-0.74) and leaf width (lew; -0.71). in pco 2, the balance between negative and positive correlations was similar, with important correlations for peduncle width (frwp; -0.77), peduncular cavity width (frscw; 0.740), leaf width (lew; -0.71), length of the apical bud (shl; 0.70), flower p edicel l ength ( flpedl; 0 .67) and lenticels (shlent; 0.62). in pco 3, the strongest positive correlations involved the maximum width of the leaf blade (lemwl; 0.65), leaf length (lel; 0.54), and apical shoot shape (shshp; 0.49). among the negative correlations in pco 3, most noticeable were the width of the apical bud (shw; -0.51) and over colour (frupcol; -0.48). in the plot that represents apple individuals in pco 1 and pco 2 (24% of variance), we detected that clonal replicates of references were closely grouped while old apple cultivars appeared separated from apple references, but no further structure was detected in the traditional pool (figure 4). despite their low structure in the plot, ‘agridulce’ (mamo1 and mamo2) and ‘hojancas’ (mhho1) were clearly distinct from the rest of the cultivars thanks to quantitative descriptors such as leaf area (learea), fruit weight (frm) and fruit width (frw). finally, ‘verde doncella’ with vdca, vdho and vdti clustered together. in the cluster analysis, the two ‘agridulce’ individuals (mamo1 and mamo2) split off very early from the rest of the individuals (figure 5). they were followed by a ‘rojillo temprano’ (rtev1), two ‘pero de aragón’ (papr2 and papu1), a ‘camueso tard́ıo’ (ctep1), a ‘pero gordo’ (pghi2), and a ‘temprano’ (tepi1). the rest of the dendrogram was structured in three clusters. the first cluster was composed of two ’peros’ (pppu2 and prbu2), two ’reineta’ (‘reineta blanca’ and repr2), ‘hojancas’ (mhho1), and one ‘camueso’ (capr1). the second cluster contained 28 individuals, including the two reference ‘verde doncella’ (vdho2). here also appeared a pool consisting of the cultivar ‘esperiega’ (espu1, espu2), ‘de chapa’ (chva1, chca1, and chca2) and ‘pepita de melón’ (peca2, peva2), as well as a group that nested the cultivars ‘rojo’ and ‘rojillo’ (rjho1, rjpu1, rjpu2, and rjpr2). this second cluster also included a ‘camuesa’ (cabu2) and the three remaining individuals of ‘pepita de melón’ (pehi1, pehi2, and peca1). the third cluster was composed by references (‘fuji’, ‘gala’, ‘golden’, and ‘granny smith’), as well as one ‘reineta’ (repr1) and two ‘pero de aragón’ (papr1 and papu2). discussion phenotypic diversity the results of the present morphological analysis of shoots, leaves and flowers were similar to other morphological studies (božović et al, 2015; hassan et al, 2017). fruit size measures were highly variable, especially fruit length (frl), fruit weight (frm) and fruit width (frw). although averages of these three descriptors were similar to results reported by other works, the registered range was larger than those described in studies by mratinić et al (2011), özrenk et al (2011), király et al (2012), božović et al (2015), pérez-romero et al (2015) and posadas-herrera et al (2018). according to pereira-lorenzo et al (2003), gaši et al (2011), božović et al (2015) and pérez-romero et al (2015), quantitative descriptors related to apple cavities (frscl, frscw, frccl, frccw) are informative because they are genetically controlled. those four descriptors detected statistically significant differences among studied cultivars, but such significance may be due to their correlation to fruit length (frl) and fruit width (frw). in fact, the depth of the calyx cavity (frccdep), a qualitative descriptor that relativizes the calyx cavity width and the calyx cavity length with the global size of the fruit, showed an intermediate diversity, since the depth of this cavity was found to be ’intermediate’ in many of our studied old apple cultivars, similarly to božović et al (2015). furthermore, no ’external’ cavity was found neither in our collections nor in božović et al (2015), being ‘sisa’ the unique old apple cultivar reported with such characteristic (zovko et al, 2010). related to fruit shape, our results agree with božović et al (2015), since their predominant shapes were ’conical’ and ’obloid’, with some presence of ’ellipsoidal’ and ’globose’. nevertheless, not all studies reported conical shapes as dominant, as pırlak et al (2003) found that the ’flat’, ’conical’ and ’spherical’ shapes were all abundant. similarly, hassan et al (2017) reported predominantly the shapes ’globose’, ’obloid’ 30 arnal et al genetic resources (2022), 3 (6), 22–37 figure 2. diversity in the morphological descriptors of fruit peduncle length (frpedl) and peduncle width (frwp): a) density model of fruit peduncle length (frpedl; left) and peduncle width (frwp; right) from each mean and variance. curves were computed with the rnorm(20000, − x, s2) code from r. red, reference cultivars (ref.); green, old apple cultivars (oac); b) significant negative correlation between both continuous descriptors. red triangles, reference cultivars; green circles, old apple cultivars. figure 3. correlation matrix plot for continuous and ordinal apple descriptors assessed on old traditional apple cultivars. shoot colour (shcol) could not be computed due to a lack of variation at a tree level. descriptors are coded as in table 3. ***, p ≤ 0.001; **, p ≤ 0.01; *, p ≤ 0.05; no asterisk, non-significant correlation. genetic resources (2022), 3 (6), 22–37 morphological characterization of apple cultivars from central spain 31 table 4. central statistics of the 32 significant descriptors analyzed by type of cultivar (references and old apple cultivars). descriptor codes are as used in table 3. c, continuous; d, discrete; n, nominal; o, ordinal; ref., reference cultivar; oac, old apple cultivar; sig., significance; ***, p ≤ 0.001; **, p ≤ 0.01; *, p ≤ 0.05. organ descriptor type mean median mode p-value sig. cohen’s d ref. oac ref. oac ref. oac shoot shcol n brown 2.69 × 10−5 *** shdia c 4.95±0.06 6.81±0.07 8.74 × 10−94 *** large shl c 6.75±0.08 5.41±0.05 1.74 × 10−41 *** large shw c 4.54±0.05 4.13±0.04 6.63 × 10−14 *** medium leaf lefold n folded 0.003 ** lemwl c 4.48±0.17 4.72±0.07 0.013 * small letip c 0.62±0.03 0.68±0.01 0.006 ** small flower fland c 10.46±0.53 9.96±0.24 0.001 ** small flpedl c 17.43±0.93 8.66±0.25 2.20 × 10−16 *** large flpetl c 23.28±1.18 21.85±0.52 0.001 ** small flpetn d five 0 *** flpetw c 16.27±0.82 15.58±0.38 0.023 * small flsepl c 7.54±0.39 7.01±0.17 0.014 * small flshp n flat 0.034 * fruit frccd c 5.88±0.22 3.81±0.13 4.26 × 10−24 *** large frccdep o strong intermediate 0.006 ** frccl c 11.93±0.38 10.44±0.26 3.47 × 10−10 *** medium frccw c 24.82±0.76 24±0.57 0.016 * negligible frdiscol o blurred uniform 0.012 * frgrocol n yellow 2.46 × 10−10 *** frl c 57.35±1.71 55.57±1.29 0.003 ** small frloc d five 2.20 × 10−16 *** frm c 131.87±4.47 124.37±3.27 0.028 * negligible frmcol n greenishyellow 6.12 × 10−05 *** frpedl c 24.36±1.01 12.54±0.38 5.57 × 10−37 *** large frrs n presence 0.017 * frscl c 2.42±0.47 3.64±0.29 7.67 × 10−20 *** medium frscw c 29.71±0.91 27.94±0.67 2.64 × 10−05 *** small frupcol n absent 2.69 × 10−06 *** frvitr n absent *** frwax o very intense 0.011 * frwp c 2.65±0.10 3.42±0.09 7.37 × 10−18 *** large and ’ellipsoid’, finding only one conical old apple cultivar. although a great morphological diversity in apple shapes is generally reported, probably some of these differences could be attributed to the high subjectivity of this descriptor (currie et al, 2000). discrepancies in shape should have affected other descriptors such as flattening (frflat), but our results were similar to those reported in other collections (božović et al, 2015; salkić et al, 2017), leading us to consider that old apple cultivars are, in general, wider than longer. we also support this conclusion, as although fruit length (frl) was larger in references, no significant differences between types of cultivars were found in fruit width (frw). apple skin colour is supposed to be a distinctive trait in apple cultivars. the fruit ground colour (frgrocol) ranged in our collection from ’greenish white’, ’green’, ’greenish yellow’ to ’yellow’, similarly to božović et al (2015), mǐsić (2002) and zovko et al (2010). regarding over colour (frupcol), apples herein described were mainly cheekless, as the most common level for this descriptor was ’absent’. therefore, the studied old apple cultivars have a more uniform colour than those reported in the collection of božović et al (2015) and šebek (2013). concerning our cheeked apples, the most common colour was ’red’, as in mratinić et al (2012) and božović et al (2015). correlations many of the significant correlations computed were logical, supporting the botanical description of the apple tree (terpó, 1981; aedo et al, 1998) and agree 32 arnal et al genetic resources (2022), 3 (6), 22–37 table 5. correlation coefficients between the first three principal coordinates (pco) and the morphological descriptors, abbreviated as in table 3. eigenvalues below -0.5 and above 0.5 are highlighted in bold. descriptor pco 1 pco 2 pco 3 descriptor pco 1 pco 2 pco 3 fland -0.59 0.08 -0.04 frrib 0.12 -0.09 -0.15 fldom -0.26 -0.45 -0.06 frrs 0.03 -0.21 -0.21 flgin -0.66 -0.34 -0.03 frscl -0.68 0.45 0.07 flpedcol -0.13 0.39 -0.22 frscw -0.74 -0.26 -0.08 flpedl -0.61 0.51 -0.30 frseed 0.10 0.55 -0.14 flpetcol -0.13 0.23 0.14 frshp -0.26 0.53 0.25 flpetl -0.60 -0.01 0.06 frupcol 0.08 0.43 -0.48 flpetn 0.24 -0.15 0.12 frvitr 0.22 -0.12 0.19 flpetw -0.69 -0.22 -0.05 frw -0.70 -0.41 -0.10 flpub -0.01 0.07 0.21 frwax -0.14 -0.12 0.22 flrpp -0.12 -0.34 -0.07 frwp 0.11 -0.77 -0.03 flsepl -0.68 0.04 0.14 learea -0.75 -0.19 0.29 flshp -0.13 -0.05 0.38 leasim -0.03 0.10 0.24 flsti -0.30 -0.03 -0.30 lebas -0.18 -0.44 -0.29 fltyp -0.16 -0.14 -0.23 lecol -0.22 -0.26 0.15 flweld 0.04 0.07 0.29 leedg -0.40 -0.09 0.24 frccd -0.55 0.07 -0.44 lefold -0.09 -0.06 -0.004 frccdep -0.15 0.18 0.20 lel -0.54 0.09 0.54 frccl -0.63 0.12 -0.05 lemwl -0.43 0.15 0.65 frccshp -0.18 -0.02 0.10 lepetl -0.04 0.24 0.38 frccw -0.52 -0.31 -0.15 lepub 0.19 -0.39 -0.16 frdiscol 0.10 0.37 -0.37 leshp -0.03 0.21 0.44 freye 0.08 -0.04 -0.29 lesti -0.49 -0.08 -0.47 frflat 0.12 -0.64 -0.36 letip -0.13 -0.22 0.17 frgrocol 0.02 0.18 0.40 letipshp -0.17 -0.21 -0.20 frhea -0.26 -0.25 -0.13 lew -0.71 -0.31 0.09 frl -0.75 0.06 0.17 shcol 0.29 -0.05 0.18 frloc -0.07 -0.07 0.16 shdia 0.10 -0.59 0.36 frm -0.76 -0.33 -0.03 shl -0.29 0.70 -0.30 frmcol -0.20 0.46 0.09 shlent -0.23 0.62 -0.19 frpedl -0.57 0.67 -0.14 shshp 0.03 -0.13 0.49 frrcc -0.21 -0.35 -0.24 shspub 0.16 0.19 0.01 frrf 0.03 0.07 -0.14 shw -0.12 -0.002 -0.51 with ganopoulos et al (2018) and farrokhi et al (2013), as the strongest correlations occurred among quantitative descriptors and in the same organ. also, we obtained a strong correlation between leaf area and descriptors related to fruit size, as mentioned by migicovsky et al (2018). a significant correlation, important in breeding, was detected between the fruit peduncle length (frpedl) and peduncle width (frwp). salkić et al (2017) consider that short peduncles are not desirables. we agree with these authors, as probably short and wide peduncles may suffer from lack of growth space, causing some injuries to the fruits and decreasing their commercial quality (figure 6). although this behaviour was not deeply studied in this morphological characterization, our observations suggest that correlation analyses help breeders select descriptors that have a lever effect on genetic improvement (chen and lübberstedt, 2010; ganopoulos et al, 2018). multivariate analysis the pcoa decomposed the variance of the morphological descriptors analyzed. the sedimentation rate along the pco is almost identical to the spanish study of pereira-lorenzo et al (2003), but it was slower compared to other collections (gaši et al, 2011; božović et al, 2015; ganopoulos et al, 2018). for example, our pco 1 only gathers 14% of the variance, whereas pco 1 from gaši et al (2011) gathered almost 30% with 18 descriptors. a slow sedimentation rate does not necessarily indicate that our collection is more diverse than others. probably, the greater the number of descriptors and accessions analyzed, the slower the sedimentation process tends to be. in fact, our study analyzed the largest number of descriptors, followed by pereiragenetic resources (2022), 3 (6), 22–37 morphological characterization of apple cultivars from central spain 33 figure 4. plot between pco 1 and pco 2 for all 67 old apple cultivar individuals and 18 reference individuals from ‘fuji’, ‘gala’, ‘golden’, granny smith’, ‘reineta blanca’ and ‘verde doncella’ based on morphological descriptors. red triangles, reference cultivars; green circles, old apple cultivars. lorenzo et al (2003), who used 49 descriptors in 350 trees. regarding pco eigenvalues, fruit descriptors were usually predominant, especially fruit weight and size. our results were very similar to other studies, such as božović et al (2015), gaši et al (2011), farrokhi et al (2013) and pereira-lorenzo et al (2003). some results reported from other collections do not totally agree with ours. this is the case of ganopoulos et al (2018) where they highlighted other types of fruit characteristics, such as the number of loculi (frloc), pulp colour (frmcol), russeting on fruit faces (frrf) and calyx opening diameter (frccd). the importance of fruit descriptors in the total variance can be explained because it is the organ where selection is performed (šebek, 2013; božović et al, 2015; dar et al, 2015; pérez-romero et al, 2015; salkić et al, 2017; posadas-herrera et al, 2018). results from pcoa are consistent with the cluster analysis, as both detect a high morphological diversity in the old apple cultivars. differences may be due to the lower number of references analyzed, as ‘gala’, ‘golden’, ‘fuji’, and ‘granny smith’ are few, but they are the most widespread varieties in spain and represent almost the whole apple production (iglesias et al, 2009). in addition, we could discriminate references from old apple cultivars and we found an early separation of ‘agridulce’, the presence of two clusters composed of ‘de chapa’, ‘esperiega’, ‘pepita’, and ‘camuesa’ and ‘pepita’, respectively, and the inclusion of ‘verde doncella’ within old apple cultivars. the closeness of ‘verde doncella’ to other traditional apples is congruent with its breeding history, as this cultivar is autochthonous to spain (iglesias et al, 2009; urrestarazu et al, 2012; pina et al, 2014). we did not find further classification in old apple cultivars, as for instance we could not separate ’camuesas’ from ’peros’. a clear separation of references from old traditional cultivars without a strong structure has been reported before (božović et al, 2015; ganopoulos et al, 2018), indicating that old apple germplasm is different from references, in contrast with posadas-herrera et al (2018) and király et al (2015), who could not differentiate between both type of cultivars. regarding descriptors, no single descriptor can distinguish among cultivars, but some of them may be informative and should be considered in cultivar classification, such as the flower pedicel length (flpedl), depth of the calyx cavity (frccdep), fruit peduncle length (frpedl) and peduncle width (frwp). difficulties in old apple cultivar classification are probably due to boundaries among old apple cultivars being more diffuse than in modern cultivars, whose 34 arnal et al genetic resources (2022), 3 (6), 22–37 figure 5. dendrogram constructed based on unweighted pair group method with arithmetic mean (upgma) cluster analysis calculated with the euclidean distance for all 67 old apple individuals and 18 reference individuals from ‘fuji’, ‘gala’, ‘golden’, granny smith’, ‘reineta blanca’ and ‘verde doncella’ based on 67 morphological descriptors. purple, references; green, old apple cultivars. accession and individuals are numbered as in table 1 and table 2. genealogy is always known (noiton and alspach, 1996; laurens, 1999). for example, ‘crisp pink’ (pink lady) derives genetically from ‘golden’ and ‘lady williams’ (iglesias et al, 2009). the lack of information on the origin of old apple cultivars is also accompanied by homonymies. probably, any morphological or sensory trait may be enough to link two cultivars not necessarily parented. for example, flattened apples may evoke a ’reineta’, as martinelli et al (2008) found that ‘reineta grigia’ was not really a ’reineta’. something similar was reported by mratinić and fotirić (2012), who informed that some accessions named ‘šerbetka’ (which means ’too sweet’) were later clustered separately. figure 6. apple damaged due to lack of growth space, possibly triggered by the presence of a short or wide peduncle. support of ssr molecular data in old apple cultivars identities morphological and dna characterization are two complementary techniques, although conclusions about diversity and parental analysis are more robust with molecular analysis (király et al, 2012). therefore, we recently published a molecular analysis of the same accessions in this study based on 13 microsatellites (arnal et al, 2020) in which we reported germplasm with breeding potential that should be further considered. interestingly, pcoa and clustering analysis between the two studies are very comparable, as both clearly differentiate references from old apple cultivars and in general no further groups could be defined. our two studies pointed out two singular old apple cultivars (‘agridulce’ and ‘hojancas’), which may derive from ’reineta’. in the morphological study, these two old cultivars (especially ‘agridulce’) showed traits that differentiated them better than microsatellites, as their differential morphology allowed us to segregate them even earlier than in the molecular study. in consequence, they could be considered for ex situ conservation and further studies. moreover, both methodologies closely related ‘camuesa’, ‘de chapa’ and ‘pepita’. also, ‘rojillo’ and ‘rojo’, which seemed synonyms in our molecular analysis, showed similar morphological profiles, as they appeared together in the multivariate analysis. in contrast, the present morphological study does not gather all triploids in a cluster, nor detect the two groups of ’peros’ found with microsatellites, as pghi2, papu1, and papu2 fell each one in different clusters and one of them (papu2) was closely related to papr1. in conclusion: 1) a great morphological diversity of old apple cultivars was detected in rural areas of central spain; 2) the presented results confirm our previous analysis with microsatellites; 3) both approaches will help to better understand spanish and global apple genetic resources; 4) the described collection contains genetic resources (2022), 3 (6), 22–37 morphological characterization of apple cultivars from central spain 35 two old apple cultivars (‘agridulce’ and ‘hojancas’) with a very distinct morphology, which may deserve further studies (such as flowering and ripening times, productivity, resistance to pests, etc.); 5) two old apple cultivars (‘de chapa’ and ‘pepita’) may be a variation of ‘camuesa’, and 6) the cultivars ‘rojillo’ and ‘rojo’ are likely synonyms. funding this study was supported by the research project fp16etnob ’prospection, characterization, ethnobotanical documentation and conservation of traditional horticultural varieties of the region of madrid’ funded by the local government of madrid (spain). the national institute for agricultural and food research and technology (inia) cofunded a. arnal predoctoral contract. acknowledgements a. arnal thanks the national institute for agricultural and food research and technology (inia) for the predoctoral contract in agrifood, yolanda gogorcena for her comments on the draft, and adrián gómez for his editorial support. author contributions a. arnal, j. tard́ıo and a. lázaro designed the research. a. arnal sampled the individuals with the help of j. tard́ıo and a. lázaro. a. arnal adapted the r code and analyzed the data. a. arnal, j. tard́ıo and a. lázaro wrote the manuscript. a. arnal, j. tard́ıo and a. lázaro revised the manuscript. conflict of interest statement the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. references aceituno-mata, l. 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(2010). pomological properties of autochthkonous cultivars in the area žepča. glasnik zastite bilja (1), 54–74. url: https://hrcak.srce.hr/index.php?show=clanak& id clanak jezik=240860&lang=en. https://doi.org/10.1080/01140671.2003.9514266 https://doi.org/10.1080/01140671.2003.9514266 https://doi.org/https://doi.org/10.35196/rfm.2018.1.49-58 https://doi.org/https://doi.org/10.35196/rfm.2018.1.49-58 https://www.r-project.org/ https://www.r-project.org/ https://doi.org/10.1080/14620316.2007.11512227 https://doi.org/10.1080/14620316.2007.11512227 https://doi.org/10.1023/a:1020691618797 https://link.springer.com/article/10.1023/b:gres.0000020857.29125.2b https://link.springer.com/article/10.1023/b:gres.0000020857.29125.2b https://hrcak.srce.hr/en/191417 https://doi.org/10.5424/sjar/2009073-442 https://doi.org/10.5424/sjar/2009073-442 https://doi.org/10.1038/nmeth.2089 https://doi.org/10.1038/nmeth.2089 https://github.com/mtorchiano/effsize https://github.com/mtorchiano/effsize https://doi.org/10.1007/s11295-012-0502-y https://doi.org/10.1007/s11295-012-0502-y https://doi.org/10.1515/sg-2014-0012 https://doi.org/10.1515/sg-2014-0012 https://doi.org/10.1002/ppp3.10211 https://doi.org/10.1002/ppp3.10211 https://github.com/taiyun/corrplot https://github.com/taiyun/corrplot https://hrcak.srce.hr/index.php?show=clanak&id_clanak_jezik=240860&lang=en https://hrcak.srce.hr/index.php?show=clanak&id_clanak_jezik=240860&lang=en introduction material and methods plant material morphological descriptors data analysis results phenotypic diversity correlations multivariate analysis discussion phenotypic diversity correlations multivariate analysis support of ssr molecular data in old apple cultivars identities funding acknowledgements author contributions conflict of interest statement original article genetic resources (2021), 2 (4), 7–20 doi: 10.46265/genresj.wuda2135 https://www.genresj.org issn: 2708-3764 local breeds and pastoral farming on the north mediterranean shore: a univocal coevolution? an example of dairy sheep farming systems in corsica (france) and thessaly (greece) lola perucho *,a, ioannis hadjigeorgiou b, anne lauvie c, charles henri moulin c, jean christophe paoli a and christina ligdad a inrae selmet-lrde, quartier grossetti, corte, 20250, france b department of nutritional physiology and feeding, faculty of animal science, agricultural university of athens, 75 iera odos, athens, 11855, greece c umr systèmes d’́elevage méditerranéens et tropicaux (selmet), inrae -cirad montpelliersupagro -univ montpellier, 2 place pierre viala, montpellier cedex 1, 34060, france d veterinary research institute, hellenic agricultural organization, thessaloniki, 57001, greece abstract: using local resources for ruminant feeding is a way to achieve agroecological production in pastoral farming systems. in north mediterranean countries, sheep farming systems have evolved towards more intensive systems in lowland and hilly areas, whereas remote and rough pastureland is abandoned and local breeds are rarely maintained; rather, they are progressively replaced by highly productive breeds and their crosses. using the examples of corsica (france) and thessaly (greece), two dairy sheep farming territories developing intensive farming systems that differ in the livestock breeds they use, we explored the hypothesis that the use of local breeds may not be systematically related to the maintenance of pastoral practices in mediterranean dairy sheep farming systems. for this purpose, three data sets based on interviews with sheep farmers of local breeds and crossbred flocks were analysed in two study areas. the results demonstrated that local breeds’ adaptive abilities can be used in crossbred flocks or purebred flocks to maintain a feeding system based on pastoral components. however, other drivers also appear to lead into the declining use of local pastoral resources. apart from the use of local breeds, year-to-year adjustments of replacement and culling rates sometimes have to be applied in order to address the inter-annual variations of the fodder on offer. this paper provides an original approach to studying the link between local breeds and the pastoral components of farming systems by combining synchronic and diachronic analyses of the practices in crossbred and purebred flocks composed of local breeds. keywords: crossbreeding, grazing, breeding practices, karagouniko sheep breed, corsican sheep breed citation: perucho, l., hadjigeorgiou, i., lauvie, a., moulin, c. h., paoli, j. c., ligda, c. (2021). local breeds and pastoral farming on the north mediterranean shore: a univocal coevolution? an example of dairy sheep farming systems in corsica (france) and thessaly (greece). genetic resources 2 (4), 7–20. doi: 10.46265/genresj.wuda2135. © copyright 2021 the authors. this is an open access article distributed under the terms of the creative commons attribution license (cc by 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. introduction the agroecological transition in livestock farming relies on decreasing farm inputs and increasing the use of ∗corresponding author: lola perucho (lolaperucho88@gmail.com) local feed resources and farm leftovers to meet the needs of the on-farm production procedure and on grazing modalities closely following ecological processes in order to ensure the renewal of resources in the long term (jouven et al, 2010; dumont et al, 2013). worldwide, in pastoral farming systems of dairy sheep and other ruminant species, feed requirements are received: 27.02.2021 accepted: 31.08.2021 published online: 04.10.2021 https://www.genresj.org https://www.dx.doi.org/10.46265/genresj.wuda2135 https://www.genresj.org https://www.dx.doi.org/10.46265/genresj.wuda2135 mailto:lolaperucho88@gmail.com 8 perucho et al genetic resources (2021), 2 (4), 7–20 covered to a varying degree through grazing of local fodder resources under a range of management systems (fao, 2001). grazing on a diversity of fodder plant species on rangelands also aids the conservation of landscapes (d’ottavio et al, 2016) and limits soil erosion resulting from continuous cropping (schnabel et al, 2009) in a way that is not competing with the use of land for other food production. moreover, grazing of local resources promotes the biological interactions between animals and their biophysical environment, providing an interesting basis to design agroecological production systems (dumont et al, 2013). despite these benefits, pastoral farming systems are disappearing, mostly in disadvantaged areas, but also in favourable sites of north mediterranean countries, leading to degradation of traditional rural landscapes (hadjigeorgiou et al, 2005; caballero et al, 2009). the disappearance of pastoral activity in many regions in recent decades has revealed the importance of sustainable livestock management for environmental conservation. the absence of pastoralism has had negative consequences on biodiversity and ecosystem services, particularly in marginal areas traditionally used for this activity (constanza et al, 1997). in many ecosystems with a deep-rooted grazing tradition, such as those of the mediterranean (hadjigeorgiou, 2011), the withdrawal of livestock farming activities has led to broad negative changes in the richness and diversity of plant species (sternberg et al, 2000). moreover, the abandonment of pastoral farming has led to modification of various interspecies interactions, affecting negatively, for instance, pollinators, herbivorous insects, parasitoids and birds (plieninger et al, 2006; dover et al, 2011). similarly, this change favoured woody vegetation encroachment, leading to the accumulation of fuel biomass and consequently raising the danger of wildfires (rosa-garćıa et al, 2012). at the same time, ruminants’ farming systems have evolved towards intensive ones and established in more accessible areas, such as lowlands and hilly areas of the mainland. examples include reports on zero grazing in castilla-leon in spain or sedentary intensive or semi-intensive systems in greece (caja and de rancourt, 2002; de rancourt et al, 2009), the abandonment of winter transhumance under agricultural pressure in the ebro valley in spain (caballero et al, 2009) or the replacement of permanent grasslands by agriculturally improved pastures in italy (porqueddu et al, 2017). in parallel, the evolution of flock composition in breeds and their crosses shows different patterns in mediterranean regions subjected to intensification of farming systems and decreased use of native grazing resources. for example, in north and central spain, as well as in greece, farms with highly productive sheep breeds and their crosses coexist with those of local sheep breeds1 (e.g. (de-la fuente et al, 2006; perucho et al, 2018). however, in other north mediterranean regions, also subjected to intensification of farming systems, local breeds remain the main genetic resources in sheep farming. in the south of france, for instance, the blackface manech and the red-face manech are traditionally raised due to their hardiness in the transhumant systems of the west pyrenees, although the red-face manech now prevails over the black-face manech in most intensive farming systems of the area (labatut, 2009; lauvie et al, 2015). likewise, in the crau region in france the merino breed is used due to its specific transhumance ability, but it is also raised in many different sedentary farming systems, including mixed farming systems with an intensive use of land, based on cereals and hay cropping (moulin et al, 2004; lauvie et al, 2015). moreover, holdings with local sheep breeds are found in many different biogeographical regions in italy under sedentary or itinerant systems often combined with cropping (caballero et al, 2009). we thus hypothesized that the use of local breeds may not systematically ensure the preservation of pastoral practices in mediterranean dairy sheep farming systems. in order to test this hypothesis, we explored holdings using different livestock breeds, as well as the evolution of their feeding systems with emphasis on pastoral practices. in particular, we focused on two production territories where dairy sheep farming systems were based on the use of different livestock breeds or their crosses, while they were subjected to similar trends towards intensification. each case study brought up features from different situations to illustrate our argumentation. material and methods choice of the study areas and specific objectives holdings with different livestock breeds considered in the study were those with a) local sheep breeds traditionally raised in pastoral systems, b) highly productive breeds (either exotic or native, raised in more intensive farming systems) and c) crosses of these breeds. the evolution of pastoral practices was analysed through two approaches, (i) a direct one: mid-term analysis of changes in feeding systems (changes in grazing lands and supplementation), and (ii) an indirect one: a study of the diversity of feeding systems found in a territory at any one time and their past trajectories of intensification. the choice of the approach depended on the genetic resources considered. the two study areas chosen to cover this diversity of situations towards livestock breeds and feeding systems were the regions of thessaly (central greece) and corsica (south of france). 1 local breeds are defined in this paper as breeds linked to a specific territory (e.g. georgoudis et al, 2001; loukovitis et al, 2016; perucho, 2018) genetic resources (2021), 2 (4), 7–20 local breeds and pastoral farming on the north mediterranean shore: a univocal coevolution? 9 the outputs of these two case studies hereinafter are successively presented in the results section. thessaly is a region of central greece composed of two central plains occupying half of the land area and bordered by mountainous chains that are traditional transhumance regions (sivignon, 1975). the lowland part of thessaly is characterized by a continental climate with cold winters, hot summers, and wide annual temperature ranges, while in the mountainous part summer temperatures are cooler. typical grazing areas both in lowlands and highlands are composed of both grasslands and shrublands. in the last few decades, the region’s holdings were subjected to an intensification trend centred on harvested feeds, encouraged by a combination of various subsidies, a favourable economic context (low price for feeds compared to the price of milk) or by the proximity of crops for animal consumption in the lowlands (perucho et al, 2015). in this context, highly productive breeds with high feed requirements were progressively adopted since the 1970s. nowadays, the breeds used by sheep farmers are multiple and include the three above mentioned categories, ranging from local breeds under recording schemes (e.g., the karagouniko breed) or conservation programmes (e.g., the kalarritiko breed) and other local animal populations, to highly productive breeds raised in purebred or crossbred flocks. several changes in the genetic composition of the flocks were taking place at the time of the study. the objective of data collection in this region was (i) to understand the farmers’ reasoning for choosing the specific breeds composing current flocks, and to identify among them the reasons linked to the feeding system in place (data set t1), and (ii) to compare recent changes of flock genetic composition and changes of feeding systems and identify possible causal relations (data set t2). corsica is an island in the south of france structured around two central mountainous ranges bordered by a narrow plain on the eastern side of the island. below an altitude of 1200 m (all sampled farms were located below this altitude), the typical rangeland vegetation is composed of shrublands and woodlands and the climate is mediterranean with a marked summer drought period and irregular rainfalls with local variations due to microclimates and altitudinal gradients (gamisans, 1999). sheep farming in corsica is considered as pastoral, but has evolved since the 1960s towards an increasing use of cultivated grasslands and supplementation levels, and a decrease of transhumance (santucci, 2010; choisis and vallerand, 1992). nowadays, in spite of this intensification process, the corsican sheep breed, traditionally raised in pastoral farming systems, is almost exclusively used in the region. dairy sheep farming thus relies on purebred corsican breed flocks, of which approximately 15% were included in the ewe breeding scheme of the corsican breed in 2015. the objective of the data collection in this region was to investigate the different feeding systems in which this local breed is currently raised and identify possible differences in the pastoral components of these feeding systems. additional information on breeding practices (culling and replacement rates) was collected at the same time in order to identify whether the feeding system could impact, if not on the reared breed, at least on its management through culling and replacement. data collection the three data sets that were considered in the study to respond to the above mentioned specific objectives are presented in table 1. data were collected between october 2014 and may 2016 (dataset t1) and in 20162017 (dataset t2 and c1) through semi-structured interviews with farmers. the sampling method for both case studies covered a diversity of feeding systems and focused on different sheep breeds. information on regional feeding systems and their geographical distribution was obtained from the existing literature for corsica (paoli et al (2014) and thessaly (goussios et al, 2014; perucho et al, 2015). in samples of c1 and t1 datasets, for purebred flocks of local breeds, attention was paid to address both flocks participating in the breeding scheme and flocks not participating in the breeding scheme. for dataset t2, interviews were focused on the changes from local breeds to their crosses with highly productive breeds and thus concerned flocks whose composition has evolved from local breed populations towards crossbred animals. the interviews performed included close-ended and open-ended questions. farmers’ responses could have different levels of detail depending on the respondent, but in all cases, interviews gave specific attention to the collection of comprehensive information on on-farm practices (kaufmann, 2011). the interviews were held face-to-face, while clarifications and additional information were subsequently obtained through telephone interviews, when needed. data collected and considered in the study are presented in table 1. in interviews c1, the management of replacement and culling rates was considered under routine situations and perturbations, including the interannual variations of fodder on offer. in interviews t1, the selection of breeds composing the flocks and the crossbreeding strategy were explained by the farmers through their reasoning for choosing or rejecting each breed with respect to its characteristics. interviews t2 were conducted on the basis of the conceptual framework of the analysis of the changes in livestock farming systems (moulin et al, 2008), and they aimed to identify non-varying objects and sequences of transformations or progressive modifications to the components of the farming activity. for this purpose, farmers informed the interviewer about the year they began to change the initial genetic composition of their flock, mostly through performing crossbreeding with highly productive breeds. data were collected for the year preceding the first introduction of a new breed in the flock and for the years during which a change occurred in these components, along with the 10 perucho et al genetic resources (2021), 2 (4), 7–20 table 1. characteristics of data collected in this study study area thessaly corsica id data set t1 t2 c1 data collection mode semi-structured interviews with farmers number of farms n=42 n=14 n=30 targeted sample diversity of flock genetic compositions diversity of feeding systems crossbred flocks with highly productive breeds (previously local breeds) diversity of feeding systems purebred flocks of corsican breed ewe diversity of feeding systems data collected and considered in the study agricultural land composition, grazing and feeding management, flock composition, breeds and crossbreeding strategy changes in: supplementation diet, grazing management, surface and use of land, breeds involved in the flock genetic composition agricultural land composition, grazing and feeding management, flock composition, replacement and culling reasons motivating the change. all relevant changes were recorded until the year of the study. definition of the pastoral components a range of definitions of pastoral systems can be found in the literature (tchakerian, 2008; fao, 2001). these definitions are mainly based on the presence or absence of the following components: a) seasonal movements of grazing animals, b) nature of grazing areas, c) proportion of the diet relying on native vegetation and d) knowledge and know-how related to grazing practices. in this paper, the term pastoral components” is used to describe the modalities of use of spontaneous vegetation (characteristics of grazing areas) and its relative importance in the feeding systems. more information on the variables considered is provided in the following section. data analysis thessaly data collected in thessaly were analysed in two steps. in the first step, using data set t2, the chronology of changes in feeding systems (supplementation levels and composition, importance of grazing and natural pastures, area for cropping animal feeds) and flock genetic composition (breeds introduced in the flock or abandoned), were described. the pastoral components of the feeding systems were defined according to the following modalities: low supplementation levels and high level of use of natural pastures. we analysed the evolution of these pastoral components by comparing the variations of supplementation levels, as well as the variations, in the use of natural pastures and crops for animal feeds over time. we either compared two values at different time points or used qualitative data on the evolution of the variables over time, (represented by triangles in figure 1). then, possible causal relations between changes in the feeding system and change in flock genetic composition were identified and addressed as follows: (i) defining the level of adaptation of the new genetic composition of the flock to the existing feeding system, as observed by the farmer, then (ii) detailing the different responses of the farmers to offset any failure in adaptation (see results). then, among the range of farmers’ responses (t2), we focused on the reintroduction of breeding animals from local rustic breeds (karagouniko breed), in order to reinstate the flock’s adaptation to the existing feeding system. among the reasons for choosing local rustic breeds, specific characteristics related to the hardiness of the breed and associated constraints of the feeding system were listed. this reasoning was compared to outputs of interviews t1 on the reasons for introducing or rejecting different breeds in the flock (see also table 4). the effect of such reintroduction was discussed by considering the evolution of the feeding system after the reintroduction of a local breed (t2). corsica in corsica, farming systems were characterised in terms of the feeding system and the breeding practices (replacement and culling). the related descriptors are presented in table 2. among the five descriptors of the feeding system presented in table 2, the variables used to describe pastoral components were, a) the percentage of flock’s energy requirements covered through grazing at the annual level, b) the part of cultivated grasslands in the total agricultural area of the holding and c) the nature of the grazing areas. the percentage of the flock’s annual energy requirements covered through grazing was derived from the total energy requirements minus the percentage covered by supplementary feed (roughage and concentrates) using feed tables (inra, 2007). descriptors of breeding practices (replacement rate in routine situations and under variations of fodder offer, see table 2) expressed the strategies implemented by the farmers to secure the necessary fodder for their flocks, under inter-annual climate fluctuations. in order to compare farms according to these two categories of variables, the different modalities were represented for each farm, on coloured and greyscale matrix (bertin, 1983). this representation allowed the differentiation of farms, with flocks of corsican sheep breed, into a range of feeding systems and the identification of specific breeding practices (aimed to genetic resources (2021), 2 (4), 7–20 local breeds and pastoral farming on the north mediterranean shore: a univocal coevolution? 11 table 2. variables built for the analysis of the data derived from the c1 interviews, subsequently used for bertin’s graphical representations category of variable variable variable code feeding system (results in figure 2) role of grazing in covering total flock energy requirements role grazing self-sufficiency in hay production hay production share of cultivated grasslands to the total agricultural land cultivated grasslands location of the farm location practice of transhumance transhumance replacement and culling (results in figure 3) routine replacement rate rep rate influence of variation of forage availability on culling rate forage var/cull rate influence of variation of forage availability on replacement rate forage var/rep rate secure fodder on offer) in groups of farms raising the same breed under different feeding systems. results comparative evolution of feeding systems and crossbreeding practices in thessaly figure 1 illustrates changes in the feeding systems (in terms of supplementation, grazing and crops for animal consumption) and the breeds introduced in the flocks of 14 dairy sheep farms in thessaly after the first crossbreeding with a highly productive breed (first yellow square of each line). among these numerous changes of breeds and feeding systems, several were justified by farmers after a failure of the new breed to adapt to the existent feeding system. starting from these specific cases, we analysed the different drivers of evolution of breeds and feeding systems in the short and long-term. introducing highly productive breeds displayed a lack of adaptation to existing feeding systems a lack of adaptation of newly introduced, highly productive breeds (or their crosses) in the flock to existing feeding strategy and grazing conditions was mentioned by nine farmers. these inconsistencies between breeds and feeding systems took three forms: (i) the breed/crossbreed was deemed not hardy enough to function under existing grazing conditions; (ii) the feeding strategy implemented for the breed was considered economically unsustainable; and (iii) the various breeds, raised simultaneously in the flock, had different feeding requirements, but it was not practical to implement a different feeding practice for each of them (table 3). three types of farmers’ responses (adjustments) to this lack of adaptation were identified. the first type was the modification of the feeding/grazing strategy towards decreasing the role of native resources in the applied production system, represented by red stars in $. the second type consisted of the rejection of the highly productive breed in use and/or the testing of a new breed: farmers adopted an exploratory behaviour towards locally available breeds, leading to several changes in breeding animals in a short time period, according to availabilities offered by the market and consultation with other farmers in the region and elsewhere. the third type of farmers’ response was the reintroduction of breeding animals from the local hardy karagouniko breed in the herd, represented by brown triangles in figure 1. in some farms, the failure of the highly productive breeds to adapt to the feeding system in place was not explicitly mentioned by the farmer, but feeding systems were modified after their introduction. some of these changes in feeding systems occurred in the general frame of the intensification of farm production means, i.e. the purchase of highly productive breeding animals (yellow squares in figure 1) came with an investment in feed quantity and quality, housing, equipment and task mechanization in order to maximize the expression of the specific breed’s productive potential. the introduction of a highly productive breed and the decrease of grazing and/or the change in diet quantity or quality generally occurred simultaneously or within a short time period after breed introduction (example of changes in farms 1, 2, 3, 4, 5 or 6 during the decade 2000-2010, figure 1). other changes in feeding systems were driven by the following four factors: (i) a change in land access (example of farm 8 since 2003, figure 1, (ii) economic or climatic perturbations, (iii) a modification of the role of sheep farming in the family’s income and (iv) the increase in average flock size. they occurred at different moments regardless of the change of breed (examples of changes in farms 9 and 13 in 1995 and 1997-1998, respectively). the above mentioned drivers of change could be combined (one perturbation implies farm intensification). for example, in mixed farming holdings, traditionally based on cash crops and sheep farming (farms 3,5 and 6 in figure 1), the combination of decreasing cotton prices (since the 2000’s) and the decoupled subsidies for cotton crops (since 2006) led to an increasing role of sheep farming in the family income, together with the search for new alternatives to cotton crops. according to farmers’ economic possibilities, subsequent changes in sheep farming activities were immediate (farms 5 and 6) or progressive (farm 3) and included the testing of different, highly productive breeds combined with high supplementation levels and 12 perucho et al genetic resources (2021), 2 (4), 7–20 figure 1. comparative evolution of the flocks’ breed compositions as well as the feeding and grazing systems in 14 dairy sheep farms in thessaly, greece. the replacement of cotton crops by crops destined for animal feeding. introduction of karagouniko local breed for hardiness was not necessarily associated with maintenance of pastoral components the reintroduction of the local karagouniko breed was operated by farmers in three of the nine farms for which inconsistencies between flock genetic composition and feeding system were mentioned. in farmers’ statements, the characteristics of the highly productive breeds/crossbreeds forcing such changes in the flock genetic composition were the following: high sensitivity to thermic stress (n=2), high cost of feeding (n=1), high cost of animal health care or high sensitivity to mastitis (n=2), inefficient performance on pasture (n=1), a lack of adaptation to transhumance (n=1), and difficulty in hand milking (n=1). likewise, the results from the t1 interviews indicated that the dairy sheep farmers of thessaly utilised local breeds in their holding in order to improve the hardiness of the flock (in terms of adaptation to pastoral conditions) and rejected highly productive breeds for their incapacity to do so. these abilities and the breeds used or rejected for their corresponding characteristics are presented in table 4. however, in the medium and long-term, the pastoral components of the feeding systems of those three farms were not necessarily maintained. in the first farm (farm 5), maintaining grazing on communal grasslands, in order to keep feeding costs low, was part of the farmer’s strategy according to his statement. reintroduction of karagouniko purebred animals aiming to keep a hardy flock, several years after the first crossbreeding, allowed the farmer to maintain this grazing practice for the following years (farm 5 in figure 1). in a second farm (farm 6 in figure 1), reintroducing the karagouniko breed, through crossing, was motivated by the low capacity of the highly productive breeds to cope with climatic constraints. however, the newly composed crossbred flock remained disappointingly sensitive to thermal stress, as well as to health risks on communal grasslands, which led the farmer to decrease grazing a few years after the introduction of the local breed. in a third farm (farm 11 in figure 1) using native grasslands (including summer pastures through transhumance) was part of the farmer’s strategy but, ultimately, the maintenance of this practice competed with workload management. in this case, crossbreeding with the local breed temporarily delayed the stoppage of transhumance. however, the introduction of machine genetic resources (2021), 2 (4), 7–20 local breeds and pastoral farming on the north mediterranean shore: a univocal coevolution? 13 table 3. components of the feeding system and the traits of the breed involved in the different forms of inconsistencies mentioned by farmers in thessaly inconsistency (number of farms) component of the feeding system at stake traits of the breed at stake (number of mentions) lack of hardiness (n=8) grazing conditions (climatic conditions) sensitivity to thermal stress (n=1) grazing conditions (duration of grazing/nature of the foraging resource) feeding mode leading to sensitivity to mastitis and ruminal pathologies (n=1) grazing conditions (presence of pathogens) sensitivity to vector-borne diseases (n=2) grazing conditions (distance and topography) walking ability (n=3) grazing conditions (open-field pastures) grazing behaviour (n=2) feeding costs (n=5) feed supply (quantities at the multiyear scale) longevity (n=2) feed supply (quantities at the yearly scale) feeding requirements (n=3) competition between breeds (n=1) feed supply (quantities at the yearly scale) feeding intake (n=1) milking led this farmer towards a sedentary system (this is not presented in the figure, but it was planned as a short-term project by the farmer at the year of the interview). it is also interesting to mention that the modalities of the introduction of the local breed in these three farms were limited to the introduction of breeding males in the specific year that the problem was observed and it was not followed by a concrete crossbreeding plan with the introduced local breed. local purebred flocks in a diversity of feeding systems in corsica currently, different feeding systems can be observed among local purebred flocks of corsica (corsican sheep breed), ranging from the most pastoral to the most intensive in feeding inputs and land use and including both transhumant and sedentary flocks. figure 2 presents a classification of five types of feeding systems (fs1 to fs5) according to the role of grazing in covering the energy requirements, the type of grazed pastures and the farmers’ strategy with respect to the provision of hay. most of the pastoral systems (characterized by less harvested feed energy used compared to that of grazed native resources) are found in southwestern and central corsica (fs1 and fs2), the most intensive feeding systems (in terms of feeding inputs and workload for fodder production) are located in the eastern coastal lowlands (fs5), and the intermediate feeding systems are found in all locations of the island (fs3 and fs4). this current situation of local breed dairy sheep farming conditions in corsica reflects a trend towards the securing of feeding systems, either by on-farm fodder production in areas with favourable agroecological conditions, or by hay purchased at the market. the diversity of land use in corsica, for the 206 dairy sheep farms using the corsican breed, has also been documented by perucho et al (2020) table 4. breeds’ traits motivating the introduction or rejection of a specific breed in the flock: the case of traits linked to pastoral farming. hardiness (or a similar term) was used by farmers to characterize the overall ability of the breed to withstand the constraints of its raising conditions. reasons for introducing or rejecting a specific breed (traits linked to pastoral farming) percentage of sampled farmers mentioning the trait (n=42 farmers) breeds used (in bold) or rejected (normal font) ability to handle transhumance 14% (n=6) kalarritiko breed low sensitivity to cold and high humidity 50% (n=21) karagouniko breed and local populations chios, lacaune, and frizarta/friesian breeds low sensitivity to heat 45% (n=19) karagouniko and awassi breeds frizarta/friesian and assaf breeds hardiness 48% (n=20) karagouniko, kalarritiko, piliou and local populations chios and lacaune breeds behaviour compatible with grazing 26% (n=11) karagouniko breed lacaune breed 14 perucho et al genetic resources (2021), 2 (4), 7–20 figure 2. diversity of the feeding systems among the local purebred flocks in corsica (results on 30 farms) genetic resources (2021), 2 (4), 7–20 local breeds and pastoral farming on the north mediterranean shore: a univocal coevolution? 15 adaptation to variations of fodder on offer through replacement and culling management in local purebred flocks of corsica in addition to the use of local breeds in purebred flocks, several of the interviewed farmers performed specific breeding practices in order to maintain the same feeding system in spite of the variations in climatic conditions and subsequent fodder offer. these breeding practices consisted of adjusting the demography of the flock according to inter-annual variations of fodder on offer. in some pastoral systems of southern and central corsica (farms 1, 2, 3 and 4; included in fs1 and fs2, figure 3, this consisted of increasing the replacement rate in years with good forage offers (forage var/rep rate, line 4 of the table in figure 3) and of reversing the practice in years of low forage offers. moreover, in pastoral systems fs1 and fs2, the observed replacement rates were generally low (less than 20%, rep rate, see line 2 of the table in figure 3). another adaptation of the flock demography consisted of increasing the culling rate in order to limit the number of animals that feed during the lambing period of the following year (farms 11, 12, and 13, forage var/cull rate, line 3 of the table in figure 3). this practice was observed in farming systems relying partly on cultivated forage (fs3) in areas of the island impacted by a dry microclimate (e.g. north western lowlands), resulting in variable fodder production from one year to another. these examples, although concerning only a small number of farmers of the sample, demonstrate that the use of a local breed might be combined with other breeding practices so that the farmer is able to maintain the same feeding system from one year to another. discussion our results indicate that the practice of crossbreeding with highly productive breeds in thessaly is often connected with the intensification of feeding by increasing the quantity of feed inputs and modifying the diet with the aim of improving its quality. similarly, the use of local breeds is usually linked with the applied pastoral practices and prevailing climatic conditions. boyazoglu and hatziminaoglou (2005) described the long-term evolution of feeding systems in the european part of the mediterranean basin, characterised by a decrease in transhumance and the use of grazing areas, while the population of small ruminants maintained its genetic composition. on the other hand, couix et al (2016) showed that, in dairy cow holdings, the replacement of the holstein breed by dual purpose local breeds in the western part of france was associated with an overall evolution of the farming systems towards decreased production costs, including the costs associated with the feeding system. in this study, a change in breed was often followed by the adoption of pasture-based feeding systems. samdup et al (2010) analysed the adoption of crossbreeding with different exotic, highly productive breeds in a range of cattle farming systems in bhutan (from extensive to intensive livestock farming systems) and the impact of crossbreeding on such farming systems four years after its implementation. they observed a lower adoption rate of crossbreeding in extensive and semi-intensive farms with the farms either keeping the local breed or rejecting one specific exotic breed (among the two introduced) due to its high feed requirements. the authors also mentioned that during lactation, crossbred cows were stall fed, resulting in reduced grazing in forest and natural grasslands in comparison to local cattle (samdup et al, 2010). the same conclusions were drawn in a study in ethiopia (roschinsky et al, 2015), where most of the cattle farmers adopting crossbreeding with exotic breeds changed their grazing and feeding management towards restricted grazing and the purchase of compound feeds or household by-products, while feed shortages and feed prices were the drivers for rejecting the practice of crossbreeding. caballero et al (2009) also mention the decline of some indigenous breeds in spain, italy and greece and the abandonment of extensive livestock farming in marginal areas. apart from diachronic studies, studies dealing with farmers’ preferences for breeds’ traits emphasize the key role of local breeds’ adaptive traits in pastoral systems (kosgey et al, 2008; tamou et al, 2018) and the preference for exotic breeds in agropastoral systems benefitting from best pastures and climatic conditions (konig et al, 2015). these results agree with the first part of our results associating, on the one hand, crossbreeding for higher productivity with feeding intensification and, on the other hand, local breeds with lower feeding inputs in pastoral systems. however, these two associations are not systematically observed, and farmers do not necessarily abandon raising local breeds following an intensification process. the example of corsica, where one local breed is raised under feeding systems with different levels of intensification, and the one of thessaly, where a variety of genetic types can be found at different stages of the intensification process, suggest that additional studies are needed in order to more convincingly conclude whether coevolution of local breeds and feeding systems is regionally univocal. the data collected through interviews should be compared with the physiological and behavioural responses of highly productive sheep breeds and their crosses in pastoral farming systems in north mediterranean countries. methods to predict breeds’ suitability to environmental conditions are proposed, in this sense, by (lozano-jaramillo et al, 2018) and (marshall, 2014), but these concern south mediterranean or tropical systems. in the case of north mediterranean countries, there are fewer pastoral components and the climatic conditions are milder, but the need for adaptive potential of the animals to similar environments is increasing in importance due to intensifying climatic changes (hoffman, 2013). in this sense, learning processes regarding grazing (meuret and provenza, 2014) and genetic selection should also be considered 16 perucho et al genetic resources (2021), 2 (4), 7–20 figure 3. adjustment of the flock demography according to the inter-annual variations of the fodder offers in corsica (c1, n=30) when assessing the suitability of north mediterranean breed types. finally, our results illustrate that defining the “pastoral dimension” of north mediterranean systems implies considering a set of pastoral components assessed not in terms of absolute value, but also in relative value: pastoral components of one farm are described with regard to the other farming systems of the area. accordingly, detailing constraints of feeding systems by pastoral components might be useful in the assessment of breed suitability. the fact that feeding systems in an area can evolve independently from animal genetic resources used there, is not analysed per se in current research, but rather is suggested by the diversity of factors known to affect the maintenance of pastoral components and the management of animal genetic resources. the evolution of small ruminant feeding systems towards decreased pastoral components is well documented in different mediterranean countries (de rancourt et al, 2009). the drivers of this decrease, as depicted in recent literature, echo the findings of the present study: the farmers wish to alleviate the labour-consuming tasks associated with the feeding system, which leads to a decrease in grazing (aubron et al, 2016). other factors such as the farmers’ mind-set and their social environment, the absence of markets for products based on grazing, as well as land fragmentation have been shown to influence the decision to graze or not to graze in european dairy cow holdings (dasselaar et al, 2020). in greece, the economic context (in terms of prices and subsidies) favoured the choice of purchasing feed outside of the farm (stefanakis et al, 2007; volanis et al, 2007; hadjigeorgiou, 2011), together with other factors such as the low quality of spontaneous forage material, the difficulty to access communal rangelands and an inadequate rangeland management system. other examples from small ruminant farming in greece also illustrate that the legislative framework can force the settlement of nomadic farmers through specific requirements for facilities to comply with milk and stock hygiene standards, animal welfare and manure management (hadjigeorgiou, 2011). finally, the trend towards agricultural intensification in easily accessible productive lands led to land use changes through abandonment of mountainous areas in southern europe and shrub encroachment in many remote areas formerly used for grazing (macdonald et al, 2000; caballero et al, 2009). this phenomenon is highly dependent on eu agricultural and natural conservation policies (tzanopoulos et al, 2011). likewise, the diversity of factors affecting the evolution of animal genetic resources in flocks has to be taken into account. according to (fao, 2015), the reported main causes of genetic erosion in 23 countries in europe and the caucasus (in response to openended questions) mostly consisted of (i) breeds not profitable/competitive or that have poor performance (48% of the countries), (ii) intensification of production or decline of traditional production systems or smallsized farms (39% of the countries) and/or (iii) the introduction/increased use of exotic breeds (35% of the countries). indeed, the introduction of exotic breeds through uncontrolled crossbreeding (failure or absence of crossbreeding programmes) greatly compromised genetic resources (2021), 2 (4), 7–20 local breeds and pastoral farming on the north mediterranean shore: a univocal coevolution? 17 the conservation of local breeds (leroy et al, 2016b). more than 20% of the above mentioned countries also mention problems in breed management linked to weak or absent management policies, programmes or institutions (fao, 2015). indeed, except for their adaptive traits, local breeds in european countries are also kept (being the first motivation) for their links with tradition and their importance in society and, unequally among countries and breeds, for the economic incentives and conservation programmes that benefit them (gandini et al, 2010). in this context, the successes or failures faced by national genetic management programmes and the associated involvement of researchers in such management (leroy et al, 2016a) directly impact the evolution of local breeds’ populations. in greece, for example, the state-run genetic management of local breeds has suffered several interruptions in financing, impacting the implementation of data recording (georgoudis and ligda, 2000). together with unfavourable dairy policies, the problems in the implementation of breed management programmes have been impacting on, for example, the breeding scheme of the karagouniko breed in thessaly (e.g. perucho et al, 2019). on the other hand, the financial support, through eu-funded agri-environmental measures, aimed at farmers raising local breeds threatened by extinction, together with the support provided to farmers in mountainous or disadvantaged areas, has succeeded in maintaining local breed populations over time, as is, for example, the case for the pastoral sheep farming systems of the kalarritiko purebred flocks (national rural network, 2019). conclusion the example of long-term changes in dairy sheep farms in thessaly, greece, indicated that dairy sheep farmers sometimes use local breeds in crossbred flocks in order to improve flock hardiness after a first crossbreeding with highly productive breeds. in this strategy grazing practices can be maintained and feeding costs reduced together with other health costs. in some cases, the shift to new exotic breeds was also considered as an alternative to local breeds in order to improve flock hardiness. however, any additional factors impacted the management of feeding systems, such as local trends towards intensification, access to production means (e.g. land, workforce, capital) and workforce management. this resulted in an evolution of feeding systems (decrease of grazing on native grasslands – increase of supplementation) not necessarily in accordance with the initial reason for the introduction of the local breed in the flock (the maintenance of pastoral components of the feeding system). likewise, although the corsican sheep breed is considered as hardy, its use could be maintained in different feeding systems including systems engaged in an intensification process. finally, farmers in corsica chose to act on flock structure to adapt to several constraints linked to the forage offer in their local purebred flocks. this result indicated that the use of a local breed in purebred or crossbred flocks should be combined with several other practices or conditions so that raising local breeds remains closely related to the pastoral activity. by demonstrating a non-univocal co-evolution between local breeds and farming systems, this study highlights the need to better characterize local and exotic breeds’ abilities as well as farmers’ strategies to cope with different perturbations of their environment. this knowledge will help maintain pastoral systems in production territories and the livestock breeds associated with these territories. acknowledgements this work was supported by the european union through era-net projects domestic (arimnet, ga: kbbe 219262) and perform (arimnet2, ga: 618127), by the department of nutritional physiology and feeding of the agricultural university of athens and the corsican regional research funding scheme (cper/ctc). the phd thesis scholarship of the first author was funded by the collectivité territoriale de corse. author contributions the authors confirm contribution to the paper as follows: study conception and design: lola perucho, anne lauvie, charles-henri moulin, jean-christophe paoli, christina ligda; data collection: lola perucho; analysis and interpretation of results: lola perucho, ioannis hadjigeorgiou, anne lauvie, charles-henri moulin, jean-christophe paoli, christina ligda; draft manuscript preparation: lola perucho; manuscript revision: lola perucho, ioannis hadjigeorgiou, anne lauvie, charles-henri moulin, jean-christophe paoli, christina ligda. conflict of interest statement the authors declare that no conflict of interest exists. references aubron, c., noël, l., and lasseur, j. 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(2007). supporting the extensive dairy sheep smallholders of the semi-arid region of crete through technical intervention. trop anim health prod 39(5), 325–334. doi: https://doi.org/10.1007/ s11250-007-9019-z https://doi.org/10.1016/j.smallrumres.2012.03.021 https://doi.org/10.1016/j.smallrumres.2012.03.021 https://doi.org/10.1017/s1751731114002079 https://doi.org/10.1017/s1751731114002079 https://doi.org/10.1016/j.livsci.2010.05.014 https://doi.org/10.1016/j.smallrumres.2006.08.006 https://doi.org/10.1016/j.smallrumres.2006.08.006 https://doi.org/10.1046/j.1365-2664.2000.00491.x https://doi.org/10.1046/j.1365-2664.2000.00491.x https://doi.org/10.1016/j.livsci.2018.02.013 https://doi.org/10.1016/j.livsci.2018.02.013 https://doi.org/10.1016/j.landusepol.2010.11.007 https://doi.org/10.1016/j.landusepol.2010.11.007 https://doi.org/10.1007/s11250-007-9019-z https://doi.org/10.1007/s11250-007-9019-z introduction material and methods choice of the study areas and specific objectives data collection definition of the pastoral components data analysis thessaly corsica results comparative evolution of feeding systems and crossbreeding practices in thessaly introducing highly productive breeds displayed a lack of adaptation to existing feeding systems introduction of karagouniko local breed for hardiness was not necessarily associated with maintenance of pastoral components local purebred flocks in a diversity of feeding systems in corsica adaptation to variations of fodder on offer through replacement and culling management in local purebred flocks of corsica discussion conclusion author contributions conflict of interest statement supplemental data for: moreira, g. r. p. (2022). conservation status of creole sheep flocks in brazil. genetic resources 3 (5), 68–74. doi: 10.46265/genresj.hfhg6814. supplemental data 1: breeder names and municipality/state, age (years since the flock creation), and size (number of ewes) of the corresponding creole sheep flocks between june and july 2021. symbols associated with the breeder names indicate institutions involved in the research (¥), formal education (§), or farmers/ parks open to visitors (ω). letters associated with the breeder names indicate juridical entities used to identify the breeders in the registration list of arco, as shown in supplemental data 2. the question marks correspond to missing data. supplemental data 2: number of animals registered by breeders per year in the creole sheep flock book from 2001 to 2020 at the brazilian association of sheep breeders (arco), including both the brazilian genealogical registration (rgb) and purebred (po) categories. active flocks are listed in black. letters associated with the breeder names indicate juridical entities that are listed in association either with the owner or manager in supplemental data 1. lines in red and orange correspond, respectively, to flocks that no longer exist or for which updated information on existence was not available. https://www.dx.doi.org/10.46265/genresj.hfhg6814 order creole breeder municipality state year size 1 adaldio castilhos novo horizonte sp 1961 100 2 adenilton marcos souza gomes ω gramado rs 2010 113 3 afonso de almeida costa e filho curitibanos sc 2001 50 4 alan castilho baraldo novo horizonte sp 1961 30 5 alexande pistoia nessy mostardas rs 1970 44 6 alvaro azevedo drummond de mello lavras do sul rs 1991 50 7 amilcar jardim matos bagé rs 2018 130 8 andré sittoni goelzer ω gravataí rs 2001 82 9 anésio reck salto veloso sc 2017 19 10 anselmo gentil souza viamão rs 2020 3 11 antonio alfredo maia § canguçu rs 2013 16 12 antonio brasanini água doce sc 2006 8 13 antonio carlos trierweiler general câmara rs 2017 30 14 antonio david farina lavras do sul rs 1994 280 15 antonio flores savian jr. são martinho da serra rs 1960 80 16 antonio rogério araújo ramos são pedro de alcântara sc 2001 18 17 arlindo maximiano drummond uberlândia mg 1981 100 18 armando bauermann andré da rocha rs 1995 100 19 arnaldo faversani campos novos sc 1981 80 20 arthur corradini são roque sp 2020 4 21 assis camargo ponte alta sc 1959 90 22 berilo de souza brum junior santa maria rs 2020 3 23 carlos josé hoff de souza¥a bagé rs 1982 400 24 carlos tadeu agrifólio vianna são francisco de paula rs 2003 35 25 cezar reiniger bage rs 2020 5 26 clara marineli silveira luiz vaz bagé rs 1846 186 27 clarissa lopes peixoto itaqui rs 2004 366 28 clarissa ramos rodrigues são martinho da serra rs 1999 63 29 clenia maria zanella muitos capões rs 2017 20 30 dário fagundes da costa filho uberlândia mg 1990 19 31 emmanuel veiga de camargo§¥d alegrete rs 2016 50 32 éberson eicholz canguçu rs 2018 5 33 éderson valmir oliveira da trindade viamão rs 2020 12 34 édson/vera martins ponte alta sc 1959 30 35 evandro cardoso minho§b viamão rs 2005 8 36 felipe ferraz de almeida boituva sp 2018 20 37 fernando beccon nerva/ana maria bridi caxias do sul rs 2020 9 38 fernando gaspar da silva itapuca rs 2006 24 39 frederico amarante esmeralda rs 1956 70 40 frederico wolf don pedrito rs 1999 80 41 gaspar-emmanuel desurmont santana do livramento rs 2016 25 42 geraldo toffanello osório rs 2019 22 43 gilberto mirandaω guarulhos sp 2020 14 44 gilson barreto hoffmann andré da rocha rs 1955 80 45 gilson rudinei pires moreira canguçu rs 1996 260 46 gladis ferreira corrêa§¥c don pedrito rs 2011 20 47 guilherme bounfiglio monteiro são bernardo do campo sp 2016 4 48 helene eicklin ponta grossa pr 2015 80 49 hermes rodrigo nicolait crespo/henrique fonseca glorinha rs 2009 35 50 hil josino cunha lagoa vermelha rs 2018 20 51 ianglio m.t.d. jacome palmeira das missões rs 2019 9 52 ieda bulcão de macedo guedes caçapava do sul rs 1906 60 supplemental data 1 53 igor schranck cristal rs 2021 10 54 ilda canova e taquara rs 2015 39 55 jayme barreto c. domingues lagoa vermelha rs 1985 12 56 jair noriler campos novos sc 2003 21 57 jesus martins de córdoba/andrino da silva córdoba cambará do sul rs 1969 26 58 joemir gassen gonçalves rio pardo rs 1927 530 59 joaci xavier muitos capões rs 1961 38 60 joão brasil fernandes lavras do sul rs 1888 80 61 joão carlos machado castro pr 2003 70 62 joão carlos paludo andré da rocha rs 2006 40 63 joão luiz bueno brum pedras altas rs 1841 13 64 joao luiz dornelles maestri pantano grande rs ? 60 65 joao vitor feijó viamão rs 2017 27 66 joao francisco andrade vieira barracão rs 2020 40 67 joao paulo neris da cruz água doce sc 1996 85 68 josé antonio maldonado pires pinheiro machado rs ? 80 69 josé carlos umpierre fernandes lavras do sul rs 2020 5 70 josé carlos guedes mazzitelli rosário do sul rs 2020 5 71 josé cardoso fonseca são francisco de paula rs 1961 100 72 julsan silveira encruzilhada do sul rs 2020 33 73 lara lutzemberg ω rio pardo rs 2018 10 74 larissa klein são francisco de paula rs 2006 60 75 leonardo gruppelli costa pedro osório rs 2007 400 76 lizandra machado andré da rocha rs 1982 29 77 luciano jardim lavras do sul rs ? 130 78 luciano ramos de souza viamão rs 2009 40 79 luiz christian pötter/leonardo salib são josé dos ausentes rs 2001 500 80 luiz fernando cirne lima bom jardim da serra sc ? 77 81 luiza caterine santos panegalliω são luiz gonzaga rs 1998 35 82 manoel marquezoti palmas pr 2003 70 83 marcio camargo curitibanos sc ? 15 84 marco giano bianchini lages sc 1960 60 85 marco castro cachoeira do sul rs 2019 16 86 maria helena e antonio fernandes lavras do sul rs 1888 70 87 maria luiza souza lavras do sul rs 1981 40 88 miqueli sturbelli schiavon santa cruz do sul rs 2012 38 89 moises de jesus machado porto alegre rs 2020 18 90 nélio roberto fonzeca guazzelli são josé dos ausentes rs 1951 55 91 nelson azevedo drummond de mello lavras do sul rs 1969 850 92 nelson beineke pantano grande rs 2021 21 93 noel gonçalves miranda quirinópolis go 2015 50 94 otávio pureza nunes canguçu rs 1986 140 95 paula moreira da silva canguçu rs 2015 27 96 paulo de almeida pradoωf itú sp 2010 18 97 patrizia ana bricarello§¥ florianópolis sc 2014 17 98 pedro leandro matias dos reis triunfo rs 2020 18 99 pery/heloisa zuffo são francisco de paula rs 2008 57 100 rafael gargione paim muitos capões rs 2016 26 101 ricardo pinto laranjeiras do sul pr 2003 40 102 ronir vincenzi laranjeiras do sul pr 2020 6 103 rosana cerioli bagé rs 2017 40 104 rui junior godinho don pedrito rs 1901 545 105 sandro maciel água doce sc 1981 80 106 sergio airton nilson general câmara rs 2019 13 107 silvano souza da silva cambará do sul rs 1966 70 108 vinicio/celmis bastos júlio de castilhos rs 2004 28 109 vinicius ruduit lorenz charqueadas rs 2018 50 110 vladimir mothci santana do livramento rs 1987 200 111 willy haas filho cachoeira do sul rs 2006 80 112* zico pellizaro g curitibanos sc 1980 30 * total of flocks ∑ = total of ewes = 8844 specimens registered/year order creole breeder (arco´s code) 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016 2017 2018 2019 2020 total 1 afonso de almeida costa & filho (8601) 36 32 18 14 25 20 11 13 169 2 agropecuaria e haras la cibeles ltda (12030) 16 16 3 agropecuaria klaus oliveira ltda (142) 4 4 4 amandio gomes bueno (9314) 21 2 5 28 5 amilcar jardim matos (19498) 2 5 7 6 ana cristina klaus oliveira (8577) 21 21 7 antonio alberto melo bertaco (11902) 7 7 8 antonio c. ponciano e outros (12519) 2 2 9 antonio carlos trierweiler & filho (19132) 2 2 5 9 10 antonio flores savian jr. (12947) 74 46 62 58 79 319 11 antonio & rafael paim (16438) 5 1 6 12 carla b. d' ancora dias & edison l. duarte dias (11780) 4 3 2 2 11 13 clara marineli silveira luiz vaz (9725) 9 159 10 175 353 14 claudio marcelo borja de almeida (8858) 4 6 17 10 13 10 60 15 cleber alexandre segato (11669) 13 13 16 condominio nelson souza piegas (775) 12 5 17 34 17 decio ribeiro dos santos & ronaldo de almeida (12571) 2 2 18 djonatan luis dos reis cannez (17677) 3 1 4 19 éderson valmir oliveira da trindade (18104) 1 1 20 edson luiz duarte dias (13305) 5 5 21 elen nunes garcia (14435) 3 2 5 22 embrapa pecuaria sul (9892) a 12 490 80 65 77 252 149 147 147 167 161 91 97 81 120 104 104 139 2483 23 escola técnica de agricultura eta (8717) b 1 1 2 1 8 1 6 3 1 5 1 1 31 24 eugenio hillig (7972) 10 75 33 118 25 eugenio hoffmann jacques (13509) 31 20 11 1 63 26 eugenio h. jacques & luiz christian potter (11588) 9 18 2 1 15 3 48 27 fazendas reunidas pansul ltda (18652) 33 33 28 fund. univ. fed. do pampa unipampa (16866) c 19 3 22 29 gabriel jorge neto & pedro nacib jorge neto (13468) 1 1 30 geraldo pires saldanha (10815) 6 6 31 gilson barreto hoffman (12742) 54 5 59 32 gilson rudinei pires moreira (8386) 51 19 45 40 78 31 33 26 30 33 24 48 7 26 26 78 595 33 gustavo pergher (11946) 2 28 53 4 30 2 119 34 helene ecklin (18558) 2 2 35 inst. fed. educ. cienc. tec. farroupilha iff (3345) d 4 6 2 10 22 36 isnil agropecuaria e transporte ltda (9740) e 13 5 22 40 37 itacumbi agricola e pastoril ltda (12900) 8 8 38 ivadi coninck de almeida (3515) 26 26 39 jackson peres longui & pablo s. longhi (9422) 8 8 40 jair noriler (1054) 9 7 29 95 43 24 12 7 15 7 12 17 8 9 294 supplemental data 2 41 joão alberto bronzato (2062) 4 3 7 42 joão augusto botelho do nascimento (10101) 3 4 2 1 10 43 joão carlos paludo (12734) 36 14 50 44 jose friederich da silva (14462) 1 4 5 45 leonardo gruppeli costa (14930) 15 16 31 46 leonardo salib dutra & luiz christian potter (17635) 50 50 47 luciano ramos de souza (19267) 10 16 16 42 48 luciano r. de souza & hermes r. n. crespo (16081) 16 24 22 28 37 127 49 luiz christian potter & mariana potter (10000) 2 21 48 60 87 8 27 61 9 323 50 luiz roberto tietbohl fonseca (12002) 12 12 51 luiz lopes burmeister (10001) 1 7 8 52 marcelo cecim (9220) 15 3 26 44 53 maria luiza ramalho e silva (9304) 32 6 38 54 monã centro de estudos ambientais (16678) 6 6 55 nelio roberto fonseca guazzelli (10594) 52 25 77 56 np empreendimentos ltda (16707) f 5 13 12 18 7 10 65 57 norbert reisinger (13300) 8 8 58 odilon martins jr. (296) 13 18 16 47 59 ory augerô do amarante (10657) 44 104 91 52 96 72 92 25 32 31 28 667 60 osni machado coninck (1108) 24 36 60 61 otavio pureza nunes (6387) 8 1 9 62 patricia pretto juchem (14432) 6 12 2 20 63 pedro monteiro lopes (6351) 4 3 3 10 7 21 13 8 1 2 72 64 pery marzullo sobrinho (16101) 10 10 65 reflora com. ind. imp. ltda (1729) g 82 26 43 2 3 6 5 167 66 renato eugenio de rezende barbosa e outros (11767) 25 1 26 67 rui junior godinho (6376) 38 41 42 62 145 69 142 218 48 40 50 895 68 vera maria villamil martins (11975) 29 21 21 8 79 69 vinicio bastos & celmis bastos (14901) 5 15 4 14 7 4 5 7 7 68 70 vinicius ruduit lorenz (18469) 9 9 71 volnei afonso merino (10002) 16 41 34 74 42 70 35 13 1 326 72 willy haas filho (16088) 34 34 73 zuleika borges torrealba (14922) 12 62 42 58 129 107 4 58 472 total (both sexes) 141 812 624 460 521 871 669 828 779 492 399 474 453 260 211 137 201 154 245 187 8918 total of males: (n) 58 71 69 93 100 (%) 42.34 35.32 44.81 37.96 53.48 68-supplemental data_1 68-supplemental data_2 blank page blank page blank page original article genetic resources (2020), 1 (1) 17–23 doi: 10.46265/genresj.2020.1.17-24 https://www.genresj.org issn: 2708-3764 swedish crop wild relatives: towards a national strategy for in situ conservation of cwr jens weibull *,a and jade phillips b a swedish board of agriculture, se-230 53, alnarp, sweden b school of biosciences, university of birmingham, edgbaston, b15 2tt, birmingham, uk abstract: in 2015, the nordic countries (sweden, denmark, finland, norway and iceland) initiated a project to help strengthen the efforts of conservation and use of crop wild relatives (cwr) across the region. policy recommendations that were put forward included creating national strategies for each nordic country and adopting and implementing complementary in situ conservation as the main approach for safeguarding cwr across the region. the present work explores in greater detail the situation for sweden. taxa rich areas and areas where potential data bias may be prevalent are located. an eco-geographic map is constructed to help determine how genetic diversity may be portioned across the country within populations of taxa. an in situ complementarity analysis accounting for taxa richness, eco-geographic richness and the protected area network in the country is also presented. possible reasons for diverging results, as compared to the regional analysis, are discussed. the document serves as a starting point for further in-depth research on cwr distribution, conservation and use within sweden. keywords: crop wild relatives, protected areas, elc-analysis, sweden citation: weibull, j., phillips, j. (2020). swedish crop wild relatives: towards a national strategy for in situ conservation of cwr. genetic resources 1 (1), 17-23. doi: 10.46265/genresj.2020.1.17-23. © copyright 2020 the authors. this is an open access article distributed under the terms of the creative commons attribution license (cc by 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. introduction already back in 1979, the nordic countries established what was probably the first regional genebank for the ex situ conservation of seeds of agricultural and horticultural plants. for many years, the (then) nordic gene bank stood as an example of foresight regarding long-term conservation and use of plant genetic resources. although occasional attempts were made to raise the issue of in situ conservation at the nordic level (blixt et al, 1992), concrete work and activities never took off. decades later, during the period 2015-2019, the nordic countries (sweden, denmark, finland, norway and iceland) joined forces and initiated two subsequent projects to help strengthen the efforts of conservation and use of crop wild relatives (cwr) across the region. whereas the first project focused on reviewing and ∗corresponding author: jens weibull (jens.weibull@jordbruksverket.se) revising previously published compilations of cwr taxa, and their prioritisation, the second one put more emphasis on developing guidelines. as a result, policy recommendations were put forward that included creating national strategies for each nordic country, and adopting and implementing complementary in situ conservation as the main approach for safeguarding cwr across the region (weibull et al, 2016). a central activity of the second project (wild genetic resources – a tool to meet climate change) included an eco-geographic land characterisation (elc) analysis. using eco-geographic diversity as a proxy for genetic diversity is a well-known technique (parra-quijano et al, 2012) that has been employed for certain nordic countries (phillips et al, 2016), but not previously for the entire nordic region. based on more than 971,000 occurrence records, and using elc and socalled complementary conservation analysis (rebelo, 1994), fitzgerald et al (2019) were able to single out those protected areas (pas) in the region harbouring the largest number of priority cwr. the number one complementary pa site was in aalborg commune in received: 25 05 2020 accepted: 25 06 2020 published online: 31 08 2020 https://www.genresj.org https://www.dx.doi.org/10.46265/genresj.2020.1.17-24 https://www.genresj.org https://www.dx.doi.org/10.46265/genresj.2020.1.17-23 mailto:jens.weibull@jordbruksverket.se 18 weibull and phillips genetic resources (2020), 1 (1) 17–23 figure 1. the taxon richness of priority cwr across sweden. denmark covering 88 target species in two elc zones. the surprising fact that the first complementary pa site in sweden firstly appeared as number 13 on the nordic list and, secondly, was represented by a pa site in the mountain region close to norway called for an extended analysis. the work presented below takes a specific swedish perspective and aims to answer the following questions: (1) how common are nordic priority taxa in sweden and how are they distributed over the country? (2) will a targeted elc analysis provide an eco-geographic map of higher resolution? (3) how well does taxon diversity and genetic proxy diversity coincide with the existing distribution of pas? (4) will we be able to pinpoint specific sites in sweden where active in situ conservation of cwr may begin? we began by locating taxa rich areas and areas where potential data bias might be prevalent and continued by constructing an eco-geographic map to help determine how genetic diversity could be portioned across the country within populations of taxa. an in situ complementarity analysis accounting for taxa richness, eco-geographic richness and the pa network in the country was also performed. we see this work as a starting point for further in-depth research on cwr distribution, conservation and use within sweden. methods and results priority cwr in sweden in line with the nordic level approach, priority cwr for sweden were identified from the regional list of priority cwr (fitzgerald et al, 2018). therefore, the swedish priority list contained 121 naturalized and indigenous taxa. data on the taxa distribution was gathered from swedish lifewatch (https://www.anal ysisportal.se/) and limited to data gathered between the years 1990-2018. distribution data was combined for duplicated taxa from the initial list, e.g. barbarea vulgaris and barbarea vulgaris var. vulgaris, to limit duplication of results. in total, 102 priority taxa were used for sweden that altogether consisted of 617 320 occurrence points. number of occurrences per taxon ranged from 29 646 (vaccinium myrtillus l.) to less than 100 (brassica nigra (l.) w.d.j. koch, lactuca quercina l., rubus allegheniensis porter, trifolium alpestre l. and trifolium pratense var. maritimum zabel). the taxa with fewer than 100 occurrences should be considered for further research and surveying efforts to confirm their distribution and levels of vulnerability. analysis of species richness and bias to identify areas of species richness and data bias the tombio tool in qgis software qgis (2020) was utilised. analysis of taxon richness (figure 1) shows clearly that the south and east of sweden, including the island öland in the baltic sea, are the areas containing the highest number of different taxa. the areas in the north of sweden appear to be the least rich in priority taxa, however these areas also have the lowest number of recorded taxon occurrences (figure 2). this is to be expected since these areas cover two thirds of the country and to a very high degree overlap with the three boreal zones (southern, middle and northern) and the alpine zone$, i.e. bio-geographical zones characterised by lower winter temperatures, shorter vegetation periods and lower habitat diversity. an exception to this general picture includes the coastal area along the bothnian gulf all the way up to the swedish-finnish border at haparanda which is characterised by slightly more favourable climate and, thus, growing conditions. although there are more cwr occurrence data in the areas in the southern third of the country, especially around large cities, this pattern is not completely reflected within the taxon richness map. historically, occurrence data tend to be collected on an ad hoc, non-systematic, basis and closer to cities due to ease of access (chapman, 2005). the pattern of cwr occurrences in figure 2 also mirrors the demography of sweden1 and, as an additional effect, the location of main educational centres (universities, colleges). therefore, any potential bias this may cause in the 1 see e.g. https://sedac.ciesin.columbia.edu/data/set/grump-v1-popu lation-density/maps/2?facets=region:europe https://www.analysisportal.se/ https://sedac.ciesin.columbia.edu/data/set/grump-v1-population-density/maps/2?facets=region:europe https://www.analysisportal.se/ genetic resources (2020), 1 (1) 17–23 in situ conservation of swedish cwr 19 figure 2. the number of occurrences of the priority taxa across sweden. results should be acknowledged. this also shows a need to further survey those areas showing gaps in occurrence data to limit any biased results in future work. more surveying in the northern and western boreal and alpine regions of sweden will help to fill in gaps in our knowledge on cwr distribution and increase the accuracy of predictive analyses using geographic information systems. developing an eco-geographic land characterization map eco-geographic maps take account of environmental variables that combined create unique adaptive scenarios for plant species. a combination of geophysical, edaphic and bioclimatic variables that have the greatest influence on abiotic adaptation of the species are then used to create an eco-geographic land characterization map (elc map). the resulting elc zones can be used as a substitute to represent genetic diversity (parra-quijano et al, 2012). thus, if populations are conserved both in situ and ex situ across their eco-geographic range (i.e. within all their elc zones), this will ensure that the full range of genetic diversity is protected. the eco-geographic map for sweden was created with the capfitogen software (parra-quijano et al, 2016) using the following environmental variables: isothermality (average temperature range/annual temperature range), elevation, aspect of slope, ‘northness’, ‘eastness’, topsoil organic carbon content, topsoil ph, and topsoil depth2. figure 3 (left) shows the elc map comprising 25 elc zones at a resolution of 1 km2 cells. the largescale pattern of elc zones agrees reasonably well with the dominating land use classes of the country (figure 3, right), which indicates that the elc analysis does provide a useful estimate of vegetation characteristics and habitat diversity. complementarity analysis the complementarity analysis is an important concept for ensuring efficient conservation of resources. as described by rebelo and siegfried (1990), the analysis uses an iterative selection approach in which the cell, or pa, with the highest taxon number is selected first. these taxa are subsequently excluded from the analysis and the location with the next highest number of different taxa is selected, upon which the procedure is being repeated until all taxa are conserved across a network of reserve locations. the complementarity analyses were created using capfitogen software (parra-quijano et al, 2016). our complementarity analysis of the priority cwr within the network of pas identified eight complementary areas that altogether conserve 101 (99%) of the priority taxa (figure 4). the pa complementary network ensures that the largest number of different taxa are protected. in sweden, the majority of suitable pas, as regards priority cwr, were found to be located in coastal zones of southern sweden. the number one priority reserve, kristianstad vattenrike – a unesco-mab biosphere reserve – is the number one priority location as it contains the highest number of unique taxa (85 of 102 taxa; table 1). with the addition of the two following pas – stora alvaret, a birds directive pa, and tjålmejaure-laisdalen, a ramsar site in lapland – 93% of the unique taxa on the swedish priority list are covered. using the eco-geographic map, we can determine which elc zones are within each of the complementary pas. this will help to determine how well represented the eco-geographic zones are within the proposed network, which may then help to determine the range of genetic diversity among populations that is captured within the network. in our study, 13 of the 24 elc categories, or 54 %, are represented within the proposed pa complementary network. grid cell complementarity analysis the grid cell complementary network takes account of the number of taxa across the whole of sweden (not 2 the dataset used in this publication was made available by the swedish forest soil inventory, with responsibility in the department of soil and environment, slu. the authors are solely responsible for the interpretation of data. url: https://www.slu.se/miljoanalys/statist ik-och-miljodata/miljodata/webbtjanster-miljoanalys/markinfo/mark info/kartor/ (accessed 2020-04-23) https://www.slu.se/miljoanalys/statistik-och-miljodata/miljodata/webbtjanster-miljoanalys/markinfo/markinfo/kartor/ 20 weibull and phillips genetic resources (2020), 1 (1) 17–23 figure 3. the swedish eco-geographic land characterization map at a 1 km2 resolution (left), and a schematic view of dominating land use classes in sweden (right). figure 4. the eight protected areas needed to conserve 101 priority cwr taxa. just within pas). the grid cell complementarity analysis revealed that to protect the same 101 priority taxa, altogether 10 (5 km2) locations are required (figure 5). the majority of these are found in the south of sweden along the coast and in the east of sweden around stockholm and uppsala. while the number one grid cell, located near stockholm, includes 76 different taxa the three top cells in sweden protects close to 90 % of the priority taxa. overlaying both the pa complementary and grid cell complementary networks shows where locations overlap. this serves to help identify which locations to investigate further for potential in situ protection of cwr. in sweden, it would be most efficient to focus initial in situ conservation efforts within those pas that are located in the south such as kristianstad vattenrike, stora alvaret and gotlandskusten. these pas are also close to priority grid cell complementary locations. discussion the rationale for carrying out this extended analysis was the fact that sweden came out rather poorly in the study by fitzgerald et al (2019). the southern genetic resources (2020), 1 (1) 17–23 in situ conservation of swedish cwr 21 table 1. protected area complementarity analysis. the ’number of taxa’ is the total number of different taxa in the protected area. the ’number of additional taxa’ is the number of unique taxa within that protected area (i.e. these taxa are not found in any of the previous protected areas). protected area designation number of taxa number of additional taxa priority cumulative % kristianstad vattenrike unesco-mab biosphere reserve 85 85 1 84,2% stora alvaret special protection area (birds directive) 71 6 2 90,1% tjålmejaurelaisdalen ramsar site, wetland of international importance 19 3 3 93,1% blekinge arkipelag unesco-mab biosphere reserve 77 2 4 95,0% gotlandskusten nature conservation area 66 2 5 97,0% stora karlsö nature reserve 43 1 6 98,0% höga kusten/ kvarkens arkipelag world heritage site 42 1 7 99,0% hummelholm nature reserve 14 1 8 100,0% total 101 parts of the country, known to have been repeatedly inventoried since the mid-1800s and whose flora is very well mapped (e.g. weimarck and weimarck, 1985; sterner, 1986; genberg, 1992; rydberg and wanntorp, 2001; fröberg, 2006; edqvist and karlsson, 2007; johansson et al, 2016; johansson and petersson, 2016), were surprisingly underrepresented as compared to the findings of other countries. when comparing our results with those of fitzgerald et al (2019), we observe some immediate differences. whereas both studies have three sites in common – höga kusten (world heritage site), gotlandskusten (nature conservation area) and hummelholm (nature reserve) – all other locations differ. in particular, we note that our two top locations – kristianstad vattenrike (unesco-mab biosphere reserve) and stora alvaret (special protection area birds directive) – were not even included in the joint nordic analysis. there may be several reasons for this, but we suggest that a main cause could be the background data upon which the analysis is based. the datasets provided by the un environment programme world conservation monitoring centre (wcmc-unep) contain the entire spectrum of pas, ranging from areas with ‘strict’ protection such as national parks, nature reserves, habitat protection areas, and wildlife and plant sanctuaries via so-called natural monuments (e.g. individual and unique trees) to world heritage sites and unesco-mab biosphere reserves. in our analysis for sweden, we deselected sites that could give bias to our analysis including, e.g., those representing different habitats or purposes of protection such as helcom areas (baltic sea pas), ospar (marine pas), and ramsar sites. in addition, natural monuments that commonly represent individual objects were also removed. in our view, these measures provide a better subset of pas on which to draw conclusions. another aspect relates to the analysis of occurrence data. while fitzgerald et al (2019) used 971,633 data points in their analysis of the entire nordic region, we based our results on 617,320 data points from sweden only (time frame 1990-2018). we argue that data robustness is absolutely essential to be able to draw proper conclusions from analyses at a higher level of resolution. the risk of bias when using large data sets of distribution records, such as those available from the global biodiversity information facility (gbif), has been shown earlier (beck et al, 2014). we certainly acknowledge the value of the broad nordic analysis, but, as shown in this study, care should be taken when drawing generic conclusions to describe the situation ‘on ground’. the large differences in number of elc zones found in the regional vs. the national analysis, respectively, may at first seem surprising. what could the reason(s) be that we observed 25 elc zones while fitzgerald et al (2019) only described 8-10 in their analysis? the fact that an analysis covering the entire nordic region per se implies a much larger geographical scale also means that the elc variables used should try to capture the landscape over a wider range of eco-geographic ‘niches’. given that the diversity of zones vary greatly from northern 22 weibull and phillips genetic resources (2020), 1 (1) 17–23 figure 5. the protected area and grid cell complementary networks. the large black numbers on the map represent the priority grid cell locations and the blue areas are the complementary protected areas. iceland to south denmark and eastern finland, it should be expected that sweden – not sharing all the same niches – would only be described by a share of all the zones. while the regional analysis is important from the point of developing joint approaches, this observation highlights the importance of also looking at domestic eco-geographic variability as a basis for selecting key pas for cwr in situ conservation. finally, the observation by fitzgerald et al (2019) that 58 % of the identified important pas for cwr conservation were situated in norway raises the notion of possible data bias, as well as the procedure by which sites for cwr diversity are being identified. firstly, while the total number of pas in norway is only 27 % and 50 % of that of finland and sweden, respectively, the vast majority (80.6 %) are classified as strict nature reserves (iucn pa category ia). finland, on the other hand, is characterised by a large proportion of category vi pas (89.5 %). the fact that such areas are “[. . . ] often established to protect particular species or habitats rather than the specific ecological aims of category ia” (iucn, 2020) points to the possibility that cwr diversity is higher in category ia areas and it is for this reason that norway takes a lead in the nordic regional comparison. secondly, the finding that well-known and diversity-rich sites in several of the countries (e.g. åland archipelago in finland and öland in sweden) did not appear in the regional analysis calls for a careful evaluation of how data points and variables are used in the analysis. fitzgerald (personal communication) noted a general problem with coastline taxa that, “depending on the coordinate points and country map boundaries [. . . ] in some cases end[ed] up in the sea and therefore [had to] be removed from the analysis.” from a national perspective, where priorities need to be made, it is essential that those sites comprising the widest taxon and eco-geographic diversity are selected. conclusion our extended analysis of occurrence data of swedish cwr has helped us to identify three major pas where in situ conservation could take off. initial steps are now being taken to proceed with concrete measures within the unesco-mab biosphere reserve kristianstad vattenrike. further work is needed, however, to ensure the long-term robustness of any cwr conservation strategy within sweden. such planned activities are framed within the established nordic cwr network that is led by nordgen, and include: genetic resources (2020), 1 (1) 17–23 in situ conservation of swedish cwr 23 • an ex situ conservation analysis to identify any gaps in the collection of material for conservation and use outside of pas, on the assumption that seed management of cwr is technically and economically feasible; • a predicted distribution analysis of how populations may move under the current climate and to help identify collecting and data bias gaps across the country; and • a climate change analysis to determine if, how and when taxa may shift their distributions as the climate changes. this will be vital in determining which in situ pas will be the most effective in the long-term conservation of swedish cwr. finally, from a european perspective, it would be worthwhile in the future to foster synergies with other genetic resource domains (e.g. forestry, animal) in terms of identifying conservation sites and needs. such an approach may help to strengthen an in situ conservation network for cwr by adding “value” to proposed in situ sites. acknowledgements financial support from the board of agriculture, through the swedish programme for diversity of cultivated plants (pom), is gratefully acknowledged. author contributions jp carried out most of the pa and elc complementary data analysis during 2019, and jw re-edited the report into the present format. conflict of interest statement the authors declare no conflicts of interest. references beck, j., böller, m., erhardt, a., and schwanghart, w. 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(1985). atlas över skånes flora [an atlas of the flora of scania] (stockholm) 640p, (in swedish). https://doi.org/10.1016/j.ecoinf.2013.11.002 https://doi.org/10.1016/j.ecoinf.2013.11.002 https://doi.org/10.15468/doc.jrgg-a190 https://doi.org/10.15468/itkype https://doi.org/10.1017/s147926211800059x https://doi.org/10.1017/s147926211800059x https://www.iucn.org/theme/protected-areas/about/protected-areas-categories https://www.iucn.org/theme/protected-areas/about/protected-areas-categories https://doi.org/10.1007/s10722-011-9676-7 https://doi.org/10.1007/s10722-011-9676-7 https://drive.google.com/file/d/0b7jxjdasv98bejnrmxjhqy1hylk/edit https://drive.google.com/file/d/0b7jxjdasv98bejnrmxjhqy1hylk/edit https://doi.org/10.1111/ddi.12470 https://qgis.org/en/site/ https://doi.org/10.1016/0006-3207(90)90039-r https://doi.org/10.1016/0006-3207(90)90039-r http://www.sverigesutsadesforening.se/data/pdf/sut 2016-2.pdf http://www.sverigesutsadesforening.se/data/pdf/sut 2016-2.pdf introduction methods and results priority cwr in sweden analysis of species richness and bias developing an eco-geographic land characterization map complementarity analysis grid cell complementarity analysis discussion conclusion author contributions conflict of interest statement short communication genetic resources (2021), 2 (4), 1–6 doi: 10.46265/genresj.fiqj8274 https://www.genresj.org issn: 2708-3764 ‘zeitoun ennour’: a new olive (olea europaea l.) cultivar in tunisia with high oil quality fathi ben amar *,a, imen guellaoui b, mohamed ayadi c, olfa elloumi a, mohamed ali triki a and mohsen boubakerd a laboratory of improvement and protection of olive genetic resources, university of sfax, olive tree institute, aeroport road, 3000, sfax, tunisia b laboratory of improvement and protection of olive genetic resources, university of sousse, high agronomic institute of chott mariem, 4042, sousse, tunisia c laboratory of sustainable olive and fruit crops in semi-arid and arid areas, university of sfax, olive tree institute, aeroport road, 3000, sfax d high agronomic institute of chott mariem, university of sousse, 4042, sousse, tunisia abstract: an olive breeding program was started in tunisia in 1993 in order mainly to improve the fatty acid composition of the local cultivar ‘chemlali sfax’. ‘zeitoun ennour’ is a new cultivar obtained from a cross between ‘chemlali sfax’ and the local dual-purpose use cultivar ‘chemchali gafsa’. the morphological study of this cultivar showed that eleven characters dealing with fruit and endocarp differed from ‘chemlali sfax’, mainly regarding to their respective weights. this new cultivar had the same sensitivity to verticillium dahliaekleb and earlier bearing than the original variety. its olive production was considered as high as for ‘chemlali sfax’ but with partial self-compatibility and late maturity. the new cultivar realized a net improvement in comparison with the original cultivar particularly regarding its fatty acid composition with very high oleic acid content (>75 %) and low palmitic and linoleic acid contents (<10 %). the new cultivar was recently released and will be available for growers as soon as possible. keywords: chemlali sfax, new cultivar, morphology, fatty acids, agronomy citation: ben amar, f., guellaoui, i., ayadi, m., elloumi, o., triki, m. a., boubaker, m. (2021). ‘zeitoun ennour’: a new olive (olea europaea l.) cultivar in tunisia with high oil quality. genetic resources 2 (4), 1–6. doi: 10.46265/genresj.fiqj8274. © copyright 2021 the authors. this is an open access article distributed under the terms of the creative commons attribution license (cc by 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. introduction attempts to develop new olive cultivars have been carried out in many olive-producing countries (italy, turkey, israel, spain, tunisia, egypt, iran, china, ukraine and turkmenistan) as reported by bellini et al (2008). most of these programs have focused on crossbreeding among the most outstanding cultivars in their respective countries. in tunisia, a breeding program has been carried out within the context of the project ‘olive breeding’ (supported by the international olive council) since 1994. in this program, the cultivar ‘chemlali sfax’ ∗corresponding author: fathi ben amar (fathibenamar@yahoo.fr) was crossed with both autochthonous and foreign olive varieties as pollen donors, yielding 1,200 seedlings. the goal of this program was to improve the acidic composition the oil, since ‘chemlali sfax’ has low oleic acid (55%) and high palmitic acid (19.6%) (zarrouk et al, 2009; bellini et al, 2008). several studies have shown the dietary importance of fatty acid composition of lipids. a healthy diet should contain a limited amount of saturated fatty acid (e.g. palmitic acid) to reduce the total cholesterol content and a high amount of monounsaturated fatty acid (e.g. oleic acid) which was shown to prevent the risk of cardiovascular diseases, reduce the insulin bodyrequirement and decrease the plasma concentration of glucose (d’imperio et al, 2007). according to zarrouk et al (2009), monounsaturated fatty acids have great received: 18.12.2020 accepted: 06.09.2021 published online: 17.09.2021 https://www.genresj.org https://www.dx.doi.org/10.46265/genresj.fiqj8274 https://www.genresj.org https://www.dx.doi.org/10.46265/genresj.fiqj8274 mailto:fathibenamar@yahoo.fr 2 ben amar et al genetic resources (2021), 2 (4), 1–6 importance because of their nutritional implication and effect on the oxidative stability of oils. according to barranco et al (2000), the low content of oleic acid for the ‘chemlali sfax’ olive cultivar is considered as a deficiency that should be resolved. high oleic acid content is also a breeding objective in sunflower (neto et al, 2016) and peanut (godoy et al, 2014). for example, breeding efforts in peanut have led to the release of two genotypes with much higher concentration of oleic acid (godoy et al, 2014). many studies were undertaken on seedlings of the ‘chemlali sfax’ breeding program regarding morphological description (laaribi et al, 2014; guellaoui et al, 2019) and acidic composition of the oil (manäı et al, 2007; rjiba et al, 2009; dabbou et al, 2010; ben-amar et al, 2019). morphological characteristics of the ‘chemlali sfax’ olive tree seedlings showed a high genetic variability. also, these studies revealed high variability in the main fatty acid concentrations and several seedlings had a chemical composition more interesting than that of the original cultivar. recently, five new cultivars obtained in the tunisian crossbreeding program were released and published in the official journal of republic of tunisia (jort, 2017). two released hybrids were already reported by guellaoui et al (2019) and ben-amar et al (2019). according to these authors, the two new cultivars (zeitoun ennwader and chemlali mhassen) were mainly characterized by better fatty acid composition regarding to oleic and palmitic acid contents than the original cultivar ‘chemlali sfax’. regarding abiotic stresses, elloumi et al (2016) studied the tolerance of several hybrids to salinity stress in comparison with the original cultivar ‘chemlali sfax’. the objective of this study was to describe the main morphological, agronomic and oil quality characters of another released olive cultivar ‘zeitoun ennour’ in comparison with the original cultivar ‘chemlali sfax’. material and methods plant material ‘zeitoun ennour’ is a new olive (olea europaea l.) cultivar obtained in a crossbreeding program in tunisia. it was derived from a cross carried out in 1993/1994 between ‘chemlali sfax’ (female) and ‘chemchali gafsa’ (male), two cultivars from different geographical origins: ‘chemlali sfax’ from sfax in the center-wast of tunisia and ‘chamchali gafsa’ from gafsa in the centerwest of tunisia. ‘chemchali gafsa’ showed better fatty acid composition when compared to ‘chemlali sfax’, the most widely adopted olive cultivar in tunisia as reported by grati-kamoun and khlif (2001). the cross was performed by pollination of flowers on bagged branches with fresh pollen grain and forced growth of seedlings was carried out in a greenhouse to shorten the juvenile period. seedlings were planted in two open fields during 1997-1998 with a density of 1250 trees/ha (4 x 2m spacing): the experimental station of the olive institute at sfax (central tunisia, lat 34◦ 44′ n, long 10◦ 46′ e) and the research station of ‘taous’, which is about 26 km from sfax (lat 34◦ 56′ n, long 10◦ 36′ e). the first crop was obtained in 2000 and seedling evaluation was carried out for three consecutive harvest seasons. the original seedling of ‘zeitoun ennour’ was selected mainly on the basis of its fatty acid composition. after vegetative propagation by semi-hardwood stem cuttings (2002 to 2004), propagated trees of ‘zeitoun ennour’ together with other selected seedlings were planted in a comparative trial since 2005 at 6 × 4m spacing and irrigated conditions at the experimental farm of ‘taous’ in the region of sfax. methodology the evaluation of ‘zeitoun ennour’ and the check ‘chemlali sfax’ in this trial was done for three trees per cultivar during three years (2013-2015) on the following characters: morphology morphological description was carried out each year according to the procedure of ioc (international olive council, 1997a) by using a total of 21 characters recorded on 40 leaves (2 characters), 40 fruits (10 characters) and their endocarps (9 characters). one sample was taken from the productive trees each year. leaves and fruits were harvested together when the fruit skin was almost yellow-purple and leaves were collected from the middle part of one-year old shoots. agronomy agronomic characters were recorded according to the ioc norms (international olive council, 1997b). bearing earliness was determined as the number of years of the first significant bearing after planting in 2005. this number was recorded for each cultivar when more than 50% of the plants were already in production. olive production was evaluated on the same three trees during the three years. the olive production per tree was usually recorded in november. the ripening index was determined according to the formula of hermoso et al (1991) based on the colour of the skin and the pulp and varied between 0 and 7. this index was determined on a sample of 100 fruits collected every week from november to january from the productive trees and the date which corresponded to the optimal index for harvest equal to 3.5 (international olive council, 1997b) was recorded. pollen compatibility was determined by choosing two floral branches for each tree and recording fruit sets obtained from cross-pollination (not bagged branch) and self-pollination (bagged branch). according to barranco et al (2000) , a cultivar is considered self-compatible when the two fruit set values are similar, partially self-compatible when fruit set from selfpollination is less than that from cross-pollination and self-incompatible when fruit set from self-pollination is zero. genetic resources (2021), 2 (4), 1–6 olive genetic improvement in tunisia 3 the tolerance test to verticillium dahliae kleb was undertaken in greenhouse conditions on five one year old plants per cultivar. the inoculation was made by wetting the plants in a conidial suspension adjusted to 106 conidia/ml. cultivars were classified into five categories according to their audpc average (area under disease progress curve) established previously by lopez-escudero et al (2004): highly resistant (hr), 0 to 10 %; resistant (r), 11 to 30 %; moderately susceptible (ms), 31 to 50 %; susceptible (s), 51 to 70 %; and extremely susceptible (e), 71 to 100 %. fatty acid composition each year, representative olive samples were harvested from the productive trees when the maturity index was approximately 3.5 (optimum maturity). olive oil was produced by grinding 2.5 kg stoned olives and extracting the oil by mechanical means, following standard methods used in oil factories, including milling and malaxation. the fatty acid composition of the oils was determined by gas chromatography (gc) as fatty acid methyl esters, using a hewlett-packard model 4890d gas chromatograph. fatty acids were identified by comparing their retention times with those of standard compounds. three major fatty acids were evaluated in this study, palmitic acid (c16:0), oleic acid (c18:1) and linoleic acid (c18:2) and their concentrations in ‘zeitoun ennour’ were compared with those of the original cultivar ‘chemlali sfax’. data analysis for each morphological character, the type having the highest mean value over three years was attributed to the cultivar. bearing earliness can be very early (< 3 years), early (3 years), medium (4 years), late (5 years) or very late (> 5 years). the ripening date corresponding to maturity index 3.5 can be in late autumn (early maturity), early winter (medium maturity) or late winter (late maturity). variance analysis was made for olive production and verticillium tolerance considering years and plants as replicates respectively. for pollen compatibility, variance analysis was undertaken for each cultivar to compare fruit sets from self-pollination and free pollination considering years as replicates. for oil chemical characters, variance analysis with years as replicates was carried out for each fatty acid. all data analyses were performed using the statistical procedures in xlstat 11.0 and the separation of means was done by duncan test at 5 % level. results and discussion morphological characterization the morphological evaluation of the new cultivar is presented in table 1 and figure 1. leaves of ‘zeitoun ennour’ were mostly of elliptic-lanceolate shape and flat longitudinal curvature similar to the original cultivar. figure 1. leaf, fruit and endocarp of ‘zeitoun ennour’ olive cultivar. scale in cm. fruits of the new cultivar were asymmetrical, with a truncated base and rounded apex, medium weight, central maximum diameter, few lenticels, and without nipple. at maturity stage, the location of colour change start was uniformly across the whole epidermis and the colour at the end of maturity was black. ‘zeitoun ennour’ had asymmetric endocarp with medium weight, elliptic shape, a round base, pointed apex, its maximum diameter toward the apex, rugose surface and regular distribution of grooves. the apex termination was without mucro. this cultivar differed from the typical of ‘chemlali sfax’ in more than half of the scored fruit and endocarp traits, (11 traits in total), especially regarding their respective weights, where ‘zeitoun ennour’ scored higher (> 2 g and > 0.3 g respectively) than ‘chemlali sfax’ according to ioc norms (international olive council, 1997a). chemlali sfax was previously shown to have low fruit and endocarp weights (barranco et al, 2000; trigui and msallem, 2002). despite the evidence of the subjectivity of the morphological description, it can be concluded that the new cultivar showed substantial genetic differences from ‘chemlali sfax’. laaribi et al (2014) reported wide genetic diversity observed within and between olive tree seedlings issued from the same tunisian breeding program. agronomic characterization an agronomic description of ‘zeitoun ennour’ and ‘chemlali sfax’ was carried out a the comparative field trial established at the experimental farm ‘taous’ of the olive tree institute (table 2). the new cultivar showed a short juvenility period (3 years) from planting till economic bearing. it can be classified with early bearing in comparison with ‘chemlali sfax’ (medium). yield traits were evaluated on 10-year-old trees during the period 2013-2015, the average yield per tree was classified as high for both cultivars similar what had previously been reported for ‘chemlali sfax’ (barranco et al, 2000; trigui and msallem, 2002). 4 ben amar et al genetic resources (2021), 2 (4), 1–6 table 1. description of the main morphological characters of olive cultivar ‘zeitoun ennour’ compared with the control ‘chemlali sfax’. pdm : position of maximum transverse diameter organ character zeitoun ennour chemlali sfax leaf shape elliptic-lanceolate elliptic-lanceolate longitudinal curvature flat flat fruit weight medium low shape ovoid ovoid symmetry asymmetric symmetric pdm central central apex rounded rounded base truncate truncate nipple absent absent start of colour change uniform apex lenticels many few maturity colour black black endocarp weight medium low shape elliptic elliptic symmetry asymmetric symmetric pdm toward apex central apex pointed rounded base rounded pointed surface rugose smooth distribution of groves regular regular apex termination without mucro with mucro ‘zeitoun ennour’ displayed late maturity with optimum ripening period occurring in late winter while ‘chemlali sfax’ displayed medium maturity. regarding pollination mode, fruit set following selfpollination (5.13 %) was significantly inferior to that following crosspollination (9.87 %). consequently, the new cultivar was found to be partially self-incompatible according to the norms of barranco et al (2000). ‘chemlali sfax’ had a self-compatible behavior in our study since fruit sets from self-pollination and crosspollination were statistically similar (11.21 and 10.45 % respectively). the same performance was reported for ‘chemlali sfax’ by trigui and msallem (2002) in the centre of origin. thus, while pollinator trees may not strictly be required in the field with this new cultivar, several pollinator trees planted in the orchards could compensate for the partial self-incompatibility, as suggested by mehri et al (2003) in these situations. in this case, a study of the flowering period for different olive varieties is necessary to identify the best pollen donor for ‘zeitoun ennour’. with respect to verticillium dahliae kleb, our results indicated that ‘zeitoun ennour’ and ‘chemlali sfax’ had similar levels of tolerance with 65 and 57 % respectively. consequently, they were susceptible to this fungus. thus, we suggest propagating this new cultivar with semi hardwood cuttings from healthy trees in order to avoid verticillium infection. morever, the best solution in the propagation of this cultivar is to follow the plant certification procedure as outlined in the eu plant health regulation (european commission, 2016) to ensure the verticillium free status of olive plants. in fact, verticillium wilt is the most destructive disease affecting olive orchards in tunisia (gharbi et al, 2020) and in the world (lópez-escudero and mercado-blanco, 2011). fatty acid composition the ‘zeitoun ennour’ cultivar had a considerably improved fatty acid composition compared to the original cultivar ‘chemlali sfax’ (table 3). table 2. description of the main agronomic characteristics of the new olive cultivar compared with ‘chemlali sfax’. means for each character followed by the same letter are not significantly different except for fruit set where the comparison was made between self-pollination and cross-pollination for each cultivar. character zeitoun ennour chemlali sfax earliness of bearing 3b 4a olive production per tree (kg) 12.2a 10.5a ripening late winter (late) early winter (medium) fruit set (self-pollination) (%) 5.13b 11.21a fruit set (cross-pollination) (%) 9.87a 10.45a verticillium tolerance (%) 65a 57a genetic resources (2021), 2 (4), 1–6 olive genetic improvement in tunisia 5 table 3. mean fatty acid concentrations (%) of the new cultivar ’zeitoun ennour’ compared with ‘chemlali sfax’ and corresponding ioc norms (international olive council, 1997b). for each fatty acid, different letters following the means indicate significant differences at 5 % level character zeitoun ennour chemlali sfax ioc norm oleic acid 76.3a 59b 55 – 83 palmitic acid 10.0a 19.6b 7.5 – 20 linoleic acid 9.7a 16.8b 2.5 – 21 in comparison with the original variety, the mean values of ‘zeitoun ennour’ for the three fatty acids were significantly better than the original variety. the concentration of oleic acid, the main monounsaturated fatty acid, was higher for the new cultivar (76.3 %) than the original cultivar (59 %), while the level of palmitic acid, the major saturated fatty acid in olive oil, was significantly lower (10 %) than ‘chemlali sfax’ (19.6 %). the content of linoleic acid, another important monounsaturated acid, for ‘zeitoun ennour’ was also lower (9.7 %) than ‘chemlali sfax’ (16.8 %). in addition, the fatty acid composition of ‘zeitoun ennour’ is within the standard norms reported by the international olive council (1997b), while ‘chemlali sfax’ practically has the lowest value of oleic acid and the maximum limit of palmitic acid. according to d’imperio et al (2007) and zarrouk et al (2009), a healthy olive oil should have high oleic acid content and low palmitic acid content. thus, we consider that the new selected cultivar ‘zeitoun ennour’ realized an important genetic gain in fatty acid composition and could be of great benefit in the tunisian olive sector (farmers, industrials and oil exporters). from the same breeding program, two other hybrids were released and characterized. zeitoun ennwader (chemlali sfax/lucques) and chemlali mhassen (chemlali sfax autopollinated) were presented, respectively, by ben-amar et al (2019) and guellaoui et al (2019). conclusion ‘zeitoun ennour’ was registered by the tunisian ministry of agriculture, hydraulic resources and fisheries (marhp) under number 191 in january 2017 (jort, 2017). the propagation of this new cultivar will be assured as soon as possible through a certification procedure. simultaneously, this new cultivar is under evaluation in rain fed conditions in tunisia. the crossbreeding program in tunisia since 1993 has allowed selection of superior olive genotypes which could increase the economic input of the oil sector. acknowledgments the authors wish to thank the ministry of agriculture, hydraulic resources and fishery (especially iresa) and the ministry of higher education and scientific research (university of sfax) for their financial support to this breeding program. author contributions imen guellaoui performed all the measurements and notations, fathi ben amar and olfa elloumi wrote the manuscript and processed the experimental data, mohamed ali triki executed fungi tests, mohamed ayadi performed oil quality tests and mohsen boubaker supervised the work. all authors read and approved the final manuscript. conflict of interest statement the authors declare no conflict of interest. references barranco, d., cimato, a., fiorino, p., rallo, l., touzani, a., castaneda, c., serafini, f., and trijillo, i. 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(2019). chemometric classification of new olea europaea l. cultivars developed through a crossbreeding program in tunisia. journal of scientific agriculture 3, 22–27. doi: https://doi.org/10.25081/ jsa.2019.v3.5453 hermoso, m., uceda, m., garcia-ortiz, a., morales, j., frias, l., and fernández, a. (1991). elaboración de aceite de oliva de calidad volume 5/91 of colección: apuntes. (sevilla: dirección general de investigación, tecnoloǵıa y formación agroalimentaria y pesquera), 36-39. international olive council (1997a). methodology for primary characterization of olive varieties. project resgen-ct (67/97). international olive council (1997b). methodology for secondary characterization of olive varieties. project resgen-ct (67/97). jort (2017). list of protected varieties object of the plant variety certificates for the year 2016. official journal of republic of tunisia, 033, 25.04.2017. laaribi, i., mezghani-aiachi, m., and mars, m. (2014). phenotypic diversity of some olive tree progenies issued from a tunisian breeding program. european scientific journal 10(6), 292–313. doi: https://doi. org/10.19044/esj.2014.v10n6p%25p lópez-escudero, f. j. and mercado-blanco, j. (2011). verticillium wilt of olive: a case study to implement an integrated strategy to control a soil-borne pathogen. plant soil 344, 1–50. doi: https://doi.org/10.1007/ s11104-010-0629-2 lopez-escudero, f. j., rio, c. d., caballero, j. m., and blanco-lopez, m. a. (2004). evaluation of olive cultivars for resistance to verticillium dahliae. eur. j. plant. pathol 110, 79–85. doi: https://doi.org/10. 1023/b:ejpp.0000010150.08098.2d manäı, h., mahjoub-haddada, f., trigui, a., daoud, d., and zarrouk, m. 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(2009). profiles of volatile compounds from nine new hybrids obtained by controlled crossings on olive ‘chemlali’ cultivar and mediterranean varieties. natural product research 23(7), 622–632. doi: https://doi.org/10.1080/14786410802155939 trigui, a. and msallem, m. (2002). “oliviers de tunisie,” catalogue des varietés autochtones et types locaux volume 1 (ministère de l’agriculture, iresa, institut de l’olivier, république tunisienne), 159p. zarrouk, w., baccouri, b., taamalli, w., trigui, a., daoud, d., and zarrouk, m. (2009). oil fatty acid composition of eighteen mediterranean olive varieties cultivated under the arid conditions of boughrara (southern tunisia). grasas y aceites 60, 498–506. doi: https://doi.org/10.3989/gya.021109 https://doi.org/10.25081/jsa.2019.v3.5453 https://doi.org/10.25081/jsa.2019.v3.5453 https://doi.org/10.19044/esj.2014.v10n6p%25p https://doi.org/10.19044/esj.2014.v10n6p%25p https://doi.org/10.1007/s11104-010-0629-2 https://doi.org/10.1007/s11104-010-0629-2 https://doi.org/10.1023/b:ejpp.0000010150.08098.2d https://doi.org/10.1023/b:ejpp.0000010150.08098.2d https://doi.org/10.1002/jsfa.2732 http://doi.org/10.1400/14142 https://doi.org/10.1590/1984-70332016v16n3a30 https://doi.org/10.1080/14786410802155939 https://doi.org/10.3989/gya.021109 introduction material and methods plant material methodology morphology agronomy fatty acid composition data analysis results and discussion morphological characterization agronomic characterization fatty acid composition conclusion author contributions conflict of interest statement supplemental data for: schultze-kraft, r., peters, m., wenzl, p. (2020). a historical appraisal of the tropical forages collection conserved at ciat. genetic resources 1 (2), 51–68. doi: 10.46265/genresj.kzew5023. supplemental table 1 recent nomenclatural changes of tropical forage species mentioned in this document under their previous names, based on the taxonomy accepted by the usda genetic resources information network (grin). [for further name changes of forage species during the past 50 years the reader is referred to cook and schultze-kraft (2015) and cook et al. (2020)]. previous name and authority new name and authority brachiaria brizantha (hochst. ex a. rich.) stapf urochloa brizantha (hochst. ex a. rich.) r.d. webster brachiaria decumbens stapf urochloa decumbens (stapf) r.d. webster brachiaria humidicola (rendle) schweick. urochloa humidicola (rendle) morrone & zuloaga brachiaria dictyoneura (fig. & de not.) veldkamp1 urochloa humidicola (rendle) morrone & zuloaga2 brachiaria ruziziensis r. germ. & c.m. evrard urochloa ruziziensis (r. germ. & c.m. evrard) crins brachiaria spp. hybrids urochloa spp. hybrids centrosema pubescens auct. pl.2 centrosema molle mart. ex benth.4 desmodium heterocarpon subsp. ovalifolium (prain) h. ohashi [syn. d. ovalifolium (prain) wall. ex merr.] grona heterocarpa subsp. ovalifolia (prain) h. ohashi & k. ohashi desmodium strigillosum schindl. grona strigillosa (schindl.) h. ohashi & k. ohashi panicum maximum jacq. megathyrsus maximus (jacq.) b.k. simon & s.w.l. jacobs pennisetum glaucum (l.) r. br. [syn. p. americanum (l.) leeke; p. typhoides (burm. f.) stapf & c.e. hubb.] cenchrus americanus (l.) morrone pennisetum purpureum schumach. cenchrus purpureus (schumach.) morrone pueraria phaseoloides (roxb.) benth. neustanthus phaseoloides (roxb.) benth. 1the name change from brachiaria dictyoneura to urochloa humidicola applies only to accession ciat 6133 (cpi 59610), released in several countries under various cultivar names (see table 5). the accession had entered the pasture research system under the erroneous classification of b. dictyoneura and appeared subsequently under that name in a considerable number of publications. it was reclassified as b. humidicola in 1994 by dr. stephen a. renvoize (royal botanic gardens kew) and its current name accepted by grin is urochloa humidicola. 2this refers to the species represented by the commercial cultivar ‘centro’ (‘common centro’) widely used for forage and as soil cover in tree plantations, originally described as centrosema molle but in the literature throughout decades erroneously referred to as c. pubescens. (there does exist, however, a species with the latter name, c. pubescens benth., accepted by grin. formerly known as c. schiedeanum r.j. williams & r.j. clem., it is the taxon to which the australian cultivar ‘belalto’ belongs.) https://doi.org/10.46265/genresj.kzew5023 https://npgsweb.ars-grin.gov/gringlobal/taxonomydetail.aspx?id=7570 https://npgsweb.ars-grin.gov/gringlobal/taxonomydetail.aspx?id=9848 https://npgsweb.ars-grin.gov/gringlobal/taxonomydetail.aspx?id=405748 https://npgsweb.ars-grin.gov/gringlobal/taxonomydetail.aspx?id=405748 https://npgsweb.ars-grin.gov/gringlobal/taxonomydetail.aspx?id=405748 https://npgsweb.ars-grin.gov/gringlobal/taxonomydetail.aspx?id=26558 https://npgsweb.ars-grin.gov/gringlobal/taxonomydetail.aspx?id=27187 https://npgsweb.ars-grin.gov/gringlobal/taxonomydetail.aspx?id=27208 https://npgsweb.ars-grin.gov/gringlobal/taxonomydetail.aspx?id=464278 https://npgsweb.ars-grin.gov/gringlobal/taxonomydetail.aspx?id=30357 https://npgsweb.ars-grin.gov/gringlobal/taxonomydetail.aspx?id=479388 supplemental table 2 plant collectors who participated in the collecting missions mentioned in table 1. year country collector 1972-73 colombia venezuela pedro j. argel, bert grof m. delia escobar 1974-75 bolivia brazil colombia venezuela l. martínez, víctor m. patiño d. balia, i. camargo, l.c. freire, b. grof, víctor m. patiño, rainer schultze-kraft, u.r. da silva guido delgadillo, víctor m. patiño adalberto j. flores, c. montes, víctor m. patiño 1976-77 brazil colombia venezuela mário s.f. dantas, ray isbell, dorival pimentel, robert reid, rainer schultze-kraft, f. beni de sousa jack r. harlan, manuel sánchez, rainer schultze-kraft m. delia escobar, adalberto j. flores 1978-79 brazil colombia panama thailand venezuela lidio coradin, rainer schultze-kraft, joão c. silva, glocimar p. da silva manuel sánchez, rainer schultze-kraft belisario castillo, carlos ortega, rainer schultze-kraft chanchai manidool, siriphong pattanavibul, rainer schultze-kraft m. delia escobar, adalberto j. flores, espedito garcía, rainer schultze-kraft 1980-81 brazil colombia venezuela don f. cameron, lidio coradin, rainer schultze-kraft, glocimar p. da silva arsenio ciprián, gerhard keller-grein, robert reid, manuel sánchez, rainer schultze-kraft adalberto j. flores, rainer schultze-kraft 1982-83 colombia malaysia papua new guinea peru thailand álvaro arias, javier belalcázar, luis e. chipiaje, rainer schultzekraft wong choi chee, siriphong pattanavibul, rainer schultze-kraft, hassan wahab bruce pengelly, m. raurela, graeme tupper césar reyes, rainer schultze-kraft, césar valles siriphong pattanavibul, rainer schultze-kraft 1984 brazil china colombia ethiopia kenya indonesia thailand venezuela lidio coradin, liana jank, m. isabel penteado, rainer schultzekraft, glocimar p. da silva, f. beni de sousa huang xunwen, jiang houming, rainer schultze-kraft libardo arévalo, javier belalcázar, luis e. chipiaje, charles mullenax, armando peralta, e. sánchez, tony russell-smith, rainer schultze-kraft gerhard keller-grein, a. tsehay gerhard keller-grein, s. mengistu rainer schultze-kraft siriphong pattanavibul, rainer schultze-kraft adalberto j. flores, gerhard keller-grein year country collector 1985 burundi colombia indonesia panama rwanda tanzania venezuela zimbabwe gerhard keller-grein álvaro arias, javier belalcázar, he chaozu, rainer schultze-kraft abdul gani, rainer schultze-kraft pedro j. argel, olmedo duque, rainer schultze-kraft gerhard keller-grein gerhard keller-grein, m. lugenja, s. mengistu adalberto j. flores, socors gonzález, rainer schultze-kraft gerhard keller-grein, j. matanda 1986 colombia costa rica indonesia mexico venezuela javier belalcázar, g. bravo, arsenio ciprián, ernesto dávila, les a. edye, o. garzón, l.a. giraldo e. salazar, rainer schultze-kraft, a. vargas abdul gani, rainer schultze-kraft, m.e. siregar juan josé aguirre, s. amaya, javier enríquez, j. maldonado, j.m. martínez, armando peralta, rainer schultze-kraft adalberto j. flores, f. gómez, socors gonzález, j.c. ortega, rainer schultze-kraft, f. serrano, r. zerpa 1987-88 brazil china colombia thailand lidio coradin, nuno s. costa, rainer schultze-kraft, glocimar p. da silva he chaozu, jiang houming, liu guodao, rainer schultze-kraft, yang zhen alvaro arias, javier belalcázar, arsenio ciprián, ernesto dávila, armando ferrufino, l.a. heredia, rodolfo heyn, m. mesa siriphong pattanavibul, rainer schultze-kraft 1989-90 cameroon colombia honduras b. mbélé, etienne t. pamo, rainer schultze-kraft javier belalcázar, ernesto dávila, carlos hernández, gustavo maldonado j.m. flores, américo rush, rainer schultze-kraft, óscar suazo, linus wege 1991-93 colombia thailand vietnam j.w. alvarado, javier belalcázar, diego chamorro, arsenio ciprián, i. leal, liu guodao, brigitte maass, g.s. naranjo siriphong pattanavibul, rainer schultze-kraft, pensri sornprasitti ha dinh tuan, nguyen phung ha, rainer schultze-kraft references: cook, b.g., schultze-kraft, r. (2015). botanical name changes – nuisance or a quest for precision? tropical grasslands-forrajes tropicales 3, 34–40. doi: 10.17138/tgft(3)34-40. cook, b.g., pengelly, b.c., schultze-kraft, r., taylor, m., burkart, s., cardoso arango, j.a., gonzález guzmán, j.j., cox, k., jones, c., peters, m. (2020). tropical forages: an interactive selection tool. 2nd and revised edn. (cali, colombia: ciat, and nairobi, kenya: ilri). www.tropicalforages.info. https://doi.org/10.17138/tgft(3)34-40 https://www.tropicalforages.info/ original article genetic resources (2021), 2 (3), 36–50 doi: 10.46265/genresj.obvw6791 https://www.genresj.org issn: 2708-3764 biodiversity assessment of african locust bean (parkia biglobosa) accessions from savanna and forest zones of nigeria as revealed by seed storage proteins and rapd markers pamela e akin-idowu *,a, ayodeji o aduloju a, omolara i akinyoola a, dorcas o ibitoye b, uterdzua orkpeh b, usifo g adebo a,c and yemisi o olagunju a a biotechnology unit, national horticultural research institute, p.m.b. 5432, jericho reservation area, idi-ishin, ibadan, nigeria b genetic resources unit, national horticultural research institute, p.m.b. 5432, jericho reservation area, idi-ishin, ibadan, nigeria c school of agriculture and food, faculty of veterinary and agricultural sciences, the university of melbourne, melbourne, australia abstract: understanding the level and distribution of genetic diversity in african locust bean (parkia biglobosa) would strengthen breeding and conservation programmes towards domestication and sustainable use of this species. sixteen accessions of p. biglobosa were assessed for variability based on seed morphology, seed protein and dna profiling. significant variation in seed characteristics were observed across locations. seed protein profiling by sds-page revealed homogeneity as most bands were found common in all accessions, indicating that the protein profiles are highly conserved. protein profiling separated the 16 accessions into four major clusters at 0.93 similarity coefficient. most accessions grouping into cluster 1 had a similarity coefficient of close to 100% and were from the derived savanna suggesting the presence of duplicates. accessions nh/2016/p14, nh/2016/p03 and nh/2016/p04 grouped into clusters ii, iii and iv; respectively. sixteen rapd markers generated a total of 256 bands of which 63.67% were polymorphic. gene diversity ranged from 0.41 to 0.93 and polymorphic information content (pic) from 0.39 to 0.93. the rapd-based dendrogram separated accessions into six groups at 0.68 similarity coefficient. based on a polymorphic seed storage protein marker a genetically distinct accession nh/2016/p04 could be exploited for breeding purposes. the homogeneity of alleles and narrow genetic base as revealed by rapd and sdspage analyses suggests possible loss of intraspecific genetic diversity. thus, intensification of germplasm collections across the different agroecological zones and characterization using specific markers will give a better understanding of diversity of p. biglobosa in order to enhance selection towards conservation, breeding and sustainable utilization. keywords: african locust bean, genetic diversity, agroecological zone, rapd, sds-page, parkia biglobosa citation: akin-idowu, p. e., aduloju, a. o., akinyoola, o. i., ibitoye, d. o., orkpeh, u., adebo, u. g., olagunju, y. o. (2021). biodiversity assessment of african locust bean (parkia biglobosa) accessions from savanna and forest zones of nigeria as revealed by seed storage proteins and rapd markers. genetic resources 2 (3), 36–50. doi: 10.46265/genresj.obvw6791. © copyright 2021 the authors. this is an open access article distributed under the terms of the creative commons attribution license (cc by 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. ∗corresponding author: pamela e akin-idowu (elohoidowu@hotmail.com) introduction in sub-saharan africa several indigenous agroforestry systems exist containing woody species known as multipurpose trees. africa locust bean [parkia biglobosa (jacq.) r. br. ex g. don] is a well-known indigenous received: 04.05.2020 accepted: 11.06.2021 published online: 29.06.2021 https://www.genresj.org https://www.dx.doi.org/10.46265/genresj.obvw6791 https://www.genresj.org https://www.dx.doi.org/10.46265/genresj.obvw6791 mailto:elohoidowu@hotmail.com genetic resources (2021), 2 (3), 36–50 genetic variation of african locust bean from selected agroecological zones in nigeria 37 agroforestry fruit tree species belonging to the subfamily mimosoideae and family fabaceae (amusa et al, 2014; houndonougbo et al, 2020). the species grows in multiple climatic zones and is widely distributed from senegal and guinea in west africa to uganda in central africa (lompo et al, 2018). parkia biglobosa is maintained in the parklands of africa for nontimber forest foods (houndonougbo et al, 2020; leakey, 2012; okoye et al, 2014). p. biglobosa is a valuable resource for the livelihoods of local people in sub-saharan africa because of its multipurpose function as a source of food, nutrition, medicine and income (matig et al, 2002; nikiema, 2005; awodoyin et al, 2015; sankhon et al, 2014; shao, 2002; sina and traore, 2002; teklehaimanot, 2004). the fruit pulp and seeds are both suitable for human consumption. the mealy pulp from the species’ fruits is a major source of energy and nutrients including carbohydrates, proteins, lipids, carotenoids, vitamins a, b, c, and oligoelements (nyadanu et al, 2017). the seeds are an important source of plant protein and essential amino acids among rural communities who often have limited access to animal proteins due to high cost (akin-idowu et al, 2018; alabi et al, 2005) and are also rich in energy value, saccharose, vitamin c, lipids, carbohydrates (orwa et al, 2010) and bioactive components such as phenolic compounds which may contribute in health promoting properties (dedehou et al, 2016; okoye et al, 2014). p. biglobosa seeds are fermented to make a food condiment called dawadawa (iru) which is rich in dietary protein and serves as an important substitute for animal protein (lamien et al, 2011; nyadanu et al, 2017). the leaves, bark, roots and flowers are also used in the treatment of many diseases such as hypertension, wound healing and malaria (dedehou et al, 2016; ouedraogo, 1995). in spite of the importance of this species in traditional agriculture, regular cultivation of the fruit tree has rarely gone beyond conservation of natural stands in situ (hopkins, 1983; oni et al, 1998). several studies on p. biglobosa population structure have shown low regeneration rates and ageing of the stands which may lead to extinction (padakale et al, 2015; ræbild et al, 2012a). overexploitation, late fruitification for most progenies and climate change are some of the factors suggested to explain the phenomenon (boffa, 1999; kwon-ndung et al, 2009; teklehaimanot, 2004). improved tree management practices for p. biglobosa, such as establishment of new trees or protection of natural regeneration are currently not sufficiently promoted (ræbild et al, 2012b). thus, african locust bean remains undomesticated despite increasing demand for its use. the conservation and sustainable use of genetic resources are critical to maintain tree resource availability, especially in the face of significant environmental changes, erosion of cultural heritages and declining cultivation and production activities of neglected indigenous species leading to genetic resources being threatened. the process of plant domestication and conservation requires characterization of genetic resources for identification of cultivars and effective utilization of germplasm. genome size variation has been identified as an associate of evolutionary divergence (dobes et al, 2019). variation in genome size and chromosome number becomes taxonomically significant if associated with some degrees of morphological and ecological differentiation (murray, 2005). sina (2006) studied genetic diversity among populations of p. biglobosa species in burkina faso using enzyme electrophoresis. dobes et al (2019) tested for linkage of relative genome size variation with geography, leaf morphology and population genetic variation in 58 individuals from 15 populations covering most of the distribution of p. biglobosa species in burkina faso. most of the variation was found within populations and there was no evidence from the karyological data for structured intraspecific taxonomic heterogeneity (dobes et al, 2019). in nigeria, genetic diversity of p. biglobosa accessions have been evaluated based on morphological characterization of trees (gbadamosi et al, 2005; okunlola et al, 2011) and seedlings (adesoye et al, 2013). however, these characters are known to be more influenced by the environment. uyoh et al (2011) reported variation in banding pattern of proteins obtained from leaf samples of three accessions of p. biglobosa collected from locations within cross river state, nigeria. adesoye and apo (2015) reported low levels of genetic diversity when 34 accessions of p. biglobosa were screened using seed protein electrophoresis. owing to the limited information on genetic variation and relationship of p. biglobosa accessions in nigeria, characterization using a combination of morphological, biochemical and molecular markers is essential for species identification and genetic variability establishment. this will facilitate the domestication, conservation and management of its genetic resources towards sustainable use. morphological characterization has been used to reveal phylogenetic relationships among crop populations, but this has its limitations as it is influenced by environmental factors (ferreira, 2006). selection, according to genetic variability using biochemical and molecular markers, has proved advantageous compared with the use of phenotypic markers (alghamdi et al, 2019). biochemical analysis, particularly electrophoresis of seed proteins has been effective in studying genetic diversity at intraspecific levels in several legumes including soybean and chickpea (durán et al, 2005; signor et al, 2005; malik et al, 2009; sammour et al, 2007a,b) and for cultivar identification (krochko and bewley, 2000; mustafa and el-kholy, 2008). the use of polyacrylamide gel electrophoresis (page) in fractionating seed proteins and evaluating genetic diversity among accessions of several legume species including groundnut, african yam bean, grass pea, buckwheat, bambara groundnut and underground vetches 38 akin-idowu et al genetic resources (2021), 2 (3), 36–50 has also proven effective (la rosa and gonzález, 2010; javaid et al, 2004; machuka, 2001; przybylska et al, 2000). molecular markers are known as one of the best approaches to study genetic material as well as to assess genetic variation in crop gene pools (badr, 2008; mondini et al, 2009; wang et al, 2016). a number of pcr based molecular markers such as random amplified polymorphic dna (rapd), inter simple sequence repeats (issr) and amplified fragment length polymporhism (aflp) have been used to assess genetic variability and cultivar identification in soybean (barakat, 2004; baránek et al, 2002; chowdhury et al, 2001; el-kholy, 2013) and other plant populations (hoque and hasan, 2012; lin et al, 2009). rapd is often used successfully to assess genetic diversity among species as it is fast, less technical and expensive and may reveal dominant molecular marker of good potential (badr et al, 2012; prasanthi et al, 2012). this study aimed to estimate the genetic diversity of p. biglobosa accessions across different agro-ecological zones in nigeria using sds-page and rapd markers. materials and methods plant material and morphological description seeds of p. biglobosa were obtained from 16 locations across seven states in nigeria (figure 1). thirteen of the sixteen accessions studied were collected from locations in the derived savanna agroecological zone (aez); two were collected from the guinea savanna and one from humid forest (figure 1,table 1). the sampling of p. biglobosa accessions was not conducted systematically across all aezs because the species is reported to be widely adapted and naturally distributed throughout the varied savannas of sub-saharan africa (lompo et al, 2016). the seed morphological characteristics of each collected accession, including seed shape, seed coat color and seed coat texture were observed. one hundred (100) seed weight in grams of each accession was also recorded. seed preparation and total protein extraction dried seeds were milled into flour and defatted with nhexane at a flour:hexane ratio of 1:10 (w/v) prior to protein extraction (de la rosa et al, 1992). seed total protein extraction was carried out by suspending defatted flour samples (100 mg) in 2.0 ml of pre-chilled tris buffer 50mm tris-hcl, ph 7.6, 1mm ddt, 150 mm nacl and 1mm edta for 2h at room temperature according to the method of zarkadas et al (2007). the resulting homogenates were centrifuged at 10,000 g for 20 min and supernatants were stored at -20ºc until used. figure 1. map of nigeria showing agroecological zones of the collection sites of sixteen parkia biglobosa accessions one dimensional sds-page sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) was performed according to the procedure of laemmli (1970). protein extracts were diluted in a sample buffer that contained 60 mm tris-hcl (ph 6.8), 2% w/v sds, 3.33% v/v βmercaptoethanol, 10% glycerol and 0.05% of bromophenol blue. the samples were heated at 98◦c for 5 min before loading onto a vertical slab gel (mini-protean ii electrophoresis cell) using a 4% (w/v) stacking and 12% gradient separating (acrylamide/bis acrylamide) gel containing 0.1% sds. the separating polypeptide bands were calibrated using a protein molecular weight marker which ranged from 11 to 130 kda. electrophoresis was done at 125 v for about 2 h. at the end of the run, the gels were stained with coomassie brilliant blue r-250 (sigmaaldrich) in methanol/water/acetic acid (40:50:10) and destained in a solution containing methanol/water/acetic acid (50:40:10). genomic dna extraction, rapd amplification and electrophoresis for the genetic analysis, ten viable seeds of each of the sixteen accessions of p. biglobosa collected from the different locations were grown in a greenhouse. prior to sowing, the seeds were immersed in concentrated sulphuric acid (h2so4) for 15 minutes to break seed dormancy, rinsed thoroughly in running water and air dried at room temperature. total genomic dna was extracted from young leaves of p. biglobosa accessions. about 0.2g of a composite of 5 samples per accession was ground in extraction buffer following the cetyltrimethyl ammonium bromide (ctab) method of dellaporta et al (1983). the dna quality was evaluated using 1% agarose gel electrophoresis with known concentrations of undigested lambda dna (sigma, st louis, mo, usa). quantification of dna was done using a spectrophotometer genetic resources (2021), 2 (3), 36–50 genetic variation of african locust bean from selected agroecological zones in nigeria 39 table 1. parkia biglobosa accessions used in this study and collection sites. accession code collection site agroecological zone geographical coordinates (dd) longitude latitude nh/2016/p01 obollo-afor, enugu state derived savanna 6.9153◦n 7.5139◦e nh/2016/p02 oju, benue state derived savanna 7.0383◦n 8.3451◦e nh/2016/p03 minna, niger state guinea savanna 9.5836◦n 6.5463◦e nh/2016/p04 vandeikya, benue state derived savanna 6.7835◦n 9.0695◦e nh/2016/p05 agyaragu, nasarawa state derived savanna 8.3471◦n 8.5679◦e nh/2016/p06 ikere, ekiti state derived savanna 7.5000◦n 5.2333◦e nh/2016/p07 orokam, benue state derived savanna 6.9786◦n 7.5572◦e nh/2016/p08 gbajimba, benue state derived savanna 7.8197◦n 8.8587◦e nh/2016/p09 kwatan-sule, benue state derived savanna 7.8112◦n 9.2429◦e nh/2016/p10 ogoja, cross river state humid forest 6.6548◦n 8.7977◦e nh/2016/p11 mbatiav, gboko, benue state derived savanna 7.2788◦n 8.7953◦e nh/2016/p12 suleja, niger state guinea savanna 9.2003◦n 7.1723◦e nh/2016/p13 garaku, nassarawa state derived savanna 8.8444◦n 8.1311◦e nh/2016/p14 iseyin, oyo state derived savanna 7.9765◦n 3.5914◦e nh/2016/p15 nihort, ibadan, oyo state derived savanna 7.4052◦n 3.8499◦e nh/2016/p16 oje market, ibadan, oyo state derived savanna 7.3891◦n 3.9092◦e (beckman coulter du530) at 260 nm. extracts were diluted to obtain dna concentrations of 25ng/µl. to generate dna profiles, 40 decamer oligonucleotide dna primers were initially screened for polymorphisms and only 16 which were polymorphic were used in the pcr reactions (table 2). to ensure reproducibility of rapd markers, rapdpcr amplification was performed in triplicate for each primer and accession. the pcr reaction contained the following reagents with the final concentrations: 1x pcr buffer, dntps (0.2 mm), mgcl2 (2.0 mm), primer (25 pmol), template dna (25ng/µl), taq polymerase (1.25 u) and nuclease-free water to 50µl. amplification was carried out in a 2400 perkin elmer gene amp pcr thermo-cycler as follows: pre-denaturation for 3 min at 94ºc; then 40 cycles each consisting of a denaturation step for 1 min at 94ºc; an annealing step for 1 min at 36ºc; an extension step for 90 sec at 72ºc. amplification was terminated by a final extension of 7 min at 72ºc. pcr products were electrophoresed on a 1.5% (w/v) agarose gel in 1x tris/borate/edta (tbe) buffer at 100 volts for 2h and visualized under uv light after staining with ethidium bromide. data analysis data on 100-seed weight of all accessions were subjected to anova and means were separated using duncan’s multiple range tests (p≤0.05). distribution frequencies of the qualitative traits were calculated using microsoft excel 2010. the protein bands and rapd amplified dna fragments were scored qualitatively, whereby (0) stands for the absence and (1) stands for the presence of bands in the profile of each accession. all values were pooled together to generate a binary data matrix, which was analyzed using the numerical taxonomy and multivariate analysis system (rohlf, 2002). genetic relationship among accessions was evaluated based on jaccard’s similarity index (jaccard, 1908). dendrograms were generated by using the unweighted pair group method with arithmetic mean (upgma). analysis of genetic diversity (gd) was calculated according to the method of nei (1987). polymorphic information content (pic) for each primer was determined from allele frequencies (nei and li, 1979). results and discussion variation in seed morphology improvement of the quality of indigenous fruit trees across sub-saharan africa involves assessment of fruit/seed traits; this has shown significant variation both among and within provenances and has enhanced selection in breeding programmes (ræbild et al, 2012a). in this study, significant differences (p≤0.05) were observed in the 100-seed weight of p. biglobosa collected from 16 different locations (table 3), which ranged from 17.93g (nh/2016/p09) to 26.11g (nh/2016/p05). the highest 100-seed weight was recorded for accession nh/2016/p05 collected from agyaragu, nassarawa state; this was significantly (p≤0.05) higher than that of all other accessions. the lowest 100-seed weight was recorded for nh/2016/p09 collected from kwatansule, benue state. the mean 100-seed weight (21.46g) of all accessions of p. biglobosa collected from the derived savanna zone was higher than the mean 100seed weight collected from the guinea savanna and humid forest (figure 2a). this variation in seed weight can be attributed to a wide range of factors such as environment (soil, climate), anthropogenic activities and phenotypic plasticity, thus promoting the presence of ecotypes. 40 akin-idowu et al genetic resources (2021), 2 (3), 36–50 table 2. list of primers used and rapd polymorphisms among parkia biglobosa accessions. primer sequence 5’ to 3’ major allele frequency allele number gene diversity polymorphic information content (pic) opb10 ctgctgggac 0.13 15 0.93 0.93 opt07 ggcaggctgt 0.13 14 0.92 0.92 opb04 ggactggagt 0.13 14 0.92 0.92 opt12 gggtgtgtag 0.13 14 0.92 0.92 opt08 aacggcgaca 0.13 13 0.91 0.91 opt06 caagggcaga 0.19 13 0.91 0.90 oph07 ctgcatcgtg 0.19 12 0.90 0.89 opb08 gtccacacgg 0.19 9 0.87 0.85 opt20 gaccaatgcc 0.25 9 0.84 0.82 oph06 acgcatcgca 0.38 9 0.80 0.78 opt09 cacccctgag 0.38 9 0.80 0.78 opt10 ccttcggaag 0.44 10 0.77 0.76 opt01 gggccactca 0.44 7 0.74 0.71 opt16 ggtgaacgct 0.56 6 0.64 0.61 opb05 tgcgcccttc 0.56 5 0.63 0.59 opt05 gggtttggca 0.75 4 0.41 0.39 mean 0.31 10 0.81 0.79 total 163 leakey (2017) reported that fruit and/or kernel size was greater in more humid sites for adansonia digitata and vitellaria paradoxa; but greater in drier sites for balanite aegyptiaca. also, results from tests of b. aegyptiaca and prosopis africana indicated that provenances from drier sites had significantly better aboveground growth than provenances from more humid sites (leakey, 2017). it is recommended that fruit trees germplasm should be collected in drier sites for future plantings in parklands, especially as this germplasm appears to be better adapted to dry conditions (leakey, 2017). the seeds of p. biglobosa accessions collected from the different locations in this study had distinct morphology and exhibited variation for seed shape, seed coat colour and seed coat texture (table 3). seeds of the sixteen p. biglobosa accessions were predominantly round oval in shape, black in colour with smooth seed coat and were mostly obtained from the derived savanna zone (figure 2b, c, d). seeds collected from the two sites in guinea savanna differed in shape and texture as seeds from one site were flat oval, black and smooth (nh/2016/p03) (figure 3b); while seeds from the other site were round oval, black and rough (nh/2016/p12) (figure 3c). seeds collected from the same agroecological zone (derived savanna) exhibited distinct variation in colour and texture, nh/2016/p15 seeds were flat oval, brown and smooth (figure 3e); while seeds of nh/2016/p04 were flat oval, black and wrinkled. the morphological variation may be due to gene flow across regions because of seed dispersal since p. biglobosa is an open pollinated tree crop having main pollinators such as bat, honeybees, rodents and humans (lassen et al, 2012; lassen, 2016; lompo et al, 2017). this variation offers great potential for future selection and domestication. lack of a clear association between genome size and morphological, geographical, or ecological patterns of differentiation has been reported for two species of artemisia (asteraceae) (dobes et al, 2019; de xaxars et al, 2016). seed storage protein analysis seed storage protein profiling has been used as an effective tool for varietal identification and determination of phylogenetic relationship in several plant species populations (emre, 2009; hameed et al, 2012; machuka, 2001). in this study, the sds-page profile of sixteen accessions of p. biglobosa showed similar electrophoretic patterns with respect to the number of protein bands and their band intensities (figure 4). the uniformity in the protein band profiles of all sixteen accessions suggests a low level of genetic diversity, which also corroborates earlier studies on p. biglobosa using seed protein electrophoresis (adesoye and apo, 2015). occurrence of a narrow genetic base from the accessions of p. biglobosa would suggest that they resulted not only from a common gene pool, but also from the outcrossing nature of the species over a long time, leading to a low level of inter-population diversity. low variation was observed in thirteen mungbean varieties using sds-page (hameed et al, 2012) and also in genetic diversity assessment of groundnut using sdspage (javaid et al, 2004). genetic resources (2021), 2 (3), 36–50 genetic variation of african locust bean from selected agroecological zones in nigeria 41 table 3. seed characteristics of parkia biglobosa accessions used in this study. values are mean ± standard error. means within the same column with different letters are significantly (p ≤ 0.05) different. accession code weight of 100 seeds (g) mean ± se shape colour seed coat texture nh/2016/p01 23.11d ± 0.18 round oval black smooth nh/2016/p02 18.59k ± 0.16 round oval brownish black smooth nh/2016/p03 21.75f ± 0.15 flat oval black smooth nh/2016/p04 18.30k ± 0.32 flat oval black wrinkle nh/2016/p05 26.11a ± 0.21 round oval black smooth nh/2016/p06 23.38d ± 0.10 flat oval brownish black smooth nh/2016/p07 19.44i ± 0.14 round oval black smooth nh/2016/p08 20.87g ± 0.19 round oval black rough nh/2016/p09 17.93l ± 0.23 round oval brownish black smooth nh/2016/p10 18.54k ± 0.13 round oval black smooth nh/2016/p11 21.46f ± 0.06 round oval black smooth nh/2016/p12 20.32h ± 0.26 round oval black rough nh/2016/p13 23.74c ± 0.23 round oval black rough nh/2016/p14 19.00j ± 0.12 flat oval brownish black rough nh/2016/p15 24.59b ± 0.11 flat oval brown smooth nh/2016/p16 22.48e ± 0.13 flat oval brownish black smooth the number of clearly visible protein bands ranged from eight to nine among the sixteen accessions of p. biglobosa with molecular weights ranging from 11 to 130 kda. two major clusters of bands were observed between 11−17 kda and 34−53 kda and one minor band was observed above 130 kda (figure 4). this is similar to results of adesoye and apo (2015) on albumin and globulin fractions of p. biglobosa in which two major bands were observed at the 16 and 50 kda regions. it has been suggested that high molecular weight proteins facilitate the development of seed hardness (coelho et al, 2007); therefore, the presence of a high molecular weight band observed above the 130 kda in this study may account for the seed hardness of p. biglobosa. the seeds of p. biglobosa are very hard and require treatment with sulphuric acid to induce germination. a polymorphic polypeptide with molecular weight of approximately 24 kda was absent in nh/2016/p04 but present in all other accessions (figure 4a) and can be used for its identification. differences in the presence or absence of bands among accessions are indicative of differences in the genes controlling the different polypeptide subunits (osanyinpeju and odeigah, 1998). also, the presence or absence of polypeptide bands has been found to be linked to the expression of certain characteristics such as wrinkled seeds and insect resistance (rao and pernolett, 1981; odeigah and osanyinpeju, 1996). the seed shape of nh/2016/p04 was observed to be wrinkled and the absence of the 24 kda band in this accession may be responsible for this seed texture as it was the only accession expressing this characteristic. seed storage proteins are non-enzymatic and have the sole purpose of providing proteins (nitrogen and sulphur source) required during germination and establishment of a new plant (hameed et al, 2012). legume seed storage proteins are mostly 7s and 11s globulins, which tend to be deficient in sulphur-containing amino acids (tang and sun, 2010). in mung bean, sds-page revealed that 11s globulin was composed of two bands: 40 kda and 24 kda, 8s vicilin was composed of 60 kda, 48 kda, 32 kda and 26 kda bands; and basic 7s globulin was composed of 28 kda, 17 kda and 16 kda bands (hameed et al, 2012; mendoza et al, 2001). in this study, peptides with molecular weights of 40 kda and 24 kda were detected that may correspond to 11s globulin, peptides with molecular weights of 48 kda and 32 kda were detected that may correspond to the 8s vicilin subunit, while 28 kda and 16 kda peptides were also detected that may correspond to the 7s globulin subunit. barakat (2004) reported low levels of protein polymorphism among six cultivars of soybean in which five major bands were identified at the 72, 36, 32, 20 and 16 kda regions. in african yam bean, sds-page separated identical globulin, albumin and vicilin patterns for all 26 accessions studied (machuka, 2001). strelec et al (2012) reported that barley varieties could be partially discriminated by albumin/globulin banding patterns using sds-page, whereas this was not possible with native page which showed more or less identical protein patterns for all varieties. through the use of sds-page on total proteins it is possible to detect a useful band polymorphism to explore the diversity of p. biglobosa as was detected in this study, albeit at a low level. cluster analysis a dendrogram was constructed based on protein banding patterns using jaccard’s similarity coefficients 42 akin-idowu et al genetic resources (2021), 2 (3), 36–50 (table 4). the dendrogram grouped the sixteen accessions into four clusters at 0.93 similarity coefficient (figure 5). the first cluster grouped together the largest number (thirteen) of accessions which consisted of nh/2016/p01, nh/2016/p02, nh/2016/p05, nh/2016/p16, nh/2016/p15, nh/2016/p06, nh/2016/p13, nh/2016/p12, nh/2016/p11, nh/2016/p10, nh/2016/p09, nh/2016/p08 and nh/2016/p07. the majority of the members of this cluster had similarity coefficient of 1.00 suggesting them to be genetically identical. cluster ii consisted of nh/2016/p14. accession nh/2016/p03, which was collected in minna, niger state and belonging to the guinea savanna agroecological zone, was separated alone into cluster iii, displaying pattern of geographic origin. cluster iv consisted of nh/2016/p04 and was the most divergent of the sixteen tested accessions. the genetic similarity coefficient was 0.85 among nh/2016/p03, nh/2016/p04 and nh/2016/p14. clustering showed pattern of geographic origin as eleven of the thirteen accessions grouped in cluster i were collected from the derived savanna agro-ecological zone. genetic distance values give some idea of the level of genetic variability among selected species or accessions. the variation in genetic diversity among the sixteen p. biglobosa accessions ranged from 0.85 figure 2. distribution frequencies of seed characteristics of parkia biglobosa across agro-ecological zones. a) 100-seed weight (g); b) seed shape; c) seedcolour; d) seed coat texture. genetic resources (2021), 2 (3), 36–50 genetic variation of african locust bean from selected agroecological zones in nigeria 43 figure 3. shapes and colour of seeds with seed coat of parkia biglobosa: a) round oval shape (nh/2016/p02), b) flat oval shape (nh/2016/p03), c) black colour (nh/2016/p12), d) brownish black colour (nh/2016/p14), e) brown colour (nh/2016/p15). to 0.92 depicting a low genetic diversity (table 4). all accessions in cluster i had a similarity coefficient of 0.92 in any pairwise comparisons with nh/2016/p03, nh/2016/p04 and nh/2016/p14. the low levels of variability observed in p. biglobosa is congruent with sina (2006) who reported that the genetic distances among p. biglobosa trees in burkina faso were low (between 0 and 0.240), indicating that the populations were similar enough to belong to the same genetic group. occurrence of a narrow genetic base from the accessions of p. biglobosa suggests that they may have resulted from a common gene pool and that fixation of variants through genetic drift has probably occurred. amusa et al (2014) had earlier reported that close natural populations like p. biglobosa exhibited high genetic similarity and low genetic distance attributed to a high rate of exchange of gene flow between populations. low genetic diversity was also observed in mung bean germplasm based on electrophoresis of seed storage proteins where a dendrogram analysis grouped the tested genotypes into three clusters at 93% homology (hameed et al, 2012). results of this study is similar to that obtained in cowpea in which seven landraces were separated into different clusters based on their geographical origin (alghamdi et al, 2019). the high level of similarity in the accessions of p. biglobosa suggests that the seed storage proteins are highly conserved. rapd polymorphism genetic polymorphism is an indication of evolutionary adaptation, which has a main role in the survival of species in a changing environment (stevens et al, 2007). in this study, random amplified polymorphic dna (rapd) amplification profiles revealed a total of 44 akin-idowu et al genetic resources (2021), 2 (3), 36–50 table 4. jaccard’s similarity coefficients of seed total protein profiles of parkia biglobosa accessions. p01 p02 p03 p04 p05 p06 p07 p08 p09 p10 p11 p12 p13 p14 p15 p16 p01 1.000 p02 1.000 1.000 p03 0.923 0.923 1.000 p04 0.923 0.923 0.846 1.000 p05 1.000 1.000 0.923 0.923 1.000 p06 1.000 1.000 0.923 0.923 1.000 1.000 p07 1.000 1.000 0.923 0.923 1.000 1.000 1.000 p08 1.000 1.000 0.923 0.923 1.000 1.000 1.000 1.000 p09 1.000 1.000 0.923 0.923 1.000 1.000 1.000 1.000 1.000 p10 1.000 1.000 0.923 0.923 1.000 1.000 1.000 1.000 1.000 1.000 p11 1.000 1.000 0.923 0.923 1.000 1.000 1.000 1.000 1.000 1.000 1.000 p12 1.000 1.000 0.923 0.923 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 p13 1.000 1.000 0.923 0.923 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 p14 0.923 0.923 0.846 0.846 0.923 0.923 0.923 0.923 0.923 0.923 0.923 0.923 0.923 1.000 p15 1.000 1.000 0.923 0.923 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 0.923 1.000 p16 1.000 1.000 0.923 0.923 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 0.923 1.000 1.000 256 alleles out of which 163 were polymorphic showing 63.67% polymorphism among the studied accessions. this relatively high polymorphism suggests a significant amount of genetic diversity among the accessions. a similar result was obtained among 11 soybean cultivars figure 4. sds-page of p. biglobosa seed total proteins. a) accessions nh/2016/p01 to nh/2016/08; b) accessions nh/2016/p09 to nh/2016/p16. m stands for protein size marker. * indicates the missing 24 kda band in nh/2016/p04. in which rapd amplification profiles revealed 61.7% polymorphism using five primers (el-kholy, 2013). alleles, ranging in size from 250 to 1500 basepairs (bp) and above, were scored for estimation of genetic relationship among the sixteen p. biglobosa accessions (figure 6). an average of 10 alleles per primer was obtained in this study ranging from a minimum of four alleles using primer opt05 to a maximum of 15 alleles using primer opb10 (table 2). genetic diversity is one of the important indices for the evaluation of genetic diversity among crop species (narzary et al, 2009; tamboli et al, 2016). the genetic diversity in this study ranged from 0.41 to 0.93, suggesting a significant amount of variation among the 16 accessions evaluated (table 2). an earlier report on genetic diversity assessment of 23 accessions of p. biglobosa from different agro-ecological zones using rapd indicated weak genetic diversity (expected heterozygosity, he= 0.05–0.18 and observed number of alleles, ona = 1.11–1.65) (amusa et al, 2014). the significance of the polymorphic information content figure 5. upgma dendrogram based on jaccard’s similarity of seed total protein profiles showing close relationship among parkia biglobosa accessions. genetic resources (2021), 2 (3), 36–50 genetic variation of african locust bean from selected agroecological zones in nigeria 45 (pic) value is such that they are used to evaluate the amount of genetic diversity and are categorized as high (pic > 0.05), medium (pic < 0.05) and low (pic < 0.25) (abedian et al, 2012). polymorphic information content ranged from 0.39 for primer opt05 to 0.93 for primer opb10 (table 2). fifteen of the sixteen polymorphic primers had pic values greater than 0.5 indicating that rapd can develop high-locus polymorphism, which is useful to assess genetic variability of the accessions. all primers showed an average pic value of 0.79 and nine out of the sixteen primers exceeded the average. this relatively high polymorphism has been observed in studies of genetic diversity on soybean cultivars by chowdhury et al (2001) and barakat (2004). cluster analysis based on upgma revealed six distinct clusters at a genetic similarity coefficient of 0.68 on the upgma dendrogram (figure 7). cluster i consisted of accessions nh/2016/p01, nh/2016/p05, nh/2016/p12, nh/2016/p13, nh/2016/p04, nh/2016/p02, nh/2016/p09, nh/2016/p14 and nh/2016/p15. all the accessions grouped together in cluster 1 were collected from derived savanna except accession nh/2016/p12 which was collected from guinea savanna. this suggests that gene flow between the different locations may have occurred as a result of movement of germplasm and exchange of genetic material among farmers and markets. cluster ii consisted of nh/2016/p06; cluster iii consisted of nh/2016/p07 while cluster iv consisted of nh/2016/p03. accessions nh/2016/p08, nh/2016/p16 and nh/2016/p11 grouping together in cluster v were collected from derived savanna. accession nh/2016/p10 collected from ogoja, cross river state in the humid forest zone grouped alone in cluster vi. thus, in the upgma analysis of the rapd profiles, accessions from same agroecological zone grouped together, while in other cases they were placed in different clusters. for most accessions in this study there was similar clustering pattern of geographically closer accessions, indicating significant association between genetic similarity and geographical distance. this does not agree with a report of adesoye et al (2013) who reported that distribution of p. biglobosa genotypes among clusters did not correspond with their geographical patterns. the lowest similarity was recorded between nh/2016/p10 and nh/2016/p06, nh/2016/p07, nh/2016/p03. the highest similarity was recorded between accessions nh/2016/p12 and nh/2016/p13, followed by accessions nh/2016/p05 and both nh/2016/p12, nh/2016/p13. accessions nh/2016/p05 and nh/2016/p13 with narrow genetic distance were collected from the same agro-ecological zone (derived savanna). this result corroborates findings of hamrick and godt (1989) in which species with small geographic ranges maintained less genetic diversity than geographically widespread species. rapd clearly distinguished some accessions by accession specific fragments (figure 6). therefore, rapd markers can successfully be used to produce variety specific fingerprints in p. biglobosa accessions and are a valuable tool for assessing genetic diversity. figure 6. rapd-pcr analysis of parkia biglobosa accessions as revealed by primer opb10. accessions are numbered as in table 1. m is the molecular size marker. conclusion the low level of variation observed among the accessions as revealed by sds-page limits the method’s applicability to distinguish accessions. nevertheless, sds-page was able to detect a polymorphic polypeptide with molecular weight of approximately 24 kda which was present in all accessions but absent in nh/2016/p04. thus, sds-page may not be used to identify accessions based on intraspecific variation; figure 7. upgma dendrogram based on rapd analysis of parkia biglobosa accessions. 46 akin-idowu et al genetic resources (2021), 2 (3), 36–50 rather it might be more suitable to identify interspecific variation. rapd-pcr markers were more informative and could be used to discriminate between p. biglobosa accessions. four rapd primers (opb10, opt07, opb04, opt12) revealed high degrees of polymorphism among the accessions. the identification of accessions with maximum genetic divergence should be optimized in breeding programmes. the rapd marker is therefore a more efficient tool to discriminate between accessions of p. biglobosa. implications of findings to the conservation and sustainable use of parkia biglobosa genetic resources • the variation observed in qualitative traits and 100-seed weight among the sixteen accessions of p. biglobosa across the three agro-ecological zones of nigeria indicates their potential for domestication through selection towards conservation and breeding. • cluster analysis of the seed protein profile identified nh/2016/p04 as genetically distinct as it was clustered alone and was the most divergent of the sixteen tested accessions. the absence of the 24kda band in accession nh/2016/p04 distinguishes it from all the other accessions. it can be adopted as parental line for heterosis crossing and should be exploited for conservation, breeding and sustainable utilization. • four selected rapd primers (opb10, opt07, opb04 and opt12) can be used to discriminate the accessions of p. biglobosa. • the highly diversified accessions of p. biglobosa indicate potential for domestication through selection in breeding programmes. • most accessions from the same agroecological zone clustered together suggesting that there is a strong correlation between genetic similarity and geographic proximity. • the homogeneity of alleles among the studied p. biglobosa accessions suggests possible loss of intraspecific genetic diversity. the weakening gene pool and diversity observed can be enhanced through more germplasm collections, particularly from the diverse agroecological zones of nigeria for better genetic characterization using more specific markers towards conservation, breeding and sustainable utilization. acknowledgements authors are grateful to the virology unit of the international institute of tropical agriculture (iita) ibadan, nigeria, for providing the facilities to carry out the sds-page analysis. author contributions p. e. akin-idowu designed the experiment. p. e. akinidowu, a. o. aduloju and o. i. akinyoola executed the experiment. o. i. akinyoola , p. e. akin-idowu and d. o. ibitoye performed the analysis. p. e. akin-idowu, o. i. akinyoola, u. g. adebo, u. orkpeh, y. o. olagunju and d. o. ibitoye wrote the manuscript. all the authors read and approved its submission. conflict of interest statement the authors declare no conflict of interest. references abedian, m., talebi, m., golmohammdi, h. r., and sayed-tabatabaei, b. e. 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(2007). assessment of the protein quality of fourteen soybean [glycine max (l.) merr.] cultivars using amino acid analysis and two-dimensional electrophoresis. food research international 40(1), 129–146. doi: https:// doi.org/10.1016/j.foodres.2006.08.006 https://doi.org/10.1016/j.crvi.2016.02.005 https://doi.org/10.1016/j.crvi.2016.02.005 https://doi.org/10.1021/jf904254f https://doi.org/10.1007/s12892-011-0043-1 https://doi.org/10.1016/j.bse.2016.04.019 https://doi.org/10.1016/j.bse.2016.04.019 https://doi.org/10.1016/j.foodres.2006.08.006 https://doi.org/10.1016/j.foodres.2006.08.006 introduction materials and methods plant material and morphological description seed preparation and total protein extraction one dimensional sds page genomic dna extraction, rapd amplification and electrophoresis data analysis results and discussion variation in seed morphology seed storage protein analysis cluster analysis rapd polymorphism conclusion implications of findings to the conservation and sustainable use of parkia biglobosa genetic resources author contributions conflict of interest statement original article genetic resources (2020), 1 (1) 24–39 doi: 10.46265/genresj.2020.1.24-39 https://www.genresj.org issn: 2708-3764 developing a methodology to balance benefit-sharing: application in the context of biodiversity beyond national jurisdiction jane eva collins *,a,b, aysegul sirakaya c,b, thomas vanagt b and isabelle huys a a faculty of pharmaceutical sciences, clinical pharmacology and pharmacotherapy, katholieke universiteit leuven, leuven, belgium b absint, bruges, belgium c department of european, public and international law, faculty of law, ghent university, ghent, belgium abstract: the effectiveness and success of benefit-sharing measures to date, particularly in contributing towards the conservation and sustainable use of biodiversity, has been questionable. this is likely related to the degree of beneficial impacts versus burden on the users and regulatory authorities in terms of administrative complexities. it is, therefore, timely to reconsider which forms of benefit-sharing may most favourably balance the associated beneficial and burdensome aspects. the aim of this paper is to develop and demonstrate a benefit-sharing balance methodology which can be used as a tool to help decision-makers to select options in an objective and transparent manner. application in the biodiversity beyond national jurisdiction context provides a useful example of how this tool can be used. results suggest that sharing of genetic sequence data and research results provide the most favourable balance in terms of non-monetary benefit sharing, whilst the most favourable monetary benefit-sharing options were associated with research funding and salaries. the benefit-sharing balance methodology presented here provides a useful tool and starting point, which can be built upon in the future, to include more detailed information gathered from expert groups to consolidate the perceived balance of beneficial impacts versus burden. in addition, the equation can be tailored according to different policy settings where different benefit-sharing factors may be more appropriate. ultimately, use of this tool could help to enhance implementation of benefit-sharing policies/legislation with greater potential to balance beneficial impacts with associated burden, thereby enhancing workability of the access and benefit-sharing system as a whole. keywords: benefit-sharing, methodology, genetic resources, marine, areas beyond national jurisdiction citation: collins, j. e., sirakaya, a., vanagt, t., huys, i. (2020). developing a methodology to balance benefitsharing: application in the context of biodiversity beyond national jurisdiction. genetic resources 1 (1), 24-39. doi: 10.46265/genresj.2020.1.24-39. © copyright 2020 the authors. this is an open access article distributed under the terms of the creative commons attribution license (cc by 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. introduction according to article 1 of the convention on biological diversity (cbd, 1992), the objective of the convention is ‘the conservation of biological diversity, the sustainable use of its components and the fair and equitable sharing of the benefits arising out of the utilization of genetic resources’1. ∗corresponding author: jane eva collins (jane.collins@abs-int.eu) 1 article 1, (cbd, 1992): ‘the objectives of this convention, to be pursued in accordance with its relevant provisions, are the conservation article 15.7 of the convention takes a step further by stating that parties shall take measures to share benefits such as results of research and development (r&d) and benefits arising from commercial and other utilization of genetic resources (cbd, 1992). however, of biological diversity, the sustainable use of its components and the fair and equitable sharing of the benefits arising out of the utilization of genetic resources, including by appropriate access to genetic resources and by appropriate transfer of relevant technologies, taking into account all rights over those resources and to technologies, and by appropriate funding.’ received: 23 04 2020 accepted: 10 08 2020 published online: 31 08 2020 https://www.genresj.org https://www.dx.doi.org/10.46265/genresj.2020.1.24-39 https://www.genresj.org https://www.dx.doi.org/10.46265/genresj.2020.1.24-39 mailto:jane.collins@abs-int.eu genetic resources (2020), 1 (1) 24–39 developing a methodology to balance benefit-sharing 25 despite attempts made by authors such as morgera (2014), there is as yet no specific, internationally agreed legal definition of the term ‘benefit-sharing’ in the context of utilising genetic resources (schroeder, 2007; morgera and tsioumani, 2010; morgera, 2014; parks, 2019). in addition, the terms ‘fair’ and ‘equitable’ are also not clearly defined (de jonge, 2011; morgera, 2014). this can lead to difficulties between stakeholder groups in terms of different interpreted definitions and requirements associated with benefit-sharing, as well as differences in motivation with respect to the notion (de jonge, 2009, 2011). nonetheless, the nagoya protocol and other genetic resource frameworks, such as the fao international treaty on plant genetic resources for food and agriculture (itpgrfa) and the who pandemic influenza preparedness (pip) framework for the sharing of influenza viruses and access to vaccines and other benefits, provide suggestions of what benefitsharing could entail (fao, 2001; nagoya protocol, 2011; world health organization, 2011). a list of types of nonmonetary and monetary benefits that can be shared are listed in the annex of the nagoya protocol (2011). negotiations for a new international legally binding instrument (ilbi) under the united nations convention on the law of the sea (unclos, 1982) for the conservation and sustainable use of marine biological diversity of areas beyond national jurisdiction (bbnj) began in 2018. negotiations address a ‘package’ of four elements and cross-cutting issues. the four elements include: marine genetic resources (mgr), including questions on the sharing of benefits; measures such as area-based management tools (abmt), including marine protected areas (mpas); environmental impact assessments (eia); and capacity-building and transfer of marine technology (unga res. 72/249, un doc. a/res/72.249, 24 december 2017, para. 2.). in order to govern mgr in areas beyond national jurisdiction (abnj) and to answer questions on the sharing of benefits, it is important to consider which benefitsharing options may be most appropriate in this particular context. under the nagoya protocol, benefits should be shared by the users of genetic resources with the provider state, in accordance with domestic legislation (art 5. nagoya protocol 2011). however, in the bbnj context, although the types of users may be similar, there will be no provider of the genetic resources, since mgr exist outside the borders of national jurisdiction. it has not yet been agreed who the beneficiaries will be, but could involve ‘developing states parties, in particular least developed countries, landlocked developing countries, geographically disadvantaged states, small island developing states, coastal african states and developing middle-income countries’ (art 11, bbnj draft text, 2019a). in addition, since the nagoya protocol applies only to areas within national jurisdiction, the same access and benefit-sharing (abs) provisions do not apply to the utilisation of genetic resources from abnj. as such, new benefit-sharing options are under consideration for adoption as part of the new bbnj agreement (bbnj draft text, 2019a)2. whilst a list of potential benefit-sharing options can be found in the annex of the nagoya protocol (2011), the effectiveness and success of these measures, and the abs system as a whole, has been questioned by stakeholders (fedder, 2013; pauchard, 2017; ruizmuller, 2018). between 1996 and 2017, a total of 217 abs agreements for commercial research and 248 for non-commercial research were concluded (pauchard, 2017). to date, there is a lack of evidence to support the assumption that benefit-sharing leads to effective conservation of biodiversity (suneetha and pisupati, 2009; pisupati and bavikatte, 2014). in addition, the burden on the users (such as universities and private companies) and regulatory authorities in terms of administrative complexity when complying with abs legislation and conducting benefit-sharing can act as a disincentive for utilisation of genetic resources, potentially limiting the benefits derived and shared (richerzhagen and holm-mueller, 2005; tvedt, 2013). further challenges exist with regards to achieving fair and equitable benefit-sharing and sustainable development (louafi, 2013; tsioumani, 2018). it is timely and appropriate, therefore, to reevaluate how the success of implementing benefitsharing options are measured and reconsider which forms may most fairly balance the positive and burdensome associated aspects (ruiz-muller, 2018). in order to understand this balance, key factors may be used to assess the overall positive influence of a benefitsharing option, such as the number of beneficiaries receiving benefits, the effect on biodiversity goals and the long-term impacts and significance of benefitsharing, as well as the overall burdensome aspects, such as the burden on the user of genetic resources and the burden on the regulator (tvedt, 2013; correa, 2017; morgera, 2018a; harden-davies and gjerde, 2019). consideration of the benefit-sharing options for adoption as part of the new bbnj agreement provides a good opportunity to identify benefit-sharing factors and assess the overall positive versus burdensome balance of different benefit-sharing options. it has been noted that of the four elements under consideration as part of the bbnj package, mgr including questions on the sharing of benefits remains the most challenging and immature element, with few detailed solutions suggested to date (voigt-hanssen, 2018). this is to be expected given the different stakeholder perspectives, goals and concerns collins et al (2020). however, review of non-monetary and monetary benefit-sharing options in light of benefit-sharing factors and understanding of which measures may provide the most fair and balanced outcome could provide a useful platform on which negotiatons can progress (bbnj, 2019a; sirakaya, 2019; collins et al, 2020). 2 see part ii, article 11 ‘[fair and equitable] sharing of benefits’, draft bbnj agreement, 2019a. 26 collins et al genetic resources (2020), 1 (1) 24–39 the first aim of this paper, therefore, is to develop a benefit-sharing methodology which can be used as a tool to understand the balance of beneficial versus burdensome aspects associated with different benefitsharing options. this will involve making use of a multi-criteria analysis technique (mca) to help decisionmakers to select options in an objective and transparent manner (de brucker et al, 2013). the main objective of the mca technique is to overcome challenges that human decision-makers experience when handling large amounts of complicated information in a consistent manner (dodgson et al, 2009). the second aim is to demonstrate how the methodology can work by applying it to the bbnj context, to review the different types of non-monetary and monetary benefit-sharing options with potential for adoption under the new agreement, in order to determine which forms may provide more balanced outcomes. the bbnj context was selected for demonstration of the methodology because these negotiations represent a relevant, current opportunity which may directly benefit from such an exercise with regards to making informed decisions in terms of the benefit-sharing options to be adopted in the treaty text. this objective was achieved by identifying the key benefit-sharing factors to consider, as well as their relative importance to stakeholders. the benefitsharing factors were then applied to different benefitsharing options through the use of the new equation, to reveal the balance of beneficial impacts versus burden associated with the different options. the authors acknowledge the limited number of interviewees involved and suggest that further interviews with a larger number of participants would be needed to draw significant conclusions in the context of ongoing bbnj negotiations, as well as also in other genetic resource abs circumstances. in addition, the authors wish to highlight the broad scope of the current paper and general nature of the factors considered. future research is needed to build on the results gathered in this study and to include more comprehensive literature review as well as more detailed information gathered from various expert groups. this would help to consolidate the perceived balance of beneficial impacts versus burden associated with benefit-sharing options. development of a benefit-sharing tool materials and methods the study began with a scoping literature review to identify benefit-sharing factors that may enable analysis of the positive versus burdensome aspects of benefitsharing options. the benefit-sharing options which could be considered for a potential governance (abs) system for mgr from abnj were also identified (fao, 2001; nagoya protocol, 2011; world health organization, 2011; bbnj, 2019b; collins et al, 2020). the literature review involved searches through pubmed, embase, eurlex, the united nations convention on the law of the sea, the united nations bbnj website3, the convention on biological diversity (cbd) and the nagoya protocol (unclos, 1982; cbd, 1992; nagoya protocol, 2011). search keywords included: benefit-sharing, abs, genetic resources, mgr, abnj, and capacity building. results benefit-sharing factors as a result of the authors’ own experience in terms of how benefit-sharing works in practice, specifically the modalities of drafting and negotiating benefitsharing contracts, coupled with literature review, five factors were identified as being the main objectives and considerations of benefit-sharing associated with utilisation of genetic resources (referred to in this paper as ‘benefit-sharing factors’, young and tvedt, 2017). listed below and in table 1 are descriptions of the benefit-sharing factors considered in this study. 1. biodiversity goals: different goals and objectives, as defined by legal biodiversity acts or treaty’s, may be attained through benefit-sharing. bbnj context: relevant goals include contributing towards conservation of marine biological diversity of abnj, promoting sustainable use of mgr from abnj, fostering scientific r&d and promoting fair and equitable benefit-sharing (art 140, unclos, 1982; cbd, 1992; harden-davies and gjerde, 2019; collins et al, 2020). 2. direct beneficiaries: the number of individuals receiving benefits will vary according to the type of benefit that is being shared. bbnj context: a greater number of individuals may have access to mgr data if this is shared, such as via online databases, compared to the number of people who may receive funding to conduct phd projects on topics related to bbnj. 3. benefit-sharing significance: the impact associated with different forms of benefit-sharing could be approximately determined in terms of the significance, value and the duration of the impact. for example, impacts could include enhanced employment and contribution towards scientific knowledge. duration of impacts may vary from weeks to months or years and could give an indication of whether future generations may be positively influenced by the benefit-sharing or not (hardendavies and gjerde, 2019). bbnj context: training courses as part of capacity building initiatives may lead to longer-term positive impacts on a group of people, perhaps linked to enhanced employability, than would be the case for access to mgr data. 4. burden on the user: users could include any organisations or individual researchers from developed or developing states. if benefit-sharing is a requirement, these users may encounter a degree of burden linked to the process of sharing. this burden may take the form of monetary cost to 3 https://www.un.org/bbnj/ accessed 08 march 2020 https://www.un.org/bbnj/ genetic resources (2020), 1 (1) 24–39 developing a methodology to balance benefit-sharing 27 generate the so-called ‘benefits’ in the first place, such as tailored training courses or a monetary payment, but also the transaction cost of sharing benefits. in addition, the generation and sharing of benefits will involve human labour costs, for example sending experts to beneficiary locations to conduct capacity-building or training initiatives. time may represent another burdensome aspect. whilst some types of benefit-sharing require only one simple transaction, other forms may require repeated actions over time. bbnj context: sharing of benefits in the bbnj context may involve burdens and costs to the user, for example to set up and organise databases in which mgr data can be stored and accessed, or sending of laboratory equipment as a form of technology transfer. 5. burden on the regulator: a regulator may encounter a degree of burden linked to the process of benefit-sharing, as well as related to the need to build capacity in the context of novel regulation. this may involve raising awareness of and enforcing compliance with applicable legislation. these burdens may be both in terms of monetary costs and human labour costs, similar to those described for the users above. however, for the regulator, these costs may be linked to monitoring and checking compliance of users with the benefit-sharing requirements. bbnj context: at present, it remains unclear whether there will be a regulator for abnj, or who this could be (mohammed, 2017; bbnj, 2019b). however, in the event that there is a regulator/regulatory body charged with regulating abs linked to mgr from abnj, and benefit-sharing is a requirement, then the regulator may face burdens associated with overseeing the process of benefit-sharing, particularly in terms of monetary costs, complexity and time. rationale for selecting these five benefit-sharing factors the reasoning behind the five factors on benefit-sharing is threefold. the first reason relates to the concept of establishing a balanced abs system as referred to in our previous research (sirakaya, 2019, 2020). for an abs system to successfully establish balance between the user and the provider (in bilateral genetic resources frameworks), so as to fairly distribute benefits arising from utilisation of genetic resources between the users of genetic resources and beneficiaries, it is crucial to ensure that such a system attends to the international biodiversity goals. the authors here specifically explore benefit-sharing options and therefore the factors pay due regard to the international biodiversity goals as identified under our current research as well as the previous research conducted on the matter (sirakaya, 2020). secondly, the five factors were selected since they clearly define and influence the modalities of benefitsharing and can be negotiated when drafting agreements (young and tvedt, 2017). the authors believe that these factors, coupled with agreement between users and providers, can promote an objective balance in benefit-sharing. multiple stakeholder groups are involved within the bbnj context and it is of utmost importance that an instrument is developed that attains the needs of all of these groups to the greatest extent possible. a stakeholder-driven mca is the best available method to achieve this (de brucker et al, 2013) (sirakaya and de brucker, personal communication). a recent study conducted by sirakaya and de brucker (under review) demonstrates how mca can been applied to design regulatory frameworks for access to genetic resources in cases which involve multiple stakeholders. as such, the third reason for the selection of the criteria refers to the importance of stakeholder consultation under the mca. according to freeman (1984), stakeholders are defined as ‘any individual or group who can affect an organisation’s performance or who is affected by the achievement of this organisation’s objectives.’ looking into regulatory issues related to benefit-sharing in abnj, taking into account freeman’s definition, five key stakeholder groups were identified: developing states, developed states, civil society, the scientific research community and the private sector (collins et al, 2020). although questions remain regarding whether there will be any form of regulatory body in the bbnj context, this could potentially involve members from both developing and developed states working together to fulfill the role. the scientific research community and private sector represent the potential users of mgr. developing states and civil society are the likely beneficiary groups who would receive benefits shared from utilisation of mgr from abnj. stakeholders’ perceptions play a crucial role in mca conducted on abs frameworks. this is due to the fact that a balanced abs system can only be established if there is a reasonable abs framework that the users would be incentivised to adhere to. the same principle applies to a potential benefit-sharing system for abnj. the users (i.e. the scientific research community and private sector) would only be incentivised if there is a foreseeable balance between the impact of being a part of the system and the burden thereof. likewise, the system would only function as intended if the cost borne to the regulator (including members from developing and developed states) of establishing and maintaining the system is considerably lower than the benefits generated through the system to be shared with beneficiaries (developing states and civil society). in line with this, harden-davies and gjerde (2019) stipulate the ‘need to strike a balance between the right to use and the responsibility to share’. therefore, by reviewing the balance of potential beneficial impacts (private, social and environmental aspects) versus burden, negotiators may be in a better position to make informed decisions regarding which benefit-sharing options may be most 28 collins et al genetic resources (2020), 1 (1) 24–39 table 1. description of the five benefit-sharing factors considered in this study. benefit-sharing factors description references biodiversity goals (g) i. contributing towards conservation of marine biological diversity of abnj ii. promoting sustainable use of mgr from abnj iii. fostering scientific r&d iv. promoting fair and equitable benefit-sharing v. inclusivity of developing states in access to and utilisation of mgr of abnj cbd (1992); collins et al (2020); bbnj (2019a); harden-davies and gjerde (2019); morgera (2018a); unclos (1982) direct beneficiaries (#) i. number of individuals impacted/receiving the benefits morgera (2014) benefit-sharing significance (s) size of impact, for example: i. duration (months or years – gives an indication of whether the impact will be beneficial to current and future generations) ii. enhanced employability/employment rates iii. meaningfulness harden-davies and gjerde (2019) burden on the users of (m)gr (u) i. monetary cost and human labour to generate and share the benefits, including costs to transfer to and distribute amongst beneficiaries. ii. opportunity cost iii. time value harden-davies and gjerde (2019) burden on the regulator (r) i. monetary cost and human labour to monitor benefitsharing ii. complexity iii. time value pisupati and bavikatte (2014) appropriate to consider including as part of a new benefit-sharing framework for abnj. in sum, these five benefit-sharing factors are crucial in establishing a balanced benefit-sharing framework for abnj with governance options that attain international biodiversity objectives, as outlined by legal acts and treaty’s, and pay due regard to stakeholder preferences (collins et al, 2020). benefit-sharing balance in order to objectively evaluate different benefitsharing options in terms of the ratio of potential positive impacts versus associated burden in a fair and consistent manner, an equation was proposed, taking into consideration the five benefit-sharing factors (see figure 1). weighted values were used in this study to incorporate consideration of the fact that some benefitsharing factors may be perceived as more important than other factors and should therefore contribute more to the final score. different methods can be used to generate the weighting values for different factors. for example, an analytic hierarchy process, case-based reasoning, simple multi-attribute rating technique, mathematical programming or interview approaches could be used (von winterfeldt and edwards, 1986; ho et al, 2010; nerini et al, 2014). in this study, the values of weightings were obtained through an interview process. benefit-sharing factors (g, #, s, u, r) were first each multiplied by an average weighting (a-e) assigned to each factor by interviewees. the three positive, weighted benefit-sharing factor scores are multiplied together, as are the two burdensome, weighted factor scores. the positive aspects are then divided by the burdensome aspects to give a benefit-sharing balance value for each benefit-sharing option. applying the benefit-sharing tool to the bbnj context materials and methods after development of the benefit-sharing methodology (see figure 1), a semi-structured, qualitative interview was prepared (see supplemental file 1). interviews were conducted with ten experts. availability and willingness to participate in the interview represented a controlling factor in the recruitment of participants, as well as the variety of stakeholder groups, and contributed to the limited numbers. figure 1. equation to determine the balance of potential positive impact versus burden associated with different forms of benefit-sharing. letters a-e represent the average weight assigned by interviewees to the five benefit-sharing factors. benefit-sharing factors: g= biodiversity goals; # = direct beneficiaries; s = benefit-sharing significance; u = burden on the user, and; r = burden on the regulator. genetic resources (2020), 1 (1) 24–39 developing a methodology to balance benefit-sharing 29 figure 2. average weight assigned by interviewees to the five different benefit-sharing factors considered. error bars indicate standard deviation of the mean. these experts are based in different locations around the world (four from developing and six from developed states) and representative of different stakeholder groups: two from the scientific research community (one from a developed and one from a developing state), three from developing states delegations, three from developed states delegations, and two from civil society (both from developed states). whilst effort was made to include private sector stakeholders in the interview process, and indeed representatives from one organisation did provide helpful feedback on some of the questions, time constraints and availability of representatives rendered this not possible in the scope of the current study. interviews were conducted during the third session of the intergovernmental conference (igc3) for bbnj in new york (between 19-28 august 2019). the interviews followed a pre-defined guide list of questions (see supplemental file 1), which started by requesting participants to rank five benefit-sharing factors (see table 1) on a scale from 1 to 5 in terms of perceived relative importance (see figure 2). participants were then asked to score a variety of nonmonetary and monetary benefit-sharing options in terms of potential impact of these on the five benefit-sharing factors (from 0 = no impact, to 5 = very high impact), according to their perspective, and to give short reasons for their decisions. these scores were used to perform a mca, whereby scores were multiplied by the average weighting assigned to each factor and inserted into an equation (see figure 1), to determine the balance of potential positive impact versus burden associated with each different form of benefit-sharing (see figure 3 and figure 4). this enabled comparison of the ratio of potential positive impacts versus burden between different benefit-sharing options, taking into account the relative importance of different key factors. interviews were audio-recorded and transcribed. microsoft excel software was used as a means to store the data. a thematic analysis of the transcripts was conducted to identify common themes in responses. all data were anonymised by grouping results into stakeholder groups. written informed consent forms were signed by all of the interviewees in this study. results weighting the benefit-sharing factors according to the average stakeholder ranking, biodiversity goals were considered as the most important factor to be taken into consideration when assessing how balanced different benefit-sharing options are. the other four benefit-sharing factors were considered approximately equal in terms of importance (see figure 2). the range of ranking given by interviewees was greatest for the direct beneficiaries factor, as indicated by the larger error bar in figure 2. reasons given by interviewees for these rankings are described below. biodiversity goals. the majority of interviewees stated that the biodiversity goals are the most critical factor from their perspective. whilst many different goals could fall under this category, interviewees most frequently referred to conservation of biodiversity as the aspect that they consider most important. this is because the goals of ‘conservation and sustainable use of marine biological diversity’ are the key overarching goals of the new agreement as a whole (bbnj, 2019b). collins et al (2020) present further detailed information regarding stakeholder goals for a potential new abs mechanism for abnj. other goals, such as promoting marine scientific research, are viewed as necessary to achieve the conservation objectives. they also indicated that biodiversity goals are more important than any of the other four benefit-sharing factors considered in this study. one interviewee noted that biodiversity goals are important for all stakeholders involved, not just for the beneficiaries of benefit-sharing. direct beneficiaries. most interviewees indicated the benefits should be shared amongst as many people as possible, and that the greater the number of people who are positively affected the better. however, three interviewees also noted that the importance of beneficiaries depends on the definitions, whether it is only the people who are directly affected, or also those who indirectly benefit. sometimes only a few people may immediately and directly benefit from a benefitsharing initiative, such as collaborative or joint venture projects, but such an effort may indirectly have a large impact on many more people and other factors, perhaps over time. for example, sharing of mgr samples and data for scientific research may only initially affect a moderately low number of people, but if this leads to development of new pharmaceutical products to treat human illness or to maintaining the health of the ocean through conservation measures, then a far larger number of beneficiaries will be encountered. benefit-sharing significance. the majority of interviewees assigned this factor a moderately high score. two interviewees stated that the duration of benefit-sharing significance is vital. however, two other interviewees gave this a moderately low score and suggested that, from their perspective and in the current context, this factor was not as important as the others considered in this study. 30 collins et al genetic resources (2020), 1 (1) 24–39 figure 3. a) average weighted scores for the seven nonmonetary benefit-sharing options according to the five benefitsharing factors, and b) scores according the benefit-sharing balance equation for the seven non-monetary benefit-sharing options. burden on the users of (m)gr. burden on the user was considered moderately important by over half of interview participants. one interviewee raised the question of whether there might be the possibility to charge a ‘handling fee’ for certain types of benefits (such as sharing of material). the degree of burden on the user may be affected by whether there is the possibility to charge a handling fee (or similar) to disseminate mgr samples and other benefits. it was suggested that sharing of benefits cannot all be for free. burden on the regulator. the majority of interviewees assigned this factor a high score. whilst it remains unclear whether there might be or who might constitute the regulatory authority in the bbnj context, it is possible that such an institution will be established. interviewees acknowledged that this is an important factor because if burden on the regulator is not taken into careful consideration, then the whole benefitsharing system, whether non-monetary or monetary, could fail. for example, if the system is overly expensive, then any potential monetary benefits may be used to fund running of the system itself rather than accruing for the benefit of beneficiaries (morgera and tsioumani, 2010; morgera, 2014; tsioumani, 2018). in addition, burden on the regulator could result in lengthy decisionmaking processes, thereby indirectly affecting potential users. non-monetary benefit-sharing options to gather perspectives on benefit-sharing, interviewees were asked to consider the potential influence of different benefit-sharing options on the five benefitsharing factors (table 1). analysis of the president’s aid to negotiations (2019) and a review of the literature lead to identification of seven non-monetary benefitsharing options which could be considered in the context of governing the utilisation of mgr from abnj (see figure 3). a description of the options considered in this study, and their significance, can be found in sirakaya (2019) and collins et al (2020). non-monetary benefitsharing options considered here include: 1. sharing of raw data: (a) metadata; (b) genetic sequence data (gsd)4,5,6 (c) biochemical information 2. sharing of research results 3. capacity building 4. technology transfer 5. research directed towards priority needs the benefit-sharing balance, as calculated using the equation described above, indicated the most favourable balance of beneficial impacts versus burden associated with sharing of gsd and sharing of research results. sharing of biochemical information, metadata and capacity building received a similar, but slightly lower score. technology transfer, and in particular research directed towards priority needs, received less favourable balances (see figure 3b). reasons given by interviewees for potential positive impacts versus burden associated with different non-monetary benefit-sharing options are described below. sharing of raw data (metadata, gsd and biochemical information). for some participants, all three types of raw data sharing are viewed together as a package. it was suggested that if raw data were to be made publicly available online, this could have a positive impact on a relatively large number of beneficiaries (figure 3a). this is because many people would then be able to access the data easily, quickly and possibly for free. however, two interviewees also acknowledged that many people, particularly in developing states, may not be able to make use of the raw data since they lack the capacity to work on it. as such, unless accompanied by capacity building, the number of beneficiaries impacted by sharing of raw data may not actually be very high. interviewees suggested that sharing of raw data may not immediately have a large effect on the number of 4 gsd is the term most commonly used in the bbnj context to refer to data/information which is described as digital sequence information (dsi) under the auspices of the cbd. according to the consortium of european taxonomic facilities (cetaf) and the society for the preservation of natural history collections (sphnc), the term gsd is in line with the concept of dsi. however, despite efforts to define dsi/gsd, there is currently no official, internationally accepted definition of the term. 5 https://www.cbd.int/abs/dsi-views/2019/cetaf-dsi.pdf accessed 09 march 2020 6 https://www.cbd.int/abs/dsi-views/2019/spnhc-dsi.pdf accessed 09 march 2020 https://www.cbd.int/abs/dsi-views/2019/cetaf-dsi.pdf https://www.cbd.int/abs/dsi-views/2019/spnhc-dsi.pdf genetic resources (2020), 1 (1) 24–39 developing a methodology to balance benefit-sharing 31 direct beneficiaries, the biodiversity goals or benefitsharing significance. however, by building up large data sets, the beneficial impact of sharing raw data on these benefit-sharing factors may grow and could become relatively high over time. this could also be in part due to the long-lived nature of data; once data is curated and stored, it can be made available and accessed for a very long time. one interviewee suggested that sharing of raw data could be one of the best things we could do to have a positive influence on the benefit-sharing significance. one interviewee explained that a lot more work is required to generate biochemical information than for generation of metadata and gsd, because the process is comparably less straightforward. in addition, one interviewee suggested that whilst it is already best practice to share metadata and gsd, they were unsure whether it is yet best practice to share all biochemical information. therefore, the burden on the user associated with sharing of biochemical information, in terms of opportunity cost, may be higher than that associated with the other forms of raw data sharing. in addition, the burden on the users related to sharing of data may depend on the stakeholder group in question and the type of data that is required to be shared. for some users, such as those in the private sector, this is likely to be very burdensome and possibly even a deterrent, particularly if mandatory and involves the obligation to share commercially important data. this is because mandatory sharing of raw data would likely change the incentive to invest. scientists in the private sector routinely conduct novel scientific research and publish results in scientific journals. they also disclose scientific information in the form of patents. however, from a commercial viewpoint, if sharing of data beyond current practice is mandatory, this may have broad implications for protection of intellectual property (ip) and for maintaining competitive market advantage. as such, mandatory sharing of valuable data may disincentivise investments in private sector projects involving mgr from abnj. interviewees indicated that the level of burden on the users and on the regulators would depend on the way in which benefit-sharing is done. under the assumption that a new system is developed and must be used to share benefits, for example sharing of gsd through a clearing house mechanism or other form of database, this could lead to significant burden on the users and regulators. one interviewee noted that in the draft treaty text (bbnj, 2019a), reference is made to a new system whereby benefits will need to be shared through a clearing house7 and raises the question of timing. 7 ‘samples, data and related information shall be made available in open access [through the clearing-house mechanism [upon access] [after [. . . ] years]]’ and ‘states parties shall publish and communicate the reports of the results of the assessments in accordance with [articles 204 to 206] [article 205] of the convention [, including through the clearing-house mechanism]’ (president’s aid to negotiations, 2019). according to the interviewee, the issue of when and how benefits will be shared could have huge impacts on the degree of burden experienced. if benefit-sharing is left according to current practices, particularly in terms of where raw data are stored, then this could have very little or even zero impact on the burden felt by users or the new regulator. however, if we proceed with a new system, requiring a change in the way that data are shared, burden may be encountered by users in terms of requiring conversion of data into a particular format to fit into a specific database. as such, if benefitsharing were to become mandatory and involves new requirements and procedures, the burden on the users will be higher than at present. the burden on the regulator could be relatively straightforward, if all they have to do is verify that data is being shared with a database, but the degree of burden will depend on how much information they need. one interviewee also noted that it may be important to consider the burden on the beneficiaries accepting raw data. this may involve requirements to develop infrastructure to receive and make use of data. sharing of research results. interviewees indicated that a relatively large number of beneficiaries could be impacted by the sharing of research results (figure 3a). however, one interviewee suggested that research results, such as a research paper, may be read by fewer people compared to the number of people who could access or use raw mgr data. sharing of research results could have a large positive effect on the overall impact of benefit-sharing, and also on the biodiversity goals such as conservation. it was suggested that this could be partly due to the long-lived nature of research results. interviewees suggested that the impact of sharing research results on the burden experienced by users would be moderate, since writing of research articles inherently involves a degree of human labour costs. however, it was also noted that this could be less burdensome for users than sharing of raw data, because this would involve a different system which may remain more similar to current practice. it was suggested that the burden on the regulator may also be moderate, but would depend on whether this was mandatory or voluntary. if the regulator was required to track that sharing of research results has been done, then this could lead to quite a heavy burden. capacity building. it was suggested that the influence of capacity building on biodiversity goals and significance would depend on how sustainable the capacity building initiatives are. for example, if scientists are trained as part of a capacity building effort and then decide to leave the country, this cannot be considered as sustainable. according to one interviewee, this is unfortunately the case when reviewing current capacity building activity, and represents a challenge faced in many developing countries. the key question here is how to ensure the sustainability of capacity building. in an ideal 32 collins et al genetic resources (2020), 1 (1) 24–39 world capacity building would be sustainable, so that effects are long-lasting. the burden on the users of mgr may be greater in association with capacity building than some of the other options (figure 3a). this is because it requires a combination of time, money and specific expertise to execute such an initiative. technology transfer. it was suggested that the number of beneficiaries who could benefit from technology transfer would be moderately low. it was proposed that this may be because only a few people will be able to make use of the technology, particularly if the technology is very sophisticated and the absorptive capacity is low. one interviewee suggested that the impact of technology transfer on the biodiversity goals will be less than that associated with other benefitsharing options (figure 3a). as with capacity building, the influence of technology transfer on the benefitsharing significance will depend on the sustainability. interviewees were of the opinion that technology transfer may have an impact for a short amount of time, but were uncertain whether this would have long-lasting effects. the burden on the users related to technology transfer may depend on the stakeholder group in question, the type of technology that is required to be shared, who pays for the transfer of technology and the means by which this is done. it was suggested that for some users, such as the scientific research community, this may be less burdensome than sharing of raw data. however, for other uses, such as those in the private sector, this is likely to be very burdensome, particularly if mandatory. this is because investors in the private sector aim to recoup investments, and mandatory, free transfer of technology would possibly discourage further investment. alternatively, one interviewee suggested that if technology transfer were to be conducted on commercial terms, this could represent less burden for the private sector. research directed towards priority needs. one interviewee stated that, although research directed towards priority needs has the potential to reach a lot of people and could have a very high impact on attending to the biodiversity goals, particularly contributing towards conservation and sustainable use, they simply did not think that it was going to happen. this is because the interviewee felt that such a system of focusing research on priority needs would be unfeasible and unworkable. as such, the anticipated number of beneficiaries and influence on biodiversity goals and significance was scored relatively low (figure 3a). in addition, it was noted that the impact of research directed towards priority needs on the benefit-sharing factors would depend on what exactly the priority needs are. the burden on the user was thought to be moderately high, unless the users are already conducting research in the priority needs area. one interviewee suggested that such priority needs could be linked to research with socially beneficial uses, including ‘health and food security’ as described in article 8 of the nagoya protocol (2011). this is because forcing scientists to change their research from one field of work to a different one, associated with the identified priority needs, would require significant monetary costs. similarly, the burden on the regulator would likely also be high. it was suggested that the regulator may experience burden associated with understanding who the developing states are, identifying their priority needs and deciding how this should be regulated. one interviewee noted that it would likely be very difficult for a regulator to monitor this, and could be very challenging if it involves a new system in which to direct their regulation towards this specific purpose. monetary benefit-sharing options analysis of the president’s aid to negotiations (2019) and a review of the literature lead to identification of eight monetary benefit-sharing options which could be considered in the bbnj context (see figure 4). a description of the options considered in this study, and why they might be important, can be found in sirakaya (2019) and collins et al (2020). monetary benefitsharing options considered here include: 1. research funding 2. salaries 3. joint ventures8 4. joint ip rights 5. royalties 6. milestone payments 7. license fee 8. access fee interviewees were asked to consider the potential impact of different monetary benefit-sharing options on the five benefit-sharing factors. according the benefit-sharing balance calculation, the most favourable monetary benefit-sharing option, in terms of balancing beneficial impacts versus burden, was research funding followed closely by salaries (see figure 4b). joint ventures were considered the next most favourable option, followed by joint ip rights. access fee, milestone payments, license fee and royalties were viewed as similar in terms of balancing the potential positive impacts versus burden. reasons given by interviewees for potential positive impacts versus burden associated with different monetary benefit-sharing options are described below. interviewees noted that their answers in this section were given under the assumption that commercialisation of mgr is successful and that there will be money to share. this is crucial, because four interviewees out of the ten indicated that they do not believe that there will be any financial profits derived from r&d on mgr, and therefore no money to share. 8 in this paper, the term ‘joint ventures’ is used in a general, broad sense to describe a collaborative project/initiative undertaken jointly by two or more entities which otherwise retain their distinct identities. the term is not used here in the corporate or legal manner. genetic resources (2020), 1 (1) 24–39 developing a methodology to balance benefit-sharing 33 figure 4. a) average weighted scores for the eight monetary benefit-sharing options according to the five benefit-sharing factors, and b) scores according to the benefit-sharing balance equation for the eight monetary benefit-sharing options. research funding and salaries. five of the ten interviewees indicated that they view research funding and salaries as similar in terms of the potential positive impacts versus burden. interviewees indicated that research funding and salaries could have a greater positive influence on the biodiversity goals and benefit-sharing significance than the other forms of monetary benefit-sharing, and may have more longterm positive effects (see figure 4a). for example, research funding could help to build capacity in developing states, potentially contributing towards enhanced employability, and could also be directly focused on long-term conservation and sustainable use of biodiversity. one interviewee noted that the level of burden on the user associated with research funding and salaries would depend on who is paying for it. the burden on the users would be low if this is to be paid by the regulator, and the burden on the regulator may be relatively high. however, vice versa may be true if the users are required to pay this. it was also suggested that since research funding is something that is already done by the scientific community, then the burden on the regulator would likely be less for research funding than other monetary benefit-sharing options, if the regulator is only required to make sure that the money has been channeled appropriately. joint ventures. most interviewees agreed that joint ventures would influence a relatively low number of beneficiaries (figure 4a). one participant stated that joint ventures will require money which cannot be shared amongst many people, and therefore the number of people affected will likely be low. the influence of joint ventures on the biodiversity goals and significance was also thought to be low, and would likely depend on the conditions. it was suggested that the level of burden experienced by the user and the regulator would be moderate, but could be low if joint ventures were considered on a voluntary rather than a mandatory basis. this is because if joint ventures were required on a mandatory basis, then perhaps users/ organisations who are not equipped with the appropriate capacity would struggle to establish such projects. joint ip rights. the majority of interviewees felt that joint ip rights (ipr) would impact a low number of beneficiaries (figure 4a). one interviewee suggested that perhaps it was possible for this number to grow over time. participants were also of the view that the influence of joint ipr on biodiversity goals and benefitsharing significance would be low, possibly with no longterm positive effects. with regards to burden on the user, interviewees suggested that this would depend on the conditions associated with the joint ipr, but was likely to be moderate to high. the burden on the regulator is likely to be high, and participants noted that they were unsure how this form of benefit-sharing could be regulated. one interviewee indicated that they did not see joint ipr as a feasible option, as it would either not be relevant or appropriate in most cases. the interviewee stated that only inventors or co-inventors can claim ipr and requesting joint ownership with entities who do not represent this would lead to an inability to fulfill the required criteria to claim ipr. the only situation where such an approach has been considered previously is in the context of traditional knowledge, where indigenous knowledge may be included in an invention. however, according the interviewee, even then there are much better approaches to follow than joint ipr in order to share benefits. nonetheless, if scientists from developing countries are included in a research project and at some point in time a new invention is developed, then joint ipr could be appropriate. it was also noted that publications have copyrights involved, which could also be considered as a form of joint ipr if multiple coauthors are involved in the effort. as such, the feasibility of these options will depend on the details regarding how this is done. milestone payments, license fee and royalties. most interviewees considered that milestone payments, license fees and royalties would lead to similar impacts on the benefit-sharing factors (see figure 4a). one reason given for this is that people find it very difficult to distinguish between the three. it was suggested that the milestone payments could have similar positive impacts on the benefit-sharing factors as an access fee (see below). as with the access fee, questions were raised regarding whether users of mgr would be able to pay the milestone payments, license fees or royalties. one 34 collins et al genetic resources (2020), 1 (1) 24–39 interviewee stated that they did not believe that a license fee would actually be workable. interviewees indicated that license fees and royalties may be more burdensome on the user and regulator than the milestone payments, but that all three would likely be more burdensome than an access fee. this is because the access fee could take the form of a one-off payment (with no subsequent costs), of a preset and limited amount, whereas the others may incur more than one fee and may be payable at times which are less clearly defined than at the point of access (collins et al, 2020). access fee. the majority of interviewees were of the opinion that an access fee would impact only a low number of beneficiaries (figure 4a). it was suggested that this may be in part because relatively little money would be generated and few users would be able to pay this if it was expensive, although research institutions could potentially seek scholarships to cover this (see collins et al, 2020) for more detail). questions were raised regarding to whom this fee would be paid. the influence of an access fee on the biodiversity goals and significance was also considered low. one interviewee noted that the influence would depend what this fee is used for, but was sceptical that it would have a positive effect on these factors. another interviewee stated that an access fee system could be attractive because it would ensure that some income is generated when mgr are collected from the high seas which could be channelled into benefit-sharing. however, the same person also acknowledged that they did not think an access fee would be feasible, because it would inflict on the ‘freedom of the high seas’ principle. therefore, from a negotiation point of view, this would likely be very difficult to implement. discussion a benefit-sharing tool development of a benefit-sharing methodology benefit-sharing in the context of existing genetic resource frameworks under the nagoya protocol (conducted in a bilateral manner) is often considered to lack transparency, clarity and accountability, particularly for monetary benefit-sharing options (fedder, 2013; pauchard, 2017; ruiz-muller, 2018). for example, when dealing with abs under the nagoya protocol, and since abs contracts are private and users are not obliged to report what is in the contract, governments do not have access to information regarding the amount of money that flows out as a result of abs. making use of a methodology, such as that described in this study, could help to achieve a more favourable balance by negating the issue of transparency, by clarifying the key factors and helping negotiators to make informed decisions. in addition, lessons learnt from the itpgrfa in terms of aspects to consider as part of an operating, multilateral abs system may also be useful here (louafi, 2013; stannard and moeller, 2013; tsioumani, 2018). ultimately, use of this tool could help to enhance understanding and implementation of benefit-sharing policies/ legislation with greater potential to balance beneficial impacts with associated burden, thereby enhancing workability of the abs system as a whole. however, challenges with the methodology have been identified during application in this study (as described in the section below) which should be addressed in order to develop a more robust tool for application in a variety of benefit-sharing contexts. lessons learnt from application of the methodology to the bbnj context accurately determining the influence of different benefit-sharing options on benefit-sharing factors, such as the significance, are difficult to measure. indeed, interviewees found it challenging to give scores for some options, partly because participants may not have fully understood or known how to measure and give an accurate answer. this is a clear aspect of the tool which could be improved in the future through consultation with a greater number of experts on that particular aspect. in addition, given the fact that these questions were asked in the context of ongoing negotiations, it is expected that some participants will have responded with strategic rather than objective answers. this may be the reason why a small minority of interviewees indicated that salaries as a form of monetary benefit-sharing would have a large positive influence on benefit-sharing factors, whilst the authors expected the impact to be moderately low compared to other options. in addition, the authors do not suggest that results collected in this study represent a comprehensive review of stakeholder perspectives on this subject matter. further interviews with a much larger number of participants would be needed to draw significant conclusion for the bbnj context. however, it is suggested that the benefit-sharing equation described in this article provides a useful tool and starting point, which can be built upon by decision makers in the future, to include more detailed information gathered from various expert groups, such as likely impacts on potential beneficiaries, to consolidate the perceived balance of beneficial impacts versus burden. in addition, the equation can be tailored according to different policy settings where different benefit-sharing factors and additional nuance may be more appropriate. benefit-sharing in the bbnj context benefit-sharing factors the most important benefit-sharing factor, according to average weights allocated by interviewees (see figure 1), was the influence on biodiversity goals. this is likely because these goals, in particular conservation and sustainable use of biodiversity, are the primary objective of existing genetic resource frameworks, such genetic resources (2020), 1 (1) 24–39 developing a methodology to balance benefit-sharing 35 as the nagoya protocol9 and the plant treaty, and as such set the precedence for similar frameworks in the future (cbd, 1992; fao, 2001; nagoya protocol, 2011; world health organization, 2011; bbnj, 2019a). in the bbnj context, it has already been suggested that fair and equitable benefit-sharing related to mgr from abnj can enable the international community to address global challenges (morgera, 2018a). these global challenges may be related to environmental protection, social objectives and private economic goals (de brucker et al, 2013; mohammed, 2017; morgera, 2018a). the particular goals of greatest importance to stakeholders are different in the bbnj context compared to areas within national jurisdiction, where aspects such as legal certainty are considered most vital (collins et al, 2020; sirakaya, 2020). legal certainty is perceived to be of particularly high importance for private sector users of genetic resources (in order to promote investment), who at present appear to view genetic resources in areas within national jurisdiction as more relevant to them than mgr in abnj (sirakaya, 2019; collins et al, 2020). as such, the benefit-sharing factors involved in the methodology, and the weighting assigned to each, are likely to vary according to the circumstances. non-monetary benefit-sharing options and balance of potential positive aspects vs burden interviewee results indicate that sharing of gsd and research results provide the most favourable balance in terms of beneficial impacts versus burden. technology transfer, and in particular research directed towards priority needs, received less favourable balances (see figure 3). it has been noted by the ad hoc technical expert group on dsi on genetic resources under the cbd10 that dsi ‘plays an important role in deepening knowledge about biodiversity, identifying and mitigating risks to threatened species, enhancing our ability to track illegal trade, identifying species and the geographic origins of products, and assisting with biodiversity planning and conservation management’. as such, the sharing of this type of information in the context of bbnj is likely to have a positive influence on the biodiversity goals, in particular the conservation and sustainable use of bbnj. sharing of gsd appears to be a relatively straightforward procedure, and it is considered best practice amongst the scientific research community (devi and pisupati, 2018). under the assumption that the bbnj agreement describes requirements to share gsd in keeping with current practices, through the same current channels (and not through other systems), then this form of benefit-sharing may not incur additional burden on the users of mgr. however, the authors acknowledge the 9 for example, according to article 9 of the nagoya protocol (2011), ‘the parties shall encourage users and providers to direct benefits arising from the utilization of genetic resources towards the conservation of biological diversity and the sustainable use of its components’. 10 https://www.cbd.int/doc/c/079f/2dc5/2d20217d1cdacac787524d 8e/dsi-ahteg-2018-01-03-en.pdf accessed 09 march 2020 contentious nature of discussions regarding gsd/dsi in multiple fora, including the cbd and the plant treaty. whilst sharing of gsd/dsi itself may be relatively uncontroversial, the potential requirement for monetary benefit-sharing associated with utilisation of gsd/dsi is highly contentious (kobayashi, 2019). sharing of research results is thought to have a similar positive effect to the sharing of gsd on enhancing the potential for conservation of marine biodiversity, by helping to build an enabling environment in which msr can flourish (harden-davies and gjerde, 2019). technology transfer represented a relatively unfavourable balance. this may be associated with a combination of relatively high expected burden on the users of genetic resources and on the regulators, together with limited potential beneficial impacts. the potential burdens on different stakeholder groups associated with technology transfer are described in collins et al (2020). it is suggested that the degree of burden, in terms of financial cost and administration, will depend on the type of technology, the conditions, how it is funded and how the transfer is managed, but on the whole, these will be more significant than for other forms of non-monetary benefit-sharing. for example, it is generally expected that the funding required for technology transfer will be greater than for other nonmonetary benefit-sharing options (collins et al, 2020). in addition, the owners of technology are often private companies or individuals (prip et al, 2015). as such, transfer of this technology will need to involve consideration of commercial aspects, such as the inherent requirement for private sector entities to generate a return on investment and maintain market competitiveness. related factors will include appropriate intellectual property arrangements and/ or private economic incentives or public funding, particularly in the circumstance of private ownership of technology (prip et al, 2015). moreover, the beneficial impacts linked to technology transfer may be limited by absorptive capacity (or enabling factors) in the recipient state. according to prip et al (2015), successful technology transfer depends on three factors: the existence of relevant technology to address particular (environmental) challenges, the relevant dissemination of technology in a manner which makes it available to entities which need this, and the application of technology in a manner which is conducive to solving the challenges in mind. as noted by interviewees involved in a study conducted by sirakaya (2019), research directed towards priority needs would involve a degree of burden associated with making sure that the research precisely benefits the provider country (or in the context of bbnj, the beneficiary state(s)), because this would require a considerable amount of time, effort and resources. this potential burden is likely to contribute to the less favourable balance for research directed towards priority needs. https://www.cbd.int/doc/c/079f/2dc5/2d20217d1cdacac787524d8e/dsi-ahteg-2018-01-03-en.pdf 36 collins et al genetic resources (2020), 1 (1) 24–39 monetary benefit-sharing options and balance of potential positive aspects vs burden according to interviewee results, research funding, followed closely by salaries, were selected as the monetary benefit-sharing options which provide the most favourable balance regarding beneficial impacts versus burden. access fees, milestone payments, license fees and royalties received less favourable balances (see figure 4). the favourable benefit-sharing balance indicated for research funding and salaries may be because there is a greater degree of perceived transparency involved in these approaches, compared to other forms of monetary benefit-sharing, in terms of where the financial resources are directed and how ‘beneficial’ this could be (altman and simera, 2010). this transparency with regards to research funding is promoted by funding organisations11,12, science foundations13, journals and international initiatives, such as the equator (enhancing the quality and transparency of health research) network, which aim to encourage transparent reporting of research, research funding processes and the use of reporting guidelines14. in addition, salaries and in particular research funding can directly support r&d on mgr, with potential to enhance conservation and sustainable use of bbnj. the less favourable balance indicated for access fees, milestone payments, license fees and royalties may, therefore, be associated with a lack of clarity and transparency in terms of how financial resources are used and how much of a beneficial impact this might have (altman and simera, 2010). indeed, interviewees involved in this study raised questions and uncertainty regarding how money could be utilised in the scope of monetary benefit-sharing options to promote biodiversity goals, such as conservation and sustainable use of biodiversity. this uncertainty may also be due to the complicated nature of the factors explored. further research, building on the current study, should be more specific in nature and would likely be simpler for interviewees to respond to, yielding increasingly insightful results. for example, it was suggested that the balance for monetary benefit-sharing options could be more favourable than currently perceived if money could clearly be linked to supporting these goals. whilst some monetary benefit-sharing options may at some stage create incentives for biodiversity conservation, this would depend on how and where financial resources are allocated. this is currently not as clear or as transparent as processes associated with research funding and salaries (altman and simera, 2010). in addition, the potential impact of benefit-sharing options 11 https://www.nihr.ac.uk/blog/how-do-you-make-research-fundingtransparent-and-fair/10991 accessed 02 march 2020 12 https://www.ukri.org/about-us/policies-and-standards/transparen cy/ accessed 09 march 2020 13 https://www.nsf.gov/od/transparency/transparency.jsp accessed 21 march 2020 14 https://www.equator-network.org/accessed 09 march 2020 on bioidiversity goals and the overall benefit-sharing balance may be affected by the approach and language adopted in the new agreement. for example, the nagoya protocol (2011) text refers mostly to ‘encouraging’ the flow of benefits towards biodiversity conservation, but there is no clear obligation15. the benefit-sharing fund of the plant treaty provides a different example, whereby the contracting parties decide for themselves the contributions that they will make (tsioumani, 2018). in order to reach a more favourable balance, it will be necessary to include language in the bbnj agreement which clearly ties benefit-sharing options to the objectives. this is an important point which should be kept in mind during the negotiation process. in this study, the authors investigated benefit-sharing options in the manner in which they are currently being addressed in accordance with existing abs frameworks under the nagoya protocol, plant treaty and in the draft bbnj text. when investigating details regarding monetary benefit-sharing options with a view to developing a methodology to balance these with various factors, it became apparent that these options are pooled together under the ‘monetary benefitsharing’ term without making the distinction between those which accumulate (such as royalties, access fees and milestone payments) and those which distribute funding (such as research funding and salaries). given that monetary benefit-sharing is already a contentious item in the realm of benefit-sharing, the fact that it is dealt with in this confusing manner is not useful and could prove problematic when translated into practice. this point represents an important finding in the present study. whilst out of scope of this paper, further research should be conducted in the future to adjust and tailor the formula according to whether the monetary benefitsharing options generate or distribute funding. a further factor which might contribute towards the less favourable balance of monetary benefit-sharing options compared to the non-monetary options is the fact that the benefits will need to be shared amongst large numbers of beneficiaries. whilst sharing of data or information with many people does not dilute or divide the benefits received by each individual/ entity (each can receive the same package of data/ information), the sharing of monetary benefits amongst many beneficiaries will likely result in limited and small amounts of money reaching the beneficiaries. this aspect may contribute towards the perception held by many delegates that non-monetary benefitsharing could represent more predictable and more significant options than forms of monetary benefitsharing (morgera, 2018b). by focusing on goals and identified capacity requirements, non-monetary benefitsharing can have a more immediate and tangible impact 15 article 9, nagoya protocol, 2011: ‘the parties shall encourage users and providers to direct benefits arising from the utilization of genetic resources towards the conservation of biological diversity and the sustainable use of its components.’ https://www.nihr.ac.uk/blog/how-do-you-make-research-funding-transparent-and-fair/10991 https://www.ukri.org/about-us/policies-and-standards/transparency/ https://www.nsf.gov/od/transparency/transparency.jsp https://www.equator-network.org/genetic resources (2020), 1 (1) 24–39 developing a methodology to balance benefit-sharing 37 on bridging the equity gaps related to r&d on mgr. this approach has been considered the principal success of the plant treaty (stannard and moeller, 2013; tsioumani, 2018). conclusion to date, benefit-sharing related to the utilisation of genetic resources has not been as effective as was hoped (ruiz-muller, 2018; pauchard, 2017). this is at least partially related to the lack of focus associated with benefit-sharing options on conservation and sustainable use of biodiversity, possible hampering of scientific research as well as the burden placed on the regulator and users of genetic resources. this has in some cases led to a loss of incentive to conduct r&d on genetic resources, with potential to further limit conservation and sustainable use of biodiversity (tvedt, 2013). the authors acknowledge that there are many other political and legal issues, in addition to the technical aspects referred to in this study, which contribute towards the challenges currently experienced with existing benefitsharing systems. these issues include, for example, the lack of objectivity in terms of approaches to agreeing appropriate benefit-sharing measures. a tool, such as the methodology presented in this study (figure 1), provides a useful means to assess the balance of different benefit-sharing options in terms of the potential beneficial and burdensome impacts, taking into account the varying relative importance of different factors. this would help policy-makers in the future when deciding which forms of benefit-sharing may be most appropriate to adopt, or when improving existing abs measures, with greater potential for meeting proposed objectives, such as conservation and sustainable use of biodiversity. use of this methodology would also likely promote transparency, objectivity, clarity and workability of the benefit-sharing system, possibly leading to enhanced generation, accrual and sharing of benefits (lindhjem et al, 2010). results in this study indicate that in terms of nonmonetary benefit-sharing options under consideration for the new bbnj agreement, sharing of gsd and research results may provide the most favourable balance. should monetary benefit-sharing be included in the agreement, research funding and salaries may represent the most fairly balanced options. in addition, in order to reach a favourable balance, it may be necessary to include language in the bbnj agreement which clearly ties benefit-sharing options to the objectives. however, further interviews with a larger number of participants would be needed to draw significant conclusion for the bbnj context. nonetheless, the benefit-sharing equation described and demonstrated in this article provides a useful tool and starting point, which can be tailored according to different policy settings where consideration of different benefit-sharing factors may be more appropriate. in addition, the methodology can be developed to include more detailed information gathered from various expert groups to consolidate the perceived balance of beneficial impacts versus burden associated with benefit-sharing options. acknowledgements many thanks to all of the interviewees for their valuable contribution towards this research. thanks also to the marie curie h2020 fund (h2020-msca-itn-etn: marpipe ga 721421) for supporting this research. supplemental data supplemental file 1: outline for interviews for ”developing a methodology to balance benefit-sharing: application in the context of biodiversity beyond national jurisdiction”. author contributions jc and tv jointly conceived the idea for the study and manuscript. jc developed the research methodology, performed data collection, data analysis and led writing and editing of the text. as assisted with developing the research methodology and co-wrote and edited the manuscript. tv also assisted with developing the research methodology and edited and reviewed the manuscript. ih edited and reviewed the manuscript. conflict of interest statement the authors declare no conflicts of interest. references altman, d. g. and simera, i. 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(2017). drafting successful access and benefit-sharing contracts (brill). url: https://doi.org/10.1163/9789004356573. https://doi.org/10.13140/rg.2.2.14108.28804 https://doi.org/10.13140/rg.2.2.14108.28804 https://doi.org/10.1016/j.ecolecon.2004.06.031 https://dx.doi.org/10.1136/jme.2006.016790 https://doi.org/10.3389/fpls.2019.01175 https://doi.org/10.1002/sd.2040 https://doi.org/10.1002/sd.2040 https://undocs.org/a/conf.232/2019/1 https://www.bioversityinternational.org/e-library/publications/detail/identifying-benefit-flows-studies-on-the-potential-monetary-and-non-monetary-benefits-arising-from-t/ https://www.cbd.int/abs/doc/unu-abs-report-2009-en.pdf https://doi.org/10.1163/15718085-12334012 http://www.who.int/influenza/pip/en https://doi.org/10.1163/9789004356573 introduction development of a benefit-sharing tool materials and methods results benefit-sharing factors rationale for selecting these five benefit-sharing factors benefit-sharing balance applying the benefit-sharing tool to the bbnj context materials and methods results weighting the benefit-sharing factors non-monetary benefit-sharing options monetary benefit-sharing options discussion a benefit-sharing tool development of a benefit-sharing methodology lessons learnt from application of the methodology to the bbnj context benefit-sharing in the bbnj context benefit-sharing factors non-monetary benefit-sharing options and balance of potential positive aspects vs burden monetary benefit-sharing options and balance of potential positive aspects vs burden conclusion supplemental data author contributions conflict of interest statement untitled untitled original article genetic resources (2022), 3 (5), 24–35 doi: 10.46265/genresj.uppq3994 https://www.genresj.org issn: 2708-3764 stylosanthes scabra: genetic variability of forage quality traits fabiana karla de araújo américo a, marcelo ayres carvalho b, juaci vitória malaquias b, allan kardec braga ramos b, gustavo josé braga b, claudio takao karia b and carlos eduardo lazarini da fonseca *,b a universidade estadual de feira de santana-uefs, bahia, brazil b embrapa cerrados, planaltina, df, brazil abstract: stylosanthes scabra vogel is a tropical legume grown in dry tropical and subtropical environments. the objective of this research was to evaluate the genetic diversity of forage quality traits for 80 accessions of s. scabra. seven plants from each accession were planted in a single-line plot with no replicates at embrapa cerrados, brazil. all plants were harvested 90 days after planting. crude protein (cp), in vitro dry matter digestibility (ivdmd), neutral detergent fibre (ndf), acid detergent fibre (adf), lignin (lig), hemicellulose (hemic) and cellulose (cellu) were estimated. data were submitted to principal component analysis (pca) and a cluster analysis was performed to identify groups of similarity. simpson and shannon–weaver diversity indices estimated the genetic diversity. the average values of cp, ivdmd, ndf, adf, lig, hemic and cellu were 220g/kg, 560g/kg, 516.8g/kg, 368g/kg, 69.4g/kg, 148.8g/kg and 298.6g/kg, respectively. there was a significant difference among collection sites for ivdmd, adf and cellu. the first two principal components accounted for 73% of the total variation. the 80 accessions resulted in four clusters, among which significant differences were observed for cp, ivdmd and adf. group iv, with 24 accessions, had the highest cp and ivdmd concentrations and the lowest adf concentration, being the highest-quality forage group. diversity indices were 0.78 and 0.81 for simpson’s and shannon–weaver’s, respectively. in conclusion, there is genetic diversity for forage quality traits among s. scabra. keywords: tropical legume, genetic resources, nutritional value, diversity index, germplasm characterization citation: de araújo américo, f. k., carvalho, m. a., vitória malaquias, j., braga ramos, a. k., braga, g. j., karia, c. t., lazarini da fonseca, c. e. (2022). stylosanthes scabra: genetic variability of forage quality traits. genetic resources 3 (5), 24– 35. doi: 10.46265/genresj.uppq3994. © copyright 2022 the authors. this is an open access article distributed under the terms of the creative commons attribution license (cc by 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. introduction brazil is a major beef producer and exporter, with an estimated herd of 171.8 million heads and about 158 million hectares of pastures (18.7% of brazilian territory), of which 70% are cultivated and 30% are natural (ibge, 2017). most of the livestock farming in brazil is still in extensive form, with the use of forage crops for livestock direct feeding into pasture (hoffmann et al, 2014). most production systems are based on the monoculture of tropical grasses of african origin. the use of high-quality forages improves the profitability ∗corresponding author: carlos eduardo lazarini da fonseca (carlos.lazarini@embrapa.br) of production systems; an option to obtaining pastures with a high nutritional value is the introduction of legumes associated with grasses. legumes play an important role in animal production due to their high protein concentration compared to grasses, in addition to soil biological nitrogen fixation (neres et al, 2012). several studies showed the associated use of legumes and grasses enables a significant increase in the nutritional value of ruminants’ diet (carvalho and pires, 2008). when working with genetic resources it is very important to evaluate the genetic diversity of a set of germplasm, which will allow genetic improvement and the efficient use of a species’ available germplasm. received: 29.11.2021 accepted: 22.02.2022 published online: 23.03.2022 https://www.genresj.org https://www.dx.doi.org/10.46265/genresj.uppq3994 https://www.genresj.org https://www.dx.doi.org/10.46265/genresj.uppq3994 mailto:carlos.lazarini@embrapa.br genetic resources (2022), 3 (5), 24–35 genetic variability in stylosanthes scabra 25 germplasm characterization include studies of ecogeographic and demographic adaptation, plant genetics, physiology and pathology, parameters of an organism’s vital cycle and yield evaluation, among other studies. breeding programmes should begin only after appropriate germplasm characterization (cameron, 1983). characterization is the best way to understand the variability in a germplasm collection and, consequently, increase its use by plant breeders. it is also important in monitoring the genetic stability of germplasm storage processes. characterization of germplasm can be based on molecular, biochemical, morphological, and agronomic features. despite the importance of forage legumes in various production systems, the adoption of tropical legume germplasm has been poor in several latin american countries (shelton et al, 2005; boddey et al, 2020). however, according to kretschmer (1988), there are about 18,000 species of forage legumes and at least 1,000–2,000 species with potential for cultivation. the genus stylosanthes includes nitrogen-fixing and drought-tolerant species economically important for perennial pasture, green manure and land recovery (marques et al, 2018). its importance as a forage crop has increased in recent years due to its potential to improve the nutritional quality of mixed pastures in tropical and subtropical regions (cameron et al, 2004; costa, 2006; rocha, 2014; epifanio et al, 2020; carvalho et al, 2020). due to its easy adaptation to acid and lowfertility soils, it has been used in pastures to minimize nitrogen deficiency in the soil, increase protein content in the diet, as well as improve pasture carrying capacity, longevity and productivity (resende et al, 2008; rocha, 2014). embrapa cerrados has a germplasm bank (agb) with thousands of accessions of the genus stylosanthes, of which 80 are from stylosanthes scabra vogel species. genetic resources conservation and characterization are of utmost importance to support breeding programmes for incorporating novel agronomic traits and developing new cultivars (wetzel et al, 2012; moreira et al, 2015). genetic resources characterization has been used to quantify genetic diversity, its magnitude, nature and distribution among and within populations (boldt, 2011; cruz et al, 2011). the evaluation of the nutritional quality diversity of s. scabra accessions can contribute to expanding the use of these genetic resources, and provides important information to high-quality forage breeding programmes (pereira et al, 2011). thus, the objective of this study was to access the genetic diversity of forage quality traits among 80 accessions of s. scabra from the embrapa cerrados active germplasm bank. materials and methods experimental setup an experiment was carried out at the embrapa cerrados research centre, in planaltina, federal district, brazil (15◦ 35’ s, 47◦ 42’ w; 993m a.s.l.), from december 2017 to april 2018, under irrigated conditions, to evaluate the genetic variability of accessions of s. scabra. the climate at the experimental site is tropical savannah according to the köppen–geiger classification (peel et al, 2007). the experiment was planted in clay soil (rhodic haplustox oxisol) with, on average: ph 5.1, organic matter concentration of 29g/kg, k concentration of 46mg/kg, al concentration of 25mg/kg and p concentration of 2.2mg/kg (mehlich-i) at 0–0.2m soil depth. seeds from all samples were treated by mechanical scraping with 100-grit sandpaper and sown in polyethene trays with 60 cells of 230cm3 filled with a commercial substrate. fifty-day old seedlings were planted in single-line plots spaced 0.5m within and 1.0m between plots. each plot consisted of seven plants from a specific accession. plant material eighty accessions of s. scabra collected in 9 different states from 4 distinct geographical regions in brazil were evaluated: 26 from the states of goiás (go), mato grosso (mt), and mato grosso do sul (ms) in the central-west region; 10 from pará (pa) and tocantins (to) in the north region; 36 from bahia (ba) and maranhão (ma) in the northeast region; and 8 from minas gerais (mg) and rio de janeiro (rj) in the southeast region. detailed information on each accession is presented in table 1. data collection and analysis all plants were harvested 90 days after transplanting to the field at 10cm stubble height. after an individual morphological plant characterization, all seven plants from each accession were pooled together, dried for 72 hours in a forced-air oven at 55◦c, ground through a 1mm-screen wiley mill (a. h. thomas co., boulder, co), stored and sealed in plastic containers, and sent to the analytical plant chemistry laboratory at embrapa cerrados. the samples were then analyzed to estimate the content of neutral detergent fibre (ndf), acid detergent fibre (adf), lignin (lig), crude protein (cp) and in vitro dry matter digestibility (ivdmd) on dry matter (dm) basis. dm content of the samples was determined by drying approximately 2g of each sample in a forcedair oven at 105◦c ± 2◦c for at least 2 hours (aoac, 1996). sequential ndf, adf and lig analysis from 0.5g initial sample weight placed in ankom f57 filter bags (van soest et al, 1991; komarek and sirois, 1993; komarek et al, 1994; vogel et al, 1999; mertens, 2002) was performed for each accession sample. for ndf and adf analysis, samples were digested in an automated fiber analyzer model ankom 2000 (ankom tech. corp., fairport, ny, usa) using a neutral detergent solution ph6.9–7.1, without α-amylase, and 1m h2so4 acid detergent solution, sequentially after drying and weighing procedures. lig determination was performed by placing the adf dry residue in a 12m h2so4 solution and incubated in a tecnal in vitro incubator system 26 de araújo américo et al genetic resources (2022), 3 (5), 24–35 table 1. passport data of stylosanthes scabra vogel accessions from the embrapa cerrados germplasm bank. embrapa cerrados, planaltina, df, 2020. cpac number, germplasm bank accession number. brazilian states: ba, bahia; go, goiás; ma, maranhão; mg, minas gerais; mt, mato grosso; mts, mato grosso do sul; pa, pará; rj, rio de janeiro; to, tocantins. brazilian geographic region: co, central-west; n, north; ne, northeast; se, southeast. access id cpac number brazilian city/state brazilian geographic region latitude longitude 1 512 rondonópolis-mt co 16◦ 18’ 00” s 54◦ 45’ 00” w 2 961 rio de janeiro-rj se 22◦ 54’ 06” s 43◦ 10’ 27” w 3 963 camaçari-ba ne 12◦ 40’ 60” s 38◦ 19’ 00” w 4 964 nazaré-ba ne 13◦ 00’ 00” s 39◦ 01’ 60” w 5 965 feira de santana-ba ne 12◦ 22’ 00” s 39◦ 06’ 00” w 6 966 cruz das almas-ba ne 12◦ 40’ 00” s 39◦ 06’ 00” w 7 967 mucuri-ba ne 18◦ 04’ 60” s 39◦ 34’ 00” w 8 968 pé de serra-ba ne 11◦ 49’ 57” s 39◦ 36’ 06” w 9 969 feira de santana-ba ne 12◦ 16’ 00” s 39◦ 01’ 60” w 10 970 santo amaro-ba ne 12◦ 31’ 88” s 38◦ 49’ 60” w 11 971 feira de santana-ba ne 12◦ 22’ 00” s 39◦ 06’ 00” w 12 972 catu-ba ne 12◦ 20’ 60” s 38◦ 23’ 60” w 13 973 conceição do almeida-ba ne 12◦ 55’ 00” s 39◦ 16’ 00” w 14 974 catu-ba ne 12◦ 20’ 60” s 38◦ 23’ 60” w 15 976 monte recôncavo-ba ne 12◦ 37’ 88” s 38◦ 37’ 00” w 16 977 valença-ba ne 13◦ 19’ 60” s 39◦ 15’ 00” w 17 978 nazaré -ba ne 12◦ 51’ 45” s 38◦ 58’ 55” w 18 979 ladeira grande-ba ne 13◦ 00’ 00” s 39◦ 01’ 60” w 19 980 barreiro-ba ne 12◦ 10’ 60” s 38◦ 23’ 60” w 20 983 chiador-mg se 22◦ 00’ 00” s 43◦ 00’ 00” w 21 985 cuiabá-mt co 15◦ 40’ 00” s 55◦ 32’ 60” w 22 986 nortelândia-mt co 14◦ 28’ 60” s 56◦ 45’ 00” w 23 988 campo verde-mt co 15◦ 39’ 00” s 55◦ 17’ 60” w 24 990 anastácio-ms co 20◦ 34’ 00” s 55◦ 36’ 00” w 25 993 coxim-ms co 18◦ 15’ 00” s 54◦ 40’ 00” w 26 997 senador canedo-go co 16◦ 37’ 60” s 49◦ 07’ 60” w 27 999 capinzal do norte-ma ne 04◦ 37’ 00” s 44◦ 22’ 00” w 28 1000 porto franco-ma ne 06◦ 19’ 60” s 47◦ 19’ 60” w 29 1001 porto franco-ma ne 06◦ 25’ 60” s 47◦ 21’ 00” w 30 1003 porto franco-ma ne 06◦ 19’ 60” s 47◦ 19’ 60” w 31 1007 alexânia-go co 16◦ 10’ 00” s 48◦ 30’ 00” w 32 1009 rio verde-ms co 18◦ 43’ 88” s 54◦ 47’ 60” w 33 1010 coxim-ms co 18◦ 30’ 00” s 54◦ 45’ 00” w 34 1013 itiquira-mt co 17◦ 08’ 88” s 54◦ 51’ 00” w 35 1016 são josé da serra-mt co 15◦ 40’ 00” s 55◦ 21’ 00” w 36 1017 cuiabá-mt co 15◦ 43’ 00” s 55◦ 43’ 60” w 37 1018 campo verde-mt co 15◦ 43’ 00” s 55◦ 17’ 60” w 38 1019 primavera do leste-mt co 15◦ 35’ 60” s 54◦ 07’ 60” w 39 1020 general carneiro-mt co 15◦ 40’ 00” s 52◦ 46’ 60” w 40 1025 lagoa da confusão-to n 11◦ 34’ 00” s 50◦ 40’ 00” w 41 1026 campo verde-mt co 15◦ 16’ 00” s 54◦ 55’ 60” w continued on next page genetic resources (2022), 3 (5), 24–35 genetic variability in stylosanthes scabra 27 table 1 continued 42 1240 belo horizonte-mg se 19◦ 55’ 17” s 43◦ 56’ 05” w 43 1243 abadiânia-go co 16◦ 07’ 88” s 48◦ 37’ 60” w 44 1246 campinorte-go co 14◦ 22’ 60” s 49◦ 10’ 00” w 45 1250 paráıso do tocantins-to n 10◦ 04’ 00” s 48◦ 49’ 60” w 46 1252 miranorte-to n 09◦ 15’ 00” s 48◦ 34’ 60” w 47 1253 wanderlândia-to n 06◦ 52’ 00” s 47◦ 51’ 00” w 48 1254 darcinópolis-to n 06◦ 45’ 00” s 47◦ 49’ 60” w 49 1256 dom eliseu-pa n 04◦ 07’ 60” s 47◦ 28’ 00” w 50 1257 nova rosalândia-to n 10◦ 31’ 00” s 49◦ 01’ 60” w 51 1259 porto nacional-to n 10◦ 58’ 60” s 48◦ 17’ 60” w 52 1260 sta rosa do tocantins-to n 11◦ 19’ 00” s 47◦ 52’ 00” w 53 1262 lavrado-to n 12◦ 07’ 00” s 46◦ 28’ 60” w 54 1264 monte alegre-go co 13◦ 15’ 00” s 47◦ 08’ 60” w 55 1265 são joão da aliança-go co 14◦ 30’ 00” s 47◦ 31’ 00” w 56 1267 planaltina-go co 15◦ 22’ 60” s 47◦ 31’ 60” w 57 1268 alexânia-go co 16◦ 07’ 00” s 48◦ 22’ 00” w 58 1270 linda vista-go co 13◦ 18’ 00” s 49◦ 07’ 60” w 59 1271 porangatu-go co 12◦ 52’ 00” s 49◦ 07’ 60” w 60 1274 goiânia-go co 16◦ 34’ 03” s 49◦ 15’ 54” w 61 4944 matias cardoso-mg se 15◦ 01’ 48” s 43◦ 51’ 04” w 62 4945 monte azul-mg se 15◦ 14’ 01” s 43◦ 03’ 32” w 63 4946 monte azul-mg se 15◦ 15’ 12” s 43◦ 02’ 60” w 64 4947 pajeú-mg se 15◦ 17’ 12” s 42◦ 54’ 39” w 65 4950 andaráı-ba ne 12◦ 48’ 39” s 41◦ 19’ 30” w 66 4951 andaráı-ba ne 12◦ 48’ 48” s 41◦ 19’ 26” w 67 4952 palmeiras-ba ne 12◦ 27’ 33” s 41◦ 29’ 51” w 68 4953 palmeiras-ba ne 12◦ 27’ 15” s 41◦ 29’ 14” w 69 4954 palmeiras-ba ne 12◦ 27’ 15” s 41◦ 29’ 21” w 70 4955 palmeiras-ba ne 12◦ 27’ 09” s 41◦ 29’ 13” w 71 4956 palmeiras-ba ne 12◦ 27’ 10” s 41◦ 29’ 24” w 72 4957 iraquara-ba ne 12◦ 22’ 07” s 41◦ 31’ 00” w 73 4959 iraquara-ba ne 12◦ 22’ 08” s 41◦ 31’ 01” w 74 4960 iraquara-ba ne 12◦ 22’ 09” s 41◦ 30’ 59” w 75 4962 utinga-ba ne 12◦ 06’ 11” s 41◦ 06’ 48” w 76 4963 morro do chapéu-ba ne 11◦ 55’ 02” s 41◦ 08’ 02” w 77 4965 cafarnaum-ba ne 11◦ 45’ 25” s 41◦ 32’ 01” w 78 4966 oliveira dos brejinhos-ba ne 12◦ 18’ 06” s 42◦ 37’ 55” w 79 4986 aracatu-ba ne 14◦ 28’ 36” s 41◦ 26’ 41” w 80 5180 francisco sá-mg se 16◦ 27’ 56” s 43◦ 25’ 54” w 28 de araújo américo et al genetic resources (2022), 3 (5), 24–35 (tecnal scientific equipments, piracicaba, sp, brazil) for 3 hours. ashing was done by placing folded filter bags in crucibles in a muffle furnace at 500◦c for at least 5.5 hours. n concentration was determined by kjeldahl method (aoac, 1996) with a tecnaltm 0365 digestion–distillation system and cp was calculated as n x 6.25. true ivdmd was determined by 48-hour ruminal fermentation at 39.5◦c in a tecnaltm in vitro incubator system using the procedure described by tilley and terry (1963), with modification by goering and van soest (1970). hemicellulose concentration (hemic) was estimated by subtracting adf from ndf and cellulose (cellu) by subtracting lig from adf. statistical analysis univariate analysis was performed through the f test to detect potential differences among sites of collection as well as similarity clusters for each quality trait. multivariate analysis was used to examine the genetic variability of the accessions. a hierarchical clustering analysis was done to sort out groups of similarity based on ward’s minimum variance method and their genetic dissimilarity were estimated by the square root of the generalized mahalanobis d2 distances (alvares et al, 2012; bapurao et al, 2018). cluster analysis allows classifying accessions into homogeneous groups based on their quality-traits values. accessions within the same group are more similar and homogeneous while accessions among different groups are more divergent and heterogeneous. a principal components analysis (pca) was performed to discriminate among accessions and group them. the goal of pca is to provide a reduced dimension model that would indicate differences among groups of similarity. it also contributes to a better understanding of the variables by describing how much of the total variance was explained by each one of them. also, pca analysis allows graphical visualization of the variables, identification of similar and divergent accessions, and highlights the variables that contribute most to the differentiation of accessions (philippeau and philippeau, 1986). all statistical analyses were done with the r software version 3.6.2 programme (r core team, 2019). the phenotypic diversity among accessions was estimated by the index of shannon and weaver (1949) corrected by the logarithm of the number of classes and simpson (1949): shannon and weaver corrected: j = h’/ ln s = ∑ pi * ln pi / ln s; and simpson: d = 1 ∑ pi 2, where j is the shannon corrected index, h’ is the richness and evenness shannon index, pi is the proportion of the class i, and ln s the natural logarithm of the total number of classes. both indices are used to express the concepts of richness, meaning the number of different accessions in a sampling effort, as well as evenness, meaning the degree of equality in the abundance of individuals, or the relative uniformity of their distribution across groups of accessions (meng et al, 1999). these indices are used to estimate diversity within populations (kosman and leonard, 2007) and range from zero to one, where one represents high genetic diversity and zero no genetic diversity. they correspond to the probability of two individuals randomly selected from a group to exhibit similar characteristics (carvalho and quesenberry, 2009). results the average concentration of cp, ivdmd, ndf, adf, lig, hemic and cellu were 220.6 ± 20.2g/kg, 560 ± 56.0g/kg, 516.8 ± 44.1g/kg, 368 ± 32.9g/kg, 69.4 ± 9.4g/kg, 148.8 ± 31.5g/kg and 298.6 ± 32.9g/kg, respectively (table 2). means of ivdmd, adf and cellu were significantly different for site of collection (brazilian states, table 3). accessions from ba, go, pa and to had higher ivdmd concentrations. pa (only one sample) and to states, had the highest ivdmd concentration, 653.4g/kg and 590.6 ± 18.5g/kg, respectively, and were significantly different from those for ms and mt states. accessions from go, ma, pa, and to had the lowest concentrations of adf and cellu, with 368 ± 32.9g/kg and 298.6g ± 32.9g/kg, respectively. lower fibre concentrations were also found for pa (only one accession) and to, with 341.4g/kg and 339.8 ± 28.3g/kg of adf and 264.5g/kg and 272.1 ± 23.2g/kg of cellu, respectively (table 3). the pca of the 80 accessions resulted in five principal components explaining all variations (table 4). the first two principal components accounted for 73% of the total variance among all accessions. the first pc was responsible for 55% and the second for 18% of the variation. variables that are correlated with pc1 and pc2 are the most important in explaining the variability in the data set. the correlation between each variable and the pc was used as the coordinate in a circle of radius equals to one, and represents the intensity in which the variable contribute to pc1 and pc2 (figure 1). traits adf, ndf, ivdmd and cp contributed most to pc1, with values ranging from 0.73 to 0.90, while only lig had an important contribution to pc2, with a value of 0.76. figure 1 displays the correlation circle of the pca. positively correlated variables point to the same side of the plot and negatively correlated variables point to opposite sides of the graph. the kelley–gardner–sutcliffe penalty function for hierarchical cluster trees was used to define the number of groups (kelley et al, 1996). the minimum of this function defines the group size. in this study, clustering of the 80 accessions resulted in four clusters, which means four diversity classes of forage nutritive values, considering cp, ivdmd, ndf, adf, and lig concentrations (figure 2). all traits had high and similar diversity values from simpson’s that ranged from 0.771 to 0.781 and shannon–weaver’s indices that ranged from 0.806 to 0.817 (table 5). discussion in australia, ’seca’ and ’fitzroy’ s. scabra cultivars have been released with high dry matter yields, adegenetic resources (2022), 3 (5), 24–35 genetic variability in stylosanthes scabra 29 table 2. mean ± standard deviation (sd), minimum and maximum values (range) in g/kg, site of collection (state) mean square (mss), and p-value of five quality traits from 80 stylosanthes scabra vogel germplasm accessions. *, ** significant differences among states at 0.05 and 0.01 α levels, respectively. trait statistics mean ± sd range mss p-value (pr>f) crude protein 220.6 ± 20.2 185.1–279.2 6.748 0.2003 in vitro dry matter digestibility 560.0 ± 56.0 427.9–718.8 71.550* 0.0127 neutral detergent fibre 516.8 ± 44.1 419.3–630.1 21.640 0.3550 acid detergent fibre 368.0 ± 32.9 288.2–456.4 31.045** 0.0014 lignin 69.4 ± 9.4 45.7–91.6 1.243 0.186 hemicellulose 148.8 ± 31.5 103.7–264.3 13.031 0.229 cellulose 298.6 ± 29.9 229.9–395.3 30.034** 0.00016 table 3. seven quality traits, estimated from 80 stylosanthes scabra vogel germplasm accessions by brazil source states. given are the number of accessions per state and values in g/kg for means ± standard deviation (mean ± sd), minimum and maximum values (min, max). brazilian states: ba, bahia; go, goiás; ma, maranhão; mg, minas gerais; mt, mato grosso; mts, mato grosso do sul; pa, pará; rj, rio de janeiro; to, tocantins.cp, crude protein; ivdmd, in vitro dry matter digestibility; ndf, neutral detergent fibre; adf, acid detergent fibre; lig, lignin; hemic, hemicellulose; cellu, cellulose. mean values for traits followed by the same superscript letter are not significantly different, based on lsd t student at 0.05 α level. state ba go ma mg ms mt pa rj to trait nº 32 11 4 7 4 11 1 1 9 cp mean 221.8 233.6 226.7 218.5 241.2 226.1 209.3 195.9 239.8 ± sd 27.7 12.9 10.5 17.0 6.5 19.9 15.6 min 185.1 218.3 213.6 196.8 235.2 185.3 209.3 195.9 215.6 max 279.2 257.0 238.7 240.8 249.6 250.2 209.3 195.9 265.5 ivdmd mean 572.1ab 563.0ab 549.5abc 558.7abc 520.0bc 509.8c 653.4a 533.0abc 590.6a ± sd 55.4 55.1 23.0 60.3 73.9 47.3 18.5 min 476.0 467.7 517.9 479.4 427.9 431.4 653.4 533.7 563.9 max 718.8 658.9 567.4 651.1 587.9 587.7 653.4 533.7 611.4 ndf mean 511.1 515.3 518.6 539.1 502.6 541.3 470.6 541.7 499.8 ± sd 39.3 52.5 46.0 58.0 21.6 38.6 47.0 min 419.3 442.6 459.9 475.8 488.2 485.7 470.6 541.7 445.8 max 627.2 624.2 572.3 630.1 534.7 594.9 470.6 541.7 597.7 adf mean 370.3ab 357.9bc 336.0c 390.0a 366.6abc 390.3a 341.4bc 420.2a 339.8c ± sd 28.8 23.3 24.4 36.1 11.8 36.5 28.3 min 371.6 368.9 335.0 392.3 364.1 385.0 341.4 420.2 346.2 max 382.3 371.7 345.4 412.4 372.9 411.7 341.4 420.2 364.8 lig mean 69.2 66.5 76.2 76.4 65.0 67.2 76.9 82.0 67.7 ± sd 9.6 8.2 13.3 6.8 5.1 9.4 9.5 min 49.9 55.8 62.3 68.7 59.5 45.7 76.9 82.0 58.5 max 91.6 85.9 91.4 87.5 70.1 81.1 76.9 82.0 86.4 hemic mean 140.7 157.3 182.6 149.1 136.1 151.0 129.2 121.4 160.0 ± sd 25.7 41.3 37.9 34.2 21.3 20.2 41.4 min 103.7 119.2 152.5 119.5 113.3 130.3 129.2 121.4 128.4 max 221.1 253.6 234.1 214.9 164.8 189.9 129.2 121.4 264.3 cellu mean 301.2ab 291.4ab 259.9c 313.5a 301.5ab 323.1a 264.5bc 338.2a 272bc ± sd 22.9 23.2 16.1 30.7 11.9 37.3 23.2 min 234.7 250.0 239.2 273.6 286.1 284.3 264.5 338.2 230.0 max 363.9 318.7 275.3 359.4 313.3 395.3 264.5 338.2 299.7 quate nutritive values, drought tolerance and persistence (edye et al, 1998). in nigeria, akinlade et al (2008) found cp concentrations ranging from 147g/kg to 151g/kg for four s. scabra cultivars. in argentina, ciotti et al (1999) evaluated 33 accessions of 6 different species of stylosanthes in which 4 s. scabra accessions had cp mean values ranging from 78g/kg to 134g/kg. in brazil, cp from stylosanthes commercial 30 de araújo américo et al genetic resources (2022), 3 (5), 24–35 table 4. principal component analysis (pca) of the 80 accessions of stylosanthes scabra vogel. cp, crude protein; ivdmd, in vitro dry matter digestibility; ndf, neutral detergent fibre; adf, acid detergent fibre; lig, lignin. pca pc1 pc2 pc3 pc4 pc5 eigen values 2,77 0,89 0,67 0,43 0,24 proportion of variance (%) 55,48 17,77 13,41 8,57 4,78 cumulative variance (%) 55,48 73,24 86,65 95,22 100,00 cp 0,73 -0,27 0,51 -0,37 -0,08 ivdmd 0,74 -0,47 0,05 0,47 -0,08 ndf -0,77 -0,09 0,55 0,18 0,24 adf -0,90 -0,07 0,16 0,05 -0,40 lig -0,54 -0,76 -0,29 -0,19 0,09 table 5. simpson and shannon–weaver diversity indices for stylosanthes scabra vogel. trait simpson shannon–weaver crude protein (cp) 0.781 0.813 in vitro dry matter digestibility (ivdmd) 0.771 0.806 neutral detergent fibre (ndf) 0.779 0.812 acid detergent fibre (adf) 0.774 0.808 lignin (lig) 0.779 0.817 cultivars from different species or a mixture of species (s. guianensis cv. mineirão, s. macrocephala cv. pioneiro, and a mixture of s. capitata and s. macrocephala cv. figure 1. correlation circle illustrating the direction and intensity of the five forage quality variables used in the principal component analysis. cp, crude protein; ivdmd, in vitro dry matter digestibility; ndf, neutral detergent fibre; adf, acid detergent fibre; lig, lignin. campo grande) are reported with concentrations ranging from 98 to 210g/kg (braga et al, 2020). the mean value obtained in this study for s. scabra was 220g/kg, higher than those from argentina and higher or similar to other studies in brazil. this cp high value reflects the species’ potential to be used as a forage in animal production systems in the tropics. the s. scabra ndf and adf values obtained in this research are similar to the ones reported for 71 accessions from five stylosanthes species collected in brazil (santana, 2010): s. angustifolia with 540g/kg of ndf and 378g/kg of adf; s. capitata with 490g/kg of ndf and 330g/kg of adf; s. macrocephala 545g/kg of ndf and 394g/kg of adf; s. pilosa with 531g/kg of ndf and 394g/kg of adf; and s. viscosa with 553g/kg of ndf and 390g/kg of adf in average. the average fibre concentration of s. scabra in this study was 517g/kg for ndf and 368g/kg for adf, which indicate that the fibre content among different stylosanthes species is similar. this fibre characteristics together with the high cp content indicate that s. scabra species may be used, as other stylosanthes species, as an important forage for seasonally dry tropical and subtropical environments in brazil. there were significant differences among sites of collection for ivdmd, adf, and cellu, pointing to the existence of phenotypic variability among accessions (table 2). the range for ivdmd, adf and cellu were 291g/kg, 168g/kg and 165g/kg, respectively. this indicates that there is potential genetic diversity among genotypes for ivdmd, adf and cellu to be successfully exploited in a breeding programme. differences among genotypes from different states were not significant for cp, ndf, lig, and hemic. thus, genetic gains from selection might be less effective when selecting for high cp and hemic, as well as low ndf and lig from this collection. the negative correlation between digestibility and fibre concentrations in forage species is well known because the carbohydrates in the ndf and adf portions of plant tissues are mostly cell walls indigestible to ruminants. thus, the lower adf and cellu concentrations from pa and to indicate that those accessions may produce segregant populations when used in breeding programmes for higher nutritive quality forage. this is only an indication, additional studies should be carried out since higher ivdmd and lower fibre contents may not be due only to genetic effects but also management practices, plant age and other environmental effects. the goal of pca is to provide a reduced dimension model that would indicate measured differences among groups. it also can contribute to a better understanding of the set of variables by describing how much of the total variance is explained by each one. the circle of correlations graphic illustrates the direction as well as the intensity of the variable vectors in the first two components (figure 1). note that cp, ivdmd, ndf, adf vectors have closer inclinations along pc1, with cp and genetic resources (2022), 3 (5), 24–35 genetic variability in stylosanthes scabra 31 figure 2. dendrogram illustrating the distribution of 80 accessions of stylosanthes scabra vogel in four clusters based on mahalanobis distance. the accessions are numbered as in table 1. ivdmd vectors pointing to the same orientation, as are ndf and adf but in opposite directions. the ivdmd and adf concentrations are the closest to the circle of correlations, indicating the better representation of the traits on the definition of pc1. yet, lig was the only trait with a better representation on pc2. variables close to the centre of the circle are less important for the first component. in brief, there was a positive correlation between cp and ivdmd and between ndf and adf, as well as negative correlations among the former traits with the latter ones. adf, ivdmd and lig were the traits with the best representations on the first two principal components. in this study, 80 accessions resulted in four clusters, which means four diversity classes of forage nutritive values, considering cp, ivdmd, ndf, adf, and lig concentrations (figure 2). concentrations of hemic and cellu were not considered in the analysis since they are highly correlated to ndf and adf concentrations, respectively, from which they are directly derived (table 4). the four mahalanobis distance clusters were plotted in the pca graphic, each in a 0.95 probability level ellipsis, to get a better view of their distribution (figure 3). scatter plots of the principal component scores were convergent with the clusters’ ellipsis, allowing the visualization and identification of locations with higher concentrations of a given trait in the forage. four out of the five original variables had high contributions to the first principal component. this first component is a measure of forage quality when ivdmd and cp increase. also, it is a measure of lack of quality when the fibre components ndf and adf increase. furthermore, the first principal component correlates most strongly with the adf (r = 0.90). thus, this first principal component may be considered primarily a measure of the adf concentration. this implies that accessions with higher values of adf tend to have a lower quality of forage. the second principal component increases with the decrease of lig concentration. this component can be viewed as a measure of forage quality as lig concentration decreases. pca analysis did separate the accessions into two dimensions, even though the traits are quantitative with a scatter distribution along pc1 and pc2. all five quality characteristics had high loading values in the first two principal components, ranging from 0.73 to 0.90, indicating their importance as s. scabra descriptors. cluster ii and iv with 28 and 24 accessions, respectively, were the largest, representing together 65% of the total accessions. cluster i with 16 accessions and cluster iii with 12 accessions represented the remaining 35% of the total number of accessions (table 6). the analyses of variance among clusters for each trait are presented in table 7. there were highly significant differences among clusters for cp, ivdmd and adf, indicating substantial genetic variability. cluster iv included accessions with the highest cp and ivdmd concentrations and the lowest adf concentration, representing the group of accessions with the higher forage quality, and may be used for hybridization in breeding programmes. clusters i and ii had the lowest cp and ivdmd, as well as the highest adf 32 de araújo américo et al genetic resources (2022), 3 (5), 24–35 figure 3. dispersion of 80 stylosanthes scabra vogel accessions in relation to the first two components and four mahalanobis similarity clusters. the accessions are numbered as in table 1 and grouped in four clusters as in table 6. concentrations, representing the lowest nutritive value cluster. cluster iii had intermediate values for all traits and its cp concentration was significantly different from all other clusters. formation of similarity groups and respective estimation of their divergence is useful when choosing accessions to be used as progenitors for superior hybrid combinations in breeding programmes. divergence estimates by mahalanobis d distances are presented in table 8. the intra-group distances were very similar for all four clusters, ranging from 3.15 to 3.37, and were consistently lower than the inter-cluster distances, which ranged from 6.92 to 12.47, as expected. these distances clearly suggest a great deal of genetic similarity within and genetic divergence among clusters. the shortest inter-cluster distance was between cluster ii, indicating lower divergence. the highest inter-group distance was 12.46 between clusters i and iii, followed by 11.99 between clusters iii and iv, and 11.98 between clusters i and iv. high divergences among groups were consistent with the anova results where cluster iv was significantly different from clusters i and iii, for cp, ivdmd and adf. in a breeding programme, crosses between accessions with maximum divergence may give rise to genetically diverse lines and the potential production of heterotic transgressive segregants. a great deal of variation in subsequent generations is expected from crosses among plants of maximum inter-class distances clusters. this may not be true between low-divergent clusters as clusters i and ii. the pattern of distribution of accessions in different clusters did not reflect the geographical diversity of the site of collection (states). accessions from each state were consistently distributed among the four clusters of forage quality (figure 4). cluster i presented accessions from five states, cluster ii from eight, cluster iii from seven, and cluster iv from six, all out of a total of nine states. thus, the observed traits diversity was somewhat similar among the different collection sites, leading to a lack of effect of the source locations on the four quality groups definition considering this germplasm collection. simpson’s and shannon–weaver’s indices expressed the diversity among accessions within each population, figure 4. geographic distribution of collection sites of the 80 stylosanthes scabra vogel accessions in brazil, coloured according to the clusters in which they were grouped. genetic resources (2022), 3 (5), 24–35 genetic variability in stylosanthes scabra 33 table 6. distribution of 80 stylosanthes scabra vogel accessions in four clusters based on the forage quality variables. embrapa cerrados, planaltina, df, 2020. cluster nº of accessions accessions id i 16 1, 3, 4, 7, 9, 13, 18, 19, 25, 27, 31, 32, 35, 36, 41, 44 ii 28 2, 5, 6, 8, 10, 11, 12, 15, 16, 17, 20, 21, 22, 23, 28, 38, 39, 48, 49, 51, 53, 56, 59, 60, 61, 66, 71, 80 iii 12 14, 3033, 34, 37, 42, 43, 52, 57, 63, 68, 69 iv 24 24, 26, 29, 40, 45, 46, 47, 50, 54, 55, 58, 62, 64, 65, 67, 70, 72, 73, 74, 75, 76, 77, 78, 79 table 7. mean concentrations ± standard deviations of cp, ivdmd, ndf, adf, and lig of stylosanthes scabra vogel accessions distributed in four clusters. ** significant differences among clusters at 0.01 α level. means followed by the same subscript letter within the same column are not different, lsd t student at 0.05 α level. cp, crude protein; ivdmd, in vitro dry matter digestibility; ndf, neutral detergent fibre; adf, acid detergent fibre; lig, lignin. cp (%) ivdmd (%) ndf (%) adf (%) lig (%) cluster i 21,58 ± 2.21c 50,35 ± 4.27c 50,98 ± 3.32 36,73 ± 2.64 a 6,13 ± 0.72 cluster ii 21,25 ± 1.35c 54,81 ± 4.40b 52,06 ± 2.99 38,36 ± 2.77 a 7,63 ± 0.63 cluster iii 22,97 ± 1.74b 55,96 ± 3.88b 58,36 ± 3.52 37,65 ± 3.66 a 7,19 ± 1.24 cluster iv 24,82 ± 1.41a 61,18 ± 3.72a 48,37 ± 2.51 34,60 ± 2.98 b 6,55 ± 0.54 mean 22.65 ± 1.72** 56,00 ± 4.12** 51.68 ± 4.37n.s 36.80 ± 3.14** 6,94 ± 0.95n.s. range 18.53–27.92 42.79–71.88 44.13–52.41 33.18–40.23 4.57–7.56 table 8. mean intraand inter-cluster mahalanobis (d) distances of 80 accessions of stylosanthes scabra vogel. embrapa cerrados, planaltina, df, 2020. cluster i ii iii iv i 3.22 ii 6.92 3.15 iii 12.47 9.71 3.37 iv 11.98 9.79 11.99 3.18 in this case, each different brazilian state where collecting was done. high values for both indices suggest a significant amount of genetic variation for forage quality traits. conclusions there is considerable genetic diversity for forage nutritive values among the s. scabra accessions from embrapa cerrado’s germplasm bank. this variability was reflected across geographic areas in brazil for in vitro digestibility, acid detergent fibre, and cellulose concentrations. independent of the site of collection (brazilian states), 24 high-quality s. scabra accessions are promising parents for breeding due to their higher digestibility and protein content, as well as lower lignin concentrations. s. scabra accessions from embrapa cerrado’s germplasm bank presented significant genetic diversity that may be useful for developing new high-quality improved forage populations in a breeding programme. author contributions all authors contributed to the study conception and design. fabiana karla de araújo américo, marcelo ayres carvalho, allan kardec braga ramos and claudio takao karia did all genetic material preparation, experiment implementation and conduction, and data collection and organization. fabiana karla de araújo américo and carlos eduardo lazarini da fonseca did the wet lab analysis. juaci vitória malaquias, marcelo ayres carvalho and carlos eduardo lazarini da fonseca did the data analysis. fabiana karla de araújo américo, marcelo ayres carvalho and carlos eduardo lazarini da fonseca wrote the first draft of the manuscript, and all authors commented on the different versions of the manuscript. marina de fátima vilela did the geographical information plots. gustavo josé braga was responsible for reviewing the manuscript. all authors read and approved the final manuscript. conflict of interest statement all authors have no affiliations with or involvement in any organization or entity with any financial interest or non-financial interest in the subject matter or materials discussed in this manuscript. funding this work was partially funded by the coordenação de aperfeiçoamento de pessoal de ńıvel superior capes (coordination for the improvement of higher education personnel, a federal agency) which awarded a scholarship to the first author. this work was part of her dissertation in partial fulfilment of the requirements of doctor of science (dsc) in plant genetic resources from the universidade federal de feira de santana. 34 de araújo américo et al genetic resources (2022), 3 (5), 24–35 references akinlade, j. a., farinu, g. o., agboola, o. o., akingbade, a. a., ojebiyi, o. o., and aderinola, o. a. 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(2022). the role of tomato wild relatives in breeding disease-free varieties. genetic resources 3 (6), 64–73. doi: 10.46265/genresj.pses6766. © copyright 2022 the authors. this is an open access article distributed under the terms of the creative commons attribution license (cc by 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. tomato wild relatives crop wild relatives (cwrs) are potential sources of allelic variation useful to overcome biotic and abiotic stresses as they often grow and reproduce in marginal habitats (ortiz, 2015; bohra et al, 2021). tomato wild relatives are native to south america, distributed from the coastal region of ecuador to northern chile, including the galápagos islands (darwin et al, 2003). these regions have extremely varied environments within short distances due to differences in altitude (andean geography) and diverse ecological habitats, which led to local adaptation and generation of large genetic diversity among (figure 1) and within wild tomato species. solanum section lycopersicon (mill.) wettst. consists of cultivated tomato (s. lycopersicum) and 12 wild relatives (ramı́rez-ojeda et al, 2021). the tomato genetics resource center (c.m. rick tgrc, university of california-davis, usa, https:// tgrc.ucdavis.edu/) hosts the largest genetic stocks of wild tomato collections, with over 900 accessions. the largest collection belongs to accessions of ∗corresponding author: hamid khazaei (hamid.khazaei@gmail.com) s. pimpinellifolium (~300) followed by s. habrochaites (~120), s. peruvianum (~100), s. chilense (~100), and s. pennellii (~50), respectively (table 1). the world vegetable center (worldveg, taiwan https:// genebank.worldveg.org/#/) and the united states department of agriculture, agricultural research service (usda-ars, https://www.ars-grin.gov/) genebanks also maintain the second and third largest wild tomato collections, respectively (table 1). however, the majority of their wild tomato collection was originally obtained from the tgrc collection. furthermore, these genebanks harbour an extensive collection of introgression lines derived from different tomato wild species (ebert and schafleitner, 2015). utilization of tomato wild relatives for biotic stress breeding domestication has increased the phenotypic diversity of cultivated tomatoes but may have narrowed their resistance to biotic and abiotic constraints as selection ensued (vu et al, 2020). tomato wild relatives germplasm harbour natural resistance to various diseases and insect pests. sources of genetic resistance to many of the biotic stresses faced by cultivated tomareceived: 05.07.2022 accepted: 08.08.2022 published online: 22.09.2022 https://www.genresj.org https://www.dx.doi.org/10.46265/genresj.pses6766 https://www.genresj.org https://www.dx.doi.org/10.46265/genresj.pses6766 mailto:hamid.khazaei@gmail.com https://tgrc.ucdavis.edu/ genetic resources (2022), 3 (6), 64–73 cwrs role in breeding disease-free tomatoes 65 figure 1. diversity in fruit colour and leaf shape of some tomato wild relatives. from left to right: s. pimpinellifolium (la1269), s. peruvianum (l00887-3), s. chilense (la2737b) and s. galapagense (vi063177). the size of the marker is 14cm × 1cm. photo credit: hamid khazaei table 1. genetic resources of tomato wild relatives maintained by the tomato genetics resource center (c.m. rick tgrc), world vegetable center (worldveg), and the united states department of agriculture, agricultural research service (usda-ars) genebanks (data accessed on june 23, 2022). species no. of accessions per genebank tgrc worldveg usda-ars s. pimpinellifolium 290 342 338 s. habrochaites 120 139 49 s. peruvianum 69 116 11 s. chilense 115 46 1 s. pennellii 47 59 5 s. corneliomulleri 53 10 12 s. neorickii 47 12 8 s. galapagense 28 27 4 s. arcanum 45 4 3 s. cheesmaniae 12 17 5 s. chmielewskii 16 11 0 s. lycopersicoides 23 5 0 s. sitiens 13 5 2 s. huaylasense 16 0 0 s. ochranthum 7 0 2 s. juglandifolium 5 1 0 toes have been identified in some accessions of wild tomato species that have been historically used to introduce resistance (r) genes into cultivated tomato varieties. it should be noticed that there is genetic diversity for biotic resistance response within tomato wild species, and only a few accessions within each species have been identified as sources of resistance (ebert and schafleitner (2015) and references). we list major r genes found in and introgressed from wild tomato species into cultivated tomatoes in table 2, along with information about their causal pathogens as footnotes. for example, the r genes/alleles conferring resistance to the begomoviruses that cause tomato yellow leaf curl diseases (called ty genes), were found in wild tomato species. most tomato advanced lines grown in tropical and subtropical regions of the world carry at least one source of ty resistance genes (ty-1/ty-3) or multiple ty genes (ty-2, ty-5, ty-6). the pyramiding of ty genes into one line leads to broad and probably more durable resistance to begomoviruses. likewise, r genes for late blight (caused by phytophthora infestans, ph genes) and fusarium wilt (caused by fusarium oxysporum, i genes) also originated from wild tomatoes (table 2). these genes are also frequently present in the modern tomato lines worldwide. some important major qtls (quantitative trait locus) such as bwr and eb, conferring bacterial wilt (caused by ralstonia spp.) and early blight (caused by alternaria linariae) resistance, were first introgressed to adapted cultivated tomato cultivars from s. pimpinellifolium and later uncovered and genetically mapped. some r genes (rx1, -2 and -3) for bacterial spot resistance (caused by several species of gram-negative bacteria in the genus xanthomonas) were also derived from s. pimpinellifolium via cultivated tomato. several r genes against important pathogens in tomato have mainly been introgressed, from the wild species s. pimpinellifolium, s. habrochaites, s. peruvianum, s. chilense and s. pennellii, into modern tomato varieties (table 2). however, most tomato breeding programmes focus on s. pimpinellifolium due to its red fruit colour (figure 1) and its close relationship to cultivated tomato, which allows breeders to easily obtain interspecific crosses. that is probably why most of the r genes have already been identified in this species. 66 khazaei and madduri genetic resources (2022), 3 (6), 64–73 table 2. list of major biotic resistance genes incorporated into cultivated tomatoes (solanum lycopersicum) from wild relatives of tomato and s. lycopersicum var. cerasiforme. causative agents for the listed diseases are included in footnotes. species r-genes’ contribution disease/insect pest references s. pimpinellifolium l. i and i-2 fusarium wilt1 stall and walter (1965); reviewed in chitwood-brown et al (2021) ph-1, ph-2, ph-3 and ph-5 late blight2 bonde and murphy (1952); gallegly and marvel (1955); avrdc (1994); foolad et al (2006) eb-5 and eb-9 early blight3∗ anderson et al (2021) bwr-6 and bwr-12 bacterial wilt4∗ wang et al (2013) rx-1, rx-2 and rx-3 bacterial spot5∗ yu et al (1995); reviewed in adhikari et al (2020) rx-4 bacterial spot robbins et al (2009) sm gray leaf spot6 parlevliet (2002) cf genes (except cf-4 and cf-5) leaf mold7 bailey (1950); reviewed in scott and gardner (2007) sw-1(a and b), sw-2, sw-3 and sw-4 tswv8 finlay (1953); roselló et al (1998); zhu et al (2017); reviewed in qi et al (2021) pto bacterial speck9 pitblado and kerr (1980) cmm genes bacterial canker10 forster and echandi (1972); sotirova et al (1994); sen et al (2021) s. habrochaites s. knapp and d. m. spooner ph-4 late blight lough (2003) cf-4 leaf mold stevens and rick (1986) ty-2 tylcv11 hanson et al (2006) tm-1 tomv12 pelham (1966) ol-1/ol-3, and ol-5 powdery mildew13 van der beek et al (1994); huang et al (2000); bai et al (2005) cmm genes bacterial canker forster and echandi (1972); francis et al (2001); coaker and francis (2004); sotirova et al (1994) rbcq genes gray mould14 ten have et al (2007); finkers et al (2007a,b) s. peruvianum l. ty-5 tylcv hutton et al (2012) sw-5 and sw-6 tswv giordano et al (2000); rosello et al (2001) mi genes root-knot nematodes15 smith (1944); reviewed in el-sappah et al (2019) tm-2, tm-22 and tm-2a tomv soost (1963); ganal and tanksley (1996) and tanksley and nelson (1996) ve verticillium wilt16 diwan et al (1999) frl fusarium crown17 vakalounakis et al (1997) s. chilense (dunal) reiche ty-1/ty-3a, ty-4, and ty-6 tylcv zamir et al (1994); ji et al (2007); ji et al (2009); gill et al (2019) sw-7 tswv stevens et al (1994) cmm genes bacterial canker sotirova et al (1994) lv powdery mildew yordanov et al (1975); chunwongse et al (1997) gray mould ten have et al (2007) continued on next page genetic resources (2022), 3 (6), 64–73 cwrs role in breeding disease-free tomatoes 67 table 2 continued r-genes’ contribution disease/insect pest references s. pennellii correll i-3 and i-7 fusarium wilt catanzariti et al (2015); gonzalez-cendales et al (2016) asc alternaria stem canker18 scott and gardner (2007) xv-4 bacterial spot astua-monge et al (2000) acylsugar-related genes a wide range of insects leckie et al (2012, 2016); schilmiller et al (2012) s. galapagense s. c. darwin and peralta wf-1 and wf-2 whiteflies19 firdaus et al (2013); santegoets et al (2021) s. arcanum peralta ol-4 powdery mildew bai et al (2005) cmm genes bacterial canker crinò et al (1995); sotirova et al (1994); sen et al (2013) s. neorickii d. m. spooner, g. j. anderson and r. k. jansen v2 verticillium wilt kanagawa agricultural technology center (1999) gray mould ten have et al (2007); finkers et al (2008) s. l. var. cerasiforme cf-5 leaf mold dickinson et al (1993); dixon et al (1998) ol-2 powdery mildew ciccarese et al (1998) 1fusarium wilt caused by fungal pathogen fusarium oxysporum (schlecht. emend. snyder & hansen). 2late blight resistance caused by the oomycete phytophthora infestans (mont.) de bary. 3early blight is caused by fungal pathogen alternaria spp. 4bacterial wilt caused by the group of soilborne bacteria in the ralstonia solanacearum species complex. 5bacterial spot caused by several species belonging to the genus xanthomonas. it can be caused by xanthomonas euvesicatoria ex doidge, x. vesicatoria ex doidge, x. perforans, and x. gardneri šutic. ∗early blight, bacterial wilt, and bacterial spot (rx-1, rx-2, and rx-3) resistance genes are most likely derived from s. pimpinellifolium via cultivated tomato. 6gray leaf spot caused by fungal pathogen stemphylium lycopersici (s. lycopersici). 7leaf mold caused by fungal pathogen cladosporium fulvum (syn. passalora fulva). 8tswv, tomato spotted wilt orthotospovirus (order bunyavirales, family tospoviridae, genus orthotospovirus) is transmitted by frankliniella occidentalis (pergande) (thysanoptera: thripidae). 9bacterial speck disease caused by pseudomonas syringae pv. tomato (pst). 10bacterial canker caused by clavibacter michiganensis subsp. michiganesis. 11tylcv, tomato yellow leaf curl virus disease. tylcv is caused by whitefly transmitted geminiviruses (begomoviruses). ty-1 and ty-3 are allelic (verlaan et al, 2013). 12tomv, tomato mosaic virus. tomv is a member of the family tobamoviridae and belongs to the genus tobamovirus. 13powdery mildew can be caused by three species of biotrophic fungal pathogens; oidium lycopersici, oidium neolycopersici (syn. pseoudoidium neolycopersici), and leveillula taurica. ol-1 and ol-3 are allelic (huang et al, 2000). 14gray mould caused by fungal pathogen botrytis cinerea (teleomorph: botryotinia fuckeliana). 15root-knot nematodes can be caused by meloidogyne incognita, m. javanica, and m. arenaria. 16verticillium wilt caused by the biotrophic fungus verticillium dahliae. 17fusarium crown rot caused by f. oxysporum f. sp. radicis-lycopersici. 18alternaria stem canker caused by fungal pathogen alternaria alternate. 19whitefly (bemisia tabaci) resistance. species 68 khazaei and madduri genetic resources (2022), 3 (6), 64–73 challenges the utilization of wild tomatoes in breeding programmes is not without a cost. cwrs generally show poor adaptation beyond their natural distribution range (bohra et al, 2021). furthermore, the introgressed gene from a wild relative into advanced lines may disrupt long-accumulated horticultural traits due to linkage drag (tanksley and nelson, 1996). for example, s. galapagense has been identified as a source of insect-pest resistance (rakha et al, 2017; vendemiatti et al, 2021). when it is crossed with cultivated tomatoes, the fruit size and setting reduce significantly, w h ich a r e undesirable traits. linkage drag can be removed by conducting backcrosses to the recurrent parents (cultivated tomato). applications of dna molecular markers (mab, marker-assisted backcrossing) allow for the monitoring of the genome around the gene/locus of interest and the genetic background, speeding up the return to the recurrent parent genome (tourrette et al, 2021). genome editing can also be used to remove the undesirable gene without having extensive backcrossing. for example, crispr-cas9-based gene editing was used to overcome a linkage drag in tomato by editing the jointless2 gene introgressed from s. cheesmaniae (roldan et al, 2017). the world vegetable center is currently testing crispr-cas9 to edit genes implicated with fruit size regulation in interspecific c r osses b e tween s . galapagense and cultivated tomato (schafleitner et al, 2022). genomics-assisted breeding tools tomato wild relatives have more to offer. bai et al (2018) stated that about 20 pathogens could be genetically controlled by resistance genes derived from a few wild species. the genome of some tomato wild species, including s. pennellii (bolger et al, 2014; schmidt et al, 2017), s. chilense (stam et al, 2019), s. pimpinellifolium (razali et al, 2018; wang et al, 2020; gramazio et al, 2020) and s. lycopersicoides (powell et al, 2022) along with the pan-genome (gao et al, 2019) have been assembled. these efforts have bolstered our knowledge and understanding of tomato wild species along with the genetics of resistance genes. recent improvements in genomic resources have enabled us to track and genetically map the wild tomato genes in commercially adapted varieties (anderson et al, 2021). the tomato community largely benefits from advanced, rich genomic resources (https:// solgenomics.net/) and phenotyping tools; however, rapid generation technology (speed breeding) has not yet been developed. robust dna markers for major genes derived from wild relatives have been developed and widely applied in private and public breeding programmes worldwide (foolad and panthee, 2012; hanson et al, 2016). in the past, sources of important disease resistance genes in wild tomato relatives have been intensively investigated. however, the majority of r genes were discovered only in a few species (listed in table 1). the other wild species that either are not easy to cross with cultivated tomatoes or are self-incompatible or allogamous have not contributed much to this journey (e.g. s. chmielewskii, s. corneliomulleri, s. huaylalloasense, s. juglandifolium, s. ochranthum, s. lycopersicoides and s. sitiens). regarding these species, the literature only represents a few accessions of s. lycopersicoides being resistant to grey mould (caused by botrytis cinerea) (davis et al, 2009) or a few accessions of s. corneliomulleri being resistant to the tylcv (yan et al, 2018), but major genes/alleles from these species are yet to be reported. for some of these species, only a few accessions have been collected or are available in genebanks (table 1). among these species, some genomic studies were performed on s. sitiens (chetelat et al, 2019) and s. lycopersicoides (powell et al, 2022), which are potential sources of genes for adaptation to abiotic stresses (i.e. drought and heat stresses). introgression lines were also developed from s. chmielewskii to study the accumulation of secondary metabolites in tomato fruit (ballester et al, 2016). conclusions now that the scientific community has access to advanced tissue culture techniques, double haploidy protocols, modern phenotyping facilities, and genomic and bioinformatic tools, tomato wild relatives could be explored even more. this may provide new sources of genetic resources and r genes that could be used to pyramid new genes into one variety leading to broad and probably more durable resistance. furthermore, ongoing advances in sequencing technology can be used to develop reference genome sequences for undiscovered tomato wild relatives, and the development of tomato pan-genomes will be a valuable strategy in harnessing the genetic diversity of these species. additionally, genome editing enables de novo domestication strategies for the targeted use of tomato relatives (zsögön et al, 2018). thus, exploring the variation in tomato wild species could be an interesting topic for future studies. conflict of interest statement the authors declare that they have no conflict of interest. author contributions original draft: hamid khazaei; review and editing: hamid khazaei and adithya madduri. all authors read and agreed to the published version of the manuscript. funding financial support was provided by the foreign, commonwealth and development office (uk) and by the long-term strategic donors to the world vegetable center, taiwan: uk aid from the uk government, the united states agency for international development (usaid), the australian centre for international agricultural research (aciar), germany, thailand, philippines, genetic resources (2022), 3 (6), 64–73 cwrs role in breeding disease-free tomatoes 69 korea, and japan. the authors thank roland schafleitner, peter hanson and ya-ping lin for their 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(2021). genetic diversity and population structure among indigenous and imported goat breeds in kenya. genetic resources 2 (3), 25–35. doi: 10.46265/genresj.eqfq1540. © copyright 2021 the authors. this is an open access article distributed under the terms of the creative commons attribution license (cc by 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. introduction goats are known to be the most adaptable and widespread species of domestic animals, thriving across various geographical conditions, ranging from mountains to deserts and the tropics, africa included. the importance of goats in supporting rural household economies in developing countries is well documented (deshingkar et al, 2008; herrero et al, 2013). they are an important source of food and nutritional security through the supply of milk and meat, income generation through sale of surplus stock, and insurance against unforeseen risks in addition to having important, non-tangible cultural values (herrero et al, 2013; mbuku ∗corresponding author: ruth w waineina (waineinaruth@gmail.com) et al, 2015; ogola et al, 2010). recent studies have shown that goat farming is one of alternative climatesmart agricultural practices that could build farmers resilience to climate change-related challenges (ojango et al, 2016). the diminishing land sizes in the medium to high potential areas for agriculture due to human population pressure, expansion of urban areas and climate change-related challenges, call for alternative farming practices such as intensive dairy goat production, which offers more multi-functionality, flexibility, and adaptability to varied production conditions (scarpa et al, 2003). in kenya, dairy goat production has mainly been supported by imported breeds such as toggenburg, anglo-nubian, german alpines, saanen and boer, and crossbreeds between imported and selected local breeds such as galla and small east african goat (ahuya et al, 2006; bett et al, 2007; krause, 2006). galla received: 29.12.2020 accepted: 27.05.2021 published online: 17.06.2021 https://www.genresj.org https://www.dx.doi.org/10.46265/genresj.eqfq1540 https://www.genresj.org https://www.dx.doi.org/10.46265/genresj.eqfq1540 mailto:waineinaruth@gmail.com 26 waineina et al genetic resources (2021), 2 (3), 25–35 in kenya is also referred to as boran/somali goat. they are indigenous in arid and semi-arid northern kenya, and pure galla are maintained in various government breeding and conservation stations in the country. their characteristics, such as resistance to dehydration, preference for browsing and a wide range of feeding habits (chenyambuga et al, 2004) have allowed them to adapt to the massive arid and semiarid regions in the country, and could potentially be advantageous traits for goat breeding programmes. the imported breeds were introduced to various parts of the country by the government of kenya and nongovernmental organizations, with the aim of increasing goat productivity through appropriate husbandry and disease interventions (peacock, 2005) and targeted breeding strategies such as crossbreeding (bett, 2009; peacock et al, 2011). the crossbreeds were kept in different geographical locations as isolated populations and subjected to separate breeding objectives for several decades. crossbreeding has been the strategy of choice to improve the productivity of goats under various production systems (ahuya et al, 2009; peacock et al, 2011). this has resulted in an increase in population sizes of crossbred goats especially in the areas the breeds were introduced (mburu et al, 2014; peacock et al, 2011). however, increase in population sizes did not necessarily correspond to enhanced productivity of the goats but rather reflected large numbers of households striving to support their livelihoods through goat farming (aziz, 2010; bett et al, 2011; mburu et al, 2014). in kenya, there has been limited technical capacity on the farmers’ side on how to manage the rather complex crossbreeding programmes, a fact that may have had a bearing on the sustainability of such initiatives in the long term (aziz, 2010; bett et al, 2011; mburu et al, 2014). the net result of this has been the unsystematic crossing of the existing population, poor flock management, lack of records to support decision making and general lack of simplified breeding programmes to guide in genetic improvement of goats in the country (kosgey and okeyo, 2007). currently, crosses of imported and local goats are reared as dairy goats in different parts of the country under different production systems. there is a huge source of genetic diversity in the current goat populations in kenya. this is a result of unsystematic crossbreeding and lack of record keeping by most of the smallholder farmers. this calls for the need to characterize, conserve and sustainably utilize goats under various production systems in kenya. it is important to determine genetic diversity in populations because it provides the basis for natural and artificial selection (qanbari and simianer, 2014). to measure and describe genetic diversity of animal genetic resources, phenotypic and molecular characterization tools are used as a starting point to understand the animal resources and make use of them sustainably (fao, 2011). characterization starts with the gathering of all information on breed origin, development, structure, population, quantitative and qualitative characteristics in defined management and climatic conditions (gizaw et al, 2011; rege and okeyo, 2011). molecular characterization, using genetic markers to detect polymorphisms in nuclear dna, is a powerful tool which can be applied in breeding programmes. for instance, it can be used to characterize the genetic variability within, and genetic distance between, populations, as well as for genomic selection, parentage verification and genetic diversity preservation (groeneveld et al, 2010). microsatellite markers and single-nucleotide polymorphisms (snp) are the most commonly used markers in animal breeding related fields (fao, 2011). microsatellite markers have several limitations, for example in the detection of null alleles (hoshino et al, 2012) and homoplasy (jarne and lagoda, 1996; anmarkrud et al, 2008), while snps have several advantages over microsatellites, including being highly reproducible and informative (vignal et al, 2002) and the fact that snps can represent either neutral or functional genetic diversity (kohn et al, 2006). a snp microarray with more than 50,000 snps (goatsnp50 bead chip, illumina, inc. san diego, ca 92122 usa), which was developed from snp loci detected by wholegenome sequencing of six different goat breeds according to tosser-klopp et al (2014) is available. this has made snp markers the most popular and advanced technology in molecular breed characterization in goats. additionally, its robustness, low genotyping costs, automatic allele calling and capability to interrogate the goat genome at high resolution (ajmone-marsan et al, 2014) demonstrate practicality in implementing genomic characterization in goats. there has been no deliberate effort to understand the genetic diversity and population admixture among the goat breed populations in kenya by use of snp makers. this study, therefore, investigated genetic diversity, population structure and admixture among goat breeds in kenya. the results from this study will facilitate management efforts in conserving and utilizing the various goat genetic resources sustainably. materials and methods study area blood samples were collected from goats in three counties in kenya: nyeri (mukurweini sub-county), meru (central imenti sub-county) and homa bay (homa bay town sub-county) located in the central, eastern and western regions of kenya, respectively (figure 1). these areas were selected because they represent the entry points of different imported dairy goat breeds in kenya. mukurweini sub-county lies in the upper midlands, also known as the main coffee zone, at an altitude of 1460-1710 metres above sea level (masl) and receives 950-1500 mm of mean annual rainfall. genetic resources (2021), 2 (3), 25–35 genetic relationships of goats in kenya 27 figure 1. map of kenya showing area sampled within the selected sub-counties central imenti is in the upper highlands, at an altitude ranging between 1830-2210 masl and has an average annual precipitation of 800-2600 mm. the homa bay town sub-county lies in the lower midlands, at 1166 masl and receives an annual rainfall of 1226 mm. animal resources and sampling goat keeping households were purposively selected based on the following criteria: 1) being a member of dairy goat farmer group and 2) having more than two mature does which matched the breed kept by the farmer group in the said county of study. the herd structures between the breeds and within the county/breed varied among the selected households. therefore, when a farm had only two mature does, only one doe was sampled. where more than two does were available, the relationship of the does was confirmed by the farmer to avoid sampling closely related does. sampling of full and half siblings was avoided. to ensure the representativeness of sampling for each breed, unrelated animals were selected from various farms across the designated counties. the galla goat breed, however, did not follow the criteria because they were from the government breeding station where breeding records were used to identify the animal to be sampled. therefore, to minimize sampling from closely related animals within the galla population, pedigree data were used to select against full and half sibling animals. a total of 96 animals including three imported breeds (31 toggenburg, 29 alpine, 24 saanen) and one indigenous breed (12 galla) were incorporated in this study. the toggenburg and alpine were found in eastern and central kenya under meru goat breeders association (mgba) and dairy goat association of kenya (dgak), respectively. saanen goats were found in homa bay under nyanza goat breeders association (ngba). all samples were collected from a total of 53 farms in the three counties, nyeri (18), meru (19) and homa bay (16). galla goats were sampled from the sheep and goats government station in naivasha. whole blood (10 ml) was collected from the jugular vein into vacutainer tubes with ethylenediaminetetraacetic acid (edta) as an anticoagulant. the blood was stored at -20◦c for two months before genomic dna extraction. the procedure of blood sampling followed fao guidelines (fao, 2012). from each animal, duplicate samples were collected and kept separately during transportation and storage. for each sample, the following information was collected: sex of the animal, basic pedigree information, size of the flock, breed, any relevant phenotypic feature, and a photograph of the goat. the study was conducted in strict accordance with the recommendations of the institute of primate research (ipr) ethical guidelines on animal care and use of laboratory animals (https://grants.nih.gov/grants/olaw/g uide-for-the-care-and-use-of-laboratory-animals.pdf). the protocol was approved by the committee on the ethics of animal experiment of egerton university of egerton in kenya (iserc/03/2020). a qualified veterinary officer collected the whole blood following fao guidelines (fao, 2012) to reduce pain and discomfort to a minimum. dna extraction and genotyping genomic dna was extracted from the whole blood using the dneasy blood and tissue kit (qiagen®, hilden, germany) according to the manufacturer’s instructions. ten µl of each ten randomly selected samples were subjected to a preliminary estimate of the dna quality and quantity on a 1 % agarose gel electrophoresis. secondary quantification and purity analysis of the dna were confirmed using one µl for each sample on both nanodrop spectrophotometer (nanodrop® nd-2000) and qubit ® dsdna br (broad-range) assay kit on the qubit 3.0 fluorimeter (invitrogen). the extraction and quality control check of genomic dna was done at kenya agricultural and livestock research organization biotechnology laboratory in kabete, kenya. the dna samples were genotyped using the goatsnp50 bead chip (illumina, inc. san diego, ca 92122 usa), developed by the international goat genome consortium (iggc), which features 53347 snps across the whole genome with inter-snp spacing of approximately 40 kb (tosser-klopp et al, 2014). the genotyping was outsourced to neogen europe limited in scotland (https://genomics.neogen.com/en/). snp quality control and data analysis the snp genotype quality control process was applied to raw reads for both merged (all breeds) and then per breed using plink v1.07 (purcell et al, 2007). https://grants.nih.gov/grants/olaw/guide-for-the-care-and-use-of-laboratory-animals.pdf https://genomics.neogen.com/en/ 28 waineina et al genetic resources (2021), 2 (3), 25–35 table 1. goat breed, number of goats and snps excluded and remaining after quality control processes on genotyping data. n, number of animals; mind, genotype missing (< 0.1), geno, snp missing (< 0.15), maf, minor allele frequency (< 0.05); hwe, hardy-weinberg equilibrium (p-value < 0.001). breed n excluded snps remaining snps remaining samples mind gen hwe maf total saanen 24 0 2453 38 3658 6149 47198 24 alpine 29 1 2496 47 2586 5129 48218 28 galla 12 0 2413 26 8249 10688 42659 12 toggenburg 31 1 2345 50 4690 7085 46262 30 merged 96 2 2235 663 644 3542 49805 94 first, individuals with a missing genotype call rate of more than or equal to 10 % were removed from further analysis using the mind function in plink with default settings. the remaining individuals were then exposed to further filtering. snps with less than 95 % call rate, minor allele frequency (maf) of less than 0.05 and p<0.001 hardy weinberg equilibrium (hwe) were excluded from downstream analyses. the snp data set used for downstream analysis is accessible from the mendeley digital repository (https://doi.org/10.17632/hhb9rhdzzt.1). basic genetic diversity indices, which include the proportion of polymorphic markers, inbreeding coefficient, observed (ho) and expected (he) heterozygosity were calculated within breeds using plink (purcell et al, 2007). the proportion of polymorphic snps (pn ) offers the fraction of the total snps that showed both alleles within each population. the pn was calculated as the proportion of snps with more than 1 % maf within each breed. the maf is the approximate frequency of the second most common allele per breed. the output from plink for observed and expected heterozygosity per animal within breed was subjected to further calculation to get an average estimate of ho and he per breed. the heterozygosity values were calculated by getting the average of all snps (that is the sum of all heterozygosity values averaged over the total number of snps passed the quality control). the population structure and relatedness were estimated by principal components analysis (pca) using the r package snprelate (zheng et al, 2012) and admixture proportion inference using model-based clustering admixture 1.3.0 software (alexander et al, 2009). the pca analysis allowed for visual investigation and solid quantitative summaries. the admixture analysis inferred the proportions of ancestry within the populations by use of prior defined k-values matching the assumed number of ancestral populations. the admixture procedure employs a maximum-likelihood based method by converging the ancestry proportions and allele frequencies that maximize the likelihood function. the most optimal population structure was determined by cross-validation error procedure (mcvean, 2009) with assumed admixture runs from k = 2 to k = 4. the k-value with the lowest cv error was selected as the optimal value. a phylogenetic tree based on reynolds genetic distances representing relationships among goat breeds was visualized using itol software (letunic and bork, 2019). results quality control procedure on the 53,347 snps included on the snp chip excluded a total of 3,542 snps retaining 49,805 snps for downstream analyses as shown in table 1. of the excluded snps, 2,235 had less than 0.1 missing per snp, 663 snps significantly deviated from hwe (p < 0.001) and 644 snps had mafs lower than 0.05. the galla breed had the highest number of snps excluded in total (10,688), whereas alpine revealed the lowest number of snps excluded (5,129). it is worth noting that some snps were left out due to more than a single criterion. genetic diversity the four indices of genetic diversity (polymorphic snps, mean allele frequency, observed and expected heterozygosity and inbreeding coefficient) were calculated within each breed (table 2). the assessment of the proportion of snps that exhibited both alleles within each breed indicated high levels of diversity. the percentage of within-breed polymorphic snps ranged from 94.6% to 80.7%. the highest values of polymorphic loci were found in alpine (94.6%) and saanen (92.2%) while the lowest proportion was found in the galla breed (80.7%). across all loci, the lowest maf was found in galla (0.291) and the highest in alpine (0.323). results revealed differences in genetic diversity between breeds. the expected heterozygosity was, in all cases, higher than the observed heterozygosity (he > ho). the alpine had the lowest observed heterozygosity (ho = 0.558 ± 0.026) while toggenburg had the highest (ho = 0.580 ± 0.032). inbreeding coefficients for all the breeds were negative and ranged between -0.013 (toggenburg) and -0.042 (galla). population structure analysis principal components analysis was used to cluster goats and explore the association among individuals and breed groups. in figure 2, the principal component 1, which accounts for 15.2% of the total variance, separated galla breed from the other three breeds. the https://doi.org/10.17632/hhb9rhdzzt.1 genetic resources (2021), 2 (3), 25–35 genetic relationships of goats in kenya 29 table 2. proportion of polymorphic snps(pn ), mean allele frequency (maf), observed (ho) and expected (he) heterozygosity and inbreeding coefficient (f) for the goat breeds. n, number of animals. breed n pn [%] maf ho ± sd he ± sd f toggenburg 30 89.9 0.297 0.580±0.032 0.580±0.001 -0.013 alpine 28 94.6 0.323 0.558±0.026 0.564±0.001 -0.015 galla 12 80.7 0.291 0.563±0.025 0.580±0.000 -0.042 saanen 24 92.2 0.311 0.559±0.019 0.573±0.001 -0.034 principal component 2 accounts for 14.1% of the total variance, split the goat breeds into four clusters (alpine, saanen, galla and toggenburg clusters). one outlier, was, however, observed for the saanen population mixing with alpine population. to examine admixture between the breeds, modelbased clustering was performed and the most likely number of genetic population (cluster or k) between the four goat breeds were deduced using admixture crossvalidation procedure (mcvean, 2009). the k-value with the lowest cv error was k = 4 and was selected as an optimal number of ancestral populations (figure 3). a population structure plot (figure 4) showed proportions of ancestral populations for all breeds (alpine, galla, saanen and toggenburg) for k = 2 to k = 4. at k = 2, galla goats were separated from the other three goat breeds (toggenburg, saanen and alpine). moreover, galla goats largely do not carry ancestral components present in saanen, alpine and toggenburg goats (shown in light blue, figure 4). at k = 3, alpine and saanen goats carry ancestral components largely absent from either the galla or toggenburg goats. at k = 4, galla had the lowest level of admixture, whereas toggenburg, alpine and saanen demonstrated some signs of admixture with galla. figure 2. principal components analysis plot based on snp array data of goat breeds figure 3. a cross-validation plot indicating the choice of optimal k-value the proportions of individuals in each breed in the four most likely clusters estimated by admixture is shown in table 3. 71 % of alpine breed were allocated to cluster one, 97 % of galla were assigned to cluster two with one percent (1 %) of its genome assigned to cluster one, three and four, 84 % percent of saanen were in cluster three with seven percent (7 %) of its genome assigned to cluster one. on the other hand, 78 % of toggenburg were assigned to cluster four with 17 %, and three percent (3 %) of its genome allocated to cluster two and one respectively. breed relationships were evaluated by computing the genetic distance between all pairwise combinations of individuals (d) from the average proportion of shared alleles. based on the calculated reynolds genetic distances, a phylogenetic tree was constructed to represent breed clustering (figure 5). the results revealed five clusters for the four populations (alpine, saanen, toggenburg and galla). some toggenburg goats were found to be grouped together with alpine, forming the fifth cluster. discussion genetic diversity livestock has been exposed to various forces that contributed to the genetic diversity underlying phenotypic dissimilarities ever since domestication. these forces include natural selection, artificial selection for specific traits, migration, genetic drift and inbreeding (andersson and georges, 2004; groeneveld et al, 2010). genetic drift plays an important role during short-term evolu30 waineina et al genetic resources (2021), 2 (3), 25–35 figure 4. population structure plots showing proportions of ancestral populations for individuals of sampled goat breeds (alpine,galla, saanen and toggenburg) for k= 2 to k = 4 tion in situations where populations are reproductively isolated (laval et al, 2002). genotyping with the goatsnp50 bead chip revealed some levels of diversity within the goat breeds in this study. in each breed, fewer than 80% of snps exhibited polymorphisms, and heterozygosity ranged from 0.558 to 0.580 (table 2). a large number of polymorphic snps were detected for alpine (94.6) and saanen (92.2) figure 5. phylogenetic tree based on reynolds genetic distances representing breed relationships among goat breeds. breeds; this was expected because sequenced data from alpine and saanen were included in the 50k snps panel discovery (tosser-klopp et al, 2014). other results from various authors using different numbers of samples and goat breeds showed >93 % of polymorphism (visser et al, 2016; onzima et al, 2018). however, it is difficult to compare and conclude the estimates of snps stated as polymorphic by other authors, because the number of samples genotyped per breed and proportion of genotyped samples used for snp discovery varied. the diversity amongst the four breeds showed galla had the lowest diversity among sampled individuals in comparison with the other breeds. the polymorphic variance is dependent on the history of each breed. as opposed to galla goats, the three introduced breeds showed greater diversity, likely as a result of crossbreeding with local goats (galla and small east african). therefore, each breed may contain genetic contributions from various breeds, thus revealing higher polymorphisms than galla goats. in contrast, galla goats sampled from the government breeding station with detailed pedigree records still maintained levels of diversity (pn = 81%). the galla population had been introduced in the naivasha sheep and goat station in the early 1970s during a sheep and goat table 3. proportion of individuals of goat breeds in each of the four clusters estimated by admixture. the diagonal indicates the inferred cluster. n, number of animals sampled predefined populations inferred clusters n 1 2 3 4 alpine 28 0.708±0.197 0.136±0.128 0.101±0.071 0.055±0.048 galla 12 0.013±0.020 0.970±0.044 0.008±0.013 0.009±0.015 saanen 24 0.073±0.095 0.049±0.073 0.838±0.149 0.040±0.039 toggenburg 30 0.030±0.047 0.173±0.142 0.015±0.019 0.782±0.193 genetic resources (2021), 2 (3), 25–35 genetic relationships of goats in kenya 31 project funded by fao (palian and racokzi, 1976). the population is registered with the galla goat breeders society of kenya (ggbsk) and the kenya stud book. the population is inspected every year by inspectors from ggbsk using the galla goat breed standards. therefore, the station has maintained pure breed galla goats which produce meat, milk and reproduce under harsh conditions while maintaining or conforming to the set standards of excellence defined by the ggbsk, where the objective is genetic improvement of target traits while controlling the level of inbreeding. the negative inbreeding coefficient in this study can effectively be taken as zero values, which means that there is no inbreeding observed in the reference populations. it could also mean that many heterozygotes were observed although the sample size for the four breeds was small. the increased heterozygosity could be due to random mating within the herd rather than random differences between herds. the observed heterozygosity was lower than the expected heterozygosity (ho < he) in the three breeds apart from toggenburg, which recorded the same value for both observed and expected heterozygosity. the difference between the observed and expected heterozygosity was small, which may not be due to inbreeding but a wahlund effect (garnier-géré and chikhi, 2013). the observed heterozygosity in the current study was from a sample of individuals from a structured population even though all sub-divisions were in hardy-weinberg equilibrium. over and above the semi and intensive production systems practised by smallholders, there is the presence of artificial selection, gene flow and non-random mating, hence not holding the law of hwe in these populations. in this study the observed and expected heterozygosity for alpine (ho = 0.558; he = 0.564), saanen (ho = 0.559; he = 0.573) and toggenburg (ho = 0.580; he = 0.580) were higher than those stated in canada for alpine (ho = 0.385; he = 0.388), saanen (ho = 0.379; he = 0.382) and toggenburg (ho = 353; he = 336) (brito et al, 2017). moreover, saanen in italy recorded the same trend as in canada (ho = 0.41; he = 0.41) (nicoloso et al, 2015). differences in effective population sizes, length of isolation, selection and breeding management practices in the various production system may be the cause of these variances. toggenburg and galla breeds had the highest expected heterozygosity. this could be explained by the types of crossbreeding programmes practised by farmers keeping toggenburg breeds resulting in an admixed population. organized breeding strategies using artificial selection are practised for galla goats under the government breeding station resulting in genetic variability and lack of inbreeding for the populations. population structure and relationship principal component and population structure analyses confirmed distinctiveness among the goat breeds (saanen, galla, toggenburg and alpine) according to their geographic regions in kenya. this can be explained by the demographic history of these breeds that have been reared for a long time in separate geographic locations (ahuya et al, 2009; peacock et al, 2011). although goats from each breed clustered separately, model-based clustering revealed some signs of admixture and genetic links between alpine, toggenburg, saanen and galla. the results (figure 4 and table 3) of this study indicate that kenyan alpine goats were the most admixed breed with about 14 % of its genome derived from galla, while ten and six percent of its genome is resulting from saanen and toggenburg respectively. it is worth noting that saanen were introduced in the sampling region (nyeri county) already before the alpine were imported in the late 1970s. therefore, the 10 % of saanen genes in the alpine genome may be a result of saanen being one of the kenyan alpine ancestors. according to waineina et al (2019), lack of breeding stock was one of the challenges alpine farmers were encountering, thus driving them to source breeding animals from local markets, friends, neighbours and commercial farms notwithstanding their undefined genetic composition. furthermore, the increase in demand for dairy goats in the country has resulted in several farms setting up nucleus flocks with a significant proportion of the crossbred flocks as a source of breeding material for distribution to lower cadre farmers (ahuya et al, 2006; bett, 2009; ogola et al, 2010). through such arrangements, most of the breed-types have migrated to other areas apart from their original entry in the country (mburu et al, 2014; peacock, 2007). toggenburg and alpine goats shared some linkage with galla goats, 17 % and 14 %, respectively. this was expected because galla goat was used as the founder population for crossbreeding with alpine and toggenburg breeds (ahuya et al, 2009; bett et al, 2011; mburu et al, 2014; peacock et al, 2011; shivairo et al, 2013). as expected, galla was the least admixed breed, in agreement with the history of this breed as the first indigenous goat for which a breed society was formed in kenya. moreover, the particular population in this study has been managed in seclusion within the government farm, and only animals registered within the society are allowed into the population. the galla breed displayed isolation by distance and seemed to be at equilibrium under dispersal and genetic drift. in comparison with the other breeds in this study, galla arrived in their current locations long before these breeds were introduced in kenya and that is why there has been sufficient time for isolation by distance to take effect and, that long distance gene dispersal is sufficiently common to prevent genetic divergence. the phylogenetic analysis categorized the breeds into five clusters (figure 5). the outcomes show a clear differentiation of galla, saanen, some toggenburg and alpine. a group of some alpine and toggenburg, however, remained clustered together, which may 32 waineina et al genetic resources (2021), 2 (3), 25–35 be attributed to the adjacent regions of the breeds (figure 1). lack of differentiation in some of alpine and toggenburg breeds signified a high level of genetic resemblance and low divergence, which may be a result of gene flow among alpine and toggenburg breeds. common ancestry, short domestication history, lack of selection pressure and movement of the goats may play a role in lack of differentiation in varied geographically separated populations. furthermore, in kenya, as well as other parts of africa, goats are also used for religious and other cultural ceremonies such as payment of dowry and gifts (herrero et al, 2013; mbuku et al, 2015; ogola et al, 2010). therefore, some of the alpine and toggenburg breeds clustering together may be a result of movement of breed animals between the communities in those two regions due to the forementioned cultural ceremonies. as mentioned earlier, one of the criteria for selecting the goat keeping households in this study was them being members of a dairy goat farmer group association (dgak, mgba, ngba). the associations are responsible for buck rotation among the group members, maintaining the purtity of the breed and providing technical backstoping. however, the results indicate a need to technically strengthen the dairy goat association of kenya for alpine and the meru goat breeders association for toggenburg, because urgent management efforts are essential to improve on breeding aspects, utilization and conservation of the various goat genetic resources. all saanen goats formed one cluster in the phylogenetic analysis. indeed, the long distance (over 450 km) between the regions where saanen and the rest of the breeds are kept may be the barrier to gene flow from other breeds. through adaptive hitchhiking, natural selection can play an essential role in shaping this variability (andolfatto, 2001). therefore, the observed genetic divergence of saanen from alpine, toggenburg and galla breeds could have been contributed by random genetic drift and natural selection for adaptation to their environment/region. genetic uniqueness can be determined from the magnitude of genetic distances and phylogenetic relationships between populations if supporting indications such as genetic history, records of production, reproduction and on adaptation are lacking (eding and laval, 1999; tosser-klopp et al, 2014; zheng et al, 2012). embracing this principle with respect to the results of this study, saanen and galla breeds seem to be the most genetically distinct among the populations sampled, and can be categorised as important genetic resources. it will be interesting to enlarge this breed level investigation in later studies through addition of all kenyan goat breeds to better appreciate the genetic relationship among them. conclusion the study revealed clear divergence between some goat breeds, which provides a wide prospect on the current genetic diversity of goats in kenya. this will be vital in planning breeding strategies for genetic resources that should be sustainably utilized and conserved. of the breeds studied, galla breed displayed isolation by distance and seemed to be at equilibrium under dispersal and genetic drift. this shows that stronger efforts of genetic conservation and sustainable management of its gene pool have been undertaken. however, further studies are required for the onfarm galla population. the most admixed breeds were alpine and toggenburg. therefore, there is need to technically strengthen the dairy goat association of kenya for urgent management efforts that are essential for genetic improvement, utilization and conservation of the various goat genetic resources. additional studies on phenotypic similarities and performance evaluation of the breeds in this study could add value to the information generated from this study to form the basis for future genetic resource conservation and improvement of goat breeds in kenya. acknowledgements the authors wish to acknowledge the african development fund (afdb), usaid and the government of kenya under the kenya climate smart agriculture project (kcsap cgs/crgs-ad-2019) for their financial support to the first author. we sincerely thank egerton university (njoro, kenya) for providing technical support to undertake the study, kenya agricultural and livestock research organizations for granting the study leave for the first author, dairy goat farmers and naivasha sheep and goat breeding station for making their animals available for sample collection. author contributions rw, kn, to and ei conceived the study, rw analyzed the data and drafted the manuscript. all authors read and approved the manuscript. conflict of interest statement the authors declare that there is no conflict of interest 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(2019). performance of dairy goat breeds in different production systems in kenya. in proceedings of the tanzania society of animal production (tsap), 42nd scientific conference, 23-25. zheng, x., levine, d., shen, j., gogarten, s. m., laurie, c., and weir, b. s. (2012). a high-performance computing toolset for relatedness and principal component analysis of snp data. bioinformatics 28(24), 3326–3328. doi: https://doi.org/10.1093/ bioinformatics/bts606 https://doi.org/10.1093/bioinformatics/bts606 https://doi.org/10.1093/bioinformatics/bts606 introduction materials and methods study area animal resources and sampling dna extraction and genotyping snp quality control and data analysis results genetic diversity population structure analysis discussion genetic diversity population structure and relationship conclusion author contributions conflict of interest statement short communication genetic resources (2023), 4 (8), 29–36 doi: 10.46265/genresj.vooz8371 https://www.genresj.org issn: 2708-3764 the genetic composition of the traditional irish horse – towards the development of a dna-ancestry test for the preservation of traditionally bred irish sport horses beatrice a mcgivney a, deirdre harty b, alison corbally b and emmeline w hill *,a,c a plusvital ltd, dun laoghaire industrial estate, pottery road, co. dublin, dun laoghaire, ireland b horse sport ireland, beech house, millennium park, co. kildare, osberstown, naas, ireland c school of agriculture and food science, university college dublin, belfield, dublin 4, ireland abstract: the traditionally bred irish sport horse, known as the traditional irish horse, is an important cultural asset to horse genetic resources in ireland. we tested the hypothesis that the irish sport horse, which was originally developed from the irish hunter, may contain a genetic background distinct from european warmblood horse populations that would be valuable to preserve. using genome-wide single nucleotide polymorphism (snp) data, the results show that traditional irish horses (with confirmed pedigrees) have lower levels of european warmblood ancestry components than other irish sport horses. these results indicate that measurement of the levels of european warmblood ancestry components in the irish sport horse may assist in the preservation of traditional irish lineages. keywords: equine, genomics, snp variation, admixture, irish sport horse, conservation, population genetics citation: mcgivney, b. a., harty, d., corbally, a., hill, e. w. (2023). the genetic composition of the traditional irish horse – towards the development of a dna-ancestry test for the preservation of traditionally bred irish sport horses. genetic resources 4 (8), 29–36. doi: 10.46265/genresj.vooz8371. © copyright 2023 the authors. this is an open access article distributed under the terms of the creative commons attribution license (cc by 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. introduction the horse has a long history in ireland, developing through millennia into several distinct native breeds, from small riding ponies to larger work horses (mccormick, 2007). the earliest archaeological evidence for domesticated horses in ireland dates to 2,400 bce in the early bronze age contexts of newgrange (bendrey et al, 2013). the introduction of the celtic horse into ireland can be traced to the 7th century, with images of ridden horses contained within the book of kells (unattributed, 800). the medieval irish horse was known as the irish hobby, resulting from crossing horses from northern spain with local animals (hendricks, 1995). the hobby was noted for speed and ∗corresponding author: emmeline w hill (emmeline.hill@ucd.ie) agility, and was mentioned in 1296 in documents relating to an irish contingent of horses brought to scotland by john de wogan, judiciar of ireland (lydon, 1954). the irish hobby was likely a founder breed for the extant native breeds (mccormick, 2007), the connemara pony and irish draught, and also likely contributed to the thoroughbred (hendricks, 1995). the irish draught was bred as a light, versatile farm, carriage, riding and hunting horse while the connemara pony was developed as a multipurpose animal for the harsher conditions of the western irish seaboard (hendricks, 1995). the thoroughbred was developed in the 17th and 18th centuries when stallions from the middle east were brought to england and crossed with the best racing mares in britain, many of which were irish or had been bred from irish stock (bower et al, 2011). received: 18.02.2023 accepted: 07.08.2023 published online: 12.09.2023 https://www.genresj.org https://www.dx.doi.org/10.46265/genresj.vooz8371 https://www.genresj.org https://www.dx.doi.org/10.46265/genresj.vooz8371 mailto:emmeline.hill@ucd.ie 30 mcgivney et al genetic resources (2023), 4 (8), 29–36 prior to the development of the thoroughbred, a centuries-old tradition of hunting in ireland led to the breeding of specialist hunters originating from the irish draught horse (hendricks, 1995). when the thoroughbred became established in ireland, it was used extensively for further improvement of the irish hunter (hendricks, 1995). the irish hunter was referred to by william youatt who noted that the irish bred “a large, long blood-horse of considerable value” (youatt, 1831). until 1922 the irish hunter was registered in the hunter stud book, but it was later redefined as the irish sport horse and has been registered in the irish sport horse studbook since 1970. the irish sport horse became the world leader in eventing and has topped the world breeding federation for sport horses eventing studbook rankings for all but two years since its inception in 1994. in the 1980s, the crossing of european warmblood stallions with irish sport horse mares gained popularity (doyle et al, 2022). in 1990, the percentage of foals registered in the irish sport horse studbook by foreignbred sires was 1.1% and by 2014 this had increased to 37.5%. this dramatic change in the breed profile of the irish sport horse population has led to a growing concern among breeders that its traditional traits are being displaced by a pan-european sport horse. demand has developed amongst breeders to identify traditionally bred irish sport horses, referred to as the traditional irish horse, and to establish conservation measures for the lineages descended from the original irish hunter. to date, there is no published study on genetic diversity in the irish sport horse population, and there are only limited published studies on genetic diversity and relatedness among the other irish horse breeds. mitochondrial dna (mtdna) analyses showed no evidence of clustering of irish draught and connemara pony mtdna haplotypes to the exclusion of other breeds, with their distribution among the most common european horse sequences. on the other hand, the kerry bog pony, a rare, recently recharacterized breed, contained an uncommon mtdna haplotype (mcgahern et al, 2006). in a study comparing british and irish native ponies, the connemara pony showed no greater divergence among breeds than between british native breeds, which may be due to recent gene flow resulting from historic migration (reilly et al, 1998). in consideration of the concerns from breeders about the dilution of irish sport horse genetics with european warmblood genetics, the aim of this study was to establish whether there are genetic differences between the traditional irish horse and european warmblood breeds by testing the hypothesis that irish sport horses with warmblood ancestry in their pedigree can be identified using admixture analysis to test for warmblood introgression. the purpose of a molecular genetics approach is to provide novel information to augment pedigree-based classifications of the traditional irish horse for the long-term preservation of traditional irish lineages. methods samples a total of 133 samples from 8 horse populations (table 1) were used in the initial analyses, including 2 native irish breeds (21 irish draught and 24 connemara pony), 23 irish sport horse (comprised of 8 certified traditional irish horse, 7 irish sport horse with known warmblood influence in their pedigree, and 8 irish sport horse with some missing pedigree), as well as 3 breeds with known ancestral influence on the irish sport horse (25 irish thoroughbred, 14 hanoverian, and 14 swiss warmblood). the results were validated using a set of 12 pedigree-certified traditional irish horses. genotyping snp genotypes, derived from the equine illumina snp50 genotyping array (snp50), were publicly available for swiss warmblood and hanoverian (petersen et al, 2013). for the other samples, genomic dna was isolated from blood or hair samples and genotyped on the affymetrix axiom equine genotyping array (snp670). quality control (qc) procedures were carried out in plink 1.9 (purcell et al, 2007; chang et al, 2015). only individuals and snps with a genotyping rate > 95% were included. a minor allele frequency threshold of > 0.05 was applied. qc criteria were performed separately on the data from the snp50 and snp670 genotyping platforms. only snps that passed qc on both platforms were included in the snp dataset. snps were then pruned on the basis of linkage disequilibrium (ld) using the parameter –indep 50 5 2 in plink. pruning of snps that are in high ld have been shown to counter the effect of ascertainment bias and to generate meaningful comparisons among breeds (molomane et al, 2018). following qc and pruning, a set of 9,015 snps was used for the analyses. population genetics analyses pairwise genetic distance (d) between all individuals was calculated using plink 1.9 (–distance 1-ibs) and was represented by constructing a neighbourjoining tree. principal component analysis (pca) was conducted using plink 1.9. pca plots were generated within the r environment (r core team, 2014). the popgenome package in r was used to calculate the pairwise population differentiation index fst . population structure was visualized using an unrooted neighbour-joining tree based on the between-population fst . for the analysis of population substructure, modelbased clustering was performed using the software package admixture (alexander et al, 2009). the model assigns ancestry based on a predefined number of k ancestral populations. individuals are assigned to k clusters based on allele frequencies and the proportion of ancestry from each population is estimated. the analysis was performed for k ranging from 2 to 8. genetic resources (2023), 4 (8), 29–36 genetics of the traditional irish sport horse 31 table 1. breeds, abbreviations, sample sizes (n) and study cohort for horses used in the study. population abbreviation n cohort connemara pony cp 24 ancestral hanoverian han 14 ancestral irish draught id 21 ancestral irish thoroughbred itb 25 ancestral irish sport horse with warmblood ish wb 8 test irish sport horse ish 7 test swiss warmblood szw 14 ancestral traditional irish horse tih 8 test traditional irish horse tih 12 validation total 133 results genetic distance within and among breeds the greatest genetic distance between individuals was between a connemara pony and a traditional irish horse (d = 0.29) and the closest genetic distance between individuals was between two irish thoroughbreds (d = 0.19). the average genetic distances among individuals within breeds ranged from 0.22 (irish thoroughbred) to 0.26 (irish draught). the pairwise population differentiation index, fst , was calculated among breeds (table 2) and used to create a neighbour-joining tree (figure 1) to illustrate the topological relationship among breeds. the traditional irish horse was identified as a distinct subset of the irish sport horse population and was more similar to the irish sport horse that did not contain foreign blood (fst = 0.006) than the irish sport horse with warmblood (fst = 0.011). the pairwise genetic difference between the traditional irish horse and irish sport horse with warmblood (fst = 0.011) was greater than between hanoverian and swiss warmblood (fst = 0.008), which are distinct breeds. to visualize the branching structure among the irish sport horse populations and warmbloods, genetic distances between individuals were used to create a neighbour-joining tree (figure 2). traditional irish horse individuals were on branches separate to the two main warmblood branches with the exception of a single individual that was observed among a predominantly swiss warmblood cluster. five of the eight irish sport horse with warmblood samples were contained in the two main hanoverian/swiss warmblood branches. to further visualize the overall population structure, a pca analysis was performed. in the pca, pc1 explained 7% of the variance with clear separation of connemara pony from the other breeds as well as divergence between irish thoroughbred and irish draught (figure 3a). in the plot created from pc2 and pc3 (figure 3b), there was considerable overlap among the hanoverian and swiss warmblood with a general european warmblood cluster observed. there was a broad distribution of the irish sport horse population centred among the recorded ancestral breed populations reflecting the varying contributions from the other breeds. population substructure in the admixture analysis, k = 4 had the lowest estimate of the standard error of the cross-validation. based on this result as well as the observations in the pca plot and knowledge of breed history, k = 4 was chosen as the most appropriate number of clusters to use to establish ancestry and quantify admixture within the irish sport horse population (puechmaille, 2016; lawson et al, 2018). unsupervised modelling was used to predict allele frequencies in four ancestral genetic lineages and each individual’s genome was partitioned and proportionally assigned to one of the lineages (figure 4). there was clear evidence of four distinct lineages – irish thoroughbred, connemara pony, irish draught and warmblood – among the five distinct breed populations. the proportion of warmblood ancestry in individual irish sport horses ranged from 0.04 to 0.88. to focus on the irish sport horse alone, the proportion of warmblood ancestry assigned to each of the 23 irish sport horse samples was used to categorize the samples (figure 5). using the proportion of warmblood admixture as an indicator, the seven irish sport horse samples with the lowest levels of warmblood lineage were classified as traditional irish horse (warmblood lineage ≤ 0.15 (15%)), the eight with the highest levels were classified as irish sport horse with warmblood influence and the remainder were assigned as irish sport horse of unknown lineage. six of the eight traditional irish horse were correctly assigned. two traditional irish horse were assigned as irish sport horse of unknown lineage, one of which was the traditional irish horse that clustered with the predominantly swiss warmblood horses in the neighbour-joining tree. one irish sport horse with missing pedigree was assigned as a traditional irish horse. all eight irish sport horse with warmblood were correctly assigned and overall, 20 of the 23 horses were correctly assigned using genotyping data from 9,015 snps. these results demonstrate a sensitivity of 32 mcgivney et al genetic resources (2023), 4 (8), 29–36 table 2. pairwise population differentiation index (fst ) among horse breeds: connemara pony (cp), hanoverian (han), irish draught (id), irish sport horse with warmblood (ish wb), irish sport horse (ish), swiss warmblood (szw), irish thoroughbred (itb), traditional irish horse (tih). cp han id itb ish wb ish szw tih cp han 0.049 id 0.041 0.031 itb 0.096 0.043 0.069 ish wb 0.049 0.012 0.027 0.040 ish 0.044 0.014 0.012 0.033 0.012 szw 0.048 0.008 0.029 0.038 0.011 0.014 tih 0.052 0.018 0.022 0.020 0.011 0.006 0.014 figure 1. neighbour-joining tree illustrating the genetic distance between breeds: connemara pony (cp), hanoverian (han), irish draught (id), irish sport horse with warmblood (ish wb), irish sport horse (ish), swiss warmblood (szw), irish thoroughbred (itb), traditional irish horse (tih). 75% and specificity of 93% for assigning irish sport horse of unknown ancestry as traditional irish horse, with an accuracy of predicting traditional irish horse from genetic data of 87.5%. admixture analysis of a validation set of 12 pedigree-certified traditional irish horse revealed similar results with warmblood lineage contributions in individuals ranging from 0.01 to 0.15. discussion there is a growing trend in equestrian breeding to move away from the traditional approach of selection within breeds, towards developing hybrid sport horses (gilbert and gillet, 2011). originally the irish sport horse was a breed that was established by crossing the irish draught with thoroughbred horses (reilly et al, 1998; alexander et al, 2009). more recently, crossbreeding the irish sport horse with european warmblood breeds has become popular with the intention of improving the quality of the horses for sport (doyle et al, 2022). this practice threatens the maintenance of genetic variation within the traditional lineages of the irish sport horse that may be beneficial to preserve. in our phylogenetic analyses, we found that the irish sport horse is genetically distinct from european warmblood populations. by contrast, there was no clear genetic distinction between the two european warmblood breeds (hanoverian and swiss warmblood) included in this study. this agrees with a microsatelgenetic resources (2023), 4 (8), 29–36 genetics of the traditional irish sport horse 33 figure 2. neighbour-joining tree illustrating individual relationships among irish sport horses (yellow) and warmbloods (blue; han, szw); including ish with no foreign blood (ish), ish with foreign blood (ish wb) and traditional irish horse (tih). lite genotype-based phylogeny in which hanoverian clustered closest to other european warmblood breeds (including the italian maremmano horse breed) on a branch most distant from other geographically proximal german coldblood breeds (felicetti et al, 2010). the irish sport horse has not previously been included in population genetic diversity studies and there are limited studies including the irish draught and connemara pony. in a microsatellite, protein and blood group marker study, hanoverian clustered with holsteiner, thoroughbred, quarter horse and irish draught, suggested to be due to the influence of thoroughbred in these breeds (luis et al, 2007), and reflected in a more recent genome-wide snp analysis of genetic diversity in which the european warmbloods (hanoverian, swiss warmblood, maremmano) clustered with recently admixed thoroughbred breeds (petersen et al, 2013). between breeds, the pairwise genetic difference (fst ) indicated that the traditional irish horse was more different from the irish sport horse with warmblood than the two european warmblood breeds (hanoverian, swiss warmblood) were from each other. these genetic results indicate that there may be grounds to identify the traditional irish horse as a distinct breed for conservation purposes, should this be desired by breeders. the term ‘breed’ is generally used to describe a population of animals with common phenotypically distinct traits, and it has been suggested that a breed 34 mcgivney et al genetic resources (2023), 4 (8), 29–36 figure 3. principal component analysis plots for pc1 and pc2 (a), and pc2 and pc3 (b) for n = 121 individuals coloured according to breed: connemara pony (cp), hanoverian (han), irish draught (id), irish sport horse with warmblood (ish wb), irish sport horse (ish), swiss warmblood (szw), irish thoroughbred (itb), traditional irish horse (tih). pc1, pc2 and pc3 explained 7%, 4% and 3% of the genetic variance, respectively. figure 4. proportion of genetic ancestry assigned to each breed where a column indicates a single horse and colours represent the four ancestry clusters. the colour of the primary cluster representing each breed ancestral component is depicted in the legend. breed identifiers are noted at the bottom: irish thoroughbred (itb), connemara pony (cp), warmblood breeds (wb), irish draught (id), irish sport horse (ish). figure 5. proportion of genetic ancestry of 23 irish sport horses assigned to each cluster where a column indicates a single horse and colours represent the four clusters. the colour of the primary cluster representing each breed ancestral component is depicted in the legend. the irish sport horses based on genetic classification (top) and pedigree classification (bottom) are indicated as warmblood (wb), irish draught (id), connemara pony (cp), irish thoroughbred (itb), irish sport horse with warmblood (ish wb), irish sport horse (ish), and traditional irish horse (tih). the vertical black lines separate the horses on the basis of pedigree classification. all except for three horses (yellow highlights) were classified correctly on the basis of genetics; two horses with traditional irish horse pedigrees were genetically assigned as irish sport horse, one horse with irish sport horse pedigree was genetically assigned as traditional irish horse. may also be a cultural concept defined by breeders (fao, 2023). however, genetic analyses now enable the examination of distinct and/or common genotypes to provide scientifically informed categorization of breeds (fao, 2023). in order to genetically differentiate the traditional irish horse from phenotypically similar irish sport horse with warmblood, here we evaluated the ancestral genetic contributions to various cohorts of the irish sport horse, defined on the basis of pedigree, to delineate the maximal ancestry component of warmblood genetics required to determine traditional irish horse status. we observed four of the eight irish sport horse with warmblood to have comparable warmblood ancestry to horses in the hanoverian and swiss warmblood cohorts; however, a distinguishing feature of the irish sport horse with warmblood was a greater irish draught influence compared to the hanoverian and swiss warmblood cohorts. in some of the irish sport horse with warmblood, there was more warmblood ancestry component than in some of the european warmblood animals. since by 2014 more than 37% of registered irish sport horses had warmblood ancestry recorded in their pedigrees, our results indicate that concerns regarding european warmblood introgression and the creation of a homogeneous pan-european sport horse population are valid. without a dedicated breeding programme for the traditional irish horse, the unique genetic heritage of the irish sport horse may be lost. current efforts to recognize and assign traditional irish horse genetic resources (2023), 4 (8), 29–36 genetics of the traditional irish sport horse 35 status to traditionally bred horses are hindered by the fact that many irish sport horses may have an unregistered dam or sire in their pedigree. this is a situation where ancestral profiling using genomicsbased technologies could provide a basis for identifying the irish sport horse with traditional lineages. the fao practical guide for genomic characterization of animal genetic resources notes that “genetic tools now allow for errors in recording of parentage to be identified and remedied; this is especially useful in the case of unknown paternity” (fao, 2023). this has immediate practical implications, since in this study there was one irish sport horse with missing pedigree that had warmblood ancestry within the range of the traditional irish horse (10%), indicating that this individual has a traditional genetic heritage. applying a cut-off of ≤ 15%, warmblood ancestry would have an accuracy of prediction of 87.5% for the traditional irish horse. conclusion considering the unique position of the irish sport horse within the international sport horse world, with traits for elite eventing abilities and versatility as a valued leisure horse, the traditional irish horse should be conserved and the genetic legacy of the irish hunter protected. the fao practical guide for genomic characterization of animal genetic resources notes that the results of genomic characterization studies “should also be used to improve the management of the angr [animal genetic resources] involved” (fao, 2023). the application of genetic ancestry testing to identify the traditional irish horse, particularly in the absence of complete pedigree information for a horse, would provide a novel route for conservation efforts and the promotion of the irish horse in equestrian sport. acknowledgments we thank the traditional irish horse association for assistance with the study. the project was funded by the department of agriculture, food and the marine under equine technical support & equine breeding schemes and by horse sport ireland with in-kind contribution from plusvital ltd. data availability data will be publicly available on the european variation archive http://www.ebi.ac.uk/eva/ authors’ contribution bam contributed to the study design, performed the data analysis and interpretation, drafted the manuscript and approved the submitted manuscript. dh conceived the project, contributed to the study design and sample acquisition. ac conceived the project, contributed to the study design and sample acquisition. ewh conceived the project, contributed to the study design, performed the interpretation, drafted the manuscript, revised the manuscript and produced the final submitted manuscript. conflict of interest bam is employed by and ewh is a shareholder in plusvital ltd. plusvital ltd funded the project in-kind with salaries and technical resources. references alexander, d. h., novembre, j., and lange, k. 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(1831). the horse; with a treatise on draught (london, uk: baldwin and cradock). https://doi.org/10.1111/j.1365-2052.2006.01545.x https://doi.org/10.1111/j.1365-2052.2006.01545.x https://doi.org/10.1111/j.1365-2052.2006.01506.x https://doi.org/10.1111/j.1365-2052.2006.01506.x https://doi.org/10.1186/s12864-017-4416-9 https://doi.org/10.1186/s12864-017-4416-9 https://doi.org/10.1371/journal.pone.0054997 https://doi.org/10.1371/journal.pone.0054997 https://doi.org/10.1111/1755-0998.12512 https://doi.org/10.1111/1755-0998.12512 https://doi.org/10.1086/519795 https://doi.org/10.1111/j.1439-0388.1998.tb00324.x https://doi.org/10.1111/j.1439-0388.1998.tb00324.x https://doi.org/10.48495/hm50tr726 https://doi.org/10.48495/hm50tr726 introduction methods samples genotyping population genetics analyses results genetic distance within and among breeds population substructure discussion conclusion acknowledgments data availability authors' contribution conflict of interest short communications genetic resources (2023), 4 (7), 68–75 doi: 10.46265/genresj.etes2274 https://www.genresj.org issn: 2708-3764 wild relatives of fruit trees in syria: genetic resources threatened by conflict munzer aldarvish *,a, anas al kaddour b, akram bourgol c, yasser ramazand, yousef hallak e, stephen cavers f and joan cottrell g a aleppo university and agriculture research centre, syria b aleppo university and general organization for seed multiplication, syria c general commission for scientific agricultural research, syria d plant protection directorate, ministry of agriculture, syria e ministry of agriculture, syria f uk centre for ecology & hydrology, bush estate, eh26 0qb, penicuik, midlothian, united kingdom g forest research, northern research station, roslin, eh25 9sy, midlothian, united kingdom abstract: wild relatives of fruit trees (wrft) are highly valued for food and tradable products by rural communities, especially in low-income countries and as such are a vital resource for tree improvement. during periods of conflict, a lack of support and protection by national authorities may make wrft vulnerable. in syria, wrft are at risk of extinction due to the ongoing crisis, which has limited efforts to conserve and propagate these unique genetic resources. we collected information about the current status and key threats to wrft in northwest syria from 50 agricultural experts using structured interviews. our results show that many sites have experienced erosion, overgrazing and drought. to initiate in situ and ex situ conservation, the locations of wrft exposed to deterioration were identified and mapped using gps, and seeds from five genotypes per species were collected from each of ten species to be used for the establishment of an in situ and ex situ wrft collection. keywords: conservation of wild fruit plants, in situ conservation, plant genetic resources, genetic erosion, crop wild relatives citation: aldarvish, m., al kaddour, a., bourgol, a., ramazan, y., hallak, y., cavers, s., cottrell, j. (2023). wild relatives of fruit trees in syria: genetic resources threatened by conflict. genetic resources 4 (7), 68–75. doi: 10.46265/genresj.etes2274. © copyright 2023 the authors. this is an open access article distributed under the terms of the creative commons attribution license (cc by 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. introduction ensuring future global food security is one of the biggest challenges facing humans today. one in nine people worldwide already suffers from poor access to food resources (fao, ifad and wfp, 2015), and with predicted human population growth, pressure on food resources is likely to increase dramatically (godfray et al, 2010; un desa, 2015). new high-yielding varieties of crops that can withstand adverse climatic conditions and new pests and diseases are urgently needed (mccouch et al, 2013). all cultivated crops derive from historic human domestication of wild ∗corresponding author: munzer aldarvish (drgeneral2015@yahoo.com) genotypes, which have been gradually selected to produce commercial varieties (engels and thormann, 2020). crop wild relatives are the wild original sources of these modern cultivars and remain the primary resource of genetic diversity for breeding new, climatechange tolerant, and higher-yielding cultivars (maxted and kell, 2009; vincent et al, 2019). as such, it is vital to ensure wild relatives are identified, protected and propagated to help secure the future of the species we depend on. globally, fruit trees are of enormous economic importance. in many places (ercişli et al, 2009; otieno, 2017), the wild relatives of fruit trees (wrft) remain essential sources of food and medicines (symphorien et al, 2016) but also provide feed for domestic animals and food additives. they also play a crucial received: 14.11.2022 accepted: 15.05.2023 published online: 08.07.2023 https://www.genresj.org https://www.dx.doi.org/10.46265/genresj.etes2274 https://www.genresj.org https://www.dx.doi.org/10.46265/genresj.etes2274 mailto:drgeneral2015@yahoo.com genetic resources (2023), 4 (7), 68–75 threats to fruit tree wild relatives in syria 69 role in maintaining ecological diversity (musayev and huseynova, 2016). notably, some of these wild relatives are known to possess traits that could greatly benefit contemporary agriculture through the breeding of new, improved cultivars. for example, wild genotypes of species in the genus prunus are characterized by adaptation to extreme climatic conditions (uğur and gündeşli, 2020). moreover, wild fruit tree genotypes are an essential source of resilient rootstock, as grafting is one of the key methods for making new fruit varieties available to growers (zhebentyayeva et al, 2019). several wild edible fruits are used by rural and tribal populations and significantly contribute to their livelihood. the use of non-cultivated foods, of which wild fruits form a part, as a dietary supplement or as a coping mechanism in times of food shortage, provides an important safety net for the rural poor, especially in low-income countries (ercişli and sagbas, 2017). the term underutilized and neglected species refers to a category of wild and cultivated plant species characterized by a low level of investment in research and development, and a recognized but untapped livelihood potential (eyzaguirre et al, 1998). widespread genetic erosion of these species has been recorded worldwide: for example, in nepal, more than half of the traditional landraces have become absent from the farmer’s fields (paudel et al, 2016). generally, despite several initiatives in recent years aimed at the conservation of such genetic resources, these wild relatives are still at risk of erosion or extinction (pilling et al, 2020). they have been almost entirely neglected in ex situ genebanks (castañeda-álvarez et al, 2016), although some efforts to change this have begun (dempewolf et al, 2013). in addition, current in situ reserves often do not meet the required management standards to maintain the wild relatives and their genetic diversity for long-term use (iriondo et al, 2012; weibull and phillips, 2020). syria lies within a region of substantial topographic and climatic diversity in the fertile crescent, which is the centre of origin and domestication of many globally important crops. syria is rich in agrobiodiversity (food, forage crops and fruit trees) like wheat, barley, lentils, chickpeas, vetch, olives, almonds, pear, plums, medic, clover, as well as other ornamental, medicinal and aromatic plants. such plants are the main stocks and heritage for farming in syria and are widely used elsewhere in the world. according to the fourth national report on biodiversity in syria (msea, 2009), the reduction in plant genetic resources was caused by urbanization, climate change, decreasing rainfall and increasing temperatures in dry seasons, which the region has experienced in the last seven years. an increasing incidence of wildfires also poses a particular threat. during the conflict period from 2012 to 2019, syria lost 20.4% of its tree cover (gaafar, 2021), much of it in idlib and lattakia governorates. the depletion of wild tree populations has been associated with several factors related to the conflict, including intense reliance on trees for heating and shelter, frequent fires in wild forests, charcoal production, illegal logging, agricultural expansion, and the weakness of state institutions in protecting natural resources and in managing environmental development. the losses of wild tree species include the cherry prunus mahaleb, oak trees, wild pines and hundreds of other plant (tree and crop) species are also threatened. unfortunately, there is no data available about the current situation of the wrft, the only available data were published by fao (1996), which clearly mentioned the deterioration of plant genetic resources in syria started before the crisis. it can be assumed that the deterioration of wrft has increased following the ongoing crisis in syria resulting from the absence of any control for the protection of these genotypes against the deterioration. moreover, there was clear evidence of plant genetic resources decline in northwest (nw) syria based on the finding of previous research about crop landraces in the same locations in nw syria (aldarvish et al, 2022) the primary causes were the spread of cultivation to new areas, the need to grow economic crops, particularly cereals, forages and food legumes, and accidental forest fires (fao, 1996; aldarvish et al, 2022). hence the wrft were already facing extinction before the syrian crisis began. since then, the absence of research authorities responsible for the conservation and propagation of genetic resources has severely exacerbated the problem. currently, no conservation activity is occurring as the conflict is preventing any access to the field by syrian botanists, in particular in mountain areas where the threat from mines and bombs is severe (valderrabano et al, 2018). as a first step towards addressing the lack of activity on these vital global resources, this study aimed to: i) collect information about the current status and critical threats to ten species of wrft in nw syria, ii) characterize and map the locations of wrft exposed to deterioration, and iii) collect seed from multiple genotypes of each species to be used for the establishment of an in situ and ex situ wrft collection. materials and methods data collection and analysis the national strategy for conservation and management of plant genetic resources for food and agriculture 2015–2035 (fao, 2015) directs national actions for wrft in syria, and our methods were designed to address these goals using guidelines recommended by valderrabano et al (2018). the study was conducted between september 2021 and february 2022 in idleb governorate (ehsem, mhambal, jisrash-shugur, badama, darkosh, janudiyeh, harim, dana, kafr takharim, qourqeena, armanaz subdistricts). this region is located within syria’s second and third agroecological zones, incorporating mountains and plains. agroecological zone 2 covers 2,473,000 ha (13.4% of the country) with an annual rainfall of 250 to 350mm 70 aldarvish et al genetic resources (2023), 4 (7), 68–75 and no less than 250mm across two-thirds of the monitored years. agroecological zone 3 comprises 1,306,000 ha (7.1% of the country) with an annual rainfall of 250 to 350mm and no less than 250mm over half of the monitored years (fao, 2003). these agroecological zones are key factors affecting the spread of the wrft, which is key information when it comes to the planning for subsequent studies on their propagation and preservation. it is worth noting that this region is currently exposed to climatic changes, especially drought, based on the amount of seasonal rain mentioned in the description of this region. to identify the location of wrft exposed to deterioration after the conflict, the researchers conducted focus group discussions with community representatives in each subdistrict to explain the aim of the study, its methods and objectives (one in each of 11 subdistricts under study during the period from 25 september 2021 to 25 october 2021). these representatives nominated 50 key informant candidates, such as agricultural engineers, senior farmers and agricultural researchers, which had considerable knowledge of the agriculture sector in their region and were targeted for data collection via interviews and a multiple-choice questionnaire (supplemental data). the questionnaire was designed to gather local knowledge on the location, usage, reasons for decline and appropriate conservation strategies for wrft. due to the outbreak of covid-19 in nw syria during data collection, 38 of the targeted informants elected to participate via online interviews, while the remaining 12 agreed to in-person interviews. it’s worth mentioning that the quality of online interviews was the same as inperson data collection. data collection started on 25 september 2021 and was completed by 25 october 2021. the data collected from the questionnaires were transferred to ms excel (microsoft office 2020) prior to analysis. data cleaning was undertaken to ensure the accuracy and integrity of the dataset. this involved identifying and rectifying any inconsistencies, outliers or missing values that could potentially skew the results. variability and dispersion within the dataset were calculated with excel formulas, including but not limited to measures such as standard deviation, to extract valuable insights. seed collection and storage the main criterion to decide whether the collected seeds were wrft or not, was their natural spread sites. more specifically, the collected wrft were collected from wild and forest locations. for species with similar phenotypes and for which phenotype is not sufficiently reliable to distinguish between genotypes, for example, ficus carica, olea europaea and vitis vinifera, the seeds were collected from wild mountainous sites far away from agricultural areas, and according to the interviewees and field team observations. the collected wrft existed in these wild sites for a long time, and their morphological growth differs from the cultivated species and landraces of the same species (especially the shape of leaves, fruits and trees). wrft fully ripe fruits were collected during the harvesting season and the fruit flesh (pulp) was removed. the extracted seeds were then spread out on paper sheets and dried in the open air for 30 days, with the seeds flipped daily to make sure they were completely dry. then they were disinfected using thiram fungicide, placed in a paper bag labelled with the species name, location, date and storage data, and stored in airtight plastic barrels with dry silica gel for future studies associated with the propagation and establishment of local wrft in situ conservation sites and ex situ collections. this conservation method was selected due to the ongoing war in syria, where electricity is not available for preserving seeds under refrigerated conditions, based on the recommendation of the millennium seed bank staff provided to the research staff through their visit to the millennium seed bank in july 2019 as an alternative method for seed conservation. moreover, ashok et al (2017) showed that this method of conservation could help to conserve seeds for several years as recommended by yoshinaga (2010). for each of the collected accessions, 100 seeds were weighed for each genotype, and the value was multiplied by 10 and expressed as g/1,000 seeds; average values per species, with standard deviation, were calculated. results focus group discussions conducted with community representatives and experts in the agricultural sector in the targeted subdistricts showed that several wrft locations had been subject to neglect after the syrian crisis and had experienced significant deterioration (figure 1). data from the 50 interviews showed that wrft were found in the following four types of locations within the study area: forests, verges along agricultural roads, rocky and neglected parts of farms, and working farms. the percentage of responses among the 50 participants for the distribution of wrft in study locations is illustrated in figure 2. the interviews revealed that the following ten wrft species are experiencing erosion: prunus mahaleb, prunus orientalis, vitis vinifera, rhus coriaria, olea europaea, crataegus azarolus, ficus carica, pistacia atlantica, prunus spinosa, and pyrus syriaca. a range of reasons was reported for wrft deterioration (figure 3). the primary causes were overgrazing, frequent droughts and climate change. the predominant conflict-related factors reported were neglect by local authorities, lack of awareness of the importance and value of wrft and deforestation resulting from the collection of wood for fuel and conversion to arable lands. several respondents mentioned the lack of interest by the younger generation in making use of local wild fruit landraces, overuse (massive unmanaged fruit gathering), desertification and expansion of building into forgenetic resources (2023), 4 (7), 68–75 threats to fruit tree wild relatives in syria 71 figure 1. study area for identification of wild relatives of fruit trees (wrft) exposed to deterioration. source: humanitarian data exchange figure 2. proportion of respondents reporting types of locations in which wild relatives of fruit trees (wrft) are found. est locations as contributing factors. floods were not considered a threat by any of the respondents. based on the assessment result with the interviewees, the reasons for erosions of wrft are equal within all areas of the study. with respect to the impact of the ongoing conflict in nw syria on wrft, all participants stated that the syrian armed conflict had intensified the deterioration due to the lack of government control leading to overgrazing and excessive cutting of trees in these locations. when questioned about conservation actions that were considered to be the most important for the protection of wrft against the current deterioration, participants responses showed the following answers: 36% suggested exchanging seeds of wrft with other farmers, 26% supported the establishment of local collections for multiplication of these species, 22% considered public awareness campaigns and 14% favoured seed collection and conservation of these species in local genebanks. only 2% considered awareness campaigns to be the most important action for the protection and preservation of these species. regarding the availability of seeds or cuttings of the wrft species, 56% of the participants stated that seeds or cuttings of these species were not available, whereas 44% stated that they have access to seeds and cuttings. this response suggests that some wrft are currently at high risk of decline and may even suffer extinction over time. collection of wrft genotypes the results of the interviews and questionnaire allowed the identification of locations where wrft had been exposed to deterioration and needed conservation. based on available resources, the researchers collected mature seeds of each wrft species from these locations as a first research and conservation step. for each of the priority wrft species identified, five genotypes were collected from areas facing particularly severe deterioration there were slight variations in seed 72 aldarvish et al genetic resources (2023), 4 (7), 68–75 figure 3. reasons given, and percentage of respondents reporting, for deterioration of wild relatives of fruit trees (wrft). from top to bottom – c: overgrazing; b: frequent droughts and climatic changes; k: consequences of syrian crisis (lack of law enforcement); i: lack of knowledge about the importance and value of these species; g: deforestation, including collection of wood for fuel; h: neglecting by local authorities; j: interest of the younger generation for local wild fruit landraces is very low; f: overuse (massive unmanaged gathering); a: spread of cultivation to new areas and need to grow economic crops; e: desertification and expanding of buildings towards forest locations and arable lands; d: floods. weight among the genotypes within species, and the species averages are given in table 1. as seed weight can influence the speed and percentage of germination and can be particularly important in challenging environments (upadhaya et al, 2007), and since maternal effects should be accounted for in any subsequent comparative testing of plants derived from the collection, seed weights for each genotype were taken. the primary purpose of presenting the weight of seeds was to know the standard deviation for the seed weight within different genotypes collected from the same species. this can be investigated in future studies (genetic fingerprinting for these genotypes) to distinguish between phenotype and genotype within the same species and to identify if these variations in seed weight result from environmental factors or are related to genetic features discussion future food security for humankind is likely to rely heavily on the sustainable availability of a wide range of plant genetic resources for food, agriculture and nutrition, amongst other needs. wrft are highly valued genetic resources for food production and to support the livelihoods of rural populations, especially in lowincome countries. wrft in syria are threatened by extinction due to conflict, which has led to increased direct pressure on the tree populations themselves, and to an absence of research authorities working in the field table 1. average seed weights (g/1,000 seeds) for each wild relative of fruit trees (wrft) species collected. wrft species average seed weight (g/1,000 seed) standard deviation ficus carica 14.82 0.38 olea europaea 720.00 144.05 prunus spinosa 309.00 20.74 vitis vinifera 100.00 7.91 prunus orientalis 506.00 20.74 pistacia atlantica 141.00 20.43 rhus coriaria 92.00 9.08 prunus mahaleb 180.00 16.96 crataegus azarolus 400.00 15.81 pyrus syriaca 165.20 2.83 of conservation and propagation of genetic resources. consequently, there is a high risk that this vital resource may be lost to future research, and its fruits to future generations. our results parallel those of previous studies, which have identified that plant genetic resources in syria are particularly exposed to deterioration arising from the ongoing conflict (gaafar, 2021; aldarvish et al, 2022). however, similar threats to wild plant genetic diversity have also been reported worldwide (iriondo et al, 2012; castañeda-álvarez et al, 2016; paudel et al, 2016). prior to this study, the most recent available information on the current status of wrft dates from 11 genetic resources (2023), 4 (7), 68–75 threats to fruit tree wild relatives in syria 73 years ago (fao, 1996) before the start of the conflict. at that time in the study area, the key locations for wrft were rich in wild genotypes because they were protected by government authorities such as the ministry of agriculture and the directorates of environmental protection. as reported by our interviewees, the absence of state protection has resulted in increased deterioration of wrft populations in these locations, as exploitation such as grazing or cutting of fuelwood for heating is now uncontrolled (gaafar, 2021). this neglect and lack of protection for wrft by state institutions has and continues to exacerbate ongoing pressure from climate change and is resulting in severe genetic erosion of these species. as government bodies have not been present in the study area for over a decade, the condition of wrft has continued to deteriorate, as reported by valderrabano et al (2018). moreover, all interviewees stated that the weakness of current state institutions, which demonstrate little interest in wild fruit trees, has led to increased genetic erosion and deterioration of these genotypes in nw syria. wrft are highly valued for use directly as food resources and as commodities that contribute to livelihoods, especially in rural populations in lowincome countries. they are also an essential source of genetic diversity for breeding new, higher-yielding, climate change-tolerant varieties. despite their high value for food and agriculture (maxted and kell, 2009; musayev and huseynova, 2016; symphorien et al, 2016; ercişli and sagbas, 2017; vincent et al, 2019; zhebentyayeva et al, 2019; engels and thormann, 2020; uğur and gündeşli, 2020), wrft have been almost entirely neglected in both ex situ and in situ conservation programmes. as they are likely to be locally adapted and genetically distinctive, wrft can provide vital novel variation for domesticated fruit species to help meet the challenge of increasingly stressful environments and climatic changes. for example, they have been used by plant breeders to develop more efficient nutrient uptake and utilization, and to integrate genes for adaptation to stressful environments such as water stress, salinity and high temperatures (ayenan et al, 2019; mishra et al, 2022). as for wrft, spiegel-roy (1986) stated that wild relatives of fruit trees are one of the essential resources for fruit tree breeding, especially for disease tolerance, rootstocks and genetic engineering. to secure the future of these vital resources, conservation protection efforts are urgently needed for the degraded areas they occupy. the priority species we have identified need to be included in plantation programmes to secure the genetic diversity that will be needed for the improvement of commercially cultivated crops. countries of the near east and the mediterranean basin have perhaps the longest tradition in fruit cultivation for historical, environmental and evolutionary reasons (barone and caruso, 1998; wolf et al, 2000; khoury et al, 2017; migicovsky and myles, 2017; bissessur et al, 2019). this study represents the first s tep i n highlighting the extremely threatened status of these genotypes. our initiative to obtain accurately mapped locations for these genetic resources can underpin future research efforts towards ex situ and in situ conservation programmes. furthermore, the seed we have collected will form the start of a local genebank collection for future studies of genetic variation and propagation, which can help to ensure resilient and sustainable agricultural development for future generations in syria and worldwide. acknowledgments this research was supported by cara (the council for at-risk academics), united kingdom. the syrian research team wishes to extend their thanks to joan cottrell and stephencavers for their valuable assistance in developing the manuscript. supplemental data interview questionnaire author contributions munzer aldarvish coordinated the research and contributed to the research design and manuscript; anas al kaddour, akram bourgol, and yasser ramazan contributed to the research design and manuscript and undertook the data analyses, yousef hallak contributed to the research design and carried out the field data collection; stephen caversand joan cottrellprovided academic guidance and support throughout the research process and contributed to the manuscript. conflict of interest statement the authors of this manuscript have no conflicts of interest to declare. all co-authors have seen and agree with the contents of the manuscript, and there is no financial i nterest t o r eport. w e c ertify t hat the submission is original work and is not under review at any other publication. references aldarvish, m., alkaddour, a., bourgol, a., alramadan, y., hallak, y., kell, s., and s (2022). survey and conservation of crop landraces in northwest syria. genetic resources 3(5), 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https://population.un.org/wpp/publications/files/key_findings_wpp_2015.pdf https://population.un.org/wpp/publications/files/key_findings_wpp_2015.pdf https://journals.tubitak.gov.tr/botany/vol31/iss1/4 https://journals.tubitak.gov.tr/botany/vol31/iss1/4 https://dergipark.org.tr/en/download/article-file/1246751 https://dergipark.org.tr/en/download/article-file/1246751 https://doi.org/10.2305/iucn.ch.2018.21.en https://doi.org/10.2305/iucn.ch.2018.21.en https://doi.org/10.1038/s42003-019-0372-z https://doi.org/10.46265/genresj.2020.1.17-24 https://doi.org/10.46265/genresj.2020.1.17-24 https://doi.org/10.17660/actahortic.2000.538.4 https://doi.org/10.17660/actahortic.2000.538.4 https://www.ctahr.hawaii.edu/ougc/downloads/guidelines_for_storage.pdf https://www.ctahr.hawaii.edu/ougc/downloads/guidelines_for_storage.pdf https://doi.org/10.1038/s41438-018-0090-6 https://doi.org/10.1038/s41438-018-0090-6 introduction materials and methods data collection and analysis seed collection and storage results collection of wrft genotypes discussion acknowledgments supplemental data author contributions conflict of interest statement ntsys-melina-appendix 1 supplemental table 1: alleles observed in each of the 23 clones of gmelina arborea. summary of the presence (1) or absence (0) of the 75 allele found for the fiftteen polymorphic microsatellite loci in each clone. this information was used to estimate genetic similaritiesfor all pairwisecomparisons among clones. clone id locus allele pc-12 pc-18 2pc-6 pc-33 pc-35 t-26 t-27 t-28 t-29 t-30 ca-4 ca-8 ca-19 ca-20 ca-24 mc-2416 mc-902 mc-1313 mc-3204 mc-814 n-15 n-1 n-5 meldi-1 114 0 0 0 0 0 0 1 0 1 0 1 1 1 1 1 0 1 1 0 1 0 1 0 meldi-1 122 1 1 1 1 1 1 1 1 0 1 1 1 1 1 1 1 0 0 1 1 1 1 1 meldi-1 130 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 0 0 0 0 1 meldi-2 299 1 1 1 0 0 1 0 1 1 1 0 0 0 0 0 0 1 0 0 1 0 0 0 meldi-2 303 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 meldi-2 307 0 0 0 0 0 0 1 0 0 0 1 1 0 1 1 0 0 0 0 0 1 0 0 meldi-2 311 1 1 1 1 1 1 1 1 1 1 0 0 0 0 0 1 1 1 1 1 0 0 0 meldi-2 317 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 1 1 0 0 0 0 0 meldi-4 214 1 8 1 1 1 1 0 0 1 0 1 1 1 0 1 0 0 0 0 0 0 0 0 meldi-4 219 0 0 0 0 0 0 0 0 1 0 1 1 1 1 1 1 1 0 0 1 0 0 0 meldi-4 225 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 0 meldi-4 231 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 1 0 0 1 0 0 1 1 meldi-4 237 1 1 1 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 meldi-4 252 1 1 1 1 1 0 1 0 0 0 1 1 1 1 1 1 1 1 1 1 1 1 0 meldi-5 283 1 1 1 0 0 0 0 0 0 0 0 0 0 0 0 1 1 0 0 0 0 0 0 meldi-5 287 0 0 0 0 0 1 1 1 1 1 0 0 0 0 0 0 0 0 0 0 0 0 1 meldi-5 291 0 0 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 meldi-5 295 0 0 0 0 0 1 0 1 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 meldi-5 299 1 1 1 1 1 0 0 0 0 0 1 1 0 0 1 1 0 0 0 0 1 0 0 meldi-5 307 0 0 0 0 0 0 1 1 1 1 0 0 1 1 0 0 1 1 1 1 0 1 0 meldi-6 292 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 meldi-6 296 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 meldi-6 300 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 1 0 1 1 0 0 0 meldi-6 304 0 0 0 0 0 1 0 1 0 1 1 1 1 1 1 1 1 0 0 1 1 1 1 meldi-6 308 1 1 1 1 1 0 1 0 0 0 0 0 0 0 0 1 1 1 0 1 0 0 0 meldi-6 316 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 meldi-6 320 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 0 0 0 0 0 0 0 0 meldi-7 352 0 0 0 0 0 0 1 0 1 0 1 1 1 1 1 1 1 0 1 1 1 1 1 meldi-7 356 0 0 0 0 0 1 0 1 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 meldi-7 360 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 meldi-7 364 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 meldi-7 368 0 0 0 0 0 1 0 1 1 1 1 1 1 1 1 1 0 0 1 1 1 0 0 meldi-7 372 0 0 0 0 0 1 0 1 1 1 0 1 0 0 1 0 0 0 0 0 0 0 0 meldi-7 376 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 meldi-10 175 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 1 0 1 0 0 0 meldi-10 179 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 1 0 1 0 0 1 0 meldi-10 187 0 0 0 0 1 0 0 0 0 0 1 1 0 1 1 1 0 0 0 1 0 0 0 meldi-10 191 1 1 1 1 1 1 1 1 1 0 1 1 1 1 1 0 1 0 1 0 1 0 1 meldi-10 195 1 1 1 1 1 1 0 1 1 1 1 0 0 1 1 0 1 0 1 1 1 1 0 meldi-10 210 1 1 1 1 1 0 0 0 1 0 0 1 0 1 1 0 1 1 0 0 1 0 0 meldi-11 168 1 1 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 meldi-11 175 1 1 1 1 1 0 0 0 0 0 1 1 1 1 1 1 1 1 1 0 1 1 1 meldi-11 182 1 1 0 0 1 1 0 1 1 0 0 0 1 0 0 1 0 0 0 0 0 0 0 meldi-11.2 140 0 1 1 1 0 0 1 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 meldi-11.2 144 0 0 0 0 0 1 1 1 1 1 1 0 0 0 0 0 0 0 1 1 0 0 0 meldi-11.2 148 1 0 0 0 0 0 0 0 0 0 1 0 1 0 0 0 0 1 1 0 1 1 1 meldi-11.2 154 0 1 1 1 1 0 0 0 0 0 0 1 0 1 1 1 1 0 0 1 0 0 0 meldi-11.2 158 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 1 1 0 0 0 0 meldi-11.2 166 0 0 0 0 0 1 0 1 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 meldi-12 206 0 0 0 0 0 0 0 0 0 0 1 1 0 0 0 1 1 0 1 0 0 0 1 meldi-12 216 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 meldi-16 213 1 1 0 1 1 1 1 0 0 0 0 0 1 1 1 0 0 1 1 0 1 1 0 meldi-16 219 0 1 1 0 0 0 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 meldi-16 225 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 meldi-17 153 1 0 0 1 0 0 1 0 0 1 0 0 0 0 0 0 1 0 0 1 0 0 0 meldi-17 161 0 0 0 1 1 0 0 1 1 1 1 1 1 0 1 1 1 0 0 0 1 1 1 meldi-17 165 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 1 meldi-17 167 0 0 0 0 1 1 0 0 0 0 1 1 1 1 1 1 0 0 1 0 1 0 0 meldi-20 205 1 1 1 0 0 1 1 0 1 0 0 0 1 1 0 0 0 0 1 1 0 0 0 meldi-20 211 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 meldi-20 215 1 0 1 0 0 0 0 1 0 0 0 0 0 1 1 0 0 0 0 0 1 1 0 meldi-20 219 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 1 0 1 1 0 0 0 0 meldi-20 227 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 meldi-20 231 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 1 0 0 0 0 0 0 meldi-21 231 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 meldi-21 235 1 1 1 1 1 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 meldi-21 244 0 0 0 0 0 0 0 0 0 0 1 1 1 1 0 0 0 0 0 0 0 0 0 meldi-21 248 0 0 0 0 0 0 1 0 0 0 1 0 0 1 1 0 0 0 0 0 1 0 0 meldi-21 262 0 0 0 0 0 0 0 0 1 1 0 1 0 0 0 0 0 0 0 0 0 1 1 meldi-22 192 0 0 0 0 0 1 0 1 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 meldi-22 210 1 1 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 1 1 0 0 0 0 meldi-22 214 0 1 1 0 0 0 1 0 1 0 0 0 0 0 0 1 1 1 0 1 0 0 0 meldi-22 220 0 0 0 1 1 0 0 0 0 0 1 0 0 1 1 0 0 0 0 0 1 0 0 meldi-22 224 0 0 0 0 0 0 0 1 0 0 0 1 1 0 0 0 0 0 0 0 0 0 0 meldi-22 228 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 supplemental data for: rocha, o. j., méndez-álvarez, d., rojas parajeles, f., murillo gamboa, o. (2020). isolation and characterization of fifteen microsatellite loci for the use in breeding of gmelina arborea roxb. (lamiaceae). genetic resources 1 (2), 23–28. doi: 10.46265/genresj.lltj1737. https://doi.org/10.46265/genresj.lltj1737 original article genetic resources (2024), 5 (9), 13–28 doi: 10.46265/genresj.rejr6896 https://www.genresj.org issn: 2708-3764 european genetic resources conservation in a rapidly changing world: three existential challenges for the crop, forest and animal domains in the 21st century françois lefèvre *,a, danijela bojkovski b, magda bou dagher kharrat c,d, michele bozzanod, eléonore charvolin-lemaire e, sipke j hiemstra f, hojka kraigher g, denis laloë e, gwendal restoux e, suzanne sharrockh, enrico sturaro i, theo van hintum f, marjana westergren g, nigel maxted j and genres bridge expert panel k a inrae, ecologie des forêts méditerranéennes, urfm, domaine saint paul agroparc, 84914, avignon, france b biotechnical faculty, department of animal science, jamnikarjeva 101, 1000, ljubljana, slovenia c laboratory of biodiversity and functional genomics, faculty of science, saint joseph university, beirut, lebanon d european forest institute, sant pau art nouveau site, carrer sant antoni m. claret, 167, 08025, barcelona, spain e gabi, agroparistech, inrae, université paris-saclay, 78350, jouy-en-josas, france f centre for genetic resources, the netherlands, wageningen university & research, radix building 107, droevendaalsesteeg 1, 6708 pb, wageningen, the netherlands g department of forest physiology and genetics, slovenian forestry institute, večna pot 2, 1000, ljubljana, slovenia h botanic gardens conservation international, descanso house, 199 kew road, richmond, tw9 3bw, uk i department of agronomy, food, natural resources, animals and the environment dafnae, università degli studi di padova, viale dell’università 16, 35020, legnaro (pd), italy j school of biosciences, university of birmingham, birmingham b15 2tt, uk k full list available at the end of the article abstract: even though genetic resources represent a fundamental reservoir of options to achieve sustainable development goals in a changing world, they are overlooked in the policy agenda and severely threatened. the conservation of genetic resources relies on complementary in situ and ex situ approaches appropriately designed for each type of organism. environmental and socioeconomic changes raise new challenges and opportunities for sustainable use and conservation of genetic resources. aiming at a more integrated and adaptive approach, european scientists and genetic resources managers with long experience in the agricultural crop, animal and forestry domains joined their expertise to address three critical challenges: (1) how to adapt genetic resources conservation strategies to climate change, (2) how to promote in situ conservation strategies and (3) how can genetic resources conservation contribute to and benefit from agroecological systems. we present here 31 evidencebased statements and 88 key recommendations elaborated around these questions for policymakers, conservation actors and the scientific community. we anticipate that stakeholders in other genetic resources domains and biodiversity conservation actors across the globe will have interest in these crosscutting and multi-actor recommendations, which support several biodiversity conservation policies and practices. keywords: agroecology, climate change, in situ conservation, multi-actor engagement, policy citation: lefèvre, f., bojkovski, d., bou dagher kharrat, m., bozzano, m., charvolin-lemaire, e., hiemstra, s. j., kraigher, h., laloë, d., restoux, g., sharrock, s., sturaro, e., van hintum, t., westergren, m., maxted, n., genres bridge expert panel (2024). european genetic resources conservation in a rapidly changing world: three existential challenges for the crop, forest and animal domains in the 21st century. genetic resources 5 (9), 13–28. doi: 10.46265/genresj.rejr6896. © copyright 2024 the authors. this is an open access article distributed under the terms of the creative commons attribution license (cc by 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. received: 26.05.2023 accepted: 20.02.2024 published online: 11.03.2024 https://www.genresj.org https://www.genresj.org https://doi.org/10.46265/genresj.rejr6896 https://doi.org/10.46265/genresj.rejr6896 14 lefèvre et al genetic resources (2024), 5 (9), 13–28 introduction genetic resources are at the crossroads of multiple policy agendas, in particular biodiversity conservation and sustainable development goals (fao (2019), e.g. p. 3 about diversity loss in production systems; ipbes (2019), e.g. spm-a6 and p. 247 about erosion of genetic resources diversity; ipcc (2019), e.g. spmb6.2; cbd (2020), e.g. aichi targets 13, 14 and 16; cbd (2022), e.g. targets 4 and 13). in the common international classification of ecosystem services (haines-young and potschin, 2018), the term ‘genetic resources’ is not used but genetic resources explicitly appear both as provisioning services under the term ‘genetic material’, as regulation and maintenance services under the term ‘gene pool’, and could also be considered as cultural services in the class of “characteristics or features of living systems that have an option or bequest value”. this classification reveals the multiple values of genetic resources: direct use value of well-characterized genetic material, option value of the genetic diversity, bequest value of biodiversity components. however, the threat to and the erosion of genetic resources diversity, both in wild populations and in production systems, is now widely documented (fao, 2019; ipbes, 2019; cbd, 2020) and the related aichi target of safeguarding genetic diversity has not yet been achieved (cbd, 2020). the kunming-montreal global biodiversity framework calls for “target 4: ensure urgent management actions [. . . ] for the recovery and conservation of species [. . . ] to significantly reduce extinction risk, as well as to maintain and restore the genetic diversity within and between populations of native, wild and domesticated species to maintain their adaptive potential including through in situ and ex situ conservation” (cbd, 2022). despite their critical importance for sustainable development, on the one hand, and the ongoing erosion of their diversity, on the other hand, genetic resources are largely overlooked by policymakers. three reasons may explain this paradox. first, the role of within-species genetic diversity remains poorly understood and appreciated in biodiversity conservation (hoban et al, 2020). second, few recognize the existential importance of withinspecies genetic diversity in sustaining continued crop, forest and animal production. third, the term ‘resources’ does not explicitly refer to the notions of diversity, which in a changing world is valued over quantity, and rather focuses on the use aspects. the important role of diversity between and within crop1, animal and forest genetic resources for maintaining production has been recognized for centuries, ∗corresponding author: françois lefèvre (francois.lefevre.2@inrae.fr) 1 in this article, the term ‘crop genetic resources’ encompasses plants used for agricultural production, including crop wild relatives and wild food plants, and is used instead of the more common ‘plant genetic resources’ to avoid confusion with the forest domain, which deals with the genetic resources of forest trees and other woody plants. but the actual term ‘genetic resources’ was only coined in 1967 at the international conference on crop plant exploration and conservation (frankel and bennett, 1970). it received a common definition a nd global consideration in the convention on biological diversity (cbd (1992), article 2): “genetic resources means genetic material of actual or potential value”. thus, genetic resources refer to genetic diversity of actual or potential use value between and within species, with a continuum from domestic gene pools (varieties, breeds, isolates) to wild populations. the evolutionary processes during domestication are mainly driven by targeted human interventions such as selection, migration and hybridization. in the case of partially anthropized systems where populations are exploited and managed through natural regeneration systems (e.g. many forests, fisheries and grazed areas), management practices indirectly shape genetic resources by interfering with natural evolutionary and ecological processes. the domestic and wild gene pools are often connected in the landscape where they develop three types of interactions: (1) competition for land (grau et al, 2013), (2) ecological interactions (pozo et al, 2021) and (3) possible gene flow between domestic gene pools and their wild relatives (ellstrand and rieseberg, 2016). thus, genetic resources conservation has to be considered in the context of social-ecological systems, where humans directly or indirectly sustain genetic resources and humankind benefits substantially from their genetic diversity maintenance and utilization. the communities working on genetic resources have historically tended to be defined by the scope of their taxonomic coverage, each specializing in crop, forestry, domesticated animal, fish, microbe or pollinator genetic diversity conservation and use, the linking of conservation with use of the conserved resource setting them apart from the broader biodiversity conservation community. the crop, forestry and domesticated animal domains have worked largely independently to develop conservation and use actions specifically designed within their respective contexts, without sharing experience and benefiting from mutually advantageous collaboration. to fill this gap, the european union’s horizon 2020 ‘genres bridge’ project brought together for the first time the european crop, forestry and domesticated animal genetic resources networks (http://www.genresbridge.eu/). three individual networks have been coordinating and facilitating genetic resources conservation and use in europe for more than 25 years within their respective domains: the european cooperative programme for plant genetic resources (ecpgr, https://www.ec pgr.org/), the european regional focal point for animal genetic resources (erfp, https://www.animalge neticresources.net/), and the european forest genetic resources programme (euforgen, https://www.eufor gen.org/). the three networks joined forces in the genres bridge project to elaborate a genetic resources strategy for europe speaking with a stronger policy ‘voice’ and mailto:francois.lefevre.2@inrae.fr genetic resources (2024), 5 (9), 13–28 genetic resources conservation in a changing world 15 facilitating more effective implementation. this strategy consists of a comprehensive overarching framework of appropriate coordinated actions to conserve and sustainably use genetic resources (genres bridge project consortium, ecpgr, erfp and euforgen, 2021), and three derived domain-specific documents accounting for respective contexts (ecpgr, 2021; erfp, 2021; euforgen, 2021). although the biological and socioeconomic contexts of conservation and sustainable use of genetic resources differ for agricultural crop, animal farming and forestry domains, from the biological point of view, the coexistence of human-directed and natural evolutionary processes are common to all domains of genetic resources. furthermore, from the socioeconomic point of view, sustainable development depends on continued access to a combined set of genetic resources from each domain and combined production systems (e.g. agroforestry). finally, genetic resources conservation and sustainable use in all domains are currently facing common challenges in the context of environmental, socioeconomic and legal changes. therefore, joining expertise from different domains, with various socialecological contexts, will help effectively address these challenges for sustainable use and conservation of genetic resources. this paper illustrates crosscutting and integrated solutions to three existential challenges for genetic resources in the 21st century: 1. how to adapt genetic resources conservation strategies to climate change 2. how to promote in situ conservation strategies (with common objectives despite diverse modalities across domains) 3. how can genetic resources conservation contribute to and benefit from agroecological systems we here provide general arguments and recommendations reusable by different genetic resources and conservation communities. methodology the three challenges were addressed during three workshops engaging a global panel of 43 invited experts on genetic resources, i.e. scientists in conservation science and practitioners, from 16 countries and one international organization, with balanced representation of the three domains. to develop policy-relevant conservation science, we first identified evidence-based statements common to all genetic resources domains, beyond biological and socioeconomic specificities. these statements were based on the reports of international agencies and platforms (fao, 2019; ipbes, 2019; ipcc, 2019; cbd, 2020, 2022) and workshop participants’ expertise. then, each evidence-based statement was deconstructed and reviewed, and key arguments and recommendations were derived for each of the three prime target audiences: policymakers, conservation actors and the scientific community. final statements and recommendations were elaborated through online collaboration. these statements and recommendations have broad general interest not only for other genetic resources domains, e.g. fisheries or industrial microbiology, but also for other biodiversity conservation programmes accounting for genetic diversity at global, regional and national levels. here, we present a list of 31 evidence-based statements, and 88 arguments and key recommendations related to the three challenges. we then briefly analyze the targeted audiences and describe how these particular statements and recommendations were considered in the genetic resources strategy for europe. finally, we propose some perspectives building on the inter-domain collaborative experience. results how to adapt genetic resources conservation strategies in the context of climate change ten statements (cc1 to cc10) and 26 recommendations on this challenge are given in table 1. the first three statements, cc1 to cc3, raise the point that, in the context of climate change, the diversity of genetic resources is both at risk while also representing a reservoir of options to sustain agriculture and forestry in the face of multiple uncertainties (koskela et al, 2007; fao, 2015). therefore, to better use genetic resources, we need to explore and characterize their diversity and potential benefits using both in situ material and ex situ collections. scientists and actors on genetic resources in all domains agree on the severe level of threats of erosion and extinction currently impacting genetic resources diversity. efforts to improve the conservation, characterization and use of genetic resources need to be actively promoted, even if there is still a lack of quantitative assessment of these threats (ipbes, 2019). a second set of statements, cc4 to cc6, stresses the need for raising awareness on genetic resources diversity, conservation and use issues, and for better sharing science-based knowledge with multiple actors and policymakers involved. this lack of knowledge sharing was identified as a limiting factor in genetic resources conservation and use. the related recommendations aim to support the ‘chain of knowledge’ from science to policy decisions, on the one hand, and to facilitate exchanges of information or material among local expert communities in genetic resources, on the other hand, both needed to adapt genetic resources conservation and use strategies in the context of climate change. 16 lefèvre et al g enetic resources (2024),5 (9),13–28 table 1. statements and recommendations on how to adapt genetic resources conservation strategies in the context of climate change. prime target audience: p, policymakers; s, scientific community; c, conservation actors (other than p and s). statements arguments and recommendations cc1 climate change poses a significant threat to genetic diversity. cc1.1 immediate conservation action is needed now to prevent loss of genetic diversity and political commitment linked to policy action is required to support this initiative. [p, c] cc1.2 the diversity of climate change-related threats needs clarification, and their mitigation demands a diversity of responses. [s] cc1.3 threats from climate change need to guide future genetic resources conservation and use strategy developments and prioritize mitigating action implementation. [c] cc2 genetic diversity provides resilience in the face of unexpected change. cc2.1 social and economic studies are required to evaluate how genetic resources diversity mitigates threats to food security and other contributions of agriculture and forests to people. [s] cc2.2 studies are required to provide concrete examples of the benefits provided by genetic diversity in the agroecosystems and the values of such ecological, social and economic benefits. [c, s] cc3 in order to deploy sources of resilience, diversity has to be identified and characterized. cc3.1 characterization of genetic resources and sharing of this information in a standardized manner are essential. [c, s] cc3.2 improved availability of more standardized scientific information on genetic and phenotypic diversity is required. [s] cc3.3 predictive characterization may also be used to speed up identification of desired traits. [s] cc4 genetic resource-related policies should be based upon relevant scientific findings. cc4.1 science provides evidence-based insights that are essential in defining effective policies. [p, s] cc4.2 increased collaboration between scientists and policymakers could improve the uptake of scientific messages in policy decisions. [p, s] cc5 awareness of the importance of genetic diversity for the survival of humankind should be raised. cc5.1 public and political support for genetic resources conservation is essential to secure appropriate funding. [p] cc5.2 the general public, but also policymakers, are rarely aware of the important role of the diversity provided by genetic resources in adaptation to the changing climate and changing demands from society. [p, c, s] cc6 cooperation between formal genetic resources conservation, breeding programmes and community-based conservation initiatives should be improved. cc6.1 community-based activities can play an important role in the identification of resilient genetic resources suitable for the changing environment. [c] cc6.2 link between the formal genetic resources management systems with local initiatives is often weak, and access to each other’s genetic resources is often limited. [c] continued on next page g enetic resources (2024),5 (9),13–28 g enetic resources conservation in a changing w orld 17 table 1 continued statements arguments and recommendations cc7 early signs of potential future needs and threats to genetic diversity and genetic resources use have to be detected. cc7.1 foresight studies (horizon scanning exercises) can produce scenarios to guide long-term strategies for genetic resources conservation and use. [c, s] cc7.2 studies should address possible relevant socioeconomic changes and technological advances. [s] cc7.3 scenarios of change should also consider biotic and abiotic hazards, as well as external contingencies. [s] cc8 periodic monitoring of the actual impacts of climate change on genetic diversity and associated organisms is required. cc8.1 given the climate crisis and associated uncertainties, regular monitoring allows tracking of changes and development of scenarios. [c] cc8.2 based on the knowledge gained from monitoring, prioritization of actions can and should be made. [c] cc8.3 monitoring should not only include genetic resources but also associated organisms, like pollinators. [c] cc9 communication between all practitioners involved in genetic resources management and use (genebank managers, in situ network managers, breeders, farmers and foresters, protected area managers, etc.), policymakers and scientists, needs to be improved. cc9.1 coordination of genetic resource-related actions will improve with better communication. [p, c, s] cc9.2 this will lead also to establishing and reinforcing collaboration between multiple actors involved in genetic resources conservation and sustainable use. [p, c, s] cc9.3 multiple actors will contribute to awareness raising on the basis of values and trade-offs between all relevant values. [p, c, s] cc10 the traits related to adaptation of genetic resources to climate change need to be given more attention in research. cc10.1 tolerance to climate-related hazards (heat, drought, etc.), and resistance to existing and emerging pests and diseases will become essential in adaptation to climate change; more knowledge about these traits will become essential to allow adaptation. [s] cc10.2 genetic resources will benefit from basic research on these traits and their use as study objects should be promoted. [s] cc10.3 the way how these traits can support adaptation of agroecosystems, or help to diversify these systems, should be assessed. [s] 18 lefèvre et al g enetic resources (2024),5 (9),13–28 table 2. statements and recommendations on how to promote in situ conservation strategies. prime target audience: p, policymakers; s, scientific community; c, conservation actors (other than p and s). statements arguments and recommendations is1 dynamic in situ conservation strategies integrate adaptation to global change into the conservation process. is1.1 genetic resources are kept in the productive environment allowing exposure to change and stress situations. [c] is1.2 in situ conserved genetic resources stay useful in a changed environment. [c] is2 in situ conservation continuously contributes to multiple ecosystem services and benefits to people. is2.1 in situ conservation with management contributes to rural development. [p, c] is2.2 in situ conservation provides a broad range of diversity to users. [p, c] is2.3 in situ conservation also contributes to regulation and maintenance as well as cultural ecosystem services. [p, c] is2.4 in situ conservation allows for better dynamic reactions to different drivers of change, including market needs and new market niche exploration. [p, c] is3 effective and efficient in situ conservation and sustainable use of genetic diversity rely on the participation of multiple actors and coordinated efforts. is3.1 key actors and potential new actors should be identified/recognized and involved in genetic resources strategies. [p, c, s] is3.2 in situ conservation programmes should be designed based on a participatory approach involving all actors. [p, c, s] is3.3 all actors need to be financially supported and incentives should rely on available scientific proofs. [p, c, s] is4 coordination of efforts by the various actors involved in dynamic in situ conservation is needed to ensure that long-term objectives are reached. is4.1 actions are needed to strengthen the links between all actors (practitioners, scientists, etc.) in in situ management of genetic resources. [c, s] is4.2 strategical recommendations, guidelines and directives should be tested by practitioners in collaboration with scientists and extension services before general implementation. [c,s] is5 coordinated and standardized national inventories of in situ genetic resources have to be prepared and made accessible. is5.1 inventories of in situ genetic resources improve our knowledge about what and where they are still maintained or cultivated, thus providing a resource from where important traits can be identified for plant and animal improvement by breeders or direct utilization by farmers. [c] continued on next page g enetic resources (2024),5 (9),13–28 g enetic resources conservation in a changing w orld 19 table 2 continued statements arguments and recommendations is5.2 inventories of in situ genetic resources are needed for planning more systematic crop-collecting missions addressing possible gaps and for designing on-farm conservation and management projects. [c] is5.3 data structure of the national inventories of in situ genetic resources should feed the appropriate european information systems. [c, s] is6 active genetic management including selective breeding for performance traits and evolution-oriented forest management can contribute to in situ conservation ‘in use’ of genetic resources. is6.1 knowledge of the qualitative and quantitative impacts of agricultural and forestry practices on evolutionary processes needs to be improved and shared with practitioners. [c, s] is6.2 information is needed on the potential role of active genetic management on performance and adaptive traits to improve self-sustainability of in situ genetic resources within the constraints of their typical characteristics. [c, s] is7 new operational tools for the in situ conservation of genetic resources have to be developed and practically applied in all domains to increase our understanding and capacity to develop more efficient strategies for genetic resources conservation and use. is7.1 there is a need for operational tools for in situ characterization, evaluation, management and monitoring of genetic resources. [s] is7.2 the development of tools is a dynamic process, both for the update and the uptake, in which the three domains could share experiences and innovations. [s] is8 a commitment and a concept for long-term genetic monitoring are needed to guide in situ conservation and sustainable use of genetic resources. is8.1 genetic monitoring is an efficient tool to characterize and detect changes in genetic diversity over time. [c, s] is8.2 the standardization and/or comparability of genetic monitoring information over time must be ensured to allow proper assessment of the changes (independently of the new tools). [s] is8.3 sufficient resources should be committed to implementing long-term genetic monitoring. [p] is9 the complementarity between in situ and ex situ techniques can contribute to increasing the systematic coverage of genetic diversity under conservation as well as the efficiency of genetic resources conservation. is9.1 there is a need to investigate explicitly the multiple advantages, and risks, of combining in situ and ex situ strategies: provide insurance and backup, facilitate access to material, provide additional material for reinforcement in situ, etc. [s] is9.2 suitable methods and tools to integrate dynamic and static conservation approaches should be developed for all domains. [s] is9.3 such integrated approaches will allow opportunities for a wider range of stakeholders, including local communities, to participate in different networks at various levels. [c, s] continued on next page 20 lefèvre et al g enetic resources (2024),5 (9),13–28 table 2 continued statements arguments and recommendations is10 opportunities for the protection with utilization and valorization of the diversity of genetic resources in various ecosystems should be promoted. is10.1 characterization of the genetic diversity of genetic resources available in cultivated areas, protected areas, rural and urban spaces, and private and public gardens, is needed. [c, s] is10.2 using these spaces for conservation of both wild and cultivated diversity should be promoted and supported. [p] is11 long-term conservation policies, strategies and programmes are needed to ensure dynamic in situ conservation of genetic resources diversity. is11.1 long-term perspective of genetic resources conservation strategies must clearly appear in the related eu policies, strategies and programmes, to support adaptive dynamics in the in situ conservation devices. [p] is11.2 long-term policy support for in situ management and monitoring is needed. [p] is12 cooperation within and across domains at the european scale to develop dynamic in situ conservation strategies of genetic resources is needed. is12.1 dynamic in situ conservation strategies of genetic resources can use very diverse methods and tools; sharing experiences and research efforts across domains, geographic areas or species, is needed. [c, s] is12.2 dynamic in situ strategies provide opportunities to combine multiple genetic resources targets in the same conservation action, including trans-domain actions. [c, s] is12.3 following an adaptive management framework, permanent upgrading should be incorporated into the strategies, including complementarity of in situ and ex situ conservation. [c, s] genetic resources (2024), 5 (9), 13–28 genetic resources conservation in a changing world 21 finally, statements cc7 to cc10 illustrate the fact that climate change is forcing us to revise our vision, tools and methods for sustainable genetic resources management particularly as genetic resources are evolving in an unpredictable and dynamic context. more than on current diversity per se, the focus should be put on its trajectory and the drivers of this trajectory. putting genetic resources management in such a dynamic perspective also requires monitoring strategies. to support innovative approaches, the recommendations provide a list of actions to develop governance and decision support tools like indicators or scenarios and new target traits of interest for research. how to promote in situ conservation strategies twelve statements (is1 to is12) and 31 arguments and recommendations on this challenge are given in table 2. the first two statements emphasize key characteristics of in situ conservation: framed in a dynamic perspective as mentioned above, genetic diversity continuously evolves in diverse and changing biotic and abiotic environments, and it combines conservation with sustained local use benefits. this integrative conservation approach illustrates the socioecological dimension of genetic resources: ecological adaptation interlaces with contributions to people. based on these statements, the european experts from the different domains derived some general ‘arguments’ to explain and promote the integrative in situ approach in various contexts, rather than specific recommendations for each domain. combining conservation and use, in situ conservation strategies systematically rely on multiple and diverse actors, either directly managing genetic resources or indirectly controlling the environment in which they are left evolving. to ensure effective and efficient in situ conservation, multiple actors must be coordinated not only locally, but also at national level to establish networks of local initiatives. these points are raised in statements is3 to is5. the related recommendations aim to create and support multi-actor engagement, coordinate their actions with appropriate science-based guiding tools and monitor jointly the development of actions and the diversity of genetic resources that result from these actions. statements is6 to is8 identify three specific aspects of in situ conservation on which knowledge, i.e. both scientific knowledge and practitioners’ expertise, must urgently be expanded. the first aspect is a quantitative assessment of the potential role that ecosystem management (agriculture or forestry practices) can play as evolutionary drivers of genetic resources diversity. the second aspect is the need to develop operational tools specifically dedicated to in situ genetic resources and actors involved. the third aspect is the need for standardized, long-term monitoring programmes applicable to all three domains, in the framework of international initiatives towards global genetic diversity monitoring (hoban et al, 2022). the related recommendations represent priority actions in these fields. statements is9 and is10 reveal other actions which in situ conservation could easily complement with great benefits: association with other genetic resources conservation approaches (i.e. ex situ conservation) or other compatible land uses (e.g. protected, cultivated or even urban areas). the related recommendations aim to support this integrative approach of in situ genetic resources conservation in a broader framework. finally, the last two statements and related recommendations raise the fact that benefiting from all integrative dimensions of in situ conservation requires longterm programmes and support, as well as large-scale cooperation. how can genetic resources conservation contribute to and benefit from agroecological systems nine statements (ae1 to ae9) and 31 recommendations on this challenge are given in table 3. the challenge here is to search for mutual opportunities between the emerging interest to apply agroecological principles in the development of agricultural and forestry systems and genetic resources conservation. during the discussions, european experts highlighted the potential benefit of genetically diverse resources in the agroecology framework (chable et al, 2020). to reach this benefit, the recommendations related to the first statement ae1 focus on the identification and contextualization of genetic resources in agroecological systems, and on sharing this information. the second statement ae2 highlights the key role of genetic resources managers in agroecology. five recommendations explain how to support these actors. the next five statements, ae3 to ae7, underline the specific relevance of the local scale (landscape, territory) to develop synergies between genetic resources conservation, agroecology, and resilience/sustainability of agriculture and forestry systems. indeed, local actors involved in agroecology have the capacity to implement integrative in situ conservation within-sites as proposed in the previous sections, while the diversity of socialecological contexts among localities contributes to maintaining between-sites diversity. this idealistic view relies on the engagement of multiple actors in multiple sites, which cannot be achieved without searching for win-win solutions: altogether, 16 recommendations related to the above statements were proposed. finally, statement ae8 stresses the need for a holistic and social-ecological approach to consider all levels of diversity together, and statement ae9 raises the particular importance of data management for this challenge. indeed, various types of data should be handled jointly: different kinds of genetic resources, multiple uses and related genetic resources characteristics, biological to socioeconomic data or regulation information, georeferencing, etc. 22 lefèvre et al g enetic resources (2024),5 (9),13–28 table 3. statements and recommendations on how genetic resources conservation can contribute to and benefit from the agroecology transition. prime target audience: p, policymakers; s, scientific community; c, conservation actors (other than p and s). statements arguments and recommendations ae1 diverse genetic resources are key elements in the agroecology framework. ae1.1 all component species and their role in each agroforestry system need to be identified. [c] ae1.2 long-term study cases should be established in different geographical and socioeconomical contexts to analyze and demonstrate the impact of genetic resources on agroecology systems across different time scales. [c, s] ae1.3 the use of a broad diversity of genetic resources, and the exchange of genetic resources and related information should be promoted. [p] ae1.4 increased knowledge of the (epi)genetic variability of genetic resources will favour their integration into agroecological systems. [s] ae2 genetic resources managers have a key role to play in the agroecological transition. ae2.1 policy support must be implemented with a long-term view and connected with public support. [p] ae2.2 ecological performance (multi-criteria performance evaluation) must be integrated into the value chain labelling process. [p] ae2.3 further research is needed on how to manage genetic resources for a transition from an intensive (standard) production system to a more ecologically oriented mode of production. [s] ae2.4 the long-term benefits of ecological performance/sustainability when all three domains are considered should be further investigated and knowledge communicated to the end-users. [c, s] ae2.5 scientists and genetic resources managers together should propose decision-making tools, identify actors and consider geographical information supporting sustainable use of genetic resources in the agroecological transition. [c, s] ae3 research, policy, managers and users’ communities on genetic resources must be connected. ae3.1 demonstrations of how useful genetic resources are for farmers, forest managers, and their respective systems, are needed. [c, s] ae3.2 the views of the users must be taken into account in the design and the analysis of study cases and in the implementation of the strategy. [c] ae3.3 research has to produce a synthesis of results and knowledge for stakeholders. [s] ae3.4 common terminology must be shared across the different communities involved. [c, s] continued on next page g enetic resources (2024),5 (9),13–28 g enetic resources conservation in a changing w orld 23 table 3 continued statements arguments and recommendations ae4 the agroecology framework provides an opportunity to look at landscape/territory scale which is also relevant for genetic resources management ae4.1 management and research activities must consider landscape/territory scale as the way to associate genetic resources with ecosystem services and identify multiple values of genetic resources. [c, s] ae4.2 maintenance of diversity rather than specific unicity of genetic resources must be supported to avoid negative side effects of decreased diversity. [p] ae4.3 conservation of diversity at local scale can be costly, so there is a need to involve multiple actors to share costs and to work on social organizations. [p] ae4.4 actions that connect across territories should be supported: locally appropriate genetic resources may be non-local, may need to be imported (incl. from ex situ genebanks); reciprocally each territory may handle genetic resources of poor local value but high value for elsewhere. [p] ae5 human dimension and local knowledge are important for sustainable use of genetic resources and cultural heritage ae5.1 consideration and characterization of local knowledge and traditional use have to be accounted for in the characterization of genetic resources. [c, s] ae5.2 participatory approaches must be supported and developed. [p, c, s] ae6 integrated genetic resources management contributes to increasing biodiversity as a factor of resilience of production systems in the agroecology framework ae6.1 case studies can be used to improve knowledge of the respective roles of the different levels of diversity in agroecological systems: from the within-crop/breed/population/species diversity to the between-crop/breed/population/species diversity. [c, s] ae6.2 traceability of genetic resources uses is needed to analyze crisis situations and document the role of diversity in buffering changes and unexpected disturbances. [c, s] ae6.3 research should further investigate the conditions where diversity can be beneficial or detrimental to productivity. [s] ae6.4 the agroecology framework should be implemented with the aim to optimize both the production and the management of diversity, either for conservation or for preserving variability for future selection. [c, s] ae6.5 scenarios of complementarity between agroecology and genetic resources conservation must be evidenced. [c, s] ae7 the agroecology framework takes advantage of local context specificities. ae7.1 there is no single solution to be applied everywhere: it is important to find a way to share experience/methods/tools from local to global level. [p, c, s] ae8 a holistic approach is needed to consider all levels of diversity and time scale from an agroecological perspective. ae8.1 implementation of management should be based on ecological considerations with an emphasis on the links between the three domains (forest, crops, animals), natural diversity (wildlife, micro-organisms, soils, etc) and human dimension. [c] continued on next page 24 lefèvre et al g enetic resources (2024),5 (9),13–28 table 3 continued statements arguments and recommendations ae8.2 implementation of an indicators system based on multicriteria including the assessment of diversity, including genetic resources, at different levels (e.g. fao grid) is recommended. [c, s] ae9 reliable and abundant data are needed to support a better valorization of the genetic resources into an agroecological framework. ae9.1 the abundant data present in individual databases should be made broadly accessible through global portals respecting fair principles. [c, s] ae9.2 data georeferencing can be implemented to favour making links among databases and information systems. [c, s] ae9.3 genetic resources managers and researchers together have to identify all relevant data related to genetic resources that can be useful for sustainable deployment and conservation of genetic resources for the agroecology transition (from biological to socioeconomic data or regulation information). [c, s] ae9.4 data about the societal impact of genetic resources need to be measured and metrics have to be defined. [c, s] genetic resources (2024), 5 (9), 13–28 genetic resources conservation in a changing world 25 all these data need to be standardized and broadly accessible by applying the fair principles (findable, accessible, interoperable, reusable https://www.go-fai r.org/fair-principles/). synthetic overview: targeted audiences and action plan each of the 88 (altogether) arguments and recommendations has one or multiple target audiences drawn from policymakers, conservation actors and scientists. in the consensus reached by the international experts of the different genetic resources domains, policymakers are called to be highly concerned by one-third of the arguments and recommendations, with a slightly higher proportion for climate change and in situ issues, while more than half of them concern practitioners and scientists (table 4). the proportion of arguments and recommendations addressed to conservation actors is higher for the in situ and the agroecology issues because addressing both issues requires engaging a broad range of local actors, not only genetic resources specialists. overall, more than two-thirds of the arguments and recommendations addressed to scientists are jointly addressed to other audiences, reflecting the need to reinforce participatory approaches, co-development and policy support activities in research. all of these arguments and recommendations are picked up by the genetic resources strategy for europe, which defines a comprehensive action plan at national and european levels with three main objectives: (1) strengthening and widening actions for genetic resources conservation and sustainable use, (2) enabling transformative change and (3) reinforcing international cooperation. each objective of the action plan is subdivided into several sections. figure 1 shows that the action plan of the strategy addresses most of the arguments and recommendations at multiple levels, and it also shows that all aspects of the action plan are needed to respond to the three new challenges reviewed for genetic resources. perspectives despite contextual differences between crop, forest and animal genetic resources, the international panel of experts drawn from these domains recognized the emergence of a new era for genetic resources conservation and sustainable use in a context of potentially existential environmental and socioeconomic changes. such changes that trigger multiple uncertainties require adaptive responses. in this era, the broad diversity of genetic resources can provide solutions to multiple issues, but only if the threats to diversity are effectively mitigated. multiple conservation actions and sustainable uses must be considered in an integrated way. for each of the three challenges under discussion, sharing expert views across domains resulted in a comprehensive list of general recommendations that are equally strategic for each genetic resources community in europe. beyond personal scientific expertise within the panel, the general arguments and recommendations made here also feed on the scientific evidence provided by the international conventions (the convention on biological diversity cbd), platforms (the intergovernmental science-policy platform on biodiversity and ecosystem services, ipbes) and organizations (the un food and agriculture organization, fao) addressing genetic resources and biodiversity conservation issues. other genetic resources communities around the world could benefit from this work and reuse these evidencebased arguments and recommendations in two ways. firstly, other communities can use them as a benchmark for the review of their own internal work plan. secondly, these tables provide an opportunity to identify possible collaborative actions involving multiple genetic resources and conservation communities together rather than each one independently. furthermore, all genetic resources activities have value through methodological application in the broader genetic and biodiversity conservation context. for instance, the crop wild relative population management guidelines (iriondo et al, 2021) and conservation planning toolkits (maxted et al, 2015; brehm et al, 2017) are equally applicable for genetic or taxon-based biodiversity wild plant or animal conservation planning and in situ implementation. therefore, these arguments and recommendations can feed the implementation and update of the fao global plan of action on genetic resources in the different domains, the international treaty on plant genetic resources for food and agriculture, and the cbd. to address the three challenges, the experts agreed on the need for innovative solutions requiring cross-cutting collaborations, multi-actor engagement and policy support for effective implementation. actions addressing genetic resources should engage conservation managers and users of genetic resources together with scientists from life sciences and social sciences addressing new research questions related to genetic resources, and decision-makers at multiple policy levels possibly benefiting from or influencing genetic resources conservation and sustainable use. key are local actors concerned with in situ genetic resources management in production systems and conservation programmes. with these recommendations, the panel of experts urges scientists to collaborate proactively with policymakers and a broad range of actors at local, national and international levels. engaging new actors will depend on the capacity of the genetic resources communities to raise awareness of genetic resources values, share academic and non-academic knowledge and expertise on threats and solutions, co-develop efficient tools and advocate for supportive regulations. the genetic resources strategy for europe and the related action plan can help address the three challenges mentioned here and many more. in turn, disseminating the recommendations to their respective audiences will help support the uptake of the strategy. the panel of experts hopes that the statements and recommendations jointly https://www.go-fair.org/fair-principles/ 26 lefèvre et al genetic resources (2024), 5 (9), 13–28 table 4. number of arguments and recommendations addressed to different target audiences for each challenge. cc, climate change challenge; is, in situ challenge; ae, agroecology context. *, for each challenge, the sum of percentages is more than 100% because each recommendation may have multiple target audiences. **, out of the 59 recommendations to scientists, 41 (69%) are jointly addressed to other audiences. target audience challenge policymakers conservation actors scientific community total cc 8 (31%)* 14 (54%) 18 (69%) 26 is 11 (35%) 22 (71%) 19 (61%) 31 ae 8 (26%) 21 (68%) 22 (71%) 31 total 27 57 59** 88 formulated by genetic resources experts in the three domains will be sufficiently integrated into future agricultural, food security, ecological, social and political policy across europe and broader global fora to influence these sectors’ policies. the implementation of the recommendations and follow-up with policymakers will require a tailored approach taking into account the specificities of each domain: this is achieved in the sectorial plant, animal and forest genetic resources strategies for europe (respectively, ecpgr (2021); erfp (2021); euforgen (2021)). this was the first time the three genetic resources communities (agricultural crop, animal and forestry domains) have come together at a continental level to investigate the similarities and dissimilarities between the three domains and to investigate if closer linkages could produce beneficial synergies. there is wide agreement within the panel of experts that the process itself has proven beneficial: it has shown that similarities outweigh differences, and that speaking with one unified voice is more effective in the policy context. the challenges posed by climate change, the benefit in this context of in situ conservation combined with sustainable use, and the need for locally adapted diversity have become so predominant, that the genetic resources communities should strive for continuous collaboration with mutual benefits. authors’ contributions fl, db, mbdk, mb, ecl, sjh, hk, dl, gr, ss, es, tvh, mw and nm organized and chaired the three genres bridge expert panel workshops and wrote the text of the manuscript; the expert panel elaborated the figure 1. main sections of the action plan of the genetic resources strategy for europe where the recommendations for the three new challenges are considered, here grouped by statement. genetic resources (2024), 5 (9), 13–28 genetic resources conservation in a changing world 27 statements and recommendations (tables 1-3) during the workshops and reviewed the manuscript. genres bridge expert panel contributors ricardo alia (instituto nacional de investigación y tecnoloǵıa agraria y alimentaria-centro superior de investigaciones cient́ıficas, spain); hysen bytyqi (university of prishtina, kosovo); montserrat castellanos moncho (ministry of agriculture, fisheries and foood, spain); joži j. cvelbar (ministry for agriculture, forestry and food, slovenia); suzana đorđevićmilošević (singidunum university, serbia); edoardo esposito (european forest institute, spain); anna-maria farsakoglou (european forest institute, spain); jesús fernández mart́ın (instituto nacional de investigación y tecnoloǵıa agraria y alimentaria-centro superior de investigaciones cient́ıficas, spain); gustavo gandini (university of milan, italy); ewa hermanowicz (forest stewardship council, germany); mervi honkatukia (farm animals, nordic genetic resource center nordgen, norway); ivan kreft (nutrition institute, slovenia); nataša lovrić (european forest institute, finland); joana magos brehm (university of birmingham, uk); daniel mart́ın-collado (centro de investigación y tecnoloǵıa agroalimentaria de aragón, spain); claudio niggli (prospecierara, switzerland); eduardo notivol (centro de investigación y tecnoloǵıa agroalimentaria de aragón, spain); lorenzo raggi (dipartimento di scienze agrarie alimentari e ambientali, università degli studi di perugia, italy); mari rusanen (natural resources institute, finland); stefan schröder (federal office for agriculture and food, germany); paul smith (botanic gardens conservation international, uk); katja kavčič sonnenschein (slovenian forestry institute, slovenia); michèle tixier-boichard (national research institute for agriculture, food and environment, france); branislav trudic (forestry division, food and agriculture organization of united nations, italy); luis pablo ureña (institute of agricultural research and training, spain); jelka šuštar vozlič (agricultural institute of slovenia, slovenia); sharon walshe (department of agriculture, food and marine, ireland); henri woelders (wageningen university and research, the netherlands); frank wolter (nature and forest agency, luxembourg). conflict of interest the authors declare no conflict of interest. acknowledgements this work has received funding from the european union’s horizon 2020 research and innovation programme under grant agreement no 817580, genres bridge project. references brehm, j. m., kell, j., thormann, i., gaisberger, h., dulloo, e., and maxted, n. 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(2021). reconciling livestock production and wild herbivore conservation: challenges and opportunities. trends in ecology and evolution 36, 750–761. doi: https://doi.org/10.1016/ j.tree.2021.05.002 https://doi.org/10.1016/j.cosust.2013.06.001 https://doi.org/10.1016/j.cosust.2013.06.001 https://cices.eu/content/uploads/sites/8/2018/01/guidance-v51-01012018.pdf https://cices.eu/content/uploads/sites/8/2018/01/guidance-v51-01012018.pdf https://doi.org/10.1111/brv.12852 https://doi.org/10.1016/j.biocon.2020.108654 https://doi.org/10.1016/j.biocon.2020.108654 https://ipbes.net/global-assessment https://ipbes.net/global-assessment https://www.ipcc.ch/site/assets/uploads/2019/11/srccl-full-report-compiled-191128.pdf https://www.ipcc.ch/site/assets/uploads/2019/11/srccl-full-report-compiled-191128.pdf https://more.bham.ac.uk/farmerspride/wp-content/uploads/sites/19/2021/07/crop_wild_relative_population_management_guidelines.pdf https://more.bham.ac.uk/farmerspride/wp-content/uploads/sites/19/2021/07/crop_wild_relative_population_management_guidelines.pdf https://more.bham.ac.uk/farmerspride/wp-content/uploads/sites/19/2021/07/crop_wild_relative_population_management_guidelines.pdf https://www.euforgen.org/fileadmin/bioversity/publications/pdfs/1216.pdf https://www.euforgen.org/fileadmin/bioversity/publications/pdfs/1216.pdf http://www.fao.org/3/a-mm564e.pdf https://doi.org/10.1016/j.tree.2021.05.002 https://doi.org/10.1016/j.tree.2021.05.002 introduction methodology results how to adapt genetic resources conservation strategies in the context of climate change how to promote in situ conservation strategies how can genetic resources conservation contribute to and benefit from agroecological systems synthetic overview: targeted audiences and action plan perspectives authors' contributions genres bridge expert panel contributors conflict of interest acknowledgements genetic resources (2022), 3 (5), 1–9 doi: 10.46265/genresj.svvl8053 https://www.genresj.org issn: 2708-3764 a survey on the performance and status of disseminated elite n’dama cattle breeding bulls at the multiplier tier of an open nucleus breeding scheme in the gambia for the period 2011–2019 arss secka*, lamin camara, momodou jeng and olawale olaniyan west africa livestock innovation centre, pmb 14, banjul, the gambia abstract: the objectives of this survey conducted in 2020 were to profile multiplier f armers, assess the performance of disseminated n’dama breeding bulls at the multiplier cattle herds, and inquire about cattle health and production challenges. thirty-three farmers living in 33 villages in 5 regions of the gambia who received 52 elite n’dama breeding bulls from the west africa livestock innovation centre (walic) nucleus tier were interviewed. the results showed that the disseminated bulls’ performance is generally satisfactorily as asserted by 28 respondents. twentyeight respondents reported that the bulls were healthier, 31 ascribed these bulls with stronger libido, 20 asserted that female offspring from these bulls produced more milk, and 26 claimed that offspring had a faster growth rate. a total of 473 calves have been sired by these bulls thus far. however, only 19 bulls are currently present at the multiplier herds due to the exit of 33 bulls arising from various causes. cattle production challenges reported by farmers included decreasing grazing lands, rampant bush fires, and inadequate watering points; whilst listed health constraints included inadequate access to veterinary drugs and trypanosomiasis disease prevalence. the multiplication of the disseminated breeding bulls at the multiplier tier has progressed well in terms of sired offspring, but further transfer to the commercial farmer tier is slow. this situation calls for more sensitization of farmers, increased visibility of the breeding programme, and utilization of other reproductive techniques such as artificial insemination to reach more cows for enhanced genetic improvement and productivity. keywords: performance, onbs, multiplier tier, n’dama cattle bulls, the gambia citation: secka, a., camara, l., jeng, m., olaniyan, o. (2022). a survey on the performance and status of disseminated elite n’dama cattle breeding bulls at the multiplier tier of an open nucleus breeding scheme in the gambia for the period 2011–2019. genetic resources 3 (5), 1–9. doi: 10.46265/genresj.svvl8053. © copyright 2022 the authors. this is an open access article distributed under the terms of the creative commons attribution license (cc by 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. introduction developing countries like the gambia have a gross deficit in both beef and milk products as increasing demand far outweighs local production. due to its tolerance to trypanosomiasis disease, n’dama cattle is the most widely used breed by farmers in mixed production systems practised in the gambia, a country which lies within the tsetse infested belt of the african region (secka et al, 2015; olaniyan et al, 2021). n’dama cattle breed tolerance to trypanosomiasis disease, as ∗corresponding author: arss secka (seckaarss@gmail.com) well as their heat tolerance, good draught power and ability to thrive on low-quality feeds, make them highly valuable in such a context. furthermore, this multidisease resistant n’dama cattle has been recommended for low-input traditional african farming systems in areas where trypanosomiasis, ticks and tick-borne diseases are constraints to livestock production (mattioli et al, 1998). improving the productivity of n’dama cattle under low-input production systems through genetic improvement complemented with optimum management is a viable strategy for increasing local meat and milk production using indigenous livestock breeds (figure 1). received: 12.07.2021 accepted: 14.01.2022 published online: 11.02.2022 short communication https://www.genresj.org https://www.dx.doi.org/10.46265/genresj.svvl8053 https://www.genresj.org https://www.dx.doi.org/10.46265/genresj.svvl8053 mailto:seckaarss@gmail.com 2 secka et al genetic resources (2022), 3 (5), 1–9 figure 1. selected n’dama cattle breeding bulls in 2019 for the multiplier tier. photo: arss secka. a genetic improvement programme using an open nucleus breeding scheme (onbs) involving three tiers was therefore established in 1994 (bosso, 2007) by the former international trypanotolerance centre (itc), now called west africa livestock innovation centre (walic). the three implicated tiers are nucleus, multiplier and commercial farmer. the breeding goal of this onbs is to increase the growth rate and milk yield of indigenous n’dama cattle without losing their trypanotolerance and other adaptive traits (jaitner and dempfle, 1998). similar breeding schemes for n’dama cattle genetic improvement are also operational in southern senegal, guinea and mali (traoré et al, 2017; camara et al, 2019; ouédraogo et al, 2021). this onbs utilized a young sire scheme to determine the breeding values of young bulls under performance testing from weaning at 12 months of age up to 36 months within a high tsetse challenge area under similar management conditions as practised by community cattle herders. an animal model called best linear unbiased prediction (blup) was used for estimating breeding values for daily weight gain based on monthly weight from 15 to 36 months of age under high tsetse challenge, and the milk yield derived from the first 100 days of lactation was measured on a weekly basis (bosso et al, 2009). the young sire scheme was found to be as effective as the half-sib scheme where bulls are selected at the age of 5.5 to 6 years and better than the progeny testing scheme (dempfle and jaitner, 2000). these authors asserted that this scheme is also much simpler to implement, and does not require storing semen or having any waiting bulls. the multiplier tier farmers who received elite breeding n’dama cattle bulls, djallonké sheep rams, and west african dwarf goat bucks around the country formed an association called gambia indigenous livestock multipliers association (gilma) in 2002, pulling together most of the farmers at the onbs multiplier tier. the main goal of this association was to coordinate multiplication activities of the disseminated breeding males at the multiplier tier and further dissemination of their male offspring to the commercial farmer tier. the association has two main branches, each with its executive committee members, membership and bank account. the branch on the south bank of river gambia is called gilma fulladou, whilst the one on the north bank is called gilma saloum. gilma associations were supported by itc and now by walic in terms of capacity building, organizational setup and management, technical backup and financial support. after a few years of activity, the gilmas became dormant in 2007 until itc commissioned their institutional diagnosis in 2014. to help revive the gilmas, itc/walic in collaboration with the west africa rural foundation (warf), the regional project for the sustainable management of endemic ruminant livestock in west africa (progebe) and the department of livestock services, conducted a thorough and exhaustive participatory institutional diaggenetic resources (2022), 3 (5), 1–9 performance and status of disseminated n’dama cattle breeding bulls in the gambia 3 figure 2. map of the gambia showing five administrative regions. source: https://gisgeography.com/gambia-map/ nosis (pid) of the association in mid-february 2014. the overall objective of the pid was to develop a pathway for revitalizing the gilmas into vibrant self-sustaining associations that would effectively carry out their roles and deliver on their responsibilities within the itc/walic breeding programme. findings showed a lack of clear vision and mission, and limited capacity of the executive committee members in the areas of institutional management, group facilitation, participatory planning, and effective strategic partnership and ownership (olaniyan et al, 2015). a participatory revitalization plan for the gilmas was drawn up during the pid exercise. warf proposed an action plan that involved strategic reflection, capacity building support and coaching/mentoring support to the associations (warf, 2014). under the warf leadership, two workshops were convened in december 2016 and august 2017 with gilma members for a strategic reflection on and elaboration of the vision, mission and annual work plan; and capacity building on organizational management and group facilitation, communications and information management, resource mobilization, financial management, and rural entrepreneurship respectively. the coaching support to both gilma saloum and gilma fulladou was carried out in november 2020. during the gilmas’ institutional management strengthening, itc/walic had also conducted four technical capacity strengthening workshops for their membership covering animal breeding and selection, management, animal diseases recognition and control, feeds conservation, pasture production, compost pen construction (itc, 2017), and hygienic milk collection and processing (itc, 2018). there has been much improvement in the gilmas’ structural organization and management as well as their technical knowledge and capacity, but the associations have not yet reached the desired level of performance as a livestock breeding association vis-à-vis their vision and mission. the existence of similar breeders’ association for the n´dama cattle breed in senegal, for zebus (azawak and fulani cattle breeds), baoulé, and baoulé x zebu crosses in burkina faso have been reported (ouédraogo et al, 2021). the dissemination of elite breeding bulls from the nucleus tier to the community multiplier tier resumed in 2011, after a six-year break (2004–2010), through the intervention of and collaboration with a regional project on sustainable management of endemic ruminant livestock in west africa, the progebe project (2008–2014), that revived the breeding programme. from 2011 to 2019, a total of 52 elite n’dama breeding bulls were disseminated to the multiplier farmers at various locations around the country. walic make regular biannual monitoring visits to maintain contact with farmers and check on the status of the disseminated breeding bulls. however, gaps exist in the overall profile of the multiplier farmers. the farmers’ level of satisfaction or dissatisfaction with the disseminated bulls’ general performance over nine years has not been reported. therefore, this survey aimed at filling the gap by profiling multiplier farmers, assessing the performance of the disseminated elite n’dama breeding bulls from 2011 to 2019, and inquiring about cattle health and production challenges confronting the farmers. materials and methods a five-day survey mission was undertaken in april 2020 to interview 40 farmers around the country who received 52 elite n’dama breeding bulls from itc/walic during the 2011–2019 period, and collect primary data on farmers’ profiles, breeding bulls’ performance, as well as cattle production and health challenges confronting the farmers. thirty-three farmers in 33 villages located in five regions of the gambia (figure 2) were interviewed during the field mission. the remaining seven target farmers were either not available or had received breeding west africa dwarf goat bucks or djallonke sheep rams whose information are not included in this article. the target group of this survey was the multiplier tier of the open nucleus breeding scheme (onbs). this second tier of the onbs consists of farmers whose cattle herds received and used elite n’dama breeding bulls originating from the on-station walic nucleus tier. as the name implies, the multiplier tier’s role is to produce more outstanding offspring breeding bulls for dissemination to the commercial farmer cattle herds around the country. https://gisgeography.com/gambia-map/ 4 secka et al genetic resources (2022), 3 (5), 1–9 two sets of questionnaires were developed for this survey (supplemental data). the first questionnaire was a checklist to collect information on farmers’ names, village, bull identification number, bull introduction date, bull status, cattle herd size, number of cows in the herd, number of cows mated, number of calves sired, bull exit date and reason for the exit. the second questionnaire was divided into three sections. the first section asked for the beneficiary farmer profile, the second section dealt with account and performance of received animals, and the last section asked for cattle health and production challenges, and their perceived proposed solutions to address these challenges. the questionnaires were written in english and administered through a face-to-face interview by the authors using common local dialects (wollof, mandinka and fulla) since most of the farmers cannot read or write in english. these dialects are understood by both the interviewers and the interviewees. no intermediary translation was required. four farmers were partly interviewed through telephone calls when some outstanding information could not be provided by members of the household met on the ground. telephone calls were also made at times to follow up or verify collected data from the interviewed farmers. all collected data were organized, sorted, analyzed, and summarized using descriptive statistics into tables and figures showing results of responses to various parameters of interest. the farmers’ telephone numbers were used to create a whatsapp group to serve as a communication platform. the information derived from the accounting of the disseminated bulls provides a record of their status and performance. the responses to the various questions were analyzed to deduce the performance of the bulls, the perceived cattle health and production challenges, and the farmers’ proposed solutions to remedy stated challenges. results profile of the multiplier tier farmers the highest number of villages and farmers implicated in this multiplier tier were found in the districts of upper fulladou west, kiang west, and niamina east. the age of the interviewed farmers ranged from 43 to 80 years. out of the 33 farmers, only 2 were female, 3 had western tertiary education (2 masters and 1 doctorate holders), and 25 had access to whatsapp either directly on their phones or a family member’s living in the same compound (table 1). status of disseminated elite n’dama breeding bulls a total of 52 elite n’dama breeding bulls selected from the nucleus herd were disseminated to 35 cattle herds in the 33 villages distributed in 11 districts and 5 regions around the gambia from 2011 to 2019 (table 2). by april 2020, only 19 bulls were present in the multiplier cattle herds around the country (supplemental table 1). thirty-three bulls have exited from the community cattle herds due to various reasons such as culling (27%), sales (15%), death (9%), accident (9%), disease (6%), low libido (6%), loss (6%), aggressiveness (6%), and slaughter (3%) (supplemental table 1). bulls are usually culled after a maximum service period of five years in the same cattle herd as the male breeder. culling reduced the chances of the bulls mating their daughters thus lowering the risk of inbreeding. four hundred and seventy-three calves were reported to have been sired by the disseminated bulls from 2011 to 2019 (table 2). performance of disseminated breeding bulls results of the questionnaire survey on the performance of breeding animals aggregated at national level are shown in figure 3, which provides the results of the questionnaire survey on the performance of breeding animals aggregated at national level. the health performance parameter measured the resilience of the disseminated bulls to endemic diseases versus other bulls from different sources. twentyeight of the 33 respondents asserted that these bulls performed better health-wise than other bulls. the breeding performance parameter compared the libido and fertility of the disseminated bulls with others from different sources. thirty-one of the 33 respondents affirmed that these bulls had stronger libido and fertility than other bulls. similarly, the growth performance and milk production of disseminated bulls’ offspring were reported faster and higher, respectively, than offspring from other sources. farmers who received disseminated breeding bulls were satisfied w ith the general performance of the bulls with their offspring as asserted by 28 respondents compared to 5 respondents that expressed dissatisfaction. however, most of the farmers had not disseminated mature offspring breeding bulls to commercial farmers’ cattle herds for breeding purposes. eight farmers had received bulls less than two years before the survey and had either not seen offspring or female offspring had not started lactating, therefore not responding to questions on offspring growth performance and milk production. challenges confronting producers at the multiplier tier data collected from the interviewed farmers focusing on challenges and proposed solutions are presented in table 3. most of the health and production challenges highlighted include trypanosomiasis, low access to veterinary drugs and services, inadequate feed and water, bush fires and reduced grazing fields. these challenges could limit the performance of the disseminated animals at the multiplier cattle herds. farmers’ proposed solutions to addressing these challenges include moving towards intensive management of fewer animals for market-oriented enterprise; improving access to veterinary drugs and services; and general public sensitization to reduce bush fire incidents. genetic resources (2022), 3 (5), 1–9 performance and status of disseminated n’dama cattle breeding bulls in the gambia 5 table 1. profile of the 33 interviewed multiplier tier farmers region district no. of villages no. of farmers interviewed no. of westerneducated farmers no. accessing whatsapp farmer’s gender age range (years) m f west coast kombo east 1 1 1 1 1 0 67 lower river kiang west 6 6 1 5 5 1 43-70 north bank upper baddibu 2 3 0 1 3 0 46–56 central river upper saloum 1 1 0 0 0 1 70 nianija 4 4 0 4 4 0 55–80 niani 2 2 0 2 2 0 47–59 upper fulladou west 8 6 0 4 6 0 45–70 lower fulladou west 1 1 0 1 1 0 70 niamina east 5 5 0 5 5 0 43–66 niamina west 1 1 0 0 1 0 60 upper river sandu 2 3 1 2 3 0 54–60 total 11 33 33 3 25 31 2 43–80 table 2. account of disseminated bulls at the multiplier tier. additional details are provided in supplemental table 1. regions districts no. of villages no. of cattle herds implicated no. of bulls supplied (2011–2019) no. of bulls present in 2020 no. of calves sired (2011–2019) west coast kombo east 1 1 2 1 24 lower river kiang west 6 6 6 2 64 north bank upper baddibu 2 3 4 2 105 central river upper saloum 1 1 1 1 0 nianija 4 4 4 0 145 niani 2 3 3 3 24 upper fulladou west 8 7 16 4 38 lower fulladou west 1 1 2 2 0 niamina east 5 5 9 2 25 niamina west 1 1 1 0 28 upper river sandu 2 3 4 2 20 total 11 33 35 52 19 473 6 secka et al genetic resources (2022), 3 (5), 1–9 figure 3. responses of 33 multiplier tier farmers to bulls breeding performance indicators table 3. challenges confronting livestock producers and proposed solutions region no. of districts no. of farmers interviewed health challenges production challenges marketing challenges proposed solutions west coast 1 1 none lack of sufficient land for free grazing of cattle none introduce techniques of intensive production using exotic breeds to increase milk and meat production lower river 2 5 access to veterinary drugs bush fires causing feed shortages during the dry season none improve access to veterinary drugs and services. sensitization activities to reduce bushfires in the dry seasons especially in kiang west district north bank 1 3 trypanosomiasis infections in dry seasons insufficient clean drinking water for the cattle during the dry season none improve access to veterinary drugs and provision of drinking points for cattle central river 6 21 access to veterinary drugs insufficient grazing lands none improved access to veterinary services and drugs. upper river 1 3 none lack of clean drinking water none provide cattle drinking points genetic resources (2022), 3 (5), 1–9 performance and status of disseminated n’dama cattle breeding bulls in the gambia 7 discussion multiplier tier farmers’ profile among the 33 farmers who were interviewed, 30 were illiterate in the english language, whilst three are highly educated professionals in the western form of education system. the presence of only 2 women out of 33 interviewed farmers show that male farmers dominate the onbs cattle multiplier tier around the country. this observation follows the national pattern of males dominating the ownership of cattle production and management in the gambia. the age of the interviewed multiplier farmers ranging from 43 to 80 years is indicative of the ages of the household heads. in most instances, the head of the family owns the disseminated elite breeding bulls. however, the daily management of the cattle herds rests on the shoulders of younger family members or contracted herdsmen. account of disseminated bulls the districts of upper fulladou west, niamina east, kiang west, and nianija received most of the disseminated bulls. this observed distribution pattern is explained by the fact that the first three districts have been participating in many itc/walic activities, particularly the genetic improvement programme. moreover, the districts of kiang west, niamina east and nianija were also intervention sites of the regional project for the sustainable management of endemic ruminant livestock in west africa (progebe) in 2008–2014, which had a component of supplying improved breeding bulls from itc/walic to their contact cattle farmers. out of the 52 disseminated bulls, as of april 2020, only 19 were present in multiplier cattle herds due to various reasons, as shown in table 2. performance of disseminated breeding bulls the findings on the breeding performance of disseminated bulls at the multiplier tier showed that the breeding programme was attaining its target breeding goal of increased growth rate and milk offtake without compromising the tolerance to endemic diseases like trypanosomiasis. both the health and breeding performance of the disseminated bulls at the multiplier tier got high scores from the respondents. therefore, it appears that the high genetic potential for higher growth rate and milk offtake contained in these bulls had been transferred to their offspring. this survey’s findings corroborate a survey conducted in 2003, which assessed the adoption and impact of the itc/walic genetic improvement programme at the multiplier tier (agyemang, 2003). findings from the 2003 survey showed that participating villages, associations and households were pleased with the benefits from the improvement programme and disseminated breeding bulls performed well under village conditions. all participating respondents believed that the use of improved n’dama bulls would improve their livelihoods. although the genetic gains at the multiplier tier had not been calculated during the 2003 survey, mattioli et al (1998) found that from 1994 to 2004 the estimated average breeding values for weight at 36 months ranged between 0 to 6.32kg at the nucleus tier. they further asserted that weight at 36 months showed the highest genetic gain with a response of 0.40kg per year. hence, it is highly probable that the bulls disseminated to the multiplier farmers from 2011 to 2019 may have had similar or higher genetic gains for weight at 36 months. siring many calves at the multiplier tier cattle herds is one of the ultimate aims of disseminating improved breeding n’dama bulls from the nucleus tier. disseminated bulls during this period have sired a total of 473 healthy and strong calves. further dissemination of bulls’ male offspring from the multiplier tier to the commercial farmer tier was found to be limited – only two offspring bulls in 2018 and one in 2019. this challenge has been recognized for some years now and efforts have been taken to remedy the situation. institutional diagnostics of gilmas in 2014 showed that there are many organizational, management, capacity and financial limitations affecting the association. activities for strengthening the gilmas have been implemented and hopefully, this situation of low transmission of breeding bulls from the multiplier tier to the commercial farmer tier will improve in the near future. it also appears that many livestock farmers are not aware of the walic breeding programme. this calls for more sensitization of farmers and increased visibility of the programme. artificial i nsemination is also a faster reproductive tool where stored semen from bulls could be used in many cows for a longer duration than live bulls. challenges confronting livestock producers the expression of disseminated bulls’ genetic values could be affected by environmental factors such as feed, management, water, changing climate and diseasecausing pathogens. various challenges on health, production and marketing were assessed during this survey. although there seem to be no marketing challenges, respondents asserted some challenges under health and production domains. access to veterinary drugs was reported as a limitation in both central river and lower river regions. this could be explained by the fact that private veterinary drug outlets operating in these regions are located mainly in towns and big villages, whilst these farmers are mostly residing in small villages. however, they could still travel to the veterinary outlets or call the public livestock officers to attend to their animals’ health needs. trypanosomiasis was mentioned as a challenge in north bank region particularly during the dry season when the animals are nutritionally stressed. n’dama cattle are trypanotolerant, hence would still survive and produce in trypanosomiasis prevalent areas (mattioli et al, 1998). 8 secka et al genetic resources (2022), 3 (5), 1–9 the major highlighted cattle production challenges were inadequate grazing lands, the occurrence of bush fires ravaging pastures and rangelands, and insufficient cattle drinking points. these challenges are very serious factors that could retard the progression of increased livestock productivity at both multiplier and commercial farmer tiers. adequate good-quality feed and water are essential for growth, maintenance and production. grazing lands are dwindling, resulting from increasing human population pressure and associated competing agro-industrial and residential activities. conflicts may arise between pastoralists and farmers during cropping seasons as access routes to the grazing areas are often blocked. this situation leads to the transhumance of cattle to other regions in the gambia or the surrounding republic of senegal. cattle drinking points are limited and these negative impacts are felt during the long dry season. many solutions were proposed by the respondents to solve these challenges. they include the use of exotic cattle breeds under intensive management for increased meat and milk production; community sensitizations to stop the harmful practice of bush fires; government support for the provision of cattle watering points; and improving access to veterinary services and drugs. embracing the proposed solutions requires capital investment from farmers, the private and public sectors, and non-governmental organizations (ngos). there are some initiatives taken up by the government and development partners to address these challenges and are registering some significant progress. conclusion although the survey has used mainly qualitative data obtained from farmers’ perceptions and recollections and not backed by longitudinal quantitative production data, it has provided some important insights about multiplier tier farmers, the performance of disseminated bulls, and constraints limiting cattle production and productivity. the survey has profiled the farmers involved in the ongoing breeding programme and the general performance of the disseminated bulls at the multiplier tier. farmers – more than 40 years old, predominantly male and very few with higher education – generally practise subsistence farming and extensive management of n’dama cattle herds combined with the cultivation of crops. farmers are generally satisfied with the health and breeding performances of the disseminated breeding bulls at the multiplier tier. they have asserted that both the growth rate and milk outputs of these bulls’ offspring are higher than those from other bulls. only three offspring bulls were disseminated from the multiplier tier farmers to the commercial farmer tier cattle herds as breeders. this figure is very low and therefore the following actions are proposed: 1) more sensitization of farmers, 2) increased visibility of the breeding programme, and 3) use of other dissemination tools such as artificial insemination that could reach more cows for enhanced genetic improvement and productivity. major health challenges mentioned by the farmers include trypanosomiasis and inadequate access to veterinary services and drugs. on production challenges, issues include insufficient grazing lands, frequent bush fires ravaging large amounts of fodder, and insufficient livestock watering points during the long dry season. many initiatives are taken by individual farmers, communities, private and public sector-led projects to address prevailing livestock production and health challenges. the gambia indigenous multipliers association (gilma) has been found weak, and activities were conducted to revamp this association to facilitate and sustain the transfer of improved genetic gains across the three-tier open nucleus breeding scheme. acknowledgements the authors are grateful to the government of the gambia for providing financial support in the form of monthly subventions that keep the walic staff personnel on board and facilitated travel costs for the collection of data during the survey period. supplemental data questionnaires used in farmer’s survey: questionnaire 1: check list for disseminated bulls questionnaire 2: performance of elite n’dama breeding bulls at the multiplier tier supplemental table. checklist data on account of disseminated bulls translated abstract in french author contributions arss secka coordinated the survey, took part in the collection, collation and analysis of data from farmers, and drafted the article manuscript. lamin camara provided inputs into the manuscript and participated in the collection, collation and analysis of data from farmers. momodou jeng provided inputs into the manuscript and participated in the data collection from farmers. olawale olaniyan provided inputs into the manuscript. conflict of interest 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(2017). production objectives, trait and breed preferences of farmers keeping n’dama, fulani zebu and crossbred cattle and implications for breeding programs. animal 11(4), 687–695. doi: https://doi.org/10.1017/s1751731116002196 warf (2014). participatory institutional diagnosis of gambia indigenous multipliers association. west africa rural foundation, senegal and international trypanotolerance centre, the gambia. http://www.lrrd.org/lrrd21/8/boss21135.htm http://www.lrrd.org/lrrd21/8/boss21135.htm https://doi.org/10.1017/s1751731118001544 https://doi.org/10.1016/s0001-706x(98)00051-5 https://doi.org/10.1016/s0001-706x(98)00051-5 https://doi.org/10.1017/s207863361500020x https://doi.org/10.1017/s207863361500020x https://doi.org/10.1007/s11250-020-02461-w https://doi.org/10.1007/s11250-020-02461-w https://doi.org/10.3390/su13042125 http://repository.au-ibar.org/handle/123456789/476 http://repository.au-ibar.org/handle/123456789/476 https://doi.org/10.1017/s1751731116002196 introduction materials and method results profile of the multiplier tier farmers status of disseminated elite n'dama breeding bulls performance of disseminated breeding bulls challenges confronting producers at the multiplier tier discussion multiplier tier farmers' profile account of disseminated bulls performance of disseminated breeding bulls challenges confronting livestock producers conclusion acknowledgements supplemental data author contributions conflict of interest statement original article genetic resources (2022), 3 (6), 38–48 doi: 10.46265/genresj.wczg9712 https://www.genresj.org issn: 2708-3764 nutritional and phenotypic variations among newly selected african eggplant (solanum aethiopicum l.) olawale olusesan. oguntolu, christian okechukw. anyaoha *, victor anozie chikaleke and joseph akindojutimi temidayo olofintoye national horticultural research institute, p.m.b 5432, jericho reservation area, idi ishin, ibadan, oyo state, nigeria abstract: african eggplant (solanum aethiopicum l.) is an important but underutilized leafy and fruit vegetable. systematic characterization of available eggplant accessions for morphological and nutritional traits is paramount to their genetic improvement. this study characterized the diversity among selected s. aethiopicum accessions from nigeria to identify promising genotypes for future eggplant breeding activities in the region. twenty new purified african eggplant accessions collected from farmers’ fields were characterized using morphological and nutritional descriptors. the accessions varied significantly in qualitative, quantitative and nutritional parameters. top performers for selected yield-contributing traits and nutritional parameters were nhepa54, nhepa39-1, nheap10, nhepa10, nhepa1, nhepa56, nhepa23 for vitamin c, iron, calcium, days to flowering, number of branches, plant height at maturity and number of fruits per plant respectively. the first four principal components accounted for 72.42% of total variability. the first principal component with the largest variation (28.77%) was loaded with number of branches, plant height at maturity, number of fruits per cluster, number of fruits per plant, and fruit width. a significant positive association was exhibited between iron and yield-increasing traits such as number of fruits per plant (r = 0.532) and number of fruits per cluster (r = 0.551). plant height at maturity positively correlated with vitamin c (r = 0.492) indicating predictable success in selecting top-performing eggplant genotypes combining high-yield potential and nutritional content. top-performing eggplant genotypes identified in this study could be deployed as donors for a hybridization programme to develop new eggplant varieties with higher yield potential and improved nutritional quality. keywords: diversity, accessions, breeding, principal component, variability, correlation citation: oguntolu, o. o., anyaoha, c. o., chikaleke, v. a., olofintoye, j. a. t. (2022). nutritional and phenotypic variations among newly selected african eggplant (solanum aethiopicum l.). genetic resources 3 (6), 38–48. doi: 10.46265/genresj.wczg9712. © copyright 2022 the authors. this is an open access article distributed under the terms of the creative commons attribution license (cc by 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. introduction the african eggplant (solanum aethiopicum l.) is one of the important indigenous fruit vegetables widely grown and consumed across most regions of tropical africa. it is the third most consumed fruit vegetable after tomato, pepper and onion both in quantity and value in the region (osei et al, 2010). mature fruits of african eggplant are eaten fresh, with fried groundnuts or used to prepare special delicacies called ’african salad’ in southern nigeria (igwe et al, 2003). a significant increase has been observed in its production across ∗corresponding author: christian okechukw. anyaoha (kriskoty@yahoo.com) sub-saharan africa from 606,672 tonnes in 1994 to 2,079,920 tonnes in 2018 (fao, ifad, unicef, wfp and who, 2018). eggplant is considered amongst the healthiest fruit vegetables for its low calories and high concentration of various macro and micro minerals essential for maintaining good health (docimo et al, 2016). they are rich sources of fibres, vitamins (a, b1, b2, b6, b12, c, d), magnesium, calcium and iron even though potassium is the most abundant mineral ranging from 200 to 600mg/100g of fresh matter (kowalski et al, 2003; nyadanu and lowor, 2015; nimenibo and omotayo, 2019). the crop has been reported to play an essential role in meeting the nutritional needs of the igbo-speaking tribe in southern nigeria received: 14.9.2021 accepted: 05.07.2022 published online: 07.09.2022 https://www.genresj.org https://www.dx.doi.org/10.46265/genresj.wczg9712 https://www.genresj.org https://www.dx.doi.org/10.46265/genresj.wczg9712 mailto:kriskoty@yahoo.com genetic resources (2022), 3 (6), 38–48 genetic variation of african eggplant 39 where consumption of fresh fruits might be of great benefit to glaucoma patients and to prevent heart disease (igwe et al, 2003; denkyirah, 2013). s. aethiopicum is used in the management and treatment of diarrhoea and hypertension (adeniji and aloyce, 2012). the high yield and nutritive value of the leaves and fruits complemented with resistance to pests and diseases endear the crop to consumers, farmers and researchers (bonsu et al, 1998; toppino et al, 2008; taher et al, 2019). eggplants belong to the solanaceae family, which encompasses three closely related cultivated species endemic to afro-eurasia. two sections exist at the subgenus level, namely melongena and oliganthes sections. the section melongena comprises two species (s. melongena and s. macrocarpon) while the oliganthes group has only one species (s. aethiopicum). s. aethiopicum has been grouped into four different ecotypes or cultivars including aculetum, gilo, kumba and shum groups as revealed by similarities in genotypic characterization through varied phenotypes (sharmin et al, 2011). aculetum is mostly used as ornamental, gilo is used for its fruits, kumba is for both fruits and leaves while shum is used for its leaves (lester and daunay, 2003). consumer preferences for an african eggplant cultivar are based on a number of traits including fruit size, form, fruit colour and taste (sweet or bitter). morphological characterization using conventional descriptors has proved useful for describing and establishing relationships among cultivar groups and accessions in scarlet eggplants (adeniji et al, 2013). the enormous morphological variability present in the eggplant family, despite being characterized by a narrow genetic base, might be attributed to new segregants emanating from natural hybridization and backcrossing (meyer et al, 2012). despite their socioeconomic significance and their role in meeting the nutritional needs of the everincreasing population across sub-saharan africa, these heirloom and indigenous adapted cultivars are becoming less popular, and the efforts to improve them for traits of interest to farmers and end-users are scarce (bationo-kando et al, 2015). continuous planting and selection of many diverse cultivars of s. aethiopicum by small-scale farmers as well as the existence of germplasm collections have helped to conserve the majority of desired traits within families over the years. the long period of selection by these poorly resourced farmers has resulted in a number of landraces exhibiting different variants with unique traits such as earliness, colour, size and taste. in essence, the african eggplant has long been neglected by formal crop improvement programmes except in breeding programmes where it is used as a source of specific traits. furthermore, they are considered neglected and underutilized crops since their nutritional and economic potentials are mostly underexploited (padulosi et al, 2019). systematic characterization of african eggplant accessions using morphological and nutritional traits is an important prerequisite toward their conservation and use in further studies and genetic improvement in the region (avrdc, 2003). unfortunately, minimal efforts have been directed to identify and select promising genotypes with a good combination of desired agronomic and nutritional qualities that could be used as parental materials for hybridization or released as new open-pollinated varieties. the process of germplasm characterization and trait screening to identify and select desired gene combinations can be challenging. this study characterized newly selected and purified eggplant accessions collected in nigeria to ascertain their mineral composition and to identify promising elite lines with the best combination of desired agronomic traits that could further be deployed for eggplant genetic improvement programmes. materials and methods experimental materials twenty new eggplant accessions (table 1) were selected from the 2019 characterization of germplasm collections from farmers’ fields across the south-west and northcentral regions of nigeria based on observable traits under field conditions. these materials have gone through two cycles of selection and selfing. experimental design and conditions the experiment was conducted at the experimental field of the national horticultural research institute (nihort), ibadan, oyo state, nigeria. nihort is located in the humid forest-savannah transition zone (210m above sea level, 7◦ 30′ n, 3◦ 54′ e) with a bimodal annual rainfall pattern of about 120–128 rainy days amounting to 1,200–1,400mm. pan evaporation is between 1,550–1,600mm. the wet season is from march through october and the dry season from november through february with an annual maximum temperature ranging between 27◦c and 34◦c and an annual minimum temperature of 20–23◦c (ogungbenro and morakinyo, 2014). the eggplant accessions were first raised in a nursery and transplanted to the field after 35 days using a randomized complete block design with three replications. the plot size was 2 x 1m with a spacing of 0.5 x 0.6m between and within rows having 10 plants per plot. manual weeding was carried out to reduce the competitiveness of soil nutrients. fertilizer was not applied while insecticides (cypermethrin) were used at the rate of 200ml/20l of water when needed to reduce damage caused by insects. phenotypic characterization phenotypic data collection was carried out on 5 uniform tagged plants out of 10 plants from each plot for the 20 accessions using 12 quantitative (number of branches, number of days to flowering, number of days to 50% 40 oguntolu et al genetic resources (2022), 3 (6), 38–48 flowering, plant height at maturity, number of fruits per cluster, number of harvested fruits, weight of harvested fruits, petiole length, fruit length, fruit width, stem girth, pedicel length) and 10 qualitative traits (fruit colour, stem colour, petiole colour, leaf hairs, sepal colour, fruit colour, fruit shape, fruit position, fruit-end shape, presence/ absence of stripes). physiochemical variables were iron, vitamin c and calcium, using the descriptor list for eggplant by the international board for plant genetic resources (ibpgr, 1990). table 1. status, collection source and states of solanum aethiopicum l. accessions collected in nigeria accessions status source states 1 nhepa01 farmers cultivar local market ogun 2 nhepa03 farmers cultivar local market ogun 3 nhepa10 farmers cultivar local market ogun 4 nhepa12 farmers cultivar farmers kogi 5 nhepa17 farmers cultivar farmers kogi 6 nhepa19 farmers cultivar farmers kogi 7 nhepa23 farmers cultivar farmers kogi 8 nhepa35 farmers cultivar farmers kogi 9 nhepa36 farmers cultivar farmers kogi 10 nhepa38 farmers cultivar farmers kogi 11 nhepa39-1 farmers cultivar farmers kogi 12 nhepa39-2 farmers cultivar farmers kogi 13 nhepa39-3 farmers cultivar farmers kogi 14 nhepa51 farmers cultivar farmers kaduna 15 nhepa52 farmers cultivar farmers kaduna 16 nhepa53 farmers cultivar farmers kaduna 17 nhepa54 farmers cultivar farmers kaduna 18 nhepa55 farmers cultivar farmers kaduna 19 nhepa56 farmers cultivar farmers kaduna 20 yalo farmers cultivar green seed company oyo calcium, iron and vitamin c determination fruit samples were dried in an oven at 600◦c for 4 hours. ashes and crucibles were previously decontaminated with a solution of 10% nitric acid at rest for a night and rinsed. then, 10ml of 5% nitric acid was added to the sample, and this mixture was heated until complete dissolution of the ash which was then filtered. after the sample had reached room temperature, the solution was put into a 25ml volumetric flask and the volume supplemented with deionized water. the determination of calcium and iron contents was performed according to aoac method 2005 using an atomic absorption spectrophotometer flame (bulks scientific® model aa 240). calibration curves for each element were plotted using standard mineral diluted with deionized water. all analyses were performed in triplicate; the results were expressed in milligrams per 100g (mg/100g) of sample on a dry basis. the amount of vitamin c in analyzed samples was determined by titration using the method described by mondal et al., (1995). about 0.5g of sample were soaked for 10 minutes in 40ml metaphosphoric acidacetic acid (2%, w/v). the mixture was centrifuged at 3,000rpm for 20 minutes and the supernatant obtained was diluted and adjusted with 50ml of bi-distilled water. ten (10)ml of this mixture was titrated to the endpoint with dichlorophenol-indophenol (dcpip) 0.5g/l (aoac. 1990). statistical analysis analysis of variances (anova) was calculated using plant breeding tools (ver.1.1.0, http://bbi.irri.org/prod uct) to determine significant variations in quantitative characters among the eggplant genotypes. the estimate of co-efficient of variation (cv) was calculated using the standard formulae (burton, 1952) and expressed in percentage. inter-species diversity pattern was analyzed through ward’s minimum variance while correlation, dendrogram clustering and principal components analysis (pca) were carried out using star software. results the frequency distribution of qualitative traits observed in all 20 accessions is presented in table 2. all the genotypes (100%) exhibited green stems, petioles and sepals. at the reproductive stage, 48.48% of the fruits had a white colour, 45.45% expressed lemon green while 3.03% were light green and 3.03% exhibited deep green fruit colour. three prominent fruit shapes were observed: oval (51.51%), long (30.30%) and round (18.18%). all accessions exhibited perpendicular fruit position with 69.69% and 30.30% of the populations having pointed and flat ends respectively. the fruits of selected eggplant accessions are presented in figure 1. table 3 lists the descriptive statistics measures of spread: mean, range, standard deviation and coefficient of variation (cv). the partitioning of the means revealed high significant variations for all traits at p ≤ 0.01. for most traits, higher variations in terms of range and cv were observed in the nutritional data compared to the phenotypic data. the highest cv was recorded for calcium (43.91%) followed by average genetic resources (2022), 3 (6), 38–48 genetic variation of african eggplant 41 table 2. qualitative traits of 20 solanum aethiopicum accessions traits modality frequency (%) 1 stem colour green 100 2 petiole colour green 100 3 leaf hairs very few 100 4 sepal colour green 100 5 fruit colour white 48.48 lemon green 45.45 light green 3.03 deep green 3.03 6 fruit shape oval 51.51 long 30.3 round 18.18 7 fruit position direct 100 8 fruit end pointed 69.69 flat 30.3 9 stripe presence present 100 figure 1. sample of fruits for selected eggplant accessions, where v04, v10, v12, v17, v19, v24, v29 and v35 represent nhepa4, nhepa10, nhepa12, nhepa17, nhepa19, nhepa24, nhepa29 and nhepa35 respectively. number of fruits per plant (37.13%) and average yield per plant (32.21%). the top performers for selected yield-contributing traits and nutritional parameters are nhepa54, nhepa39-1, nhepa10, nhepa10, nhepa1, nhepa56, nhepa23, for vitamin c, iron, calcium, days to flowering, number of branches, plant height at maturity and number of fruits per plant, respectively, while nhepa54 was outstanding for high yield potential and vitamin c content. accession nhepa10 was the top performer for calcium and days to flowering while average number of fruits per cluster had nhepa17, nhepa19, nhepa23 as the top performers. principal component analysis (pca), which is a statistical technique used to emphasize variation and bring out strong patterns in data sets, was performed to show the traits that best contributed to the observed genetic variation. the eigenvalues and proportion of accounted variance for each variable are shown in table figure 2. distribution of 20 eggplant accessions for the first two principal components based on 14 quantitative traits. dtf, days to flowering; nob, number of branches; ph, plant height at maturity; nofpc, average number of fruits per cluster; nof, average number of fruits per plant; yld, average yield per plant; fl, fruit length; fwd, fruit width; sd, stem diameter; pet.l, petiole length; ped.l, pedicel length. numbers 1-20 represent genotypes: 1, nhepa01; 2, nhepa03; 3, nhepa10; 4, nhepa12; 5, nhepa17; 6, nhepa19; 7, nhepa23; 8, nhepa35; 9, nhepa36; 10, nhepa38; 11, nhepa39-1; 12, nhepa39-2; 13, nhepa39-3; 14, nhepa51; 15, nhepa52; 16, nhepa53; 17, nhepa54; 18, nhepa55; 19, nhepa56; 20, yalo. 4. pc1 had an eigenvalue of 4.027 while pc2, pc3 and pc4 had eigenvalues of 2.889, 1.861 and 1.363, respectively. the first four principal component axes (pca) accounted for 28.77%, 20.64%, 13.29%, 9.73% of the total variation individually and, cumulatively, 72.42% of the total variability while the first two pcs contributed 49.41% (figure 2). the first pc axis, which accounted for the highest proportion (28.77%) of the variability, was dominated by traits with relatively high factor scores (> 2.60) corresponding to number of branches, plant height at maturity, number of fruits per cluster, number of fruits per plant, and fruit width. the second pc axis was dominated by days to flowering, fruit length, stem diameter, petiole length and pedicel length. also, the third pc axis was dominated by average yield per plant, fruit length, stem diameter, petiole length and pedicel length while the fourth pc axis was dominated by days to flowering, number of branches, fruit length, fruit width and petiole length. correlations between pairs of quantitative variables are recorded in table 5. there was no significant association between the nutritional parameters except for a negative moderate significant association between vitamin c and iron (r = -0.50, p < 0.05). iron content correlated positively with number of branches (r = 42 oguntolu et al genetic resources (2022), 3 (6), 38–48 ta bl e 3. n ut ri ti on al an d ph en ot yp ic da ta fo r re le va nt yi el dco nt ri bu ti ng tr ai ts on 20 a fr ic an eg gp la nt ac ce ss io ns , in cl ud in g de sc ri pt iv e st at is ti cs m ea su re s of sp re ad . m ea ns va ry si gn ifi ca nt ly fo r al lt ra it s at p = 0. 05 .d tf ,d ay s to flo w er in g; n ob ,n um be r of br an ch es ;p h ,p la nt he ig ht at m at ur it y; n of pc ,a ve ra ge nu m be r of fr ui ts pe r cl us te r; n of ,a ve ra ge nu m be r of fr ui ts pe r pl an t; yl d ,a ve ra ge yi el d pe r pl an t; fl ,f ru it le ng th ; fw d ,f ru it w id th ; st em d ,s te m di am et er ; pe t. l, pe ti ol e le ng th ; pe d. l, pe di ce ll en gt h; c v, co ef fic ie nt of va ri at io n; sd , st an da rd de vi at io n. n u tr it io n al da ta (m g/ 10 0g ) ph en ot yp ic da ta a cc es si on s v it .c ir on c al ci u m d t f n ob ph (c m ) n of pc n of y ld (g ) fl (m m ) fw d (m m ) st em d (m m ) pe t. l (m m ) pe d. l (m m ) n h ep a 01 3. 11 0. 43 7. 73 10 9. 12 7. 13 57 .6 6 2. 70 58 .5 5 13 70 .1 0 65 .9 0 37 .3 7 19 .5 0 23 .0 8 20 .0 1 n h ep a 03 2. 02 0. 47 4. 47 10 4. 00 5. 79 48 .5 8 4. 98 74 .6 1 16 99 .1 7 67 .3 1 32 .4 5 11 .8 4 18 .5 7 15 .1 7 n h ep a 10 2. 90 0. 50 14 .2 4 97 .9 1 4. 39 46 .3 1 4. 49 81 .0 3 13 20 .7 2 58 .5 4 22 .7 3 10 .4 4 19 .9 6 19 .0 6 n h ep a 12 2. 90 0. 25 8. 88 10 3. 04 5. 01 54 .5 8 4. 49 81 .6 8 16 73 .4 1 50 .4 9 36 .3 2 12 .5 7 20 .2 4 15 .0 6 n h ep a 17 3. 15 0. 43 4. 86 10 9. 44 4. 70 50 .6 9 5. 79 65 .2 9 17 26 .4 8 45 .3 7 32 .6 6 17 .5 8 14 .7 2 14 .8 6 n h ep a 19 3. 99 0. 50 10 .6 9 10 6. 56 5. 48 52 .4 7 5. 79 76 .3 8 15 22 .6 0 44 .9 1 33 .0 5 13 .7 6 12 .9 3 11 .7 8 n h ep a 23 4. 81 0. 46 4. 42 10 7. 52 6. 42 58 .8 0 5. 79 11 1. 71 17 31 .0 5 45 .6 2 26 .8 4 19 .1 2 13 .9 6 12 .5 8 n h ep a 35 3. 78 0. 25 7. 53 10 0. 79 3. 92 60 .0 9 3. 34 36 .8 7 16 66 .9 1 78 .1 8 41 .8 8 13 .0 7 26 .6 3 22 .5 4 n h ep a 36 4. 24 0. 18 5. 62 10 4. 96 5. 48 60 .4 2 3. 51 71 .5 6 17 47 .0 9 57 .1 6 31 .4 9 15 .5 5 18 .0 9 15 .8 4 n h ep a 38 3. 15 0. 42 4. 10 10 4. 00 6. 26 57 .3 4 4. 16 78 .1 4 16 82 .9 5 54 .8 1 32 .1 1 14 .5 6 19 .4 2 15 .1 8 n h ep a 39 -1 3. 53 0. 51 12 .7 7 10 2. 08 6. 16 52 .8 0 4. 65 77 .6 6 20 19 .6 8 76 .4 4 30 .4 2 16 .0 2 11 .5 1 17 .7 7 n h ep a 39 -2 3. 92 0. 43 6. 85 10 2. 72 5. 84 62 .5 2 4. 11 45 .5 4 13 09 .3 4 51 .0 3 64 .2 1 12 .7 7 15 .5 0 14 .4 3 n h ep a 39 -3 5. 80 0. 33 11 .2 7 10 3. 36 5. 32 61 .1 2 4. 21 34 .9 4 98 9. 17 53 .5 2 60 .3 0 14 .9 1 13 .6 0 14 .1 2 n h ep a 51 4. 28 0. 36 7. 53 99 .1 9 5. 01 60 .6 9 2. 48 36 .2 2 20 35 .7 1 47 .5 6 38 .8 4 12 .2 6 15 .0 6 14 .0 4 n h ep a 52 4. 84 0. 21 5. 95 10 5. 28 4. 80 60 .8 0 3. 13 35 .2 6 16 66 .9 1 56 .5 2 45 .6 2 16 .1 1 21 .6 0 12 .1 0 n h ep a 53 4. 95 0. 37 5. 45 10 3. 68 3. 66 62 .5 2 2. 59 47 .1 5 24 20 .5 5 54 .4 2 51 .0 3 18 .1 1 16 .0 1 14 .2 6 n h ep a 54 6. 01 0. 25 3. 31 10 4. 00 4. 70 58 .5 3 3. 56 53 .8 9 28 05 .3 9 75 .1 1 36 .0 8 15 .3 7 15 .6 8 13 .2 1 n h ep a 55 2. 58 0. 45 7. 04 10 2. 08 5. 43 59 .0 6 3. 24 64 .8 1 15 22 .6 0 56 .9 4 59 .6 1 14 .0 7 21 .0 2 13 .7 8 n h ep a 56 3. 50 0. 36 4. 29 10 4. 96 4. 70 66 .9 6 3. 56 44 .2 6 23 35 .0 3 37 .0 3 51 .9 4 16 .5 8 20 .9 9 14 .3 4 ya lo 5. 05 0. 21 4. 11 10 4. 00 3. 61 57 .0 1 1. 88 21 .4 5 21 9. 29 47 .0 8 70 .8 6 11 .8 7 16 .1 8 16 .3 0 m ea n 3. 93 0. 37 7. 06 10 3. 93 5. 19 57 .4 5 3. 92 59 .8 5 16 73 .2 1 56 .2 41 .7 9 14 .8 17 .7 4 15 .3 2 m in 2. 02 0. 18 3. 31 97 .9 1 3. 61 46 .3 1 1. 88 21 .4 5 21 9. 29 37 .0 3 22 .7 3 10 .4 4 11 .5 1 11 .7 8 m ax 6. 01 0. 51 14 .2 4 10 9. 44 7. 13 66 .9 6 5. 79 11 1. 71 28 05 .3 9 78 .1 8 70 .8 6 19 .5 0 26 .6 3 22 .5 4 c v 27 .2 3 29 .7 3 43 .9 1 2. 78 17 .9 2 8. 95 28 .8 3 37 .1 3 32 .2 1 20 .1 2 32 .3 0 17 .2 3 21 .7 6 17 .6 2 sd 1. 07 0. 11 3. 10 2. 89 0. 93 5. 14 1. 13 22 .2 2 53 8. 91 11 .3 1 13 .5 2. 55 3. 86 2. 70 genetic resources (2022), 3 (6), 38–48 genetic variation of african eggplant 43 table 4. eigenvalues and the proportion of accounted variance for each trait across 20 accessions of eggplant for the first four principal components (pc). dtf, days to flowering; nob, number of branches; ph, plant height at maturity; nofpc, average number of fruits per cluster; nof, average number of fruits per plant; yld, average yield per plant; fl, fruit length; fwd, fruit width; stemd, stem diameter; pet.l, petiole length; ped.l, pedicel length. variables pc1 pc2 pc3 pc4 vitamin c 0.2777 -0.2423 0.1295 -0.3933 iron -0.3759 0.0049 0.1498 0.0992 calcium -0.1837 0.3163 0.2492 -0.1366 dtf -0.0896 -0.4322 -0.1783 0.3571 nob -0.2992 -0.1570 -0.1479 0.2603 ph 0.3553 -0.2406 -0.1429 0.0086 nofpc -0.4063 -0.1331 0.1653 -0.0500 nof -0.4371 -0.0926 -0.0639 -0.0384 yld -0.0591 -0.1850 -0.3516 -0.5664 fl -0.0488 0.2823 -0.4034 -0.3045 fwd 0.3804 -0.0242 0.2446 0.2843 stemd -0.0412 -0.4246 -0.3557 0.0549 pet.l 0.1243 0.2639 -0.4563 0.3129 ped.l -0.0135 0.4255 -0.3351 0.1488 proportion of variance 0.2877 0.2064 0.1329 0.0973 cumulative proportion 0.2877 0.494 0.6269 0.7242 eigenvalues 4.0274 2.8891 1.8605 1.3625 0.487, p < 0.05), average number of fruits per plant (r = 0.532, p < 0.05) and number of fruits per cluster (r = 0.551, p < 0.05). plant height had a negative but moderate significant association with iron (r = -0.461, p < 0.05) and calcium (r = -0.407, p < 0.05) but was positively correlated with vitamin c (r = 0.492, p < 0.05). the strongest and most persistent correlation was recorded for association between fruit width and plant height (r = 0.574, p < 0.01) and number of fruits per plant (r = -0.737, p < 0.01); stem diameter with days to flowering (r = 0.734, p < 0.01). a positive significant correlation was observed between pedicel length and fruit length (r = 0.561, p < 0.05), and between pedicel length and petiole length (r = 0.528, p < 0.05). based on variation in the phenotypic parameters the 20 eggplant accessions were clustered into four unique groups (figure 3). clusters i and ii contained seven and three accessions, respectively, while clusters iii and iv both had five accessions. means of variables, ranges and standard deviation for each cluster are presented in table 6. cluster iii was unique in having accessions with high vitamin c content and high yield potential while clusters ii was characterized by accessions with high iron content, an increased number of fruits per plant and a higher number of fruits per cluster. clusters i and iv were characterized by early maturing accessions dominated by top-performing accessions in fruit-related traits (fruit length and fruit width respectively). discussion the success of genetic improvement programmes in enhancing desired traits of interest to farmers and breeders depends on the magnitude of genetic variability available in the germplasm and the extent to which the desirable traits are heritable. the high significant variation observed for most qualitative and quantitative traits considered in this study establishes the feasibility of imposing selections towards the improvement of desired traits of interest in african eggplant. frequency distribution among the qualitative traits with a preponderance of fruits characterized by white to cream colours and lemon green suggests that the majority of the accessions belong to the s. aetihiopicum group. this supports earlier reports by osei et al (2010) that eggplant accessions belonging to s. aethiopicum had mixtures involving cream white to light yellow fruits; thus, fruit colour combined with fruit shape might be considered a strong phenotypic marker in characterizing eggplant taxa in africa. the high cvs and range for some of the quantitative characters could be attributed to genetic variations from natural crossings and ecogeographical factors. the maximum and minimum mean values could present a rough estimate of the variation in magnitude of variability present among genotypes. traits such as average number of fruits per plant and fruit width that exhibited a high range of variation had more scope for improvement in the eggplant population. the principal component analysis identified traits that contributed the most to observed variations within a group of entries (sneath and sokal, 1973; grittins, 1975). the first four principal component axes in the current study accounted for 72.42% of the total variability measured. the first principal component analysis had the highest discriminating ability (contributing 28.77% out of 79.47% of variability from the first four axes) and was dominated by traits with relatively high factor scores (> 2.60) corresponding to number of branches, plant height at maturity, number of fruits per cluster, number of fruits per plant, and fruit width. this is in agreement with clifford and stephen (1975) who reported that the first principal component axis was the most important in reflecting the variation patterns among accessions and that the characters highly associated with these should be used in differentiating the accessions. furthermore, this is in line with the findings of iezzoni and pritts (1991) and chikaleke (2018) who reported that the implication of principal components can be accessed from the contribution of the different variables to each principal component (pc). correlation analysis is used to identify the relationship between variables (anshori et al, 2018) and to facilitate the identification of elite traits to rely on in selection exercises of a breeding programme. the positive significant association between iron content, number of branches, number of fruits per cluster and number of fruit per plant; number of fruit per plant with number 44 oguntolu et al genetic resources (2022), 3 (6), 38–48 ta bl e 5. c or re la ti on co ef fic ie nt s fo r nu tr it io na la nd ph en ot yp ic pa ra m et er s of th e2 0 eg gp la nt ac ce ss io ns ev al ua te d. *, si gn ifi ca nt at p ≤ 0. 05 ,* *, si gn ifi ca nt at p ≤ 0. 01 ,d tf ,d ay s to flo w er in g; n ob ,n um be r of br an ch es ;p h ,p la nt he ig ht at m at ur it y; n of pc ,a ve ra ge nu m be r of fr ui ts pe r cl us te r; n of ,a ve ra ge nu m be r of fr ui ts pe r pl an t; yl d ,a ve ra ge yi el d pe r pl an t; fl ,f ru it le ng th ;f w d ,f ru it w id th ;s te m d ,s te m di am et er ;p et .l ,p et io le le ng th ;p ed .l ,p ed ic el le ng th . tr ai ts v it .c ir on c al ci u m d t f n ob ph n of pc n of y ld fl fw d st em d pe t. l ir on -0 .5 00 * c al ci um -0 .1 62 0. 37 7 d tf 0. 06 4 0. 02 8 -0 .4 46 n ob -0 .3 08 0. 48 7* 0. 09 1 0. 37 0 ph 0. 49 2* -0 .4 61 * -0 .4 07 * 0. 05 1 -0 .1 00 n of pc -0 .2 79 0. 55 1* 0. 22 5 0. 29 3 0. 37 0 -0 .5 37 * n of -0 .4 18 0. 53 2* 0. 15 1 0. 20 0 0. 54 5* -0 .5 10 * 0. 73 0* * yl d 0. 06 3 0. 04 0 -0 .2 33 -0 .0 01 -0 .0 07 0. 21 3 0. 08 6 0. 19 3 fl -0 .0 45 -0 .0 50 0. 18 1 -0 .2 60 0. 09 7 -0 .2 29 -0 .1 27 0. 00 5 0. 21 7 fw d 0. 31 9 -0 .3 16 -0 .2 00 -0 .0 85 -0 .3 39 0. 57 4* * -0 .5 54 * -0 .7 37 ** -0 .4 10 -0 .2 55 st em d 0. 23 9 0. 06 5 -0 .3 03 0. 73 4* * 0. 33 6 0. 35 0 0. 06 8 0. 17 4 0. 36 3 -0 .0 58 -0 .1 25 pe t. l -0 .3 94 -0 .3 15 -0 .1 42 -0 .1 23 -0 .1 33 0. 15 4 -0 .3 87 -0 .1 86 -0 .0 32 0. 25 6 0. 02 9 -0 .0 99 pe d. l -0 .3 40 -0 .0 13 0. 31 3 -0 .2 75 -0 .0 61 -0 .2 12 -0 .2 36 -0 .0 95 -0 .2 12 0. 56 1* -0 .1 47 -0 .1 47 0. 52 8* genetic resources (2022), 3 (6), 38–48 genetic variation of african eggplant 45 figure 3. cluster dendrogram showing the relationships among the 20 eggplant accessions with cluster tree cut value at 7.3. of branches and number of fruits per cluster; fruit width and plant height at maturity; stem diameter and days to flowering; pedicel length with fruit length and with petiole length will facilitate selection of eggplant accessions with a good combination of these traits. the significant positive correlation displayed by these traits is in agreement with dhaka and soni (2013) who reported a positive significant association between yield and yieldrelated traits. however, where the traits had significant negative correlation coefficients (vitamin c and iron content, plant height at maturity and iron content, plant height at maturity and calcium content, number of fruits per cluster and plant height at maturity, number of fruits per plant and plant height at maturity, fruit width with number of fruits per cluster and with number of fruits per plant) indicates that an increase in one trait might lead to a decrease in the other trait or vice versa. this is in agreement with mazer et al (1999) and nyadanu and lowor (2015) who reported that traits with significant inverse relationships could be improved independently among eggplant accessions in ghana. however, selecting tall plants in this eggplant population might result in an indirect selection for low calcium content, while favouring increased iron content will lead to selecting genotypes with low vitamin c. selection pressure can be deployed for an increased number of fruits per plant to simultaneously increase iron content and number of branches. similar observations were reported by arivalagan et al (2013) and nyadanu and lowor (2015) in their earlier works on mineral composition and morphological characterization of eggplant. the cluster analysis emphasized further the relative contribution of various quantitative parameters to the total variability. the grouping of accessions in each cluster based on quantitative descriptors could be attributed to the fact that these accessions share some similarities. the high-yielding accessions in cluster iii (nhepa54) expressing high vitamin c and iron content could be deployed as progenitors to combine with eggplant genotypes from cluster i and create a new gene combination with improved calcium content and yield potential. creating new eggplant varieties with high-yield potential and increased vitamins and minerals (iron, calcium and vitamin c) will not only increase farmers’ income but will also help to reduce health challenges associated with hidden hunger among the 46 oguntolu et al genetic resources (2022), 3 (6), 38–48 table 6. means and standard deviations for various traits in different clusters. the clusters with the highest values for each trait are highlighted in bold font. dtf, days to flowering; nob, number of branches; ph, plant height at maturity; nofpc, average number of fruits per cluster; nof, average number of fruits per plant; yld, average yield per plant; fl, fruit length; fwd, fruit width; stemd, stem diameter; pet.l, petiole length; ped.l, pedicel length. trait cluster min max mean stddev trait cluster min max mean stddev vit.c i 2.02 3.78 3.06 0.56 nof i 36.87 81.68 69.79 16.50 vit.c ii 3.15 4.81 3.98 0.83 nof ii 65.29 111.71 84.46 24.24 vit.c iii 3.50 6.01 4.71 0.93 nof iii 35.26 71.56 50.42 13.58 vit.c iv 2.58 5.80 4.33 1.22 nof iv 21.45 64.81 40.59 16.03 iron i 0.25 0.51 0.40 0.11 yld i 1,320.72 2,019.68 1,633.28 233.03 iron ii 0.43 0.50 0.46 0.04 yld ii 1,522.60 1,731.05 1,660.04 119.05 iron iii 0.18 0.37 0.27 0.09 yld iii 1,666.91 2,805.39 2,194.99 480.25 iron iv 0.21 0.45 0.36 0.10 yld iv 219.29 2,035.71 1,215.22 674.45 calcium i 4.10 14.24 8.53 3.84 fl i 50.49 78.18 64.52 10.53 calcium ii 4.42 10.69 6.66 3.50 fl ii 44.91 45.62 45.30 0.36 calcium iii 3.31 5.95 4.92 1.10 fl iii 37.03 75.11 56.05 13.50 calcium iv 4.11 11.27 7.36 2.56 fl iv 47.08 56.94 51.23 4.14 dtf i 97.91 109.12 102.99 3.44 fwd i 22.73 41.88 33.33 6.08 dtf ii 106.56 109.44 107.84 1.47 fwd ii 26.84 33.05 30.85 3.48 dtf iii 103.68 105.28 104.58 0.69 fwd iii 31.49 51.94 43.23 9.10 dtf iv 99.19 104.00 102.27 1.86 fwd iv 38.84 70.86 58.76 12.00 nob i 3.92 7.13 5.52 1.13 stemd i 10.44 19.50 14.00 3.02 nob ii 4.70 6.42 5.53 0.86 stemd ii 13.76 19.12 16.82 2.76 nob iii 3.66 5.48 4.67 0.65 stemd iii 15.37 18.11 16.34 1.10 nob iv 3.61 5.84 5.04 0.85 stemd iv 11.87 14.91 13.18 1.28 ph i 46.31 60.09 53.91 5.03 pet.l i 11.51 26.63 19.92 4.62 ph ii 50.69 58.80 53.99 4.26 pet.l ii 12.93 14.72 13.87 0.90 ph iii 58.53 66.96 61.85 3.19 pet.l iii 15.68 21.60 18.47 2.74 ph iv 57.01 62.52 60.08 2.11 pet.l iv 13.60 21.02 16.27 2.82 nofpc i 2.70 4.98 4.12 0.81 ped.l i 15.06 22.54 17.83 2.89 nofpc ii 5.79 5.79 5.79 0 ped.l ii 11.78 14.86 13.07 1.60 nofpc iii 2.59 3.56 3.27 0.42 ped.l iii 12.10 15.84 13.95 1.40 nofpc iv 1.88 4.21 3.18 1.01 ped.l iv 13.78 16.30 14.53 1.01 rural and urban populace in the region. selection and hybridization of genotypes from clusters i and iv such as nhepa35 and yalo will produce new segregants characterized by bigger fruits with increased iron concentration. conclusion this study successfully characterized 20 eggplant accessions for phenotypic and nutritional traits of interest and identified top-performing new eggplant accessions with unique traits that could be deployed in crosses to facilitate the step-wise creation of new eggplant varieties with the best combination of desired traits. furthermore, selection in favour of yieldincreasing traits such as number of fruits per plant and number of fruits per cluster that showed a significant positive correlation with iron, will lead to selecting genotypes with increased iron content and higher yield potential simultaneously. top-performing accessions for iron (nhepa39-1), calcium (nhepa39-1) and vitamin c content (nhepa54) identified in this study should be deployed for hybridization to create new eggplant varieties with improved nutritional content. author contributions olawale olsesan oguntolu: study design, execution, drafting. christian okechukw anyaoha: study design, data analysis and interpretation, drafting, revision. victor anosie chikaleke: drafting and revision. olofintoye temidayo joseph a, study design and execution. conflict of interest statement the authors declare no conflict of interest. all authors approved the final manuscript. acknowledgements the authors are grateful to the internal management of national horticultural research institute (nihort) genetic resources (2022), 3 (6), 38–48 genetic variation of african eggplant 47 ibadan, oyo state nigeria for their support in carrying out this research work. references adeniji, o. t. and aloyce, a. 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(2019). soutenir une agriculture axée sur la nutrition grâce aux espèces négligées et sous-utilisées: cadre opérationnel. bioversity international and ifad, rome, italy, 978-92. sharmin, d., khalil, m. i., begum, s. n., and meah, m. b. (2011). ”molecular characterization of eggplant crosses by using rapd analysis”. international journal of sustainability and crop production 6(1), 22–28. sneath, p. m. and sokal, p. p. (1973). numerical taxonomy. the principle and practice of numerical classification (freeman; san francisco) . taher, d., rakha, m., ramasamy, s., solberg, s., and schafleitner, r . ( 2019). s ources o f r esistance for two-spotted spider mite (tetranychus urticae) in scarlet (solanum aethiopicum l.) and gboma (s. macrocarpon l.) eggplant germplasms. hortscience horts 54, 240–245. doi: https://doi.org/10.21273/ hortsci13669-18 toppino, l., vale, g., and rotino, g. l. (2008). inheritance of fusarium wilt resistance introgressed from solanum aethiopicum gilo and aculeatum groups into cultivated eggplant (s. melongena) and development of associated pcr-based markers. molecular breeding 22, 237–250. doi: https://doi.org/ 10.1007/s11032-008-9170-x https://doi.org/10.21273/hortsci13669-18 https://doi.org/10.21273/hortsci13669-18 https://doi.org/10.1007/s11032-008-9170-x https://doi.org/10.1007/s11032-008-9170-x introduction materials and methods experimental materials experimental design and conditions phenotypic characterization calcium, iron and vitamin c determination statistical analysis results discussion conclusion author contributions conflict of interest statement supplemental data for parfenchyk, m., lemesh, v., lagunovskaya, e., sakovich, v., buloichik, a., guzenko, e., khotyleva, l. (2024). identification of genetically plastic forms among belarusian ancient flax (linum usitatissimum convar. elongatum vav. et ell.) varieties using the linum insertion sequence lis-1. genetic resources 5 (9), 45–60. doi: 10.46265/genresj.dbno8764. supplemental table 1. characteristics of flax varieties for machine learning modelling. variety lis-1 ciliation of septa plant natural height, cm technical stem length, cm number of capsules per plant number of seeds per capsule anthocyanin pigmentation 624_6222 1 hh (60) 70 57 10 9 missing 624_6222 1 hh (45) 65 45 10 8 missing 624_6222 1 hh (60) 79 62 11 9 missing 624_6222 1 hh (50) 72 48 9 8 missing 624_6222 1 hh (50) 61 53 4 7 missing 624_6222 1 hh (45) 62 43 4 7 missing 624_6222 1 hh (45) 60 35 10 9 missing 624_6222 1 hh (30) 47 32 5 8 missing 624_6222 1 hh (45) 70 58 4 8 missing 624_6222 1 hh (50) 60 48 6 8 missing 624_6222 1 hh (45) 70 50 7 8 missing 624_6222 1 hh (50) 70 56 9 9 missing 624_6222 1 hh (45) 57 50 4 8 missing 624_6219 2 hh (0) 64 40 16 10 missing 624_6219 0 hh (0) 60 50 14 9 missing 624_6219 2 hh (0) 63 42 10 9 missing 624_6219 0 hh (0) 65 47 10 9 missing 624_6219 1 hh (0) 67 53 18 10 missing 624_6219 2 hh (0) 61 53 12 9 missing 624_6219 0 hh (0) 58 42 7 8 missing 624_6219 2 hh (0) 61 50 9 9 missing 624_6219 2 hh (0) 57 43 9 9 missing 624_6219 2 hh (0) 60 49 7 9 missing 624_6219 0 hh (20) 72 55 10 7 missing 624_6219 0 hh (40) 70 55 11 8 missing 624_6219 1 hh (20) 62 35 5 8 missing 624_6219 1 hh (0) 58 43 7 9 missing 624_6219 0 hh (0) 57 43 5 8 missing 624_786 1 hh (0) 67 58 21 9 missing 624_786 1 hh (0) 65 56 21 9 missing 624_786 1 hh (0) 69 60 20 10 missing 624_786 1 hh (0) 60 52 7 8 missing 624_786 1 hh (0) 65 56 17 9 missing 624_791 0 hh (0) 60 45 11 10 missing 624_791 0 hh (0) 55 43 10 9 missing 624_791 0 hh (0) 50 37 6 9 missing 624_791 0 hh (0) 44 37 6 8 missing 624_791 0 hh (0) 45 36 7 10 missing 624_6215 1 hh (0) 57 47 10 9 weak 624_6215 1 hh (0) 55 43 9 8 weak 624_6215 1 hh (0) 51 38 11 9 weak 624_6215 1 hh (0) 52 42 7 9 weak 624_1044 1 hh (50) 58 43 11 9 missing https://doi.org/10.46265/genresj.dbno8764 624_1044 1 hh (50) 64 47 9 9 missing 624_1044 1 hh (45) 54 35 11 9 missing 624_1044 1 hh (60) 65 45 15 7 missing 624_1044 1 hh (50) 50 41 7 8 missing 624_1044 1 hh (60) 48 40 5 7 missing 624_1044 1 hh (45) 50 40 6 8 missing 624_1044 1 hh (45) 51 43 13 9 missing 624_1044 1 hh (60) 49 40 5 8 missing 624_789 1 hh (0) 60 51 9 9 weak 624_789 1 hh (0) 57 41 10 8 weak 624_789 1 hh (0) 60 51 14 9 weak 624_789 1 hh (0) 57 43 8 8 weak 624_789 1 hh (0) 59 42 13 7 weak genetic divergence study on growth, yield and quality traits in pink brinjal (solanum melongena l.) in the subtropical plains of jammu, india genetic divergence study on growth, yield and quality traits in pink brinjal (solanum melongena l.) in the subtropical plains of jammu, india vishwash bandhral ​✉ 1 , anil bhushan 1 , ravinder kumar samnotra 1 , sonali sharma 1 , diksha rani 1 1 division of vegetable science, sher-e-kashmir university of agricultural sciences and technology (jammu), jammu­ 180009, india abstract in 2022, field investigation was conducted to examine the genetic diversity among 30 pink brinjal (eggplant, solanum melongena l.) genotypes originating from india. mahalanobis d2 analysis was performed to analyze the data for eighteen growth, yield and quality characters. among the traits examined, the total fruit yield per plant made the most significant contribution towards diversity. thirty genotypes were meaningfully grouped into eight clusters. cluster i, the largest, had eight genotypes, followed by cluster ii and cluster iv. there was no direct correlation observed between the geographical distribution and genetic divergence. among all clusters formed, cluster vii exhibited maximum intra-cluster distance followed by cluster vi. observing the inter-cluster distances, the maximum divergence was noted between cluster iii and cluster iv suggesting that genotypes within these clusters could serve as valuable parents for hybridization programmes aimed at producing highly heterotic hybrids and identifying transgressive segregants in the f2 generation. keywords hybridization, genetic diversity, intra­cluster distance, inter­cluster distance, eggplant, solanum melongena l, d2 analysis introduction brinjal or eggplant, scientifically known as solanum melongena l., with a chromosome number of 2n=2x=24, is a significant vegetable plant within the solanaceae family. in india, the second largest producer worldwide (fao, 2021), brinjal is mostly cultivated in west bengal, odisha, gujarat, bihar and madhya pradesh. although it is a perennial by nature, it is predominantly grown and harvested as an annual crop for its young, unripe fruits, commonly utilized in a variety of cooked dishes. it is considered to be a rich source of abundant nutrients and is the complete set of minerals, vitamins, nutritional fibre, protein, and antioxidants, along with some phytochemicals like caffeic acid, chlorogenic acid (phenolic components) glucoside, delphinidin, and nasunin (flavonoids) that have scavenging activities (bhaskar, ramesh, & p, 2015; noda, kaneyuki, igarashi, & mori, 2000). as per (zeven & zhukovsky, 1975), brinjal's primary origin is traced back to india, while china is considered a secondary centre of its origin. a large indigenous biodiversity exists in eggplant in the indian subcontinent region, due to the place being its centre of origin, and there exists a great variation in plant type, stem colour, leaf size, leaf tip, midrib colour, fruit shape, fruit size, fruit colour, fruit yield, cooking quality, fruit quality, and tolerance to pests and diseases (ullah, ijaz, shah, najeebullah, & niaz, 2014). understanding the genetic diversity within breeding materials is crucial for plant breeders to effectively select parent plants for crossing schemes, which is a prerequisite in breeding programmes. genetic diversity serves as important criterion for the selection of diverse parents who are expected to produce high hybrid vigour (harrington, 1940). crossing parents chosen for their genetic divergence, is likely to yield transgressive offspring, and subsequent selection can be applied in segregating generations. the mahalanobis d2 technique, as recommended by (rao, 1952) is a recognized method based on multivariate analysis, offering a reliable measure of genetic diversity. the objective of this study was to investigate the genetic diversity among 30 diverse brinjal genotypes in the subtropic plains of jammu, india to assist the breeder in identifying prospective parents that exhibit promising traits and possess genetic diversity to achieve the desired improvement. one of the primary goals in brinjal breeding is achieving early flowering and early harvest, as these traits enable farmers to catch the early market, leading to higher returns. plant spread, plant height and number of branches per plant are other important traits that directly impact on number of fruits and, consequently, the yield, leading to higher returns to farmers. fruit length, fruit diameter and fruit weight are directly linked to fruit yield. marketable fruit yield and unmarketable fruit yield based on fruits affected by biotic (disease-infected and pest-infested) and abiotic stresses (high and low temperature/freezing injury etc.) are also important traits in brinjal breeding. the ultimate goal of any crop improvement programme is to increase economic yield which is measured as fruit yield per plant and fruit yield per hectare. germination percentage is a parameter used to measure seed viability, and crop stands in the field primarily depend on this percentage, ultimately determining the final yield. seed vigour index is another important trait in crop improvement as seeds with a higher vigour index produce an early and uniform stand in the field. ascorbic acid content in brinjal fruit is an important biochemical character associated with increased nutritive value of the fruits which promotes better retention of colour and flavour (sasikumar, 1999). total phenol content helps determine resistance against fruit and shoot borer incidence in brinjal. the higher the phenol content, the lower the incidence of fruit and shoot borer (jat & pareek, 2003; shinde, warade, & kadam, 2009). in addition, higher phenolic levels influence antioxidant content and fruit culinary quality (stommel, whitaker, haynes, & prohens, 2015). keeping in view the importance of the above traits in brinjal breeding, these were taken into consideration in the present study. materials and methods thirty genotypes (table 1) of pink brinjal grown across various regions of jammu and kashmir were gathered, tested and assessed at the experimental farm of the division of vegetable science, sher-e-kashmir university of agricultural sciences and technology of jammu. the material encompasses local landraces, advanced breeding lines and hybrids and is conserved at the institute. seeds were sown in raised nursery beds of 3m × 1m size on 4 august 2022 in lines spaced 5cm apart. proper care was taken to water the beds and remove weeds for raising healthy seedlings. seedlings were ready for transplanting after four and five weeks. seedlings were transplanted on 2 september 2022 in a randomized complete block experimental design with three replications with a plot size of 3m × 1m and spacing of 90cm × 75cm. all the prescribed cultural practices were followed throughout the growth and development period of the crop to cultivate a healthy crop as given in package and practices for vegetable crops (anonymous, 2020). table 1: list of genotypes of pink brinjal (solanum melongena l.) used in this study and their sources. no. genotype biological status source 1 sjpb-22-01 advanced breeding line skuast-jammu, india 2 sjpb-22-02 advanced breeding line skuast-jammu, india 3 sjpb-22-03 advanced breeding line skuast-jammu, india 4 sjpb-22-04 advanced breeding line skuast-jammu, india 5 sjpb-22-05 advanced breeding line skuast-jammu, india 6 sjpb-22-06 advanced breeding line skuast-jammu, india 7 sjpb-22-07 advanced breeding line skuast-jammu, india 8 sjpb-22-08 advanced breeding line skuast-jammu, india 9 sjpb-22-09 advanced breeding line skuast-jammu, india 10 sjpb-22-10 advanced breeding line skuast-jammu, india 11 sjpb-22-11 advanced breeding line skuast-jammu, india 12 sjpb-22-12 advanced breeding line skuast-jammu, india 13 sjpb-22-13 advanced breeding line skuast-jammu, india 14 jammu sel-01 local landrace gajansoo, jammu, india 15 jammu sel-02 local landrace marh, jammu, india 16 shalimar hybrid-1 public sector hybrid skuast-kashmir, india 17 baramulla local sel-01 local landrace parihaspura, kashmir, india 18 baramulla local sel-02 local landrace pattan, baramulla, india 19 shalimar local local landrace dargah, kashmir. india 20 ganderbal local sel local landrace ganderbal, kashmir, india 21 long special private sector hybrid jyoti agritec, jammu, india 22 kashmiri long private sector hybrid jyoti agritec, jammu, india 23 long kashmiri private sector hybrid gulshan seeds, jammu, india 24 lal gulab private sector hybrid rajdhani seeds, jammu, india 25 pink long private sector hybrid shatabdi seeds, jammu, india 26 ppl-1823 private sector hybrid sultan seeds, jammu, india 27 manjhi private sector hybrid kalash seeds, jammu, india 28 pink raja private sector hybrid truegenic seeds, jammu, india 29 nisha private sector hybrid hm clause, jammu, india 30 brinjal no. 704 private sector hybrid mahyco, jammu, india five plants were selected from each plot to record observations on days to first flowering, days to first harvest, plant height (cm), number of branches per plant, plant spread (cm2), number of fruits per plant, fruit length (cm), fruit diameter (cm), fruit weight (g), marketable fruit yield per plant (kg) unmarketable fruit yield per plant (kg), total yield per plant (kg), fruit yield per hectare (q/ha), number of seeds per fruit and germination percentage as per descriptors of the international board for plant genetic resources (ipbgr, 1990). the seed vigour index was calculated by using the formula given by (abdul & anderson, 1973). ascorbic acid content (mg/100g) was assayed as described byrangana (1976) and total phenol content (mg/100g) as per procedure given bythimmaiah (1999). the d2 statistic, as introduced by (mahalanobis, 1936), was employed to evaluate genetic divergence among genotypes for both quantitative and qualitative traits. d2 analysis is a valuable tool for determining the degree of genotypic divergence between biological populations and determining the relative contributions of different components to the total divergence, both within and between clusters. the grouping of genotypes was carried out utilizing tocher’s method as outlined by rao (1952). statistical analyses were done using indostat software. results analysis of variance for various characters revealed significant differences for all parameters under study. mean performance of 30 genotypes for all traits are presented in supplemental table 1. after calculating the d2 values for every potential pair, the 30 genotypes were categorized into eight groups based on their genetic configurations. this clustering highlighted considerable genetic diversity among the genotypes (table 2) and (figure 1). cluster i constituted the largest group with eight genotypes, followed by cluster ii and iv, which each encompassed six genotypes. cluster vi had four and cluster vii had three genotypes. clusters iii, v and viii comprised only one genotype each (mono-genotypic clusters). table 2: clustering of 30 genotypes of pink brinjal (solanum melongena l.) based on d2 statistics cluster no. of genotypes genotypes i 8 sjpb-22-01, sjpb-22-02, sjpb-22-04, sjpb-22-05, sjpb-22-07, shalimar local, kashmiri long ii 6 sjpb-22-03, sjpb-22-12, sjpb-22-13, ganderbal local sel, jammu sel-01, jammu sel-02 iii 1 sjpb-22-09 iv 6 lal gulab, manjhi, long kashmiri, sjpb-22-08, sjpb22-11, brinjal no. 704 v 1 pink raja vi 4 baramulla local sel-01, pink long, shalimar hybrid-01, baramulla local sel-02 vii 3 sjpb-22-06, sjpb-22-10, long special viii 1 nisha figure 1: dendrogram representing clustering pattern of 30 genotypes of pink brinjal (solanum melongena l.). the numbers correspond to genotypes used in this study (see table 1). average interand intra-cluster distances in pink brinjal genotypes interand intra-cluster distances were calculated using the formulae described by singh and chaudhary (1977), serving as indicators of genetic diversity among clusters, as shown in table 3. inter-cluster distances surpassed intra-cluster distances, indicating a substantial level of genetic diversity among the studied genotypes. the highest intra-cluster distance was observed in cluster vii (42.78), with clusters vi and iv following closely at 40.27 and 39.57, respectively. clusters iii and iv exhibited the greatest inter-cluster distance (103.96), followed by clusters iii and vi (98.96), and clusters iii and vii (93.04). the proximity between clusters i and iii, as evidenced by the minimum inter-cluster distance of 40.17, suggests a stronger relationship among the genotypes within these clusters. table 3: mean intra-cluster distance (highlighted in bold) alongside the inter-cluster distance values (d2 values) for eight clusters among 30 genotypes of pink brinjal (solanum melongena l.). cluster i ii iii iv v vi vii viii i 28.47 46.14 40.17 85.92 40.31 72.69 90.16 42.50 ii 33.33 71.96 69.31 58.99 56.55 92.26 52.63 iii 0.00 103.96 51.53 98.96 93.04 46.59 iv 39.57 80.81 60.29 62.55 64.29 v 0.00 61.75 78.35 45.61 vi 40.27 88.34 70.29 vii 42.78 64.53 viii 0.00 cluster means for 18 characters in pink brinjal genotypes table 4 displays cluster means for each of the 18 characters studied. the single genotype in cluster iii had the maximum cluster mean value for germination percentage (87.33%) and seed vigour index (1115.67). the maximum cluster mean value for fruit length (15.81cm), unmarketable fruit yield per plant (0.32kg), fruit yield per hectare (303.8q/ha and number of seeds per fruit (275.11) was observed in cluster iv. the minimum cluster mean value for days to first flowering (37.00) and days to first harvest (62.33) was observed in cluster v, whereas desirable cluster mean values for marketable fruit yield per plant (2.05kg) and total fruit yield per plant (2.36kg) were observed in cluster vi. cluster vii exhibited the highest mean values among clusters for fruit weight (125.22g), as well as for ascorbic acid content (12.41mg/100g) and total phenol content (2.22mg/100g). cluster viii had the maximum cluster mean value for plant height (70.00cm), number of branches per plant (7.33), plant spread (70.00cm2), number of fruits per plant (31.00) and fruit diameter (5.23cm). table 4: cluster mean values for different characters in pink brinjal (solanum melongena l.). the lowest values are highlighted in bold, and the highest values are in italic. dff, days to first flowering; dfh, days to first harvest; ph, plant height (cm); npb, number of branches per plant; ps, plant spread (cm2); nfp, number of fruits per plant; fl, fruit length (cm); fd, fruit diameter (cm); fw, fruit weight (g); mfyp, marketable fruit yield per plant (kg); umfyp, unmarketable fruit yield per plant (kg); tfyp, total fruit yield per plant (kg); fyh, fruit yield per hectare (q/ha); nsf, number of seeds per fruit; g (%), germination (%); sv, seed vigour index; aa, ascorbic acid content (mg/100g); tpc, total phenol content (mg/100g). characters i ii iii iv v vi vii viii dff 41.17 40.28 38.00 40.33 37.00 38.92 41.67 41.33 dfh 67.38 67.11 64.67 66.83 62.33 63.75 70.44 63.00 ph 63.83 67.56 61.00 63.78 65.33 57.00 59.56 70.00 nbp 5.71 5.06 6.67 6.28 7.00 6.50 6.11 7.33 ps 63.83 67.56 61.00 63.78 65.33 57.00 59.56 70.00 nfp 23.21 19.89 26.67 26.56 29.33 27.75 25.67 31.00 fl 11.58 13.94 8.83 15.81 9.00 14.50 11.22 10.00 fd 3.86 3.97 3.67 4.31 3.63 4.63 3.63 5.23 fw 63.04 77.50 60.00 117.22 69.00 86.00 125.22 70.00 mfyp 1.30 1.36 1.33 1.71 1.78 2.05 1.26 1.42 umfyp 0.14 0.17 0.07 0.32 0.24 0.31 0.29 0.17 tfyp 1.44 1.53 1.40 2.05 2.02 2.36 1.56 1.60 fyh 212.91 226.04 207.40 303.85 299.74 249.56 230.61 236.53 nsf 224.79 256.44 207.33 275.11 209.33 260.50 222.56 250.00 g (%) 84.46 83.72 87.33 80.67 82.33 79.17 78.11 85.00 sv 1,030.33 998.39 1,115.67 891.61 919.67 848.25 841.33 1,050.00 aa 10.06 11.05 12.33 11.73 10.22 10.31 12.41 10.94 tpc 1.29 1.70 2.04 1.87 1.15 1.26 2.22 2.01 relative contribution of various characters towards divergence the percentage contribution to genetic divergence by all 18 characteristics is provided in table 5. observations revealed that the highest contribution to total divergence was from total fruit yield per plant (33.56%), followed by the number of seeds per fruit (21.38%), seed vigour index (7.94%), marketable fruit yield per plant (7.62%), fruit yield per hectare (3.61%), number of branches per plant (2.52%), days to first flowering (2.25%), plant spread (2.24%), unmarketable fruit yield per plant (2.21%), fruit diameter (2.07%), fruit length (1.89%), fruit weight (1.84%), total phenol content (1.61%), days to first harvest (1.13%), germination percentage (1.61%) and ascorbic acid content (0.69%). table 5: percentage contribution of various traits towards genetic divergence in pink brinjal (solanum melongena l.) rank characters percentage contribution (%) 1 total fruit yield per plant (kg) 33.56 2 number of seeds per fruit 21.38 3 seed vigour index 7.94 4 marketable fruit yield per plant (kg) 7.62 5 fruit yield per hectare (q/ha) 3.61 6 number of fruits per plant 3.34 7 plant height (cm) 2.99 8 number of branches per plant 2.52 9 days to first flowering 2.25 10 plant spread (cm2) 2.24 11 unmarketable fruit yield per plant (kg) 2.21 12 fruit diameter (cm) 2.07 13 fruit length (cm) 1.89 14 fruit weight (g) 1.84 15 total phenol content (mg/100g) 1.61 16 days to first harvest 1.13 17 germination (%) 1.05 18 ascorbic acid content (mg/100mg) 0.69 discussion the highest intra-cluster distance was observed in cluster vii while the greatest inter-cluster distance was observed between clusters iii and iv. the inter-cluster distance was larger than intra-cluster distance suggesting that genotypes within and between clusters were homogenous and diverse, respectively (pawar, prajapati, sawant, & patil, 2013). the data clearly showed that the clustering of genotypes was not influenced by their geographical distribution. overall, the distribution pattern of genotypes from different regions into distinct clusters appeared to be random. several studies (anbarasi & haripriya, 2021; chaitanya, 2022; kaur, sidhu, & dhatt, 2021; mangi, hotakar, jamkhandi, & karadi, 2020; mohanty, mishra, & barik, 2021; silambarasan, eswaran, senthilkumar, thangavel, & thirugnanakumar, 2020; verma, yadav, kumar, & verma, 2021) have all documented similar findings in their respective research concerning brinjal. one potential explanation may be that it is very challenging to establish the precise place of origin of a genotype. the frequent and open exchange of genetic material among breeders within the country complicates the maintenance of a genotype’s true identity. the lack of correlation between genetic diversity and geographical distance suggests that factors beyond geographical origins, like genetic exchange, drift, mutation, variation, and selection, could account for the observed genetic diversity. an alternative explanation could be that the diversity estimates derived from the characters examined in this study might not fully capture the variability influenced by additional physiological or biochemical characteristics, which could be significant in representing the overall genetic diversity within a population. consequently, the selection of genotypes for hybridization should prioritize genetic diversity over geographical divergence. out of eight clusters, cluster viii had the highest mean value for plant height, number of branches per plant, number of fruits per plant and fruit diameter, implying that genotypes in this cluster can be selected directly on the basis of these features and employed in hybridization programmes. cluster vi also showed the highest mean value for some important traits, namely marketable fruit yield per plant and total fruit yield per plant. the genotypes belonging to this cluster can be used in breeding programmes to develop high fruit-yielding hybrids. cluster v had the lowest value for days to first flowering and days to first harvest, implying that the genotypes in this cluster are likely to be early maturing types. hence, these genotypes can be directly employed for developing early crops. cluster vii exhibited the highest mean value for ascorbic acid and total phenol content along with fruit weight. this implies that this cluster’s genotype should be rich in nutritive value, and these genotypes should be employed in quality breeding. the genotypes in cluster iii had the highest cluster mean value for germination percentage and seed vigour index, indicating that genotypes from this cluster exhibit rapid and uniform crop establishment and growth across diverse environmental conditions. a similar comparison of clusters based on the range of mean value of each character was done by earlier works, namely (balasubramaniyam, haripriya, kumar, & elangaimannan, 2021; banerjee, singh, & verma, 2018; chaitanya, 2022; kaur et al., 2021; ravali, reddy, saidaiah, & shivraj, 2017; silambarasan et al., 2020; sindhuja et al., 2019; verma et al., 2021) . relative contribution of various characters towards divergence the highest contribution to genetic divergence was observed from total fruit yield per plant. many researchers including (balasubramaniyam et al., 2021; bhushan, samnotra, & kumar, 2018; chaitanya, 2022; mohanty et al., 2021; ravali et al., 2017; silambarasan et al., 2020; sindhuja et al., 2019) confirmed the substantial contribution of fruit yield per plant to divergence. the results indicate significant potential for developing new varieties of pink brinjal with increased yield and enhanced economically important attributes by utilizing this elite germplasm. in crop improvement initiatives, effective outcomes could be achieved through interbreeding among genotypes displaying exceptional mean performance for these traits. conclusion analysis of 18 traits using the mahalanobis d2 statistic demonstrated significant diversity among 30 pink brinjal genotypes, resulting in their classification into eight separate clusters. mahalanobis d2 proved to be an effective tool in clustering genotypes phenotypically and geographically. the genotypes within clusters iii and iv, which exhibit maximum inter-cluster distance, hold potential for utilization as parents in recombination breeding programmes as these are very diverse from each other. they could be employed to develop highly heterotic f1 hybrids or to generate a broad range of transgressive segregants within populations, thereby facilitating the development of high-yielding varieties of pink brinjal. for recovering improved progenies for yield and quality characters, crosses can be attempted between the genotypes belonging to clusters vi and vii as they show the highest cluster mean value for yield and quality parameters. supplemental data supplemental table 1. mean values of 18 different characters for 30 genotypes of pink brinjal (solanum melongena l.) described in this study. acknowledgments for providing the essential research facilities, the authors are grateful to the division of vegetable science, sher-e-kashmir university of agricultural sciences and technology of jammu. author contributions vishwash bandhral played a crucial role in drafting the research programme and objectives along with dr. anil bhushan, collecting different genotypes of pink brinjal from different sources, proper sowing and transplanting of field trial, recording field and lab parameters, analyzing data and concluding the results. dr. anil bhushan assisted in drafting the problem of genetic divergence and formulating the objective, dr. r.k. samnotra reviewed the trail of divergence, diksha rani helped with data collection, and sonali sharma assisted with data analysis. conflict of interest authors have declared that no competing interests exist. references abdul, b. and j. d. a. 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(1975). dictionary of cultivated plants and their centres of diversity (wageningen, netherlands), 219. retrieved from https://edepot.wur.nl/318076 original article genetic resources (2023), 4 (7), 20–31 doi: 10.46265/genresj.dlox8174 https://www.genresj.org issn: 2708-3764 phenotypic variations among okra (abelmoschus esculentus (l.) moench) genetic resources in sudan ibrahim mohamed el tahir * agricultural plant genetic resources conservation and research centre, agricultural research corporation, wad medani, sudan abstract: a total of 366 accessions of okra (abelmoschus esculentus (l.) moench) obtained from the agricultural plant genetic resources conservation and research centre (apgrc) in sudan were characterized for a number of morphological characters using a descriptor list derived from the list published by the international board for plant genetic resources in 1984. those accessions, which were collected from different regions of sudan, were grown in the apgrc research farm in wad medani town in central sudan during ten different seasons within the period between 2000 and 2019. phenotypic variations were observed among and within the different accessions for plant, stem, leaf, inflorescence and fruit characters. up to 59% of accessions were found to be heterogeneous for different traits. the descriptor states observed ranged from very rare in 5% or less of the accessions, to abundant in more than 90% of the accessions. substantial phenotypic variation was observed for okra fresh fruits, the main organs used for food, in terms of shape, colour, pubescence and number of ridges. accessions carrying fruits preferred in local or foreign markets were identified making them good candidates for further breeding to produce new cultivars for both markets. the cluster analysis resulted in 13 subclusters at a similarity level of 60%. when comparing the subclusters with collection sites, no direct relation was detected indicating that okra germplasm has been spreading all over the country resulting in diversified materials across different regions. keywords: okra, genetic resources, characterization, abelmoschus, sudan citation: el tahir, i. m. (2023). phenotypic variations among okra (abelmoschus esculentus (l.) moench) genetic resources in sudan. genetic resources 4 (7), 20–31. doi: 10.46265/genresj.dlox8174. © copyright 2023 the authors. this is an open access article distributed under the terms of the creative commons attribution license (cc by 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. introduction okra (abelmoschus spp.) is widely cultivated for its edible green fruits, which are harvested when immature and are famous for their slimy mucilage. it is widespread in tropical, subtropical and warm temperate regions. common okra (abelmoschus esculentus (l.) moench) has been reported in the whole of tropical africa, whereas west african okra (abelmoschus caillei (a.chev.) stevels) is restricted to the humid and perhumid climates of africa (siemonsma and kouamé, 2004). abelmoschus esculentus (usually 2n = 130) is probably an amphidiploid (allotetraploid), derived from abelmoschus tuberculatus pal & h.b.singh (2n = 58), a wild species from india, and a species with 2n = 72 chromosomes (possibly abelmoschus ficulneus (l.) wight ∗corresponding author: ibrahim mohamed el tahir (eltahir81@yahoo.com) & arn. ex wight) (siemonsma and kouamé, 2004). the exact origin of okra is unknown, however, one putative ancestor (a. tuberculatus) is native to uttar pradesh in north india, suggesting that a. esculentus originated in india. another evidence is based on the plant’s cultivation in ancient times and the presence of another putative ancestor (a. ficulneus) in east africa, suggesting also northern egypt or ethiopia as the geographical origin of a. esculentus (kumar et al, 2011). okra (abelmoschus spp.) is the most popular traditional vegetable in sudan, where both wild and cultivated types of okra are known. it is used in almost all parts of the country. it is cooked either after being dehydrated or as fresh pods (mohamed, 1991). some of the wild types seem to belong to the cultivated species a. esculentus while others belong to other species such as a. ficulneus. in sudan, there is a late 19th-century record of its occurrence in the wild along the white nile (singh et al, 1975). schippers (2002) reported that a. ficulneus received: 29.08.2022 accepted: 31.01.2023 published online: 30.03.2023 https://www.genresj.org https://www.dx.doi.org/10.46265/genresj.dlox8174 https://www.genresj.org https://www.dx.doi.org/10.46265/genresj.dlox8174 mailto:eltahir81@yahoo.com genetic resources (2023), 4 (7), 20–31 okra phenotypic variations in sudan 21 l. was found in sudan and other regions of the sahel (mali, chad) and east africa, but more so in south and south east asia. this wild species, of which both fruit and leaves are eaten in sudan is one of the two ancestors of the common okra. schippers (2002) also indicated that a range of primitive landraces could still be found in sudan, where people collect fruit from wild plants for drying purposes. for example, the dehydrated okra locally called ‘waika’, is collected from the wild from rain-lands in the central region and the southern blue nile, kordofan and darfur (geneif, 1984). okra is cultivated in different parts of sudan, where farmers depend almost completely on the use and production of traditional farmers’ varieties. apart from some introduced varieties such as clemson spineless and pusa swani, there are a number of farmers’ varieties obtained from local types, which in many cases have names relevant to the localities, where they might have evolved and are usually produced. they include a spiny type called khartoumia and other local varieties such as karrari, kassala, medani, sinnar and others (ahmed and mohamed, 1995). such variation in cultivated and wild okra varieties constitutes a wealth of indigenous genetic resources in sudan that should be given the necessary attention to ensure their conservation against any possible threats, as well as to enhance their sustainable use. therefore, germplasm collection efforts by the plant genetic resources (pgr) programme of the agricultural research corporation (arc) since the 1980s have resulted in the collection of more than 600 accessions of okra from different regions in sudan with remarkable variation in plant and fruit characters. seeds of such accessions are conserved in the genebank of the agricultural plant genetic resources conservation and research centre (apgrc) of the arc, which is located in wad medani town in gezira state in central sudan. batches of seed samples are preserved under longterm seed storage conditions, which are realized in deep freezers adjusted at around -20◦c, with seed moisture content within a 4–7% range. they are packaged in laminated aluminium foil packets, which are hermetically sealed before being deposited in the freezers. this paper reports on efforts made and results obtained in sudan under the apgrc for the enhancement of the use of okra genetic resources through morphological characterization from 2000 to 2019. materials and methods germplasm materials materials used and reported in this paper were genetic resources of okra (abelmoschus esculentus) of which sudan is a country of origin. 366 a. esculentus accessions, which were obtained by either direct collecting from different regions of the country or acquiring from other sources, such as okra breeders, were used in this work (table 1, supplemental table 1). passport data on the accessions are documented in the genebank documentation system of the apgrc, and partially in the genesys database, including georeferenced data of origin for some of them (figure 1). all accessions were conserved in the apgrc seed genebank under long-term seed storage conditions. these accessions were grown and characterized in batches across ten different cultivation seasons during the period from 2000 up to 2019 as shown in table 2. germplasm characterization samples of the 366 okra accessions were grown under full irrigation in the apgrc field for morphological traits characterization. the apgrc field is located within the research farm of the gezira agricultural research station at wad medani town, at latitude 14◦ 24’ n and longitude 33◦ 29’ e and altitude of 406.9m a.s.l. the climate of the area is hot semi-arid, and the soil is vertisol with clay content of 40–65%, and ph value ranging from 8 to 9.6, with less than 1% organic carbon, 300ppm total nitrogen and 406 to 700ppm total phosphorus (ishag and said, 1985). seed samples were obtained from the apgrc genebank and directly sown in the field in different seasons with sowing dates varied across seasons between the last week of june and the second week of july. each accession was grown in a one-line plot on a ridge that was 80cm apart from others with in-row spacing of 20cm between plants. each entry was represented by ten plants, at maximum, grown as one plant per hole. routine cultural practices including manual removal of weeds, irrigation and raising of ridge sides were practised when required at intervals of around 7–15 days. plants were nitrogen-fertilized using urea at a rate of 238kg of urea per hectare containing a total of 109.5kg of pure nitrogen. this total amount of urea was split into two doses; the first dose (119kg of urea per ha) was applied one month after sowing, while the second equal dose was applied one month later. plants of all accessions were phenotypically characterized using 17 qualitative descriptors on plant, stem, leaf, inflorescence and fruit traits derived from the international board for plant genetic resources descriptor list for abelmoschus (charrier, 1984) (supplemental table 2). all traits were fully described in the field by recording all descriptor states that were observed within each accession. statistical analysis the frequency level of different descriptor states was calculated as the percentage of accessions showing homogeneously only the same specific descriptor state within the 366 accessions characterized. accessions showing different states for a descriptor were considered heterogeneous for that specific descriptor and their frequency percentages were calculated as well. accordingly, the descriptor states were considered to be occurring at different levels including very rare, rare, moderate, common and abundant. they were categorized as very rare when occurring with a frequency level of 5% or less, 22 el tahir genetic resources (2023), 4 (7), 20–31 rarely when occurring with a frequency level > 5% and ≤ 30%, moderately when occurring with a frequency level > 30% and ≤ 60%, commonly when occurring with frequency levels > 60% and≤ 90% and abundantly when occurring with frequency levels > 90%. multivariate analysis was run on the data obtained through the hierarchical cluster analysis with complete linkage using the software genstat twelfth edition (genstat release 12.1). table 1. number of accessions collected from different geographical regions in sudan and used in this study. administrative state total accessions south kordofan (including the present south kordofan and west kordofan states) 73 west darfur 50 northern state 38 west kordofan 31 south darfur 27 blue nile 25 red sea 24 gedarif 21 north kordofan 17 sinnar 17 kassala 13 river nile 11 central darfur 6 khartoum 5 white nile 5 gezira 4 unknown 29 total 366 table 2. number of okra accessions characterized in different cultivation seasons season of cultivation total number of accessions characterized 2000–2001 43 2006–2007 86 2007–2008 35 2008–2009 39 2009–2010 33 2011–2012 16 2013–2014 15 2015–2016 12 2017–2018 33 2018–2019 54 total 366 results a total of 60 descriptor states were observed for the 17 descriptors covering plant, stem, leaf, inflorescence and fruit characters across the 366 okra accessions studied (supplemental table 3). a total of 150 accessions (41%) were observed to be homogeneous for all descriptors, while the remaining 59% (216 accessions) were heterogeneous for one or more descriptors. however, the level of heterogeneity for each descriptor, calculated as the frequency of accessions showing different descriptor states for the same descriptor within the same accession, ranged between 3% being the lowest level of heterogeneity for the descriptor of fruit peduncle length, and 34% being the highest level of heterogeneity for the leaf shape descriptor (table 3). the frequency level of different descriptor states ranged between less than 1% and 100% as shown in table 3. some of the descriptor states were observed to be abundant in more than 90% of the accessions characterized such as the linear shape of epicalyx segments (91%), or the yellow flower colour (100%). on the other hand, some descriptor states were very rarely observed in only 5% or less of the accessions such as the procumbent general aspect of plant (2%), purple stem colour (3%), pendulous fruits on main stem (< 1%) or fruits that were red or green with red patches (< 1%, and 4% respectively). otherwise, phenotypic variations were observed at different levels across the different plant organs of the okra accessions. phenotypic variations related to plant vegetative characters variations were observed in the vegetative characters that included general plant aspect, branching, leaf and stem characters, as shown in table 3. general plant aspect erect, medium and procumbent plant aspects were observed. half of the characterized accessions had medium aspect, while 23% had erect plants, and the procumbent aspect was observed very rarely in only 2% of the accessions. the rest of the accessions (25%) were heterogeneous for this descriptor. branching different descriptor states of branching were observed among the okra accessions studied including orthotropic, medium and strong branching. while medium branching was observed in the majority of the accessions with a frequency of 51%, orthotropic and strong branching were rarely observed (19% of the accessions for either). the rest of the accessions were heterogeneous for this descriptor. genetic resources (2023), 4 (7), 20–31 okra phenotypic variations in sudan 23 figure 1. collection sites in sudan of some okra accessions described in this paper, for which georeferenced data was available from apgrc. figure 2. descriptors for leaf shapes used for characterization purposes (charrier, 1984) 24 eltahir g enetic resources (2023),4 (7),20–31 table 3. phenotypic variations observed in the 366 characterized okra accessions. occurrence level: very rare ( ≤ 5%), rare ( > 5%, ≤ 30%), moderate ( > 30%, ≤ 60%), common ( > 60%, ≤ 90%), and abundant ( > 90%).1)leaf shape and some fruit characters are depicted in figure 2 and figure 5. descriptor occurrence level of each descriptor state and its frequency percentage abundant common moderate rare very rare heterogeneous general aspect erect (50%) medium (23%) procumbent (2%) 25% branching medium (51%) orthotropic (19%) 11% strong (19%) stem pubescence slight (45%) glabrous (22%) 26% conspicuous (7%) stem colour green (49%) green with red patches (22%) purple (3%) 26% leaf shape1) 3 (7%) 5 (< 1%) 34% 4 (24%) 6 (< 1%) 7 (24%) 8 (1%) 10 (7%) 9 (2%) leaf colour green with red veins (48%) green (24%) 28% number of epicalyx segments 8–10 (42%) 5–7 (2%) 16% > 10 (40%) shape of epicalyx segments linear (91%) lanceolate (9%) petal colour yellow (100%) red colouration of petal base both sides (76%) inside only (17%) 7% position of fruit on main stem erect (85%) horizontal (6%) pendulous (< 1%) 9% fruit colour green (47%) yellowish green (23%) green with red patches (4%) 26% red (< 1%) continued on next page g enetic resources (2023),4 (7),20–31 o kra phenotypic variations in sudan 25 table 3 continued descriptor occurrence level of each descriptor state and its frequency percentage abundant common moderate rare very rare heterogeneous fruit length at maturity 8–15 cm (40%) < 8cm (11%) 26% > 15cm (23%) length of peduncle 1–3 cm (84%) > 3cm (13%) 3% fruit shape1) 1 (21%) 2 (< 1%) 25% 3 (23%) 5 (< 1%) 4 (16%) 8 (< 1%) 6 (6%) 10 (< 1%) 11 (< 1%) 12 (< 1%) 14 (5%) 15 (< 1%) number of ridges per fruit 5–7 (70%) < 5 (9%) > 10 (1%) 10% 8–10 (10%) fruit pubescence downy (29%) 27% slightly rough (17%) prickly (27%) 26 el tahir genetic resources (2023), 4 (7), 20–31 table 4. number of subclusters at similarity level of 60% among which accessions from each state are distributed. state total accessions no. of subclusters northern state 38 10 south darfur 27 10 gedarif 22 9 south kordofan 73 9 red sea 24 7 north kordofan 17 6 sinnar 17 6 blue nile 25 5 west darfur 50 5 white nile 5 4 gezira 4 3 kassala 13 3 river nile 11 3 central darfur 6 1 khartoum 5 1 unknown 29 9 total 366 leaf characters leaf shape was among the characters with maximum variations recorded in the okra accessions characterized. eight descriptor states were observed in terms of leaf shapes. consequently, all descriptor states observed for shapes of leaf were found to be either very rarely or rarely occurring with frequency levels of less than 1% and up to 24%. however, the most common leaf shapes were shape 4, with a five-lobed leaf with smooth margins (24%); and shape 7, with a five-lobed leaf with non-smooth undulating margins (figure 2) (24%). on the other hand, the least occurring leaf shapes were shapes 5, 6, 8 and 9, which were very rarely occurring at frequencies of less than 1% to 2%. the rest of the accessions (34%) were heterogeneous for this descriptor. leaf colours were green (24%) and green with red veins (48%), in the accessions that were homogeneous for this character. the rest of the accessions were heterogeneous for this descriptor. stem characters stems of most accessions were either green or green with red patches with frequencies of 49% and 22%, respectively, among the homogeneous accessions characterized. on the other hand, purple stems were very rarely observed in only 3% of the accessions. stems were either glabrous, slight or with conspicuous pubescence. most of the accessions were either glabrous (22%) or had slight hairs on their stems (45%). otherwise, only 7% of the accessions had conspicuous hairs, while the rest of the accessions were heterogeneous for this character. variations in inflorescence characters the only characters that were abundantly dominating in the studied germplasm were the linear shape of epicalyx segments, and the yellow flower colour, which were occurring in 91% and 100% of the accessions, respectively. red colouration was commonly found at both sides of the petal base in the majority of the accessions (76%), while only 17% of the accessions produced flowers with red colouration only inside the petal base. the rest of the accessions were heterogeneous for this character. forty two percent of accessions had between 8 and 10 epicalyx segments, and 40% had more than 10 segments with a moderate level of occurrence. on the other hand, 5–7 segments were observed very rarely, in only 2% of the accessions. the rest of the accessions were heterogeneous. variations in fruit characters variations were observed among the okra accessions on different fruit-related characters (table 3). position of fruit on main stem erect position of fruits on main stem was a common character observed in the majority of the accessions at a frequency of 85%. the horizontal and pendulous positions of fruits on main stems were rarely or very rarely observed, with frequencies of 6% and less than 1%, respectively. the rest of the accessions were heterogeneous for this character. fruit colour the majority of fruits produced were green (47% of the accessions) or yellowish green (23%). fruits that were red or green with red patches were very rarely produced by fewer than 1% and 4% of the accessions, respectively. the rest of the accessions were heterogeneous for this character. exceptionally, it was observed that remarkable greener ridges were observed on green or yellowish green fruits in some accessions (figure 3). fruit length at maturity forty percent of the characterized accessions produced fruits that were 8–15 cm long when mature. fruits shorter than 8cm or longer than 15cm were produced by 11% and 23% of the accessions, respectively (figure 4). the rest of the accessions were heterogeneous for this character. length of peduncle the majority of the accessions had fruits with peduncles that ranged between 1 and 3cm long in 84% of the accessions, while 13% of the accessions were producing fruits with peduncles longer than 3cm or were heterogeneous for this character (3%). fruit shape fruit shape was among the characters with maximum variation in terms of the number of descriptor states observed within the germplasm collection studied, genetic resources (2023), 4 (7), 20–31 okra phenotypic variations in sudan 27 figure 3. variations in fruit colour as observed on some accessions. figure 4. variations and frequency percentages of mature fruit lengths among the characterized accessions. recording 12 descriptor states at different frequencies that ranged between less than 1% and 23%. the most common fruit shapes observed in 16–23% of the accessions were shapes 1, 3 and 4, which are characterized by elongated ridged capsules that were slender and moderately curved in shape 1, but straight or very slightly curved for shapes 3 and 4, with a wider base in shape 4 (figure 5). nine fruit shapes were observed very rarely, occurring in less than 1% to 6% of the accessions, while 25% of the accessions were heterogeneous for this descriptor (figure 5). number of ridges per fruit the number of ridges per fruit ranged between five and seven in the majority (70%) of the accessions characterized. fruits with fewer than five ridges or with eight to ten ridges were rarely observed, in 9% and 10% of accessions, respectively. fruits with more than ten ridges were very rarely observed (less than 1%). ten percent of accessions were heterogeneous. fruit pubescence most of the accessions characterized were homogeneous for fruit pubescence producing either downy, slightly rough or prickly pubescent fruits occurring at rare or moderate levels with frequencies ranging between 17% and 29%. results of cluster analysis cluster analysis resulted in a clustering pattern composed of two major clusters at similarity level of 30%. these major clusters were further grouped into 13 subclusters at a level of similarity of 60% including 4 subclusters under the first major cluster, and 9 subclusters under the second (figure 6). when comparing the clustering pattern with the collection sites of the accessions, no direct relation was detected (table 4). the only exceptions were the accessions from central darfur and khartoum states as they clustered in only one group indicating a limited variation among them. however, this could be attributed to the limited number of accessions obtained and studied from these two states (six from central darfur and five from khartoum). discussion the okra accessions described in this study were collected and obtained from different regions and sources in sudan. the majority of the accessions were heterogeneous, which is normal in a crop for which farmers’ varieties are mainly used, as is the situation in 28 el tahir genetic resources (2023), 4 (7), 20–31 figure 5. descriptor states of fruit shapes (charrier, 1984), and some of the observed fruit shapes and their frequency percentages as shown by some accessions. genetic resources (2023), 4 (7), 20–31 okra phenotypic variations in sudan 29 figure 6. dendogram of clustering patterns of 366 characterized okra accessions showing the two major clusters (a and b) and the four subclusters under a (a1-a4) and the nine subclusters under b (b1-b9). sudan. hamon and van sloten (1989) indicated that a homogeneous accession is a rare occurrence in okra as it is reproduced by seeds and is not strictly autogamous. this study has shown that the sudanese okra germplasm is rich in terms of variation for a number of morphological traits. among them are traits significant for yield and quality of the okra fruits, which are consumed by people. a considerable number of the okra accessions in this study had erect growth habit and medium to strong branching, which could be indication for high yielding potential, as described in a similar study that recorded strong erect stems and dense branching for abelmoschus species at production sites with extended harvest throughout the year (omonhinmin and osawaru, 2005). it is interesting to note from this study that fruit shape was among the characters with the highest level of observed variation in sudan with 12 descriptor states out of the 15 recommended in the ibpgr descriptors for abelmoschus (charrier, 1984). this was similar to the results reported by oppongsekyere et al (2011) on okra germplasm from ghana, where fruit shape showed the greatest diversity among the okra accessions, from short and triangular to long straight or long curved. however, only three shapes were the most frequently observed in this collection, which were actually closely similar to each other as all of them were of elongate fruit type with variation in thickness and curvature. in fact, this is the type of okra fruits that are most commonly consumed and sold in the vegetable markets in the country. such phenomenon indicates the 30 el tahir genetic resources (2023), 4 (7), 20–31 trend among the okra farmers in sudan to select for this type of elongate fruits to meet the market demand and the consumers’ preference. a number of characters observed seem to be very rarely or rarely present in the described collection indicating a higher risk to lose them. among those are characters such as procumbent aspect of plant, purple stems, pendulous fruits on main stems, fruit that were red or green with red patches, and a number of fruit shapes other than the elongate ones. more attention should be paid to conserve accessions carrying such characters in order to preserve, multiply and evaluate them for traits other than those related to the preferred fruit quality and yield potential such as resistance to biotic and abiotic factors. however, some of these rare characteristics were also observed with similar frequency in a recent study conducted in neighboring ethiopia where only 19.44% of accessions had red or purple stem colour (temam et al, 2021). the majority of the accessions produced fruits that were either green or yellowish green, while red fruits or green fruits with red patches were rarely produced in about 5% of the accessions. this also refers to the tendency of the okra farmers in sudan to select for greenish fruits that are usually preferred by the consumers. on the other hand the results of the study in ethiopia by temam et al (2021) showed that fruits that were green with red patches were the most highly represented in their collection at a frequency of 55.56%. while sudanese consumers do not tend to use green fruits with red patches, it seems this fruit colour is preferred by the consumers of okra in at least some regions of ethiopia. on the opposite the ethiopian consumers may not like fruits that were yellowish green as only 2.78% of the characterized ethiopian accessions produced such type of fruits, while 23% of the sudanese accessions in the present study had yellowish green fruits. okra consumers in sudan also tend to prefer the spiny okra fruits, as they are locally believed to be more slimy and tasty than others. the results obtained from this study showed that a considerable number of characterized accessions (44%) had spiny texture by being either prickly or slightly rough. however, downy fruits with smooth texture were also recorded for a considerable number of accessions (29%) indicating the potential of the okra germplasm in sudan for use to produce varieties that are more likely to be suitable for exportation outside the country where downy green fruits seem to be more preferred. the abundant occurrence of linear epicalyx segments in the collection described with very rare cases of lanceolate segments and the full absence of triangular segments confirm that the accessions characterized belong to the species of the common okra abelmoschus esculentus that is common in east africa. in contrast to this the species a. caillei, which is known as the west african okra, is more common in west africa. as mentioned by siemonsma and hamon (2004) a. esculentus differs in several respects from a. caillei, but the epicalyx offers the best discriminating characteristic: the width of the epicalyx segments is 4–13mm in a. caillei and 0.5–3mm in a. esculentus. generally, the okra genetic resources from sudan have considerable phenotypic variations among them as shown by the clustering pattern of the accessions described in this study reaching to 13 sub-clusters at 60% level of similarity. the variation is also detected among the okra accessions collected from each of the geographical states as they were mostly clustering in more than one group reaching to 10 sub-clusters in some states. such trend of variations in okra genetic resources means that okra germplasm has been spreading in sudan from different sources resulting in diversified phenotypes all over the country and in each of the different regions. conclusion this study has shown that the germplasm collection of okra from sudan held by the apgrc, which is mostly composed of farmers’ varieties, is diverse in a number of characters including those important for yield and quality for both local and foreign markets. such diversity in this collection makes it highly promising for breeding for improved okra cultivars through purification, selection and evaluation for superior lines. however, more germplasm collection of okra genetic resources is needed with emphasis on regions and geographical pockets that are poorly represented in the materials collected and studied so far. further characterization for morpho-agronomic traits is also necessary as well as evaluation of such germplasm for desirable traits including resistance and adaptability to biotic and abiotic stresses. acknowledgements the author is very thankful to the agricultural research corporation of sudan for providing the necessary budget to conduct this study across different seasons. supplementary funding was also obtained from the eastern africa plant genetic resources network (eapgren) during some years from a project funded by the swedish international development cooperation agency (sida) to both of which the author is very grateful. thanks and appreciation are also extended to the staff of the apgrc including researcher assistants, technicians and labourers, who had exerted substantial efforts to extract the seed samples from the genebank, establish the plants in the field, take and record data in the field and document the data in the office. conflict of interest statement the author declares that there is no conflict of interest. genetic resources (2023), 4 (7), 20–31 okra phenotypic variations in sudan 31 supplemental data • supplemental table 1: list of all okra accessions characterized, their sites of collection and characterization seasons • supplemental table 2: okra descriptors and descriptor states used in the study • supplemental table 3: characterization results recorded on the different okra accessions references ahmed, m. k. and mohamed, e. i. (1995). indigenous vegetables of sudan: production, utilization and conservation. in traditional african vegetables, ed. guarino, l., (rome, italy: institute of plant genetics and crop plant research, gatersleben/international plant genetic resources institute), volume 16, 29-31. charrier, a. (1984). genetic resources of the genus abelmoschus med. (okra) (rome, italy: ibpgr), 61p. geneif, a. a. (1984). tapping natural genetic variability of okra in the sudan. in acta horticulturae 143, eighth african symposium on horticultural crops, wad medani, sudan, 20-24 march 1983, 175-181. hamon, s. and van sloten, d. h. (1989). characterisation and evaluation of okra. in the use of plant genetic resources, ed. brown, a. h. d., marshall, d. r., frankel, o. h., and williams, j. t. (cambridge university press), 173-196. ishag, h. m. and said, m. f. (1985). groundnut production in irrigated vertisols of gezira: achievement and problems. proceeding of fifth international soil classification workshop (khartoum, sudan) 323-329. kumar, s., dagnoko, s., haougui, a., ratnadass, a., pasternak, d., and kouame, c. (2011). okra (abelmoschus spp.) in west and central africa: potential and progress on its improvement. african journal of agricultural research 525(25), 3590–3598. url: http://oar.icrisat.org/168/ mohamed, e. i. (1991). okra genetic resources in sudan. in ibpgr. report of an international workshop on okra genetic resources, international crop network series 5, international board for plant genetic resources, rome, 34-35. omonhinmin, c. a. and osawaru, m. e. (2005). morphological characterization of two species of abelmoschus esculentus and abelmoschus caillei. plant genetic resources newsletter 144, 51–55. url: https://hdl.handle.net/10568/104205. oppong-sekyere, d., akromah, r., nyamah, e. y., brenya, e., and yeboah, s. (2011). characterization of okra (abelmoschus spp. l.) germplasm based on morphological characters in ghana. journal of plant breeding and crop science 3(13), 367– 378. url: https://academicjournals.org/journal/ jpbcs/article-full-text-pdf/cc378699610 schippers, r. r. (2002). african indigenous vegetables, an overview of the cultivated species. natural resources international limited. siemonsma, j. s. and hamon, s. (2004). abelmoschus caillei (a.chev.) stevels. in in plant resources of tropical africa 2. vegetables, ed. grubben, g. j. h. and denton, o. a. (prota foundation, wageningen, netherlands / backhyus publishers, leiden, netherlands / cta, wageningen, netherlands), 21-25. siemonsma, j. s. and kouamé, c. (2004). abelmoschus esculentus (l.) moench. in plant resources of tropical africa 2. vegetables, ed. grubben, g. j. h. and denton, o. a. (prota foundation, wageningen, netherlands / backhyus publishers, leiden, netherlands / cta, wageningen, netherlands), 25-29. singh, h. b., swarup, v., and singh, b. (1975). three decades of vegetable research in india. icar tech. bull., (new delhi: icar). temam, n., mohammed, w., and aklilu, s. (2021). variability assessment of okra (abelmoschus esculentus (l.) moench) genotypes based on their qualitative traits. international journal of agronomy 2021. doi: https://doi.org/10.1155/2021/6678561 http://oar.icrisat.org/168/1/nset4.pdf https://hdl.handle.net/10568/104205 https://academicjournals.org/article/article1380015636_oppong-sekyere\%20et\%20al.pdf https://academicjournals.org/article/article1380015636_oppong-sekyere\%20et\%20al.pdf https://academicjournals.org/article/article1380015636_oppong-sekyere\%20et\%20al.pdf https://doi.org/10.1155/2021/6678561 https://academicjournals.org/journal/jpbcs/article-full-text-pdf/cc378699610 https://www.genresj.org/index.php/grj/article/view/genresj.dlox8174/suppdata105 https://www.genresj.org/index.php/grj/article/view/genresj.dlox8174/suppdata105 https://www.genresj.org/index.php/grj/article/view/genresj.dlox8174/suppdata105 https://www.genresj.org/index.php/grj/article/view/genresj.dlox8174/suppdata105 introduction materials and methods germplasm materials germplasm characterization statistical analysis results phenotypic variations related to plant vegetative characters general plant aspect branching leaf characters stem characters variations in inflorescence characters variations in fruit characters position of fruit on main stem fruit colour fruit length at maturity length of peduncle fruit shape number of ridges per fruit fruit pubescence results of cluster analysis discussion conclusion acknowledgements conflict of interest statement supplemental data original article genetic resources (2022), 3 (5), 36–50 doi: 10.46265/genresj.kpil8781 https://www.genresj.org issn: 2708-3764 phenotypic characterization of gesha horses in southwestern ethiopia amine mustefa *, aweke engdawork, seble sinke and abebe hailu ethiopian biodiversity institute, addis ababa, ethiopia abstract: fifteen qualitative and 21 morphometric variables on a total of 394 adult horses (282 stallions and 112 mares) from three selected districts were recorded to characterize the horse populations in southwestern ethiopia. general linear model, frequency, and multivariate analysis procedures of statistical analysis software (sas 9.0) were used to analyze the data. sex and location significantly affected the studied traits. stallions were larger than mares, and the gesha horse population was the tallest, longest, and largest among the studied populations. the majority of the studied horses possess plain body colour patterns with red-coloured medium hair size. a higher frequency of white-coloured horses was observed with increasing age. stepwise discriminant function analysis revealed that pelvic width, cannon bone length, and height at croup were the top three morphometric variables to discriminate the populations while head length, head neck circumference, chest width, cannon bone circumference, and croup length had the lowest discriminatory power. the results of discriminant function analysis showed advanced classification (76.7%) of the studied horses into their respective populations/locations. finally, canonical discriminant function analysis categorized the horse populations into three distinct categories. the gesha horse population was different from masha and telo horse populations while having a relatively higher relationship with the masha horse population. however, the distances calculated in this study show only the relative size differences between each population. such differences might not necessarily be due to breed (genetic) differences. therefore, diversity studies through further genetic characterization are recommended to design conservation and breeding programmes. keywords: ethiopia, horse, gesha, phenotypic characterization citation: mustefa, a., engdawork, a., sinke, s., hailu, a. (2022). phenotypic characterization of gesha horses in southwestern ethiopia. genetic resources 3 (5), 36–50. doi: 10.46265/genresj.kpil8781. © copyright 2022 the authors. this is an open access article distributed under the terms of the creative commons attribution license (cc by 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. introduction horses are among the most important livestock species in the highlands of ethiopia. in rural areas, horses are the main source of transportation, both for humans and agricultural goods. they are used in public events including social and cultural festivals, and are the most culturally respected and highly valued domestic animals in the country in general, and in southern and southwestern ethiopia in particular (kefena et al, 2012). the highlands of keffa and sheka zones in southwest ethiopia are also among the most benefitted areas from the indigenous horses (kefena et al, 2012). in these areas, horses were also used for traditional racing shows. ∗corresponding author: amine mustefa (aminemustefa32@gmail.com) ethiopia is reported to possess 2.1 million horses (central statistical agency, 2020). however, in terms of standard characterization and documentation, the equine sector has received little attention. until now, only one country-wide general study by kefena et al (2012) was performed to phenotypically characterize the country’s horse breeds, their geographical distribution and production environments. accordingly, eight breeds (abyssinian, bale, boran, horro, kafa, kundido feral horse, ogaden/wilwal and selale horse) were officially reported to exist in the country (kefena et al, 2012; ebi, 2016). however, due to different reasons, the study by kefena et al (2012) did not cover or characterize three horse breeds (boran, kundido feral horse and ogaden/wilwal horses) out of the total eight breeds. additionally, the lack of qualitative morphological data in the study, and the small sample size taken (95–106 received: 23.09.21 accepted: 11.03.22 published online: 06.04.22 https://www.genresj.org https://www.dx.doi.org/10.46265/genresj.kpil8781 https://www.genresj.org https://www.dx.doi.org/10.46265/genresj.kpil8781 mailto:aminemustefa32@gmail.com genetic resources (2022), 3 (5), 36–50 characterization of ethiopian horses 37 horses per breed) can be noted as limitations of the study. similarly, the selected sampling sites were too narrow to represent the horse populations of the area. for example, the horse populations of southwestern ethiopia were represented by a sample from a single site (masha district). a preliminary study by a team of livestock experts from keffa zone hinted at the presence of an unstudied unique horse population in gesha district. according to the results of this preliminary study, gesha horses are said to be typical riding horses of the keffa zone highlands. however, in the countrywide study by kefena et al (2012), this population was represented by horses from the neighbouring masha district. therefore, further characterization studies were required to better understand the horse populations and quantify the level of relationships among them, thus providing a clear country-wide picture. hence, the current study was designed to characterize the horse populations in southwestern ethiopia using both quantitative morphometric measurements and qualitative morphological characteristics. materials and methods locations this study was conducted in keffa and sheka zones of the southern nations nationalities and peoples regional state (snnpr), ethiopia. three locations were selected for the current study (table 1, figure 1). gesha and masha districts were sampled purposively: gesha district (one of the ten districts in keffa zone) is the location of the horses which were supposed to be unique and unaddressed before, while masha district (one of the three districts in sheka zone) is where the samples were taken for the previous country-wide study by kefena et al (2012). telo district was sampled randomly from keffa zone to study the relationship of its horses with gesha horses. the sampling frame was defined after collecting available background information (origin, distribution, population size, and unique features) of the unstudied horse population through focus group discussions with livestock keepers and experts. additionally, information regarding the sampling sites of the country-wide study was also taken from the reports of kefena et al (2012). data collection quantitative and qualitative data were recorded from a total of 394 adult horses (282 stallions and 112 mares) based on the data collection procedures outlined in fao (2012) and the previous country-wide study by kefena et al (2012). studied horses were carefully handled by their owners and trained personnel. data were collected when the animals were calm and standing in an upright position on flat ground and early in the morning of the day before feeding and watering. to minimize measurement error, data were not taken from aggressive horses that did not stand properly. similarly, to minimize subjectivity error, measurements and data recording were performed by the same researchers throughout the study. a centimetre-unit textile measuring tape was used for the morphometric measurements. twenty-one quantitative morphometric measurements (table 2) and 15 qualitative characteristics (hair size, body colour pattern, colour of the body, head, muzzle, tail and hoof, presence/absence of stripe at dorsal body, shoulder and leg, profile of the face, back and croup, length of the tail and mane) were collected. the following body measure indices were calculated from morphometric measurements (adapted from bodó and hecker (1992); cabral et al (2004); druml et al (2008); bene et al (2013)). • body index = (body length/thorax girth) x 100 • quadratic index = (height at withers/body length) x 100 • caliber index = (thorax girth/height at withers) x (cannon circumference/height at withers) x 1000 • overbuilt index = (height at croup/height at withers) x 100 • chest index = (chest width/thorax girth) x 100 • conformation index = (thorax girth2/height at withers)/100 data analysis data entry and management were performed using microsoft excel© worksheet. analysis of the quantitative traits was performed separately for stallions, mares and sex-aggregated by fitting location and age as fixed variables. univariate procedure of statistical analysis software (sas) 9.0 was used to detect outliers and test the normality of morphometric data (sas institute, 2002). data on qualitative traits were subjected to chisquare (χ2) tests of the frequency (freq) procedure of sas 9.0 software. quantitative morphometric and body measure indices data were analyzed using the general linear model (glm) procedure of sas 9.0 software, with adjusted tukey-kramer test to separate the least square means (lsm). data analysis was performed using the following model: yijk = µ + si + lj + ak + eijk where yijk is an observation, µ is the overall mean, si is the fixed effect of ith sex (i = stallion, mare), lj is the fixed effect of jth location (j = telo, gesha, masha), ak is the fixed effect of kth age (k = 4–11), and eijk is the random error attributed to the nth observation. the sex effect was removed from the class variables when the analysis was done separately for each sex. morphometric traits that better discriminate the horse populations from different locations were identified using the forward selection method of the stepwise discriminant function analysis (stepdisc) procedure of sas 9.0. the discriminant function analysis (discrim) procedure of sas 9.0. was also used to assign observations to locations and evaluate probabilities of misclassifications. linear combination of morphometric variables that provide maximal separations between locations was performed using the canonical discriminant function analysis (candisc) procedure of sas 9.0. the scored canonical variables were used to plot 38 mustefa et al genetic resources (2022), 3 (5), 36–50 table 1. climatic and agroecological features of the studied areas. data from bezabih (2012), assefa et al (2013), gebrmichael (2019). climate factors telo gesha masha altitude (m) 2,436–2,451 1,501–3,000 1,700–3,000 temperature (◦c) 17–25 15.1–20 16.7 rainfall (mm) 1,278 2,001–2,200 2,192 agroecology highland midland and highland midland and highland figure 1. map of the sampled locations and districts pairs of canonical variables to get visual interpretation of location differences. pairwise squared mahalanobis distances between locations were computed as: d2 (i|j) = (xi − xj) ′ cov−1 (xi − xj). where d2 (i|j) is the distances between locations i and j, cov−1 is the inverse of the covariance matrix of measured variables, xi and xj are the means of variables in the ith and jth populations. results morphometric measurements and body measure indices the effect of sex on the studied morphometric variables is presented in table 3. most measurements were higher for stallions than mares while ear length and barrel length measurements of the mares were higher than the stallions. on the other hand, body length and back length measurements were not significantly affected by sex. to have a clear picture of the differences among locations, the analysis was performed separately for both sexes. the effect of location on the morphometric measurements of the stallions is presented in table 4. all stallions’ measurements were affected significantly by their location. gesha stallions had significantly the highest values for most of the measurements except for cannon bone length where telo stallions had higher values. masha stallions had relatively higher measurement values than their counterparts from telo district, and these populations shared more similarities. on the other hand, chest width, shoulder depth, body genetic resources (2022), 3 (5), 36–50 characterization of ethiopian horses 39 table 2. description of the collected quantitative measurements. adapted from fao (2012); kefena et al (2012). no. morphometric measurements explanation of the measurements 1 head length distance from the nape to the alveolar edge of the incisors i of the upper jaw 2 head width distance between the upper side of the eyes measured perpendicularly to the head length 3 ear length distance from the tip of the ear to the connection point with the head 4 head neck circumference circumference of the neck at the connection point to the head 5 neck length distance from the highest point of the withers to the nape with the neck in a relaxed position 6 neck body circumference circumference of the neck at the connection point with the body 7 chest width distance between two outer points of the humeral bones from the front 8 shoulder depth distance from the withers to the shoulder joint 9 thorax depth distance from the withers to the sternum 10 thorax width distance between two hypothetical vertical parallel lines drawn at the thorax sides and along the withers’ height line 11 thorax girth measured in the place of the saddle girth 12 cannon bone length distance from the lateral tuberculum of the os metacarpale iv to the fetlock joint 13 cannon bone circumference smallest circumference of the forelimb’s cannon bone 14 height at wither distance from the highest point of the processus spinalis of the vertebra thoracic to the floor 15 height at back distance from the deepest point of the back to the floor 16 height at croup distance from the croup (rump) to the floor 17 body length distance from the most cranial point of the shoulder joint to the most caudal point of the pin bone (scapulo-ischial length) 18 back length distance from the caudal point of the shoulder joint perpendicular to the wither to the most cranial point of the hip joint measured in the saddle place 19 pelvic width distance between the right and left coxal tubers of the ilium 20 croup length distance between the sacral tuber (the highest point of croup) and ischiatic tuber (most posterior point of ischium or point of buttock or seat bone) 21 barrel length distance from the most caudal point of the scapula to the most cranial and dorsal point of the point of the hip length and back length measurements of telo stallions were higher than masha stallions. the effect of location on the morphometric measurements of the mares is presented in table 5. most of the mares’ measurements were affected significantly by their location except ear length, neck length, chest width and barrel length. gesha mares were the biggest and heaviest among the studied populations: their circumferences of head–neck, neck–body and thorax, and heights at withers, back and croup, and pelvic width were significantly larger than telo or masha. the effect of location on the morphometric measurements of the studied horse populations (sex-aggregated) is presented in table 6. all the morphometric measurements of the studied horse populations were affected significantly by their location. significantly, the gesha horse population had the highest values for most of the measurements except for cannon bone length, which was higher in telo horses. pearson correlation coefficients of the morphometric measurements of the horses (both sexes) from different locations are presented in table 7. the majority of the traits were positively correlated. higher positive correlation was observed between height at withers and height at back while lower positive correlation was observed between ear length and head neck circumference. negative correlation was observed between thorax width and cannon bone length. the effect of location on body measure indices of the studied horse populations (separately for each sex) is presented in table 8. all the body measure indices of the studied horse populations were significantly affected by sex. similarly, most of the body measure indices were significantly affected by their location. multivariate analysis stepwise discriminant function analysis revealed the order of importance of the studied morphometric variables in discriminating the horse populations (table 9). the results were also confirmed by wilk’s lambda test (table 9) where all the selected variables had a highly significant (p < 0.0001) contribution in discriminating the horse populations. pelvic width, cannon bone length and height at croup were the first three important traits used in discriminating the studied horse populations. however, some morphometric variables like head length, head neck circumference, chest width, cannon bone circumference and croup length had the lowest dis40 mustefa et al genetic resources (2022), 3 (5), 36–50 table 3. least-square means ± standard errors of quantitative body measurements (cm) of the horse populations by sex. traits stallions mares p-value n 282 112 head length 53.2 ± 0.16 52.1 ± 0.23 < 0.0001 head width 21.5 ± 0.06 21.2 ± 0.09 0.0015 ear length 15.0 ± 0.09 15.5 ± 0.13 0.0033 head neck circumference 58.9 ± 0.24 54.7 ± 0.36 < 0.0001 neck length 59.4 ± 0.27 57.3 ± 0.40 < 0.0001 neck body circumference 91.4 ± 0.37 84.3 ± 0.55 < 0.0001 chest width 25.9 ± 0.13 24.3 ± 0.20 < 0.0001 shoulder depth 53.4 ± 0.18 51.1 ± 0.27 < 0.0001 thorax depth 61.8 ± 0.23 59.8 ± 0.33 < 0.0001 thorax width 34.2 ± 0.16 32.9 ± 0.24 < 0.0001 thorax girth 143.0 ± 0.44 138.6 ± 0.65 < 0.0001 cannon bone length 24.0 ± 0.09 23.6 ± 0.14 0.0066 cannon bone circumference 16.4 ± 0.06 15.6 ± 0.09 < 0.0001 height at withers 131.8 ± 0.29 127.8 ± 0.43 < 0.0001 height at back 129.0 ± 0.28 125.7 ± 0.41 < 0.0001 height at croup 132.1 ± 0.28 129.1 ± 0.42 < 0.0001 body length 125.0 ± 0.38 124.1 ± 0.56 0.1796 back length 70.0 ± 0.26 70.2 ± 0.38 0.6114 pelvic width 40.4 ± 0.16 39.5 ± 0.24 0.0016 croup length 39.7 ± 0.18 38.6 ± 0.27 0.0020 barrel length 67.0 ± 0.29 69.4 ± 0.43 < 0.0001 table 4. means and pairwise comparisons of morphometric measurements of the stallions from different locations. means within a row bearing different superscripts are significantly different; a indicates the largest value. traits least square means (lsm ± se) mean ± se cv p-value telo gesha masha n 94 136 52 head length 52.4 ± 0.27b 54.2 ± 0.23a 53.1 ± 0.36b 53.2 ± 0.16 4.5 < 0.0001 head width 21.0 ± 0.11b 21.9 ± 0.09a 21.6 ± 0.15a 21.5 ± 0.07 4.6 < 0.0001 ear length 14.5 ± 0.15b 15.1 ± 0.13a 15.4 ± 0.20a 14.9 ± 0.08 8.9 0.0002 head neck circumference 58.1 ± 0.43b 60.8 ± 0.36a 57.9 ± 0.57b 59.3 ± 0.24 6.4 < 0.0001 neck length 59.3 ± 0.48b 60.6 ± 0.40a 58.4 ± 0.63b 59.4 ± 0.27 7.1 0.0049 neck body circumference 88.8 ± 0.63b 95.9 ± 0.53a 89.9 ± 0.83b 92.2 ± 0.41 6.1 < 0.0001 chest width 26.0 ± 0.25ab 26.7 ± 0.21a 25.2 ± 0.33b 26.0 ± 0.14 8.5 0.0008 shoulder depth 53.5 ± 0.32b 54.6 ± 0.27a 52.1 ± 0.42c 53.6 ± 0.18 5.3 < 0.0001 thorax depth 59.6 ± 0.40c 63.7 ± 0.33a 61.9 ± 0.52b 61.9 ± 0.25 5.7 < 0.0001 thorax width 32.8 ± 0.30c 35.5 ± 0.25a 34.2 ± 0.39b 34.1 ± 0.18 7.7 < 0.0001 thorax girth 141.5 ± 0.78b 149.0 ± 0.65a 138.8 ± 1.02b 143.6 ± 0.53 4.8 < 0.0001 cannon bone length 24.8 ± 0.16a 23.5 ± 0.14b 23.6 ± 0.21b 24.0 ± 0.09 6.0 < 0.0001 cannon bone circumference 16.34 ± 0.11b 16.9 ± 0.10a 16.1 ± 0.15b 16.5 ± 0.07 6.1 < 0.0001 height at withers 130.7 ± 0.51b 135.2 ± 0.43a 129.6 ± 0.67b 132.2 ± 0.31 3.4 < 0.0001 height at back 127.8 ± 0.48b 132.4 ± 0.41a 127.0 ± 0.63b 129.5 ± 0.30 3.3 < 0.0001 height at croup 131.3 ± 0.50b 135.8 ± 0.42a 129.6 ± 0.7b 132.8 ± 0.31 3.3 < 0.0001 body length 125.3 ± 0.67b 127.5 ± 0.56a 122.4 ± 0.88c 125.3 ± 0.38 4.7 < 0.0001 back length 70.7 ± 0.44a 71.1 ± 0.37a 68.2 ± 0.58b 70.1 ± 0.25 5.6 0.0003 pelvic width 38.7 ± 0.29c 42.3 ± 0.24a 40.5 ± 0.38b 40.5 ± 0.19 6.3 < 0.0001 croup length 38.7 ± 0.33b 41.1 ± 0.28a 39.3 ± 0.43b 39.7 ± 0.18 7.3 < 0.0001 barrel length 66.1 ± 0.51b 67.7 ± 0.43a 67.1 ± 0.66ab 66.8 ± 0.28 6.7 0.0287 genetic resources (2022), 3 (5), 36–50 characterization of ethiopian horses 41 table 5. means and pairwise comparisons of morphometric measurements of the mares from different locations. means within a row bearing different superscripts are significantly different; a indicates the largest value. traits least square means (lsm ± se) mean ± se cv p-value telo gesha masha n 29 47 36 head length 51.9 ± 0.49ab 53.0 ± 0.38a 51.3 ± 0.45b 52.1 ± 0.25 4.9 0.0128 head width 20.6 ± 0.18b 21.5 ± 0.14a 21.5 ± 0.17a 21.2 ± 0.10 4.4 0.0001 ear length 15.2 ± 0.27 15.4 ± 0.21 15.9 ± 0.25 15.5 ± 0.13 9.1 0.1536 head neck circumference 53.2 ± 0.70b 56.7 ± 0.53a 54.3 ± 0.64b 54.9 ± 0.37 6.6 0.0002 neck length 56.7 ± 0.75 58.4 ± 0.57 56.5 ± 0.69 57.2 ± 0.38 6.8 0.0597 neck body circumference 81.3 ± 1.20b 89.4 ± 0.91a 81.7 ± 1.10b 84.7 ± 0.71 7.3 < 0.0001 chest width 23.9 ± 0.32 24.8 ± 0.24 24.0 ± 0.29 24.3 ± 0.16 6.8 0.0535 shoulder depth 51.0 ± 0.54ab 52.2 ± 0.41a 49.9 ± 0.49b 51.0 ± 0.30 5.4 0.0015 thorax depth 58.7 ± 0.63b 61.1 ± 0.48a 59.7 ± 0.58ab 59.9 ± 0.34 5.4 0.0069 thorax width 31.4 ± 0.44b 34.1 ± 0.34a 33.2 ± 0.41a 32.9 ± 0.27 7.0 < 0.0001 thorax girth 135.9 ± 1.26b 144.4 ± 0.96a 134.6 ± 1.16b 138.6 ± 0.81 4.7 < 0.0001 cannon bone length 24.1 ± 0.27a 22.9 ± 0.21b 23.6 ± 0.25ab 23.5 ± 0.14 6.0 0.0014 cannon bone circumference 15.6 ± 0.15ab 15.9 ± 0.11a 15.4 ± 0.13b 15.6 ± 0.08 4.9 0.0067 height at withers 127.3 ± 0.81b 130.3 ± 0.62a 125.4 ± 0.75b 127.9 ± 0.45 3.3 < 0.0001 height at back 124.8 ± 0.84b 128.4 ± 0.64a 123.4 ± 0.77b 125.8 ± 0.46 3.5 < 0.0001 height at croup 128.3 ± 0.81b 132.1 ± 0.61a 126.3 ± 0.74b 129.3 ± 0.47 3.2 < 0.0001 body length 124.5 ± 1.13ab 126.6 ± 0.86a 121.0 ± 1.04b 124.0 ± 0.65 4.7 0.0004 back length 70.6 ± 0.80ab 71.4 ± 0.61a 68.1 ± 0.74b 70.0 ± 0.44 6.0 0.0033 pelvic width 38.6 ± 0.43b 41.4 ± 0.33a 38.4 ± 0.40b 39.6 ± 0.27 5.7 < 0.0001 croup length 38.8 ± 0.52ab 39.6 ± 0.39a 37.7 ± 0.47b 38.7 ± 0.27 6.9 0.0120 barrel length 68.8 ± 0.89 70.2 ± 0.68 69.4 ± 0.82 69.3 ± 0.47 6.7 0.4347 table 6. means and pairwise comparisons of morphometric measurements of the horses (both sexes) from different locations. means within a row bearing different superscripts are significantly different; a indicates the largest value. traits least square means (lsm ± se) mean ± se cv p-value telo gesha masha n 123 183 88 head length 52.0 ± 0.24b 53.6 ± 0.20a 52.2 ± 0.27b 52.9 ± 0.14 4.6 < 0.0001 head width 20.8 ± 0.10b 21.7 ± 0.08a 21.6 ± 0.11a 21.4 ± 0.06 4.6 < 0.0001 ear length 14.8 ± 0.14b 15.3 ± 0.11a 15.7 ± 0.15a 15.1 ± 0.07 8.9 < 0.0001 head neck circumference 55.8 ± 0.38b 58.7 ± 0.31a 56.0 ± 0.41b 58.1 ± 0.23 6.5 < 0.0001 neck length 58.1 ± 0.42b 59.5 ± 0.34a 57.4 ± 0.46b 58.8 ± 0.23 7.0 0.0004 neck body circumference 85.1 ± 0.58b 92.5 ± 0.48a 86.0 ± 0.64b 90.1 ± 0.39 6.4 < 0.0001 chest width 25.0 ± 0.21b 25.7 ± 0.17a 24.6 ± 0.23b 25.5 ± 0.12 8.1 < 0.0001 shoulder depth 52.3 ± 0.28b 53.4 ± 0.23a 51.0 ± 0.31c 52.8 ± 0.17 5.3 < 0.0001 thorax depth 58.9 ± 0.35c 62.6 ± 0.29a 61.0 ± 0.39b 61.3 ± 0.21 5.7 < 0.0001 thorax width 32.1 ± 0.25c 34.8 ± 0.21a 33.7 ± 0.28b 33.8 ± 0.15 7.5 < 0.0001 thorax girth 138.9 ± 0.68b 146.6 ± 0.56a 136.9 ± 0.75b 142.2 ± 0.46 4.8 < 0.0001 cannon bone length 24.5 ± 0.14a 23.2 ± 0.12b 23.6 ± 0.16b 23.9 ± 0.08 6.0 < 0.0001 cannon bone circumference 15.9 ± 0.09b 16.4 ± 0.08a 15.7 ± 0.10b 16.2 ± 0.06 5.8 < 0.0001 height at withers 128.8 ± 0.45b 132.8 ± 0.37a 127.7 ± 0.50b 131.0 ± 0.28 3.4 < 0.0001 height at back 126.1 ± 0.43b 130.4 ± 0.36a 125.5 ± 0.48b 128.4 ± 0.26 3.4 < 0.0001 height at croup 129.7 ± 0.44b 134.0 ± 0.36a 128.2 ± 0.48b 131.8 ± 0.27 3.3 < 0.0001 body length 124.8 ± 0.59b 127.0 ± 0.49a 121.7 ± 0.65c 124.9 ± 0.33 4.7 < 0.0001 back length 70.8 ± 0.40a 71.2 ± 0.33a 68.2 ± 0.44b 70.1 ± 0.22 5.7 < 0.0001 pelvic width 38.4 ± 0.25c 41.8 ± 0.21a 39.6 ± 0.28b 40.3 ± 0.16 6.3 < 0.0001 croup length 38.4 ± 0.29b 40.4 ± 0.23a 38.6 ± 0.32b 39.4 ± 0.16 7.2 < 0.0001 barrel length 67.4 ± 0.45b 69.0 ± 0.37a 68.3 ± 0.50b 67.5 ± 0.25 6.6 0.0113 42 mustefa et al genetic resources (2022), 3 (5), 36–50 table 7. pearson correlation coefficients between each morphometric measurement (above diagonal) and level of significance (below diagonal) of the horses (both sexes) from the three locations. hl, head length; hw, head width; el, ear length; hnc, head neck circumference; nl, neck length; nbc, neck body circumference; cw, chest width; sd, shoulder depth; td, thorax depth; tw, thorax width; tg, thorax girth; cbl, cannon bone length; cbc, cannon bone circumference; haw, height at withers; hab, height at back; hac, height at croup; bol, body length; bal, back length; pw, pelvic width; cl, croup length; brl, barrel length. *, p < 0.05; **, p < 0.01; ***, p < 0.0001; ns, not significant. traits hl hw el hnc nl nbc cw sd td tw tg cbl cbc haw hab hac bol bal pw cl brl hl 0.48 0.16 0.35 0.22 0.48 0.41 0.49 0.51 0.38 0.55 0.08 0.41 0.59 0.56 0.56 0.39 0.36 0.42 0.28 0.29 hw *** 0.24 0.37 0.30 0.44 0.39 0.41 0.46 0.44 0.48 0.06 0.36 0.42 0.42 0.41 0.36 0.30 0.42 0.35 0.31 el ** *** 0.001 0.14 0.07 0.14 0.08 0.17 0.20 0.11 0.03 0.10 0.11 0.11 0.11 0.11 0.16 0.19 0.20 0.23 hnc *** *** ns 0.34 0.74 0.49 0.57 0.46 0.48 0.67 0.12 0.53 0.57 0.52 0.52 0.43 0.19 0.49 0.47 0.11 nl *** *** ** *** 0.49 0.35 0.42 0.39 0.39 0.53 0.13 0.44 0.53 0.53 0.54 0.47 0.32 0.38 0.40 0.23 nbc *** *** ns *** *** 0.55 0.66 0.62 0.56 0.78 0.06 0.61 0.70 0.67 0.67 0.49 0.31 0.60 0.52 0.16 cw *** *** ** *** *** *** 0.56 0.45 0.41 0.59 0.23 0.47 0.47 0.41 0.43 0.41 0.40 0.45 0.40 0.25 sd *** *** ns *** *** *** *** 0.57 0.47 0.73 0.20 0.55 0.69 0.65 0.66 0.56 0.39 0.49 0.51 0.29 td *** *** ** *** *** *** *** *** 0.56 0.67 0.06 0.52 0.62 0.61 0.59 0.42 0.32 0.56 0.44 0.30 tw *** *** *** *** *** *** *** *** *** 0.66 -0.04 0.49 0.54 0.54 0.52 0.47 0.30 0.57 0.48 0.33 tg *** *** * *** *** *** *** *** *** *** 0.09 0.66 0.78 0.76 0.76 0.67 0.47 0.72 0.60 0.36 cbl ns ns ns * * ns *** *** ns ns ns 0.26 0.16 0.14 0.15 0.15 0.22 0.002 0.09 0.05 cbc *** *** ns *** *** *** *** *** *** *** *** *** 0.63 0.60 0.61 0.51 0.39 0.51 0.44 0.20 haw *** *** * *** *** *** *** *** *** *** *** ** *** 0.96 0.94 0.60 0.45 0.60 0.55 0.28 hab *** *** * *** *** *** *** *** *** *** *** ** *** *** 0.93 0.57 0.43 0.57 0.54 0.28 hac *** *** * *** *** *** *** *** *** *** *** ** *** *** *** 0.63 0.47 0.59 0.56 0.31 bol *** *** * *** *** *** *** *** *** *** *** ** *** *** *** *** 0.51 0.54 0.50 0.58 bal *** *** ** ** *** *** *** *** *** *** *** ** *** *** *** *** *** 0.41 0.29 0.37 pw *** *** ** *** *** *** *** *** *** *** *** ns *** *** *** *** *** *** 0.59 0.35 cl *** *** *** *** *** *** *** *** *** *** *** ns *** *** *** *** *** *** *** 0.26 brl *** *** *** * *** ** *** *** *** *** *** ns *** *** *** *** *** *** *** *** table 8. body measure indices of the studied horse populations traits least square means (lsm ± se) mean ± se p-value telo gesha masha stallions body index 88.71 ± 0.40a 85.60 ± 0.34b 88.33 ± 0.53a 87.36 ± 0.24 < 0.0001 quadratic index 104.5 ± 0.47b 106.2 ± 0.40a 106.0 ± 0.62ab 105.7 ± 0.25 0.0095 caliber index 135.4 ± 1.16ab 138.0 ± 0.98a 132.8 ± 1.53b 135.5 ± 0.65 0.0119 overbuilt index 100.4 ± 0.16 100.5 ± 0.14 100.0 ± 0.21 100.4 ± 0.09 0.2523 chest index 18.40 ± 0.15a 17.89 ± 0.13b 18.17 ± 0.20ab 18.1 ± 0.08 0.0178 conformation index 1.53 ± 0.014b 1.65 ± 0.011a 1.49 ± 0.018b 1.56 ± 0.009 < 0.0001 mares body index 91.67 ± 0.75a 87.75 ± 0.58b 90.00 ± 0.69a 89.57 ± 0.40 0.0002 quadratic index 102.3 ± 0.79 103.2 ± 0.60 103.9 ± 0.72 103.4 ± 0.42 0.3730 caliber index 130.7 ± 1.57 135.3 ± 1.20 131.4 ± 1.44 132.1 ± 0.89 0.0580 overbuilt index 100.8 ± 0.25 101.4 ± 0.19 100.7 ± 0.23 101.0 ± 0.13 0.0617 chest index 17.61 ± 0.21ab 17.23 ± 0.16b 17.88 ± 0.19a 17.53 ± 0.10 0.0379 conformation index 1.45 ± 0.02b 1.60 ± 0.02a 1.45 ± 0.02b 1.50 ± 0.01 < 0.0001 both sexes body index 90.01 ± 0.37a 86.71 ± 0.30b 89.07 ± 0.41a 87.99 ± 0.21 < 0.0001 quadratic index 103.3 ± 0.42b 104.8 ± 0.34a 105.05 ± 0.46a 105.1 ± 0.22 0.0036 caliber index 133.5 ± 0.97b 136.6 ± 0.80a 131.9 ± 1.07b 134.5 ± 0.53 0.0005 overbuilt index 100.7 ± 0.14 100.9 ± 0.12 100.4 ± 0.16 100.6 ± 0.07 0.0579 chest index 18.02 ± 0.13a 17.55 ± 0.10b 17.99 ± 0.14a 17.95 ± 0.07 0.0019 conformation index 1.50 ± 0.01b 1.62 ± 0.01a 1.47 ± 0.01b 1.54 ± 0.01 < 0.0001 genetic resources (2022), 3 (5), 36–50 characterization of ethiopian horses 43 criminatory power and were not used in discriminating the horse populations. the values and significant levels of different statistical tests used in the discriminant function analysis are shown in table 10. all the statistical tests were significant showing the appropriateness of the model used in discriminating the horse populations. outputs of the canonical discrimination analysis including eigenvalues and class means under the first two canonical structures are presented in table 11. similarly, table 11 also presents raw canonical coefficients used in constructing the two canonical variables (can 1 and can 2). accordingly, the first canonical structure (can 1) explained the majority (65.7%) of the total variability among the three horse populations. it also produced a greater eigenvalue and multiple correlation (0.70) between the classes (locations) and the morphometric measurements than the second canonical structure (can 2). these results show the higher power of can 1 compared with can 2 in separating the horse populations from the studied locations. however, can 2 also separated one-third of the population, which can 1 is unable to separate. accordingly, can 1 separated telo horses from the others while can 2 separated masha horses from the others. discriminant function analysis classified each individual observation into a known population/location (table 12). accordingly, an average of 76.7% of the sampled animals were classified into their respective population/location. the highest classification of individual horses into their respective locations was observed in the telo horse population (79.7%) with a small error rate (20.3%). on the other hand, a high error rate (26.1%) was detected in the masha horse population. the priors (33.3%) show the chance of every individual observation to be classified into the given three populations/locations. pairwise squared mahalanobis distances between locations are shown in table 13. all distances were significant. gesha and masha horse populations are closely related, while their distance from the telo horse population is large. a plot of the first two canonical structures discriminating the studied horse populations is presented in figure 2. accordingly, can 1 separates the telo horse population from the others, while can 2 discriminates the masha horse population from the others. overall, the analysis categorized the horse populations into three distinct categories. therefore, the gesha horse population is different from the masha and telo horse populations. furthermore, the gesha horse population has more relationship with the masha than the telo horse population. qualitative characteristics chi-square and cramér’s v statistical values and level of significance for the effect of the class variables on the qualitative characteristics of the studied horse populations are presented in table 14. all the traits were significantly affected by the location of the horse populations except body colour pattern and shoulder stripe. on the other hand, only five traits were significantly affected by the horses’ sex and age. face and back profile of the studied horse populations were found to be highly associated with location while the level of relationship of shoulder stripe with location was insignificant. a higher level of relationship between the horses’ sex and age with their head colour was also observed. the majority of the studied horse populations possess a plain body colour pattern with red, medium hair size, and long tail and mane with a mainly black muzzle, tail and hoof (tables 15 and 16, figure 3). all horses had sloppy croup with the absence of leg stripe. short hair size, convex face and straight back profiles were observed more frequently on stallions than mares. the majority of the gesha horses had red body and head (figure 3, c and d) while white-striped red head was also frequently observed. white body and head colour were observed more frequently on telo horses. around half of the horse population from masha district had black and white hoof, which was rarely observed in the other horse populations. the effect of age on the colour-related qualitative characteristics of the studied horse populations is presented in figure 4. little effect of age on the colourrelated qualitative characteristics was observed. as the age of the studied horses increased, the proportion of horses with white body colour showed a significant increase (p < 0.0001), while the proportion of the other colours decreased. similarly, the proportion of horses with white head colour showed a significant increase (p< 0.05) with age, while the proportion of horses with grey head colour decreased. the proportion of the others (red and red with white stripe) remained constant. finally, older horses also showed a higher proportion of white tail colour (p < 0.01) while the proportion of horses with black tail decreased. the proportion of the others (red and grey) remained the same. the majority of the gesha horses had a dorsal stripe and slightly convex face profile, which can be considered their unique characteristics (table 9). a curved back profile was predominantly observed in telo horses, which distinguished them from the others. a slight effect of sex on the qualitative characteristics was observed: shorter hair, a slightly convex face and a straight back profile were observed mainly in stallions. discussion morphometric measurements the studied morphometric measurements produced reliable information to characterize and differentiate the three horse populations phenotypically. besides studying the main effect (location), the effects of age and sex were also analyzed to see if they could cause a significant difference. the effect of age was not significant, 44 mustefa et al genetic resources (2022), 3 (5), 36–50 table 9. summary of the stepwise discriminant function analysis. traits are listed in ascending order used in discriminating the horse populations from different locations. step variables entered partial r-square f value pr > f wilks’ lambda pr < lambda 1 pelvic width 0.2214 55.60 < 0.0001 0.7785 < 0.0001 2 cannon bone length 0.1561 36.06 < 0.0001 0.6570 < 0.0001 3 height at croup 0.1362 30.68 < 0.0001 0.5675 < 0.0001 4 head width 0.0888 18.91 < 0.0001 0.5171 < 0.0001 5 body length 0.0574 11.79 < 0.0001 0.4874 < 0.0001 6 ear length 0.0500 10.16 < 0.0001 0.4630 < 0.0001 7 thorax depth 0.0381 7.62 0.0006 0.4454 < 0.0001 8 shoulder depth 0.0531 10.76 < 0.0001 0.4218 < 0.0001 9 neck body circumference 0.0393 7.84 0.0005 0.4052 < 0.0001 10 back length 0.0308 6.07 0.0025 0.3927 < 0.0001 11 barrel length 0.0336 6.63 0.0015 0.3795 < 0.0001 12 thorax width 0.0272 5.32 0.0053 0.3692 < 0.0001 13 thorax girth 0.0306 5.99 0.0028 0.3578 < 0.0001 14 height at withers 0.0191 3.67 0.0264 0.3510 < 0.0001 15 height at back 0.0227 4.39 0.0131 0.3430 < 0.0001 16 neck length 0.0200 3.83 0.0225 0.3362 < 0.0001 head length 0.0029 0.55 0.5754 head neck circumference 0.0002 0.05 0.9555 chest width 0.0028 0.52 0.5947 cannon bone circumference 0.0019 0.36 0.6946 croup length 0.0011 0.20 0.8210 table 10. values and significant levels of different statistical tests. df, degrees of freedom. statistic value f value num df den df pr > f wilk’s lambda 0.3362 17.03 32 752 < 0.0001 pillai’s trace 0.8298 16.71 32 752 < 0.0001 hotelling-lawley trace 1.4280 17.35 32 668.29 < 0.0001 roy’s largest root 0.9718 22.90 16 377 < 0.0001 figure 2. plot of the first two canonical structures discriminating the three horse populations. genetic resources (2022), 3 (5), 36–50 characterization of ethiopian horses 45 table 11. canonical correlations, eigenvalues, and class means. can 1 can 2 multivariate statistics canonical correlation 0.7020 0.5805 eigenvalue 0.9718 0.5083 proportion 0.6566 0.3434 class (location) means telo -1.4394 0.1662 gesha 0.7827 0.5109 masha 0.3841 -1.2949 raw canonical coefficients head width 0.3332 -0.2810 ear length 0.1426 -0.1680 neck length -0.0526 -0.0230 neck body circumference 0.0552 0.0332 shoulder depth -0.1501 0.0289 thorax depth 0.0693 -0.1063 thorax width 0.0581 -0.1371 thorax girth -0.0134 0.0875 cannon bone length -0.3522 -0.1375 height at withers -0.1633 -0.0871 height at back 0.1627 -0.0541 height at croup 0.0777 0.2509 body length -0.0567 0.0267 back length -0.0606 0.0513 pelvic width 0.1924 -0.0206 barrel length 0.0350 -0.0723 which might be due to the nature of the sampling, which included adult horses only. on the other hand, sex significantly affected the studied traits. stallions had higher values than mares on most morphometric measurements, in line with rensch’s rule (rensch, 1950). according to rensch (1950), males of a given species are usually larger than females. such differences between stallions and mares may be ascribed to levels of testosterone secreted by stallions, which leads to larger muscle mass and skeletal development (baneh and hafezian, 2009). similar results were also reported by kefena et al (2012), ghezelsoflou et al (2018) and sadek et al (2006) on ethiopian, iranian turkoman and arabian horses, respectively. according to kefena et al (2012), selale horses (the tallest and typical riding horses in ethiopia) had values of 131.2 ± 0.4, 125.6 ± 0.4, and 131.7 ± 0.5cm for heights at withers, back and croup, respectively. the current study revealed that gesha horses are the tallest horses in ethiopia with a value of 132.8 ± 0.37, 130.4 ± 0.36, and 134.0 ± 0.36cm for heights at withers, back and croup, respectively (table 6). however, these values were much lower than the reports of zechner et al (2001) for lipizzan horses studied in different locations in europe, and ghezelsoflou et al (2018) for iranian turkoman horses in iran. the tall and big body of the gesha horse population in ethiopia indicates that they figure 3. a, telo stallion; b, masha stallion; c, gesha stallion; d, gesha mare. photo: amine mustefa, ebi can be categorized as typical saddle horses. this is in line with the study by kristjansson et al (2016) in iceland, which showed a higher riding ability as the horses’ height increased. traditionally, gesha horses, which are known for their aggressiveness, are also known and recognized as typical riding horses. the barrel and neck lengths, and cannon bone length and circumference for all the populations from the current study are comparable with the reports of kefena et al (2012) on all ethiopian horse populations. the 46 mustefa et al genetic resources (2022), 3 (5), 36–50 table 12. number and percentage of observations classified into locations. from location telo gesha masha total telo 98 (79.7%) 14 (11.4%) 11 (8.9%) 123 (100%) gesha 19 (10.4%) 140 (76.5%) 24 (13.1%) 183 (100%) masha 7 (7.9%) 16 (18.2%) 65 (73.9%) 88 (100%) total 124 (31.5%) 170 (43.1%) 100 (25.4%) 394 (100%) error rate 0.203 0.235 0.261 0.233 priors 0.333 0.333 0.333 table 13. squared mahalanobis distance between locations; output of the multivariate analysis calculated using the quantitative measurements. *** shows the significance of the distance calculations at p < 0.0001. from location telo gesha masha telo 0 gesha 5.06*** 0 masha 5.46*** 3.42*** 0 body length of gesha horses (127.0 ± 0.49cm) is figure 4. effect of age on colour characteristics of horse populations. a) body colour; b) head colour; c) tail colour. lower than the reports of kefena et al (2012) for all ethiopian horse populations. on the other hand, the head and back lengths of gesha horses (53.6 ± 0.20 and 71.2 ± 0.33cm, respectively) is higher than all ethiopian horse populations (kefena et al, 2012). such wide disagreement might be due to differences in points of measurement. the thorax girth of gesha horses (146.6 ± 0.56cm) is comparable with selale (146.6 ± 0.8cm), bale (145.3 ± 0.7cm), and horro horses (145.5 ± 0.6cm) while it was higher than abyssinian horses (140.4 ± 0.5cm) and lower than keffa horses (152.6 ± 0.7cm) (kefena et al, 2012). body measure indices the body index shows the length of the animal. a long animal is best suited for speed, a short animal for strength (torres and jardim, 1981). long animals have a body index value greater than 90, while a value less than 85 indicates that the animal is short (torres and jardim, 1981). according to table 8, the telo and masha mares were categorized as long horses. however, in reality, gesha stallions are known for their speed. the caliber index, which shows the overall size of the horse, increases with age and size (kaps et al, 2005). kaps et al (2005) observed its increase from 119.1 to 135 in lipizzan horses from 6 to 36 months of age. the current findings show the comparably big size of gesha stallions. the overbuilt index of a horse indicates the proportion of its height at withers and at croup. a horse with downhill conformation (height at croup higher than height at withers) is indicated as the best riding horse by padilha et al (2017), since stronger muscles in the hind limbs and taller hind limbs indicate greater power for jumping and the ability to give a solo performance. in line with the current findings, mcmanus et al (2005) in campeiro horses, rezende et al (2014) in brazilian sport horses and mariz et al (2015) in quarter horses reported a slightly downhill conformation. however, uphill conformation was reported as an important characteristic by lucena et al (2015) in marchador horses and kristjansson et al (2016) in icelandic horses. according to torres and jardim (1981), a riding horse must have a conformation index value of 2.1125. a value above this threshold shows the suitability of a horse for work. the conformation index values found in the current study were between 1.47 and 1.65 (table 8), genetic resources (2022), 3 (5), 36–50 characterization of ethiopian horses 47 table 14. statistical values for chi-square and cramér’s v, and level of significance (probabilities) for the effects of location, sex and age on the qualitative characteristics of the studied horse populations: aggregate sex. χ2, chi-square; prob., probabilities; *, < 0.05; **, < 0.01; ***, < 0.0001; ns, not significant. qualitative traits location sex age χ2 value cramér’s v prob. χ2value cramér’s v prob. χ2value cramér’s v prob. body colour 43.1 0.234 *** 6.4 0.127 ns 95.2 0.201 *** head colour 34.8 0.210 * 19.4 0.222 * 90.5 0.432 * muzzle colour 37.1 0.217 *** 5.9 0.122 ns 42.2 0.164 * tail colour 23.7 0.173 ** 9.6 0.156 * 58.1 0.192 ** hoof colour 55.8 0.266 *** 1.9 0.069 ns 38.2 0.220 ** hair size 21.3 0.233 *** 12.4 0.178 ** 9.7 0.157 ns body colour pattern 8.7 0.105 ns 0.07 0.014 ns 8.3 0.103 ns dorsal stripe 16.5 0.205 ** 0.2 0.021 ns 10.5 0.163 ns shoulder stripe 1.8 0.068 ns 1.6 0.064 ns 4.8 0.111 ns face profile 52.9 0.367 *** 4.1 0.102 * 4.3 0.105 ns back profile 52.8 0.366 *** 4.0 0.101 * 2.6 0.081 ns tail length 28.4 0.190 *** 4.2 0.103 ns 17.4 0.149 ns mane length 52.8 0.259 *** 2.5 0.080 ns 10.0 0.112 ns table 15. percentages of colour-related qualitative traits of the horses (both sexes) from different locations. colour-related qualitative traits location sex telo gesha masha stallions mares body colour red 30.1 50.8 35.2 42.2 37.5 brown 20.3 13.1 21.6 14.5 24.1 gray 16.3 15.8 20.5 18.1 14.3 white 20.3 13.1 18.2 17.4 14.3 tan 0.8 5.5 4.6 3.5 4.5 black 9.8 1.1 0.0 3.2 4.4 red and white 2.4 0.6 0.0 1.1 0.9 head colour white 30.9 19.7 28.4 26.9 20.5 gray 18.7 12.0 13.6 15.3 12.5 red 21.1 26.2 18.2 24.5 18.8 red with white stripe 5.7 21.9 12.5 14.9 14.3 black 14.6 9.8 9.1 10.3 13.4 black with white stripe 0.8 1.6 4.5 1.8 2.7 brown 5.7 4.9 9.1 3.5 12.5 brown with white stripe 1.6 0.6 1.1 0.7 1.8 tan 0.8 1.1 0.0 1.1 0.0 tan with white stripe 0.0 2.2 3.4 1.1 3.6 muzzle colour black 51.2 36.6 37.5 39.4 46.4 white 26.8 19.7 12.5 22.7 14.3 red 10.6 25.7 18.2 19.1 10.6 gray 11.4 9.3 21.6 13.5 10.7 white and black 0.0 8.7 10.2 5.3 8.9 tail colour black 52.0 53.0 36.4 48.6 50.0 gray 26.0 19.7 26.1 25.2 17.9 white 13.8 8.7 12.5 11.7 9.8 red 4.9 14.8 12.5 11.0 11.6 brown 3.3 3.8 12.5 3.5 10.7 hoof colour black 91.9 74.9 52.3 74.1 77.7 black and white 4.9 21.8 47.7 22.7 21.4 white 3.2 3.3 0.0 3.2 0.9 48 mustefa et al genetic resources (2022), 3 (5), 36–50 table 16. percentages of qualitative traits of the horses (both sexes) from different locations. qualitative traits location sex telo gesha masha stallions mares hair size short 42.3 43.2 15.9 42.2 23.2 medium 57.7 56.8 84.1 57.8 76.8 body colour pattern plain 95.9 99.4 100 98.6 98.2 pied 1.6 0.6 0.0 0.7 0.9 shaded 2.4 0.0 0.0 0.7 0.9 dorsal stripe absent 67.5 44.3 57.9 53.9 56.3 present 32.5 55.7 42.1 46.1 43.7 shoulder stripe absent 99.2 99.4 97.7 98.6 100 present 0.2 0.6 2.3 1.4 0.0 face profile straight 86.2 45.4 65.9 59.6 70.5 slightly convex 13.8 54.6 34.1 40.4 29.5 back profile straight 44.7 76.5 87.5 72.0 61.6 curved 55.3 23.5 12.5 28.0 38.4 tail length short 2.4 0.0 0.0 1.1 0.0 medium 40.7 22.4 14.8 28.7 20.5 long 56.9 77.6 85.2 70.2 79.5 mane length short 4.9 0.0 0.0 2.1 0.0 medium 48.0 16.9 39.8 31.9 31.3 long 47.1 83.1 60.2 66.0 68.7 with gesha stallions having the highest conformation index values among the studied populations. multivariate analysis stepwise discriminant function analysis selected and ranked the morphometric variables according to their importance in discriminating the studied horse populations. the inclusion of height at croup and body length within the top five discriminatory variables is comparable with the reports of kefena et al (2012), who classified them among the top four variables to discriminate ethiopian horse populations. the results of discriminant function analysis showed an advanced classification (76.7%) of the studied horses into their respective populations/locations. this high value shows the dissimilarity among the studied populations. canonical discriminant function analysis revealed the higher power of can 1 than can 2 to separate the horse populations. this shows the separation of gesha and masha horses from telo horses while differences also occur between gesha and masha populations. however, the distances showed only the relative size differences between each population. such differences might not necessarily be due to breed (genetic) differences (zechner et al, 2001). therefore, a diversity study through further genetic characterization is recommended to design conservation and breeding programmes. qualitative characteristics besides their aggressiveness and top-riding ability, the examined qualitative characteristics clearly differentiated the gesha horse population from the other studied populations. the majority of gesha horses possess red body colour, red and white-striped red head colour, striped dorsal body, slightly convex face and long mane while some similarities were observed with the adjacent masha horses. a slight effect of sex and age on the qualitative characteristics was observed. shorter hair, a slightly convex face and a straight-back profile were observed predominantly in stallions than mares. the current study revealed the level of relationship between age and body colour. as age advanced, the proportion of horses with white (body, head and tail) colour increased while the proportion of horses with grey and brown colours decrease, which might be due to the progressive depigmentation of the coat’s hairs (locke et al, 2002). at birth, grey horses may have any colour but over time, white hairs begin to appear and become gradually more dominant as white hairs become intermixed with hairs of other colours. at a later age, most horses of this type ultimately become completely white, though some retain intermixed light and dark hairs (locke et al, 2002). this is due to the presence of a greying allele of the kit gene, which inhibits the hair follicles from producing melanin. the coat takes on a ’dappled’ pattern that increasingly becomes white. however, grey horses with a totally white coat can be distinguished from white horses by their underlying black skin, particularly around the eyes, muzzle, and genital area (locke et al, 2002). conclusion the studied phenotypic traits (morphometric measurements and qualitative characteristics) had produced reliable information in characterizing and differentiating gesha, masha and telo horse populations. gesha horses genetic resources (2022), 3 (5), 36–50 characterization of ethiopian horses 49 were the tallest, longest and largest among the studied horse populations. besides their size, the most important characteristics of gesha horses are their aggressiveness, top-riding ability, red-dominated body colour, whitestriped red head colour and slightly convex face. these results were also supported by the multivariate analysis, which differentiated the gesha horse population from the masha and telo horse populations, and showed a relatively higher relationship with masha horses. further genetic characterization is recommended to confirm the above results and design conservation and breeding programmes. acknowledgments the authors are highly indebted to the ethiopian biodiversity institute (ebi) for covering all the budget needs of the work. our special appreciation also goes to the animal owners for providing their animals for this work for free. we also take this opportunity to thank the animal science experts and development agents in the districts for their endless help during data collection. a special word also goes to our friend and work partner mr tadesse hunduma for mapping the study area. author contributions all authors contributed to the study conception and design. material preparation and data collection were performed by amine mustefa, aweke engdawork, and seble sinke. amine mustefa performed the data analysis and wrote the first draft of the manuscript. all authors commented on previous versions of the manuscript, and read and approved the final manuscript. conflict of interest statement the authors declare that they have no conflict of interest. references assefa, a., demissew, s., and woldu, z. 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https://www.researchgate.net/publication/284178649_morfometria_corporal_de_equinos_utilizados_em_trabalho_esporte_e_lazer_em_tres_municipios_do_mato_grosso_do_sul_morphometry_body_of_equines_used_in_work_sport_and_leisure_in_three_cities_of_mato_gross https://www.researchgate.net/publication/284178649_morfometria_corporal_de_equinos_utilizados_em_trabalho_esporte_e_lazer_em_tres_municipios_do_mato_grosso_do_sul_morphometry_body_of_equines_used_in_work_sport_and_leisure_in_three_cities_of_mato_gross https://www.researchgate.net/publication/284178649_morfometria_corporal_de_equinos_utilizados_em_trabalho_esporte_e_lazer_em_tres_municipios_do_mato_grosso_do_sul_morphometry_body_of_equines_used_in_work_sport_and_leisure_in_three_cities_of_mato_gross https://www.researchgate.net/publication/284178649_morfometria_corporal_de_equinos_utilizados_em_trabalho_esporte_e_lazer_em_tres_municipios_do_mato_grosso_do_sul_morphometry_body_of_equines_used_in_work_sport_and_leisure_in_three_cities_of_mato_gross https://doi.org/10.1111/j.1439-0388.2006.00618.x https://doi.org/10.1111/j.1439-0388.2006.00618.x https://www.sas.com/en_us/home.html https://doi.org/10.1016/s0301-6226(00)00254-2 https://doi.org/10.1016/s0301-6226(00)00254-2 introduction materials and methods locations data collection data analysis results morphometric measurements and body measure indices multivariate analysis qualitative characteristics discussion morphometric measurements body measure indices multivariate analysis qualitative characteristics conclusion acknowledgments author contributions conflict of interest statement review genetic resources (2020), 1 (1) 4–16 doi: 10.46265/genresj.2020.1.4-16 https://www.genresj.org issn: 2708-3764 global status of genetic resources for food and agriculture: challenges and research needs dafydd pilling, julie bélanger *, stefano diulgheroff, jarkko koskela, grégoire leroy, graham mair and irene hoffmann food and agriculture organization of the united nations (fao), rome, italy abstract: plant, animal, forest, aquatic, micro-organism and invertebrate genetic resources are vital to food security, nutrition, livelihoods and the resilience and adaptability of global agricultural production systems. despite increasing efforts in recent years, much remains to be done to improve the management of these resources. many are at risk of extinction or erosion and many have been overlooked in terms of use and development. there is an urgent need to address these deficiencies, both within the individual sectors of food and agriculture and in terms of how genetic resources management can be better integrated across sectors. these efforts will need to include action to address the multiple knowledge gaps that constrain improvements to management. they will also need to include the creation of policy and institutional frameworks that promote collaboration and stakeholder participation and allow sustainable management strategies to be implemented effectively at appropriate scales. keywords: genetic resources, food and agriculture, sustainable development goals, global assessments, knowledge gaps citation: pilling, d., bélanger, j., diulgheroff, s., koskela, j., leroy, g., mair, g., hoffmann, i. (2020). global status of genetic resources for food and agriculture: challenges and research needs. genetic resources 1 (1), 4-16. doi: 10.46265/ genresj.2020.1.4-16. © copyright 2020 the authors. this is an open access article distributed under the terms of the creative commons attribution license (cc by 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. genetic resources − the foundation of food and agriculture genetic resources for food and agriculture (grfa) are vital to food security, nutrition, livelihoods and the productivity, resilience and adaptability of production systems in the crop, livestock, forest, fisheries and aquaculture sectors. they are key resources in efforts to achieve the sustainable development goals (sdgs). this paper presents an overview of the state of grfa and their management, drawing largely on the findings of the monitoring activities overseen by the commission on genetic resources for food and agriculture (commission) of the food and agriculture organization of the united nations (fao) (boxes 1 and 2) and highlighting future management challenges, with an emphasis on knowledge gaps. ∗corresponding author: julie bélanger (julie.belanger@fao.org) status and trends uneven, with worrying declines knowledge of the status and trends of grfa varies across sectors. the following subsections present short overviews. selected key facts and figures on the status and trends of genetic resources and their management, at global level, are presented in table 1. plant genetic resources for food and agriculture (pgrfa) more than 6,000 plant species have been cultivated for food (leibniz institute of plant genetics and crop plant research (ipk), 2020), but today nine species (sugarcane, maize, rice, wheat, potatoes, oil palm, soybean, cassava and sugar beet) provide 67 percent of crop production by weight (fao, 2020b). the precise status and trends of within-species genetic diversity is difficult to assess. received: 18 06 2020 accepted: 06 07 2020 published online: 31 08 2020 https://www.genresj.org mailto:julie.belanger@fao.org www.doi.org/10.46265/genresj.2020.1.4-16 www.doi.org/10.46265/genresj.2020.1.4-16 www.doi.org/10.46265/genresj.2020.1.4-16 www.genresj.org g enetic resources (2020),1 (1) 4–16 g lobalstatus ofg rfa 5 table 1. selected facts and figures on the state and trends of genetic resources and their management at global level categories plant genetic resources for food and agriculture animal genetic resources for food and agriculture forest genetic resources1 aquatic genetic resources for food and agriculture2 micro-organism and invertebrate genetic resources for food and agriculture3 total number of known species estimated 391,000 plant species4 more than 17,000 avian and mammalian species5 more than 60,000 tree species6 more than 160,000 aquatic species unknown number of species and subspecies groups (i.e. varieties, breeds, etc.) used for food and agriculture 6,000 species7 unknown number of varieties8 around 40 species9 over 8,700 breeds10 8,000 species of trees, shrubs, palms and bamboo reported by countries 1,800 species targeted by capture fisheries 694 commercially farmed species items few well-established improved farmed types unknown species concentration in food and agricultural production 9 species provide 67% of global crop production11 8 species provide 97% of global meat production11 2,400 species reported as actively managed for products and services 10 species provide 50% of global aquaculture production12 not applicable status and trends of species and within-species genetic diversity reported decreases in crop diversity in farmers’ fields, but situation variable and complex8 many species of crop wild relatives under threat8,13,14 28% of local breeds at risk, 10% not at risk, 62% unknown risk status10 57% of species (34,204) have a conservation assessment: 38% of these are threatened globally15 no systematic global monitoring system for within-species diversity loss of genetic diversity in commercially important species a concern. limited information below species level increase of species diversity in aquaculture, but increased emphasis on production of a few species limited information available evidence indicates widespread declines continued on next page 6 pilling et al g enetic resources (2020),1 (1) 4–16 table 1 continued categories plant genetic resources for food and agriculture animal genetic resources for food and agriculture forest genetic resources1 aquatic genetic resources for food and agriculture2 micro-organism and invertebrate genetic resources for food and agriculture3 breeding 443 crop species reported by 28 countries with active public pre-breeding and breeding programmes (81 species reported as being used in private programmes)16 well-organized breeding programmes and use of advanced techniques largely restricted to developed regions and focused on a limited range of mostly temperate breeds9 700 species reported to be included in breeding programmes 55% of farmed species reported to be subject to some kind of genetic management few invertebrate and micro-organism species are subject to genetic improvement activities in situ conservation among 30,000 in situ conservation sites reported in 39 countries, 9% had management plans for crop wild relatives and wild food plants16 in situ conservation activities widely reported, but many gaps in coverage9 of 8,000 species used for various purposes, about 1,000 are included in in situ conservation programmes aquatic protected areas and effectively managed fisheries contribute to in situ conservation of aquatic genetic resources limited action specifically targeting these groups ex situ conservation 5.4 million accessions from more than 50,000 species conserved in over 700 genebanks in 103 countries and 17 regional and international research centres17 of 7,760 local breeds (including extinct ones), 258 reported to have genetic material stored in genebanks, 79 of these with sufficient material stored to allow them to be reconstituted10 1,800 species reported as being conserved ex situ 159,579 accessions reported globally 290 species, almost 200 of which considered threatened at national or international levels, are maintained in 690 ex situ collections 791 culture collections, containing over 3 million microbial cultures of 50,875 species and subspecies, in 78 countries and regions, are registered with the wfcc18 sources: 1 fao (2014b), unless noted otherwise; 2 fao (2019c), unless noted otherwise;3 fao (2019d), unless noted otherwise; 4 royal botanic gardens kew (2016); 5 birdlife-international (2018) burgin et al (2018); 6 beech et al (2017); 7 leibniz institute of plant genetics and crop plant research (ipk) (2020); 8 fao (2010); 9 fao (2015); 10 fao (2020a); 11 fao (2020b); 12 fao (2020c); 13 magos-brehm et al (2017); 14 bilz et al (2011); 15 global tree assessment (gta) (2020); 16 fao (2020e), data refer to reporting period 2012−2014; 17 fao (2020e), data refer to 2019; 18 world federation of culture collections (http://www.wfcc.info/ccinfo/statistics/). http://www.wfcc.info/ccinfo/statistics/ genetic resources (2020), 1 (1) 4–16 global status of grfa 7 there are no comprehensive figures available for the status of crop varieties across the world’s production systems and there is as yet no standardized way of assessing their risk status. however, the available evidence indicates that, overall, the crop diversity present in farmers’ fields has declined (fao, 2010). many farmers’ varieties and landraces have disappeared or become rarer. the situation is, however, complex, with new varieties sometimes being grown in addition to, rather than in replacement of, traditional ones. the state of genetic vulnerability (“the condition that results when a widely planted crop is uniformly susceptible to a pest, pathogen or environmental hazard as a result of its genetic constitution, thereby creating a potential for widespread crop losses” (fao, 1997a)) is also difficult to assess. however, many countries have reported significant genetic vulnerability in their production systems (fao, 2010). crop wild relatives are key resources in plant breeding and are widely under threat (bilz et al, 2011; fao, 2010; magos-brehm et al, 2017). threats to domesticated pgrfa include changes to production systems that lead to declines in the use of traditional varieties (fao, 2010). crop wild relatives are affected by pressures on their habitats, including those related to climate change and to land-use changes associated, inter alia, with agriculture (magos-brehm et al, 2017; bilz et al, 2011; fao, 2010). animal genetic resources for food and agriculture (angr) among the more than 17,000 known avian and mammalian species, (burgin et al, 2018; birdlifeinternational, 2018), only about 40 have been domesticated for use in food and agriculture (fao, 2015). production is very concentrated among a few species, with eight (pig, chicken, cattle, sheep, goat, turkey, duck and buffalo) providing 97 percent of global meat production in 2018; four of these (cattle, buffalo, goat and sheep) accounted for almost 100 percent of global milk production, and chickens alone accounted for 93 percent of egg production (fao, 2020b). a total of 8,719 livestock breeds are recorded by fao as of 2020; 26 percent of these are classified as at risk of extinction, 13 percent as not at risk, 6 percent as extinct and 55 percent as being of unknown risk status (fao, 2020a). sdg indicator 2.5.2 is “proportion of local breeds, classified as being at risk, not-at risk or unknown level of risk of extinction” (“local breeds” are breeds found in only one country). as of 2020, 62 percent of local breeds are classified as being of unknown status, 28 percent as at risk and 10 percent as not at risk (the figures exclude extinct breeds) (fao, 2020a). in all regions other than europe and the caucasus and north america, more than 80 percent of local breeds are of unknown risk status. improving reporting is thus a major challenge. angr are threatened by a range of factors. immediate threats include breed substitution, poorly managed cross-breeding and the decline of livestockkeeping livelihoods, all driven in turn by a variety of economic, social and environmental factors, exacerbated by weak policies and institutions (fao, 2015). acute events such as disease outbreaks can be a threat to small, geographically concentrated breed populations (ibid.). forest genetic resources (fgr) there are over 60,000 tree species in the world (beech et al, 2017). most of these are wild species that have not been subject to any form of domestication. the country reports submitted for the state of the world’s forest genetic resources (sow-fgr) (fao, 2014b) listed nearly 8,000 species of trees, shrubs, palms and bamboo, of which about 2,400 were being actively managed for the products and/or services they supply and over 700 were included in breeding programmes. information on the status of tree species remains incomplete. the global tree assessment, which aims to assess the conservation status of all known tree species by 2020, reports as of march 2020 that 34,204 species (57 percent of all tree species) have been assessed and that 12,237 (36 percent of the assessed species) are threatened globally (global tree assessment (gta), 2020). there is no systematic global monitoring system in place for intraspecific diversity in tree species, but loss of genetic diversity in commercially important species has long been a concern among forest managers (fao, 2014b). fgr are threatened, inter alia, by land-use change, particularly conversion of forests to cropland and grazing land, overexploitation, selective harvesting and climate change (ibid.). forests cover 31 percent of the global land area (4,060 million hectares), but they continue to be lost at an alarming rate despite efforts to promote natural regeneration and tree planting (fao and unep, 2020). between 2015 and 2020, the rate of forest expansion was 5 million hectares per year, while the rate of deforestation was 10 million hectares per year, meaning that the net loss of forests was about 5 million hectares per year (ibid.). aquatic genetic resources for food and agriculture (aqgr) there are more than 160,000 species of fish and aquatic crustaceans, molluscs and plants in the world (fao, 2019c). of these, around 1,800 species or species items (a species item is a category of aquatic animal or plant at the species, genus, family or higher taxonomic level) are targeted by capture fisheries (ibid.). the total number of farmed species items recorded in aquaculture production by fao, as of 2018, was 622, corresponding to 466 individual species, 7 interspecific hybrids of finfish, 92 species groups at genus level, 32 species groups at family level and 25 species groups at order level or higher (fao, 2020d). however, the state of the world’s aquatic genetic resources for food and agriculture (sow-aqgr) (fao, 2019c) indicated that such production figures underestimate the number of cultured species, reporting farming of 694 species or 8 pilling et al genetic resources (2020), 1 (1) 4–16 box 1. the work of the commission on genetic resources for food and agriculture the commission, a permanent intergovernmental body currently comprising 178 countries and the european union, was established in 1983 as the commission on plant genetic resources for food and agriculture. it negotiated the legally binding international treaty on plant genetic resources for food and agriculture, adopted in 2001 (fao, 2009). in 1995, its mandate was extended to cover all components of biodiversity of relevance to food and agriculture. the commission regularly oversees country-driven global assessments of particular categories of genetic resources. the first of these, the state of the world’s plant genetic resources for food and agriculture (fao, 1997a), was followed by the state of the world’s animal genetic resources for food and agriculture (fao, 2007a), the state of the world’s forest genetic resources (fao, 2014b) and the state of the world’s aquatic genetic resources for food and agriculture (fao, 2019c), the latter covering farmed aquatic species and their wild relatives within national jurisdiction. the commission has also overseen a global assessment covering all components of biodiversity of relevance to food and agriculture, the state of the world’s biodiversity for food and agriculture (fao, 2019d). the global assessments are repeated at intervals of approximately ten years, meaning that second reports on plant and animal genetic resources have been published (fao, 2015, 2010) and that the second assessment on forest and the third on plant genetic resources are ongoing. the first global assessments for plant, animal and forest genetic resources were followed by the adoption of global plans of action for the respective sectors (fao, 2014a, 2007b, 1997b). in the case of plants, a second global plan of action was adopted in 2011 (fao, 2011). a global plan of action for aquatic genetic resources is currently under negotiation (fao, 2019b). the commission has overseen the development of a number of codes, standards and guidelines to support the implementation of the global plans of action. in 2019, the commission adopted a work plan for the sustainable use and conservation of micro-organism and invertebrate genetic resources for food and agriculture (ibid.). species items. in asia, approximately twice as many species are reported farmed as in other continents. the report also records over 200 species that are farmed in countries where they are not native. aquaculture is, for the most part, a relatively new activity and the sector has few well-established improved farmed types equivalent to the varieties and breeds of terrestrial crops and livestock (fao, 2019c). farmed aquatic organisms are often very similar to their wild counterparts, which are sometimes used as broodstock or seed. little information is available on the status of aqgr below the species level. as noted in box 2, fao is currently developing a prototype registry for these “farmed types”. micro-organism and invertebrate genetic resources for food and agriculture (migr) micro-organisms and invertebrates contribute to food and agriculture in a multitude of ways, including in pollination, pest control, nutrient cycling, food processing, and digestion in ruminant animals. the status and trends of micro-organisms and invertebrates, including those that contribute to food and agriculture, are generally less well monitored than those of plants and vertebrate animals. however, at the level of broad taxonomic and functional groups the available evidence indicates worrying declines (e.g. (fao and itps, 2015; fao, 2019d; ipbes, 2019; ipbes, 2016). the habitats upon which useful micro-organisms and invertebrates depend are often in decline (fao, 2019d). while the overall number of honeybee colonies worldwide has increased over recent decades, some countries have experienced substantial falls in colony numbers or have required extra efforts on the part of beekeepers to maintain production (fao, 2019d; ipbes, 2016). there are big knowledge gaps on the state of soil biodiversity, but there are grounds for serious concern in all regions of the world (fao, 2019d; fao and itps, 2015). threats to migr include habitat destruction, inappropriate use of pesticides and other agricultural inputs and the effects of climate change (fao, 2019d). management strengthened, but progress patchy management of grfa is taken here to include use and conservation. each of the three existing global plans of action (gpas, see box 1) sets out priorities in each of these areas. implementation is monitored via periodic rounds of country reporting and via the information systems mentioned in box 2. the following subsections provide overviews based on these and other sources. it needs to be borne in mind, however, that use and conservation are multifaceted and interlinked fields of activity and that their boundaries are not clearly defined. definitions and approaches to monitoring vary across sectors, as does the significance of specific management activities (e.g. in situ vs. ex situ conservation). space precludes a detailed discussion of the state of the art in management or of the status of implementation of management activities around the world. readers are directed to the “state of the world” reports (box 1) for additional information. plant genetic resources for food and agriculture higher level composite indices for the implementation of the second global plan of action for plant genetic resources for food and agriculture (second gpapgrfa) were calculated for the period 2012 to 2014 based on data provided by 69 countries (fao, 2020e). scores for actions related to the sustainable use of pgrfa were generally at a medium level (averaging approximately 4.3 out of a maximum possible 8). a genetic resources (2020), 1 (1) 4–16 global status of grfa 9 box 2. fao’s information systems on genetic resources for food and agriculture fao operates global information systems for plant and animal genetic resources for food and agriculture, both of which are used for monitoring progress towards sustainable development goal (sdg) target 2.5. the domestic animal diversity information system (dad-is)a provides tools that can be used to monitor national breed populations and to support informed decision-making on the management of animal genetic resources for food and agriculture. it provides access to official data for monitoring progress towards the animal component of sdg target 2.5. the world information and early warning system on plant genetic resources for food and agriculture (wiews)b provides access to official data for monitoring progress towards the plant component of sdg target 2.5 and on the implementation of the 18 priority activities of the second global plan of action for plant genetic resources for food and agriculture. in 2019, the commission on genetic resources for food and agriculture requested fao to initiate the development of a new global information system on forest genetic resources (fao, 2019b). work is also underway to develop a global information system for aquatic genetic resources for food and agriculture, including a prototype registry of farmed types based on standardized terminology (mair and lucente, 2020). in the absence of such a system, aqgr are largely excluded from the monitoring of progress towards sdg 2.5. these new information systems will be fundamental to the implementation and monitoring of the global plans of action in the respective sectors. a http://www.fao.org/dad-is/en/ b http://www.fao.org/wiews/en/ preliminary study conducted on a smaller sample of country reports (fao, 2016) indicated several positive developments in the field of characterization, evaluation and further development of specific collection subsets to facilitate use, with many genebank accessions reported as having been assessed and distributed for use. in the field of plant breeding, genetic enhancement and basebroadening, again a range of activities were reported, focused mainly on major crop species. international and regional networks of genebanks were reported to be widely involved in the supply of germplasm. about one-third of the reported activities in this field aimed to address constraints relevant to the production systems of small-scale farmers or local communities. genetic enhancement and pre-breeding activities mainly targeted local cultivars and landraces. actions promoting diversification of crop production and broadening crop diversity received a relatively low average score. however, several initiatives were reported, including the introduction of a number of new crops or wild species into cultivation. countries reported a range of laws, policies, programmes and projects promoting the development and commercialization of crop varieties. actions related to supporting seed production and distribution received the highest average scores, with vegetables and cereals being the crop groups most widely reported to be targeted1. the state of ex situ conservation for pgrfa is monitored under sdg target 2.5 (box 2). over the past 24 years, the number of pgrfa accessions stored under medium or long-term conditions has steadily increased (by approximately 100,000 accessions per year) reaching 5.4 million − held in over 700 genebanks in 103 countries and 17 regional and international 1 all the findings presented here from the preliminary study were confirmed by the analysis of the larger sample of countries (fao, 2020e). centres − in 2019 (fao, 2020e). these figures are lower than previously published estimates (e.g. fao, 2010) as current wiews data comply with sdg 2.5.1 prescriptions for avoiding duplication in the reporting of collections and accessions within national inventories. between 2000 and 2018, the number of species conserved in these collections more than doubled, increasing from about 24,000 to over 51,000 (ibid.). while the highest rate of increase occurred during the first 10 years, on average about 700 new species were added to ex situ collections worldwide annually during the period from 2014 to 2018. these increases were the result both of collecting missions and of improved taxonomic classification of already conserved materials. as of december 2019, 290 genebanks around the world held almost 96,000 samples from over 1,700 species listed in the international union for conservation of nature’s categories of major global concern2 (fao, 2020e), including relatives of crops particularly important for global and local food security. despite the progress made, the global response in terms of preserving crop diversity in ex situ facilities compliant with genebank standards is likely to be insufficient to respond to the alarming pace of the growth of the threats posed by climate change, particularly of the case for crop wild relatives, wild food plants and neglected and underutilized crop species. species in these groups continue either to be absent from genebank collections or have their intraspecific diversity poorly represented. reporting on the implementation of the second gpa-pgrfa between 2012 and 2014 indicated that increased attention was being given to the in situ conservation of crop wild relatives. among the 30,000 in situ conservation sites reported in 39 countries, 9 percent had management plans addressing crop wild 2 extinct in the wild, critically endangered, endangered, vulnerable, near threatened and data deficient (iucn, 2020). http://www.fao.org/dad-is/en/ http://www.fao.org/wiews/en/ 10 pilling et al genetic resources (2020), 1 (1) 4–16 relatives and wild food plants (fao, 2020e). however, indicator scores for this area of management were low. overall, in situ conservation and on-farm management (comprising priority activities in the fields of surveying and monitoring, supporting on-farm management and improvement, assisting farmers in disaster situations to restore crop systems, and promoting in situ conservation and management of crop wild relatives and wild food plants) underperformed as compared to ex situ conservation and other areas of pgrfa management (ibid.). animal genetic resources for food and agriculture the third round of country reporting on the implementation of the global plan of action for animal genetic resources (gpa-angr) took place in 2019. analysis of the 104 country progress reports submitted is ongoing at the time of writing, but broadly speaking they reveal that many countries have continued to strengthen their activities related to sustainable use and development. however, the level of implementation and the extent to which progress has been made since the adoption of the gpa vary greatly both across regions and across countries within regions, with higher levels reported in europe and the caucasus and north america than elsewhere. in 2014, when the previous round of country reporting took place, strategic priorities targeting sustainable use and development were at low to medium levels of implementation, with global average scores of between 0.5 and 1 out of a maximum of 2 (fao, 2014c). actions related to breeding programmes scored slightly better than those related to ecosystem approaches and support for local and traditional production systems. sustainable use policies scored lowest, with averages dragged down by the underdeveloped state of access and benefitsharing policies in many countries (ibid.). as with pgrfa, the state of ex situ conservation of angr is monitored under sdg target 2.5 (box 2). out of 7,760 local breeds (including extinct ones), 258 are reported to have some genetic material stored, and 79 are reported with sufficient material stored to allow them to be reconstituted (fao, 2020a). the 2019 progress reports on the implementation of the gpaangr indicate that conservation actions have continued to be strengthened over recent years in many countries. the previous round of country reporting again indicated low to medium levels of implementation of strategic priorities in this field (fao, 2014c). in situ conservation scored relatively well compared to ex situ conservation (ibid.), although it needs to be borne in mind that in situ activities and their impacts are difficult to monitor because of a lack of detailed data and differences in the way the term is used in different countries. country reporting for the second report on the state of the world’s animal genetic resources for food and agriculture (fao, 2015) indicated that at least some in situ conservation activities were being implemented in most countries, with a wide variety of different approaches reported, including those related to breeding programmes, to market development and to other forms of support for farmers and herders raising rarer breeds. however, it also clearly indicated that levels of implementation were far below those that countries considered necessary to provide an adequate degree of protection for their angr (ibid.). as of may 2020, 223 (11 percent) of the 1,808 local breeds recorded in dad-is (box 2) as “critical” or “endangered” were listed as “maintained”, meaning that “active conservation programmes are in place or populations are maintained by commercial companies or research institutions” (fao, 2020a). forest genetic resources the first round of country reporting on the implementation of the global plan of action for the conservation, sustainable use and development of forest genetic resources (gpa-fgr) took place in 2018 (fao, 2019a). the response rate was quite low (44 countries) and hence it is not possible to draw comprehensive conclusions. across the gpa-fgr as a whole, reporting countries had on average achieved 67 percent of action points and had initiated efforts to achieve a further 10 percent. only four had achieved all 15 action points. a total of 1,145 tree and other woody plant species (including hybrids) were included in the 44 country progress reports. with regard to the state of use, a total of 531 tree species were reported to be included in national tree seed programmes. the numbers reported by individual countries varied greatly, from zero in several cases up to 114. a total of 288 species were reported to be included in tree-breeding programmes, with the numbers reported per country ranging from zero to 55. however, many more species are used in forestry; for the sow-fgr, countries reported about 2,400 species as being actively managed for products or services in forestry and more than 700 as being included in tree improvement programmes (fao, 2014b). information on the status and trends of in situ conservation activities − the main approach to fgr conservation − is limited. in 2018, only 568 species were reportedly included in in situ conservation programmes and 647 in ex situ programmes. however, the country reports submitted for the sow-fgr listed nearly 8,000 species of which about 1,000 were reportedly conserved in situ and 1,800 ex situ (fao, 2014b). only 625 out of 2,260 priority species listed were reported to be subject to any kind of ex situ conservation, with maintenance in field collections, including clone banks and provenance trials, much more frequently reported than storage in seed or in vitro collections (ibid.). aquatic genetic resources for food and agriculture as a gpa for the sector has yet to be adopted, aqgr management has no global monitoring system equivalent to those existing in other sectors. however, genetic resources (2020), 1 (1) 4–16 global status of grfa 11 some relevant data are available. for example, metian et al (2020) report the use of a large and increasing range of species in aquaculture, particularly in asia, and argue that this enhances the resilience of the sector by improving capacity to adapt to change. while new species are being developed for aquaculture and the list of cultured species continues to expand, global aquaculture production is increasingly dominated by a few key species, with the top ten species accounting for 50 percent of global production (fao, 2020c), a trend which, if it continues, may erode resilience to challenges such as disease and climate change. as noted above, genetic improvement activities are relatively underdeveloped in the aquaculture sector. among the species listed as being farmed in the country reports submitted for the sow-aqgr only 55 percent were reported to be subject to any kind of genetic management (fao, 2019c). while studies indicate that there is potential for major gains in productivity via selective breeding of farmed aquatic species (ibid.), 45 percent of countries reported that genetic improvement was yet to have any significant impact on their aquaculture production, and the report identified an important need to increase the adoption of genetic programmes, especially for lower-value species important to food security. the report highlights the need to set an appropriate balance between investment in the diversification of species used in aquaculture and the application of genetic technologies to better adapt existing cultured species to diverse culture environments. in situ conservation of aqgr relates mainly to the protection of wild species, for example via the establishment of protected areas, management and regulation of fishing and other habitat-protection measures, although “on-farm” conservation to prevent the loss of farmed-type genetic resources is also required. both aquaculture and capture fisheries have an important role to play and conservation objectives need to be integrated into aquaculture development and fisheries management strategies. countries that contributed to the sow-aqgr generally considered protected areas to be an effective means of conserving the genetic resources of wild relatives of farmed aquatic species (fao, 2019c). seventy-five percent of the 92 reporting countries indicated the implementation of ex situ conservation activities for aquatic organisms of national relevance falling within the scope of the report. approximately 290 different species, almost 200 of which were considered to be threatened at national or international levels, were being maintained in a total of 690 ex situ collections. finfish accounted for 90 percent of the species concerned, with the other 10 percent accounted for by macro-invertebrates and aquatic micro-organisms such as rotifers and microalgae. most ex situ conservation is in vivo. about 38 percent of reporting countries indicated the existence of in vitro conservation of aqgr (farmed species and wild relatives), involving a total of 133 different species. because of the difficulty of preserving the eggs and embryos of aquatic organisms, most in vitro conservation involves cryopreservation of sperm. micro-organism and invertebrate genetic resources for food and agriculture many micro-organisms and invertebrates of importance to food and agriculture are not actively managed in any way by producers. however, many approaches that involve introducing them into production systems or managing habitats to encourage their presence, for example in the context of integrated pest management, pollination management or integrated plant nutrition management, are becoming more widely implemented globally (fao, 2019d). few species are subject to genetic improvement. however, there are a substantial number of commercial honey-bee breeding companies around the world that implement genetic improvement programmes, with the main goals being higher honey production, greater docility, reduced swarming and, particularly in recent years, better disease tolerance (ibid.). micro-organisms used in food processing and in agroindustrial processes are subject to a variety of geneticimprovement strategies (alexandraki et al, 2013; chatzipavlidis et al, 2013). some genetic improvement is also being conducted in micro-organisms used in plant nutrition, biological control and food preservation (fao, 2019d). micro-organisms and invertebrates are conserved in situ along with other components of biodiversity in protected areas. they also benefit from the adoption of biodiversity-friendly management practices in the food and agriculture sector and elsewhere. however, the number of species specifically targeted is limited, as is information on the coverage and effectiveness of conservation measures (ibid.). micro-organisms can be stored under laboratory conditions in a range of different ways. existing culture collections are, however, far from representing the full range of micro-organisms of relevance to food and agriculture (ibid.). various invertebrates of importance to food and agriculture are raised in captivity by commercial companies or by research institutes. however, there are few systematic ex situ conservation programmes, even for high-profile groups of invertebrates such as pollinators. some work has been done on the cryoconservation of bee semen, although the technique has not become widely used (ibid.). knowledge gaps a key constraint knowledge gaps are a major constraint to the effective management of grfa. as discussed above, population status and trends are inadequately monitored across most categories, hindering the planning of conservation efforts. the following subsections briefly outline key knowledge gaps by sector and related to cross-sectoral integration. 12 pilling et al genetic resources (2020), 1 (1) 4–16 plant genetic resources for food and agriculture monitoring pgrfa diversity in situ and on-farm to predict and minimize loss of interand intra-specific genetic variation is a major challenge, particularly in vulnerable groups such as crop wild relatives, wild food plants and underutilized crops. national conservation planning would greatly benefit from the development of indicators that could be widely used to quantify genetic erosion and monitor changes in the extent and distribution of individual species and populations at various scales. research on the characteristics of the above-mentioned vulnerable groups, including on their reproductive biology, agronomic and nutritional properties, traditional and potential uses, and contributions to the health of agro-ecosystems, is vital to efforts to improve their conservation and sustainable use. knowledge of their geographical distribution also needs to be improved. efforts to integrate in situ and on-farm management and conservation of pgrfa with the work of national, regional and international genebanks and research institutes need to be documented and widely publicized. knowledge gaps on recalcitrant seed physiology and behaviour in neglected species, along with a lack of standardized protocols for their in vitro conservation and cryopreservation − and a lack of alternative low-cost conservation methods – is often a severe constraint to national ex situ conservation programmes. other key knowledge gaps relate to breeding systems, reproductive biology, dormancy mechanisms and technical problems associated with regeneration practices for “unconventional” species. the use of molecular methods, biochemical assays and high-throughput phenotyping in germplasm characterization and evaluation to identify useful genes, understand their expression and variation, and in particular understand their roles in adapting to climate change, increasing nutritional values and strengthening ecosystem services, has been limited to a few major crops in developed countries. further work is also needed on development and harmonization of standards for the exchange of data on in situ germplasm and the documentation of ethnobotanical information on farmers’ varieties, landraces and underutilized species. animal genetic resources for food and agriculture the genomic revolution has led to impressive progress both in terms of improving knowledge of angr and in terms of genetic improvement. however, it has also widened gaps between developed and developing countries and between the relatively few international transboundary breeds that increasingly dominate high-input production systems globally and the mass of breeds adapted to more extensive systems. there are clear knowledge gaps in terms of the characterization of phenotypes (especially functional and adaptive traits) and their relations to production environments. as characterization is a prerequisite for effective implementation of genetic improvement programmes (leroy et al, 2016), these knowledge gaps are to some extent hindering the realization of the opportunities offered by genomics. one of the most important challenges in angr management relates to the difficulty of developing governance systems that fully integrate livestock keepers from developing regions (leroy et al, 2017). systems of this kind are vital to the implementation of characterization studies, breeding programmes and market development (gowane et al, 2019). experiences in this field need to be documented and publicized, although success will also depend on the provision of adequate institutional, technical and financial support over the long term (mueller et al, 2015). forest genetic resources priorities in the field of fgr management include improving knowledge of the amount and distribution of genetic diversity in forest trees and of how well current efforts to conserve fgr in situ are maintaining this diversity in the long term (fao, 2014b). there is also a need to enhance the production of seed and other forest reproductive material, especially for many native tropical and subtropical tree species, to meet demand for restoration and for establishing new forests and tree-based production systems (fao, 2014b; fao and unep, 2020). furthermore, recent advances in forest genomics need to be translated into practical applications for conserving and using fgr and for increasing our understanding of the adaptation of forest trees to climate change (e.g. holliday et al, 2017). aquatic genetic resources for food and agriculture characterization and monitoring of aqgr suffers from a lack of knowledge of genetic resources below the level of species and a lack of standardization and harmonization of terminology and nomenclature. the prototype registry being developed by fao for farmed types (box 2) will help address this issue by promoting the collection and sharing of key information on the availability and properties of aqgr. a variety of genetic technologies can be used to develop and improve farmed types for use in aquaculture. however, a clear understanding of the risks and benefits of these technologies is often lacking. aquaculture stands to benefit greatly from effective implementation and uptake of well-managed breeding programmes, with a focus on selective breeding. many governments consider this a role for the public sector, but such programmes often fail to deliver tangible and long-term increases in production. there is a need to identify mechanisms for effective engagement of the private sector in such programmes, for example through public−private partnerships. finally, cryopreservation clearly has a role to play in ex situ conservation of aqgr, but further genetic resources (2020), 1 (1) 4–16 global status of grfa 13 research is needed into methods for cryopreservation of eggs and embryos. micro-organism and invertebrate genetic resources for food and agriculture there are enormous knowledge gaps related to migr. in every taxonomic and functional group, many species remain to be identified and characterized. the roles of migr in the supply of ecosystem services, how they are affected by environmental changes and how they can be managed to support food and agricultural production need to be much better understood. knowledge of the significance of micro-organism and invertebrate diversity at within-species level to food and agriculture is very limited. integrated management integrated use of the various “sectoral” categories of genetic resources can give rise to a range of synergies and complementarities that can help increase productivity in a sustainable way and make production systems more resilient (dawson et al, 2018; duval et al, 2018; fao, 2019d). there is a need for research into how integrated management can be made more effective at a range of scales, from the individual plot to the landscape. this needs to include research into how genetic resources management can contribute, for example via appropriate choice of combinations of species, varieties, breeds, etc. for use in particular integrated systems and via appropriate genetic improvement strategies. time to step up action despite some positive developments in various aspects of grfa management, much remains to be improved. progress towards sdg 2.5 has been minimal overall. action clearly needs to be urgently stepped up across all sectors. however, there is also a vital need to improve cross-sectoral cooperation. for example, many drivers of loss of grfa affect more than one sector of food and agriculture and in many cases also affect species and ecosystems that are priorities for the nature conservation sector. habitat destruction is a major driver of loss of forest, aquatic, invertebrate and micro-organism genetic resources, as well as of wild relatives of crops and livestock and of biodiversity in general. climate change is a severe threat across all categories of grfa. threats of this kind need to be addressed in a comprehensive and cross-sectoral way, with the food and agriculture sector recognizing its role as a major contributor to biodiversity loss. for domesticated plants and animals, changes in consumption patterns and production systems that lead to declines in the use of diverse grfa are a major threat. this threat can to some extent be addressed by ensuring that diversity is utilized as fully as possible in the interests of livelihoods and food security, for example via the production benefits of raising species, breeds, varieties and farmed types that are well adapted to local conditions, the nutritional significance of diversity in the food supply and the marketing opportunities associated with unique products provided by specific grfa. however, there is a need to recognize that the maintenance of genetic resources for the long term is a public good and that interventions specifically aimed at supporting producers in this role will, in some cases, be necessary. the challenge is to maximize synergies and manage trade-offs among the various demands placed on production systems in terms of supporting and improving local livelihoods and in terms of the reliable supply of a broad range of ecosystem services, including genetic resources conservation. within a given landscape or seascape, this may require cooperation among stakeholders from the crop, livestock, forest, aquaculture, fisheries and nature-conservation sectors (among others). approaches that effectively combine ex situ conservation with in situ conservation, and conservation with sustainable use, need to be promoted. these activities need to ensure that they target a sufficiently wide range of genetic resources to meet the needs of producers and other stakeholders across a range of diverse and changing production systems and, in the longer term, the needs of future generations. in this regard, there is a need to increase efforts to raise awareness among policymakers (and other stakeholders, including consumers) of the importance of neglected and underutilized grfa. more generally, awareness raising with respect to the significance of all types of grfa and the need to manage them sustainably remains a key priority. across all sectors (including in the context of integrated management), the numerous knowledge gaps that constrain effective management of grfa need to be urgently addressed. where research is concerned, there is again a need to ensure that activities are sufficiently broad based in terms of the genetic resources and production systems targeted. attention needs to be given to how new technologies and existing good practices can be scaled up and adapted for implementation in different contexts. enabling policy, legal and institutional frameworks for sustainable management need to be put in place at all levels, including mechanisms for ensuring active and equitable stakeholder participation and collaboration. stakeholder organizations and networks of various kinds have important roles to play, and their establishment or strengthening should be promoted where necessary. although not a topic focused on in this paper, problems with the implementation of access and benefit-sharing mechanisms also remain to be addressed in many countries. at global scale, the existing gpas have provided a valuable framework for planning and monitoring actions across the various fields of grfa management, helped to raise awareness and promoted international cooperation. over the coming period, the commission will be working to finalize a global plan of action 14 pilling et al genetic resources (2020), 1 (1) 4–16 for aqgr and a global plan of action or other policy response for biodiversity for food and agriculture as a whole. the convention on biological diversity is in the process of developing a global framework for all biodiversity for the post 2020 period (convention on biological diversity, 2018). there is an urgent need for the international community to engage fully in these processes and in the implementation of their outcomes and those of the existing gpas. research has an essential role to play in informing both policy development and the implementation of agreed actions. acknowledgements the work of the commission on genetic resources for food and agriculture is supported by the governments of france, germany, norway, spain and switzerland. their contributions to the work underpinning this article are gratefully acknowledged, as is the government of france for providing for the secondment of dr gregoire leroy to fao. author contributions d.p. and j.b. contributed to the conception and design of the submitted manuscript. d.p. drafted the manuscript. all authors contributed to data gathering and analysis and to the drafting, revision and final approval of the submitted manuscript. the views expressed in this publication are those of the authors and do not necessarily reflect the views of the food and agriculture organization of the united nations. references alexandraki, v., tsakalidou, e., papadimitriou, k., and holzapfel, w. 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(uk: royal botanic gardens, kew), 80p. url: https://stateo ftheworldsplants.org/2016/report/sotwp 2016.pdf. http://www.cropwildrelatives.org/conservation-toolkit/ http://www.fao.org/3/ca8302en/ca8302en.pdf https://dx.doi.org/10.1111/raq.12374 https://dx.doi.org/10.1111/jbg.12136 https://stateoftheworldsplants.org/2016/report/sotwp_2016.pdf genetic resources the foundation of food and agriculture status and trends uneven, with worrying declines plant genetic resources for food and agriculture (pgrfa) animal genetic resources for food and agriculture (angr) forest genetic resources (fgr) aquatic genetic resources for food and agriculture (aqgr) micro-organism and invertebrate genetic resources for food and agriculture (migr) management strengthened, but progress patchy plant genetic resources for food and agriculture animal genetic resources for food and agriculture forest genetic resources aquatic genetic resources for food and agriculture micro-organism and invertebrate genetic resources for food and agriculture knowledge gaps a key constraint plant genetic resources for food and agriculture animal genetic resources for food and agriculture forest genetic resources aquatic genetic resources for food and agriculture micro-organism and invertebrate genetic resources for food and agriculture integrated management time to step up action author contributions box 1. the work of the commission on genetic resources for food and agriculture box 1. fao’s information systems on genetic resources for food and agriculture original article genetic resources (2021), 2 (4), 72–84 doi: 10.46265/genresj.ndfm2712 https://www.genresj.org issn: 2708-3764 morphometric and morphological characterization of chicken resources adapted to pastoral and agropastoral areas of southern ethiopia amine mustefa *,a, hizkel kenfo b, teklewold belayhun a, abebe hailu a and abraham assefa a a ethiopian biodiversity institute, addis ababa, ethiopia b ethiopian biodiversity institute, hawassa biodiversity center, hawassa, ethiopia abstract: thirteen qualitative and six quantitative variables taken from 303 adult chickens (95 cocks and 208 hens) from three locations/districts were used to phenotypically characterize the indigenous chicken populations in pastoral areas of south omo zone, ethiopia. the studied traits were influenced by the effect of location and sex, where chicken populations from hamer district and females of all districts were the smallest and lightest. qualitative characteristics of the studied chicken populations such as normal feather morphology and distribution, plain plumage pattern, flat head shape, triangular body shape, and dominant red eye, earlobe and plumage colour suggest that they constitute previously undescribed populations. chest circumference, wingspan and body length were the three most important morphometric traits used in discriminating the studied chicken populations. on average, 61% of the sampled populations were classified correctly into their respective locations. the multivariate analysis results discriminate the chicken populations into two groups: the hamer group and the omo group (chickens from bena tsemay and male districts). however, such grouping should be confirmed and advanced to ecotype level using further genetic characterization studies as the observed phenotypic differences might be due to genetic or environmental variations. such confirmation is important to design breeding programmes (for sustainable utilization) specific to each ecotype. keywords: characterization, chicken, discrimination, ethiopia, indigenous, multivariate citation: mustefa, a., kenfo, h., belayhun, t., hailu, a., assefa, a. (2021). morphometric and morphological characterization of chicken resources adapted to pastoral and agropastoral areas of southern ethiopia. genetic resources 2 (4), 72–84. doi: 10.46265/genresj.ndfm2712. © copyright 2021 the authors. this is an open access article distributed under the terms of the creative commons attribution license (cc by 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. introduction ethiopia, which is believed to be the main gate for most of the indigenous animal genetic resources from asia to africa, is one of the countries that possess a large number of livestock populations across the globe (ebi, 2016). chicken are the most widely distributed livestock species in ethiopia (ebi, 2016) and worldwide (fao, 2012). the estimated chicken population size of the country in 2018/2019 was 59.42 million (6 million cocks) among which 85.68% were indigenous (central statistical agency, 2019). ∗corresponding author: amine mustefa (aminemustefa32@gmail.com) poultry production in ethiopia is one of the key livestock subsectors which plays an important role in terms of creating employment, subsidizing women, and improving the nutritional content of food. due to the relatively low investment costs and the small quantity of land required for starting and running poultry production, it has become a suitable business for low-income farmers (fao, 2019). however, this subsector is not contributing with its full capacity due to poor productivity of and less attention given to indigenous chickens. therefore, many exotic chicken breeds were disseminated in the country with the objective of increasing production and productivity, becoming the major threat to diversity due to the dilution of indigenous genetic resources and yet providing minimum effect on production and productivity. received: 04.05.2021 accepted: 11.11.2021 published online: 20.12.2021 https://www.genresj.org https://www.dx.doi.org/10.46265/genresj.ndfm2712 https://www.genresj.org https://www.dx.doi.org/10.46265/genresj.ndfm2712 mailto:aminemustefa32@gmail.com genetic resources (2021), 2 (4), 72–84 ethiopian chicken genetic resources characterization 73 genetic improvement and conservation as well as sustainable utilization of the resources can also be achieved through the application of within-breed selection-based breeding programmes (mcdowell, 1972). identification, characterization and documentation of the rich ethiopian chicken genetic resources are a prerequisite for genetic improvement, breed conservation and sustainable utilization through designing suitable breeding programmes (fao, 2012). outputs of such studies will provide insight into variation within and between populations, which is one of the key contributions to conservation and genetic improvement programmes; it is more likely to maintain diversity and bring genetic improvement in a population with high variability. according to ebi (2016), ethiopia possesses seven indigenous chicken ecotypes: the farta, horro, jarso, konso, mandura, tepi and tillili. the small number of indigenous chicken ecotypes reported in the country can be attributed to the lack of comprehensive identification and characterization work on the diversity and potential of ethiopian chicken populations. in addition, limitations in methodology and a lack of concrete conclusions in terms of population differentiation were observed in most of the earlier characterization studies. for example, the region-wide chicken characterization study by melesse and negesse (2011) did not measure the most important linear measurements (wingspan, body length, chest and shank circumference) and failed to provide concrete conclusions. measuring, recording and reporting values without strong comparisons, discriminations and conclusions cannot be taken as an input in diversity studies of any given animal breed or ecotype. therefore, there is an urgent need for continued characterization of indigenous chicken genetic resources to understand their potential and reveal the relationships within and among populations. a short pilot survey performed by a team of ethiopian biodiversity institute (ebi) researchers and south omo zone and district livestock experts hinted at the presence of unique chicken populations, in south omo zone, ethiopia, that was not described before. thus, the present study was aimed at characterizing the chicken populations found in the area based on the fao (2012) guidelines for characterization of animal genetic resources. materials and methods study areas this study was conducted in south omo zone, which is found in the southern nations nationalities and peoples regional state (snnpr) and situated in the southern part of ethiopia (figure 1). the capital of south omo zone, jinka, is 750 km south of the capital city of the country (addis ababa). the zone has a total area of 2,241,731.598 ha. the majority of the land is fertile and cultivated, with trees and bushes covering the remaining area. the zone falls in the arid and semi-arid category of the agroecological zone. chickens are the most important animals for south omo zone pastoralists and agropastoralists next to ruminant animals (tesfahun et al, 2017). this study was conducted in three randomly selected pastoral and agropastoral districts (bena tsemay, hamer and male) of the eight districts found in the zone. moreover, nine sampling sites (kebeles), three from each sampled district were selected based on the chicken population size data (table 1). two to three chicken were randomly selected for measurement from each randomly selected household within each kebele. study ecotypes in literature, chicken populations of the current study area were generally classified as konso chicken ecotypes (dana et al, 2010). however, during the classification, samples were not taken from areas near the current study areas. moreover, the results of our short pilot survey in south omo zone hinted at the presence of unique local chicken populations, which are different from konso and were not described before. furthermore, the studied chicken genetic resources are indigenous/local, producing, reproducing and surviving under the local environment since many years. data collection the fao guidelines for animal genetic resource characterization (fao, 2012) were used to set the overall data collection procedures including the male to female ratio within the sampled chickens. the sampled chickens were randomly selected from flocks of representative households. eleven qualitative traits (feather morphology and distribution, head shape, comb type and size, body shape, and colours of plumage body, earlobe, skin, shank, and eye), and six quantitative measurements (wingspan, body length, chest circumference, shank length, shank circumference, and live body weight) (table 2) were recorded from 303 adult chicken (95 cocks and 208 hens) under the effect of district and sex. during the measurements, animals were carefully handled by trained laborers and made to stand properly with parallel legs. the measurements were carried out by two researchers – one measuring and one recording data. at the same time, two other researchers handled the qualitative data recording. to minimize subjective error, all the measurements were taken by the same researcher throughout the study. quantitative measurements were taken early in the morning before the animals were fed and watered using textile tape measures. body weight was taken using sensitive digital weighing balances to the nearest of 0.05 g. data analysis data entry and management were performed using microsoft excel© worksheets. analysis of the quantitative measurements was carried out separately for both sexes and sex aggregated by fitting district as class variable. the univariate procedure of statistical analy74 mustefa et al genetic resources (2021), 2 (4), 72–84 figure 1. map of the studied areas. the three study districts bena tsemay, hamer and male are located in south omo, a zone within the southern nations nationalities and peoples regional state (snnp) in the southern part of ethiopia. sis software (sas) 9.0 (sas institute, 2002) was used to detect outliers and test the normality of the quantitative measurements data. data on quantitative measurements and qualitative characteristics were analyzed using the general linear model (glm) and the frequency (freq) procedures of sas 9.0 software respectively. least square means (lsm) of the linear measurements were separated using the adjusted tukey-kramer test (sas institute, 2002). quantitative and qualitative data were analyzed using the following model: yi = µ+ai+ei where yi is an observation, µ is the overall mean, ai is the fixed effect of district and ei is the random error attributed to the nth observation. forward selection procedure of the stepwise discriminant function analysis (stepdisc) was used to find out the quantitative variables that better discriminate populations from different districts. the canonical discriminant function analysis (candisc) was also used to find out linear combination of quantitative variables that provide maximal separations between the districts. pairwise squared mahalanobis distances between locations were computed as:d2 (i|j) = (xi − xj) ′ cov−1 (xi − xj). where d2 (i|j) is the distances between locations i and j, cov−1 is the inverse of the covariance matrix of measured variables, xi and xj are the means of variables in the ith and jth populations. the scored canonical variables were used to plot pairs of canonical variables table 1. land use, climatic factors and chicken population size of the three studied districts. data from hidosa et al (2020); hidosa and tesfaye (2018); gezahegn and bamud (2018); derib (2017). variables bena tsemay hamer male land use agropastoralism pastoralism agropastoralism altitude (m) 500 – 1800 450 – 1765 600 – 1500 temperature (oc) 17.3 – 28.9 29 – 38 18 – 35 annual rainfall (mm) 1,167 400 800 – 1200 chicken population size 94,056 54,288 226,904 genetic resources (2021), 2 (4), 72–84 ethiopian chicken genetic resources characterization 75 table 2. six quantitative measurements definition and description of measuring devices used to characterize chicken populations. adapted from fao (2012). no. linear traits definition of quantitative morphological variables unit measuring device 1 wingspan length between tips of right and left wings after both are stretched out in full cm measuring tape 2 body length length between the tip of the rostrum maxillare (beak) and that of the cauda (tail, without feathers); the bird’s body should be completely drawn throughout its length cm measuring tape 3 chest circumference taken at the tip of the pectus (hind breast) cm measuring tape 4 shank length length of the shank from the hock joint to the spur of either leg cm measuring tape 5 shank circumference taken at the middle of the shank of either leg cm measuring tape 6 body weight live body weight g digital balance to get visual interpretation of district differences. percentage assignment of observations to known geographical locations (districts) and probabilities of misclassifications were evaluated by discriminant function analysis (discrim). results quantitative measurements level of significance (p values) outputs for the effect of district and sex on the quantitative measurements analyzed for the chicken populations as a whole and separately for each sex are presented in table 3. results of the overall analysis show a significant effect of sex on the studied traits with dominance of cocks on all measured traits. similarly, all studied traits were affected significantly by district in the sex-aggregated analysis. however, effect of district on wingspan and shank circumference was variable due to individuals’ sex, where the cocks’ wingspan and the hens’ shank circumference were not significantly different across districts. least square means with the respective standard errors (lsm±se) for the effect of district on the quantitative measurements of the chicken populations as a whole and separately for each sex are presented in table 4. cocks from hamer district have the lowest values except for wingspan, while their counterparts from bena tsemay and male districts were not significantly different in the measured traits. body length, chest circumference, shank length and body weight of the hamer hens were also lower than their counterparts from the other districts. qualitative characteristics the hypothesis whether the qualitative characteristics of the studied chicken populations differ across districts and sexes was tested using chi-squared analysis. the results presented in table 5 showed a significant effect of district and sex on most of the qualitative characteristics. the colour-related traits except earlobe colour of the cocks were significantly affected by district. the percentages of qualitative characteristics in each district are presented in table 6 and table 7. accordingly, silky feather morphology was observed on bena tsemay cocks. the majority of bena tsemay cocks possess single comb while one-fourth of the male and hamer cocks have a double comb type. the rectangular body shape was observed on almost half of the bena tsemay cocks, while the triangular body shape was dominant on cocks from other districts. all hamer hens possess triangular body shapes while one-fourth of the bena tsemay hens had rectangular body shapes. the majority of the studied chicken populations have been characterized by normal feather morphology and distribution, plain plumage pattern, flat head shape, single comb type, and triangular body shape. the comb size of the studied chicken populations was sex-dependent; the majority of hens had small combs while cocks had medium-sized combs. the spur was also absent in the majority of hens from all districts. red plumage, earlobe and eye colour combined with yellow skin and shank colour, were dominantly observed in cocks from all districts (table 7). on the other hand, red earlobe and eye colour with yellow shank colour were observed in the majority of hens in all districts. the majority of chickens in the bena tsemay and male districts had yellow shank colour, while chicken populations from hamer district had a large proportion with grey shank colour. similarly, the skin colour of most of the chicken population from this study were yellow and white, with grey skin colour observed only in hamer hens (18.6%). the plumage body colour of male and hamer hens was dominated by grey colour while brown, red and black were observed in the majority of bena tsemay hens. multivariate analysis for discrimination of chicken populations stepwise discriminant analysis six quantitative measurements for both sexes were separately subjected to the stepdisc procedure of sas 9.0. accordingly, all measurements in cocks and five measurements in hens were identified as the best discriminatory variables. these results were confirmed by wilk’s lambda test (table 8) where all selected variables had highly significant (p<0.0001) contribution 76 mustefa et al genetic resources (2021), 2 (4), 72–84 table 3. level of significance for the overallanalysis and separately for each sex. ws = wingspan, bl = body length, cc = chest circumference, sl = shank length, sc = shank circumference, bw = body weight, *p<0.05, **p<0.01, ***p<0.0001, ns = not significant, cv = coefficient of variation. traits overall chickens cocks hens district sex cv district cv district cv ws ** *** 8.35 ns 7.63 * 8.76 bl *** *** 6.88 *** 6.49 *** 7.08 cc *** *** 9.25 *** 9.85 *** 8.79 sl *** *** 10.16 ** 8.45 ** 11.05 sc ** *** 10.56 ** 12.19 ns 9.27 bw *** *** 21.60 ** 22.93 *** 20.34 table 4. pairwise mean comparison (least square means and standard errors (lsm±se)) for the effect of district within each sex. means within a column bearing different superscripts are significantly different; a is given to the highest value. n = number of observations, ws = wingspan, bl = body length, cc = chest circumference, sl = shank length, sc = shank circumference, bw = body weight. traits district bena tsemay male hamer cocks n 37 30 28 ws (cm) 41.14±0.52 41.13±0.58 42.86±0.60 bl (cm) 42.30±0.44a 42.00±0.49a 39.00±0.51b cc (cm) 30.05±0.48a 31.37±0.53a 27.36±0.55b sl (cm) 9.92±0.14a 10.37±0.15a 9.39±0.16b sc (cm) 4.92±0.10a 5.00±0.11a 4.50±0.11b bw (g) 1695.89±62.92a 1859.63±69.88a 1428.86±72.33b hens n 95 70 43 ws (cm) 36.06±0.32ab 35.50±0.38b 37.23±0.48a bl (cm) 38.09±0.27a 36.94±0.31b 35.02±0.40c cc (cm) 27.91±0.25a 27.67±0.29a 25.79±0.37b sl (cm) 8.28±0.09a 8.26±0.10a 7.67±0.14b sc (cm) 4.17±0.04 4.20±0.05 4.04±0.06 bw (g) 1373.87±27.78a 1400.16±32.36a 1124.23±41.29b both sexes n 132 100 71 ws (cm) 38.67±0.29b 38.27±0.33b 40.02±0.38a bl (cm) 40.24±0.24a 39.34±0.27b 37.06±0.32c cc (cm) 29.06±0.24a 29.28±0.27a 26.67±0.31b sl (cm) 9.14±0.08a 9.25±0.09a 8.54±0.11b sc (cm) 4.53±0.04a 4.58±0.05a 4.30±0.06b bw (g) 1543.70±28.31a 1610.42±31.98a 1282.62±37.06b in discriminating the chicken populations into separate groups. the stepwise discriminant function analysis also revealed that chest circumference, wingspan and body length were the three most important morphometric traits used in discriminating the chicken populations from different districts. however, body length, wingspan and body weight were the three most important variables used to discriminate the hens from different districts. shank circumference was found to be less useful in discriminating the overall chicken populations due to its lowest discriminatory power (table 8). discriminant analysis the multivariate statistics and f approximations used in discriminating the studied chicken populations (cocks, hens and both sexes) are presented in table 9. accordingly, all the statistics used in discriminating the populations were significant. higher f and den df values were calculated as the number of observations increased. results of a discriminant function analysis (table 10) show the overall classification of individuals into a known location/district. accordingly, the correct genetic resources (2021), 2 (4), 72–84 ethiopian chicken genetic resources characterization 77 table 5. chi-square values and probabilities for the effect of classes on qualitative characteristics for the overall analysis and separately for both sexes. x2 = chi-square value; p = probabilities; *p<0.05, **p<0.01, ***p<0.0001, ns = not significant. qualitative traits overall cocks hens district sex district district x2 p x2 p x2 p x2 p feather morphology 7.93 * 13.40 ** 10.04 ** feather distribution 7.91 ns 3.00 ns 3.20 ns 5.05 ns plumage pattern 5.25 ns 1.85 ns 4.85 ns head shape 8.65 ns 3.19 ns 2.66 ns 6.79 ns comb type 25.35 ** 22.53 ** 17.34 ** 12.99 ns comb size 3.73 ns 135.2 *** 5.43 ns 7.48 ns body shape 27.72 *** 8.41 * 11.06 * 24.14 *** spur presence 3.16 ns 118.6 *** 2.51 ns 7.32 * plumage colour 36.13 *** 50.70 *** 22.18 * 26.09 ** earlobe colour 17.38 ** 20.24 ** 6.55 ns 16.57 * skin colour 49.06 *** 17.87 *** 6.71 * 49.90 *** shank colour 52.07 *** 17.73 ** 27.69 ** 40.17 *** eye colour 80.30 *** 5.50 ns 30.03 *** 57.02 *** table 6. percentages for the qualitative characteristics of the chicken populations from different districts. qualitative traits cocks hens bena tsemay male hamer bena tsemay male hamer feather morphology normal 83.8 100 100 100 100 100 silky 16.2 0 0 0 0 0 feather distribution normal 94.6 100 100 91.6 94.3 97.7 naked neck 5.4 0 0 6.3 1.4 2.3 crest 0 0 0 2.1 4.3 0 plumage pattern plain 100 100 100 95.8 100 100 barred 0 0 0 3.2 0 0 barring 0 0 0 1.0 0 0 head shape flat 100 96.7 92.9 92.6 98.6 95.4 crust 0 0 0 5.3 1.4 0 snake 0 3.3 7.1 2.1 0 4.6 comb type single 86.5 63.3 53.6 75.8 58.6 62.8 pea 2.7 0 14.3 8.4 18.6 16.3 rose 10.8 16.7 10.7 12.6 20.0 18.6 v-shape 0 0 0 3.2 0 0 double 0 20.0 21.4 0 2.8 2.3 comb size small 35.1 20.0 21.4 85.3 97.1 93.0 medium 37.9 53.3 64.3 13.7 2.9 7.0 large 27.0 26.7 14.3 1.0 0 0 body shape blocky 0 3.3 0 2.1 0 0 rectangular 45.9 16.7 17.9 26.3 7.1 0 triangular 54.1 80.0 82.1 71.6 92.9 100 spur presence absent 27.0 43.3 42.9 89.5 98.6 97.7 present 73.0 56.7 57.1 10.5 1.4 2.3 78 mustefa et al genetic resources (2021), 2 (4), 72–84 table 7. percentages of some colour-related qualitative traits of the chicken populations from different districts. qualitative traits cocks hens bena tsemay male hamer bena tsemay male hamer plumage colour black 5.4 0 0 20.0 11.4 9.3 brown 5.4 0 0 25.3 17.2 9.3 grey 10.8 30.0 17.9 15.8 30.0 44.2 red + white 8.1 10.0 25.0 1.0 7.1 11.6 red 46.0 56.7 53.6 25.3 18.6 16.3 white 24.3 3.3 3.6 12.6 15.7 9.3 earlobe colour white 5.6 6.7 3.6 15.0 18.6 32.6 red 88.8 66.6 71.4 77.5 72.8 51.1 red + white 5.6 26.7 25.0 3.8 4.3 16.3 black 0 0 0 3.7 4.3 0 skin colour grey 0 0 0 0 0 18.6 white 43.2 16.7 21.4 63.2 31.4 48.8 yellow 56.8 83.3 78.6 36.8 68.6 32.6 shank colour yellow 91.9 96.7 50.0 56.8 70.0 37.2 black 2.7 0 10.7 20.0 12.9 16.3 white 5.4 3.3 17.9 20.0 4.3 9.3 grey 0 0 21.4 3.2 12.8 37.2 eye colour red 62.2 100 92.9 73.7 95.7 72.1 blue 0 0 7.1 0 0 7.0 brown 0 0 0 1.0 2.9 18.6 white 5.4 0 0 3.2 0 2.3 yellow 32.4 0 0 22.1 1.4 0 classification of cocks into their location/district ranged from 51% in bena tsemay to 75% in hamer. the overall average error rate was 39%, and 61% of the individuals were classified correctly. an average of 64% and 58% of the sampled cocks and hens were classified correctly into their corresponding districts, respectively. a higher error rate of 55% was observed in bena tsemay hens, while a lower error rate (23%) was obtained from the classification of hamer district hens. canonical discriminant analysis different multivariate statistics (including canonical correlation and eigenvalues), the coefficient values for each trait used, and class mean outputs using the first two canonical structures are shown in table 11. the first canonical structure (can 1) explains the majority (85.4%) of the variability among the three districts (84.5% for cocks and 84.3% for hens). can 1 also produces the greatest multiple correlation (60.2%) with the classes that was achieved by using the linear combination of the quantitative traits; the values were 72.7% for cocks and 55.5% for hens. the results revealed that can 1 separates the chicken populations (class means) from different districts better than can 2. the pairwise squared mahalanobis distances between locations/districts for analysis groups presented in table 12 were highly significant (p<0.0001). the shortest distance (0.6) was calculated between the chicken populations of bena tsemay and male districts; 1.36 for cocks and 0.46 for hens. on the other hand, the chicken populations from hamer district were more distantly related to the others (2.87 and 3.71 from bena tsemay and male districts, respectively). discussion quantitative measurements the quantitative measurements taken have produced reliable information on characterization, evaluation and differentiation of the studied chicken populations. in line with most reports, these measurements were significantly affected by the location sampled. the longest wings and smallest body size of the hamer chickens make them significantly different from the chicken populations of the other two locations. these long wings stretched from the small body size can be recorded and reported as the unique characteristics genetic resources (2021), 2 (4), 72–84 ethiopian chicken genetic resources characterization 79 table 8. summary of the stepwise discriminant function analysis; ascending order of traits used in discriminating the chicken populations from different districts. group step variable entered partial r-square f value p>f wilks’ lambda p<lambda cocks 1 chest circumference 0.2353 14.15 <0.0001 0.7647 <0.0001 2 wingspan 0.1907 10.72 <0.0001 0.6189 <0.0001 3 body length 0.1886 10.46 <0.0001 0.5022 <0.0001 4 shank length 0.1267 6.46 0.0024 0.4386 <0.0001 5 body weight 0.0568 2.65 0.0764 0.4137 <0.0001 6 shank circumference 0.0542 2.49 0.0884 0.3912 <0.0001 hens 1 body length 0.1661 20.42 <0.0001 0.8339 <0.0001 2 wingspan 0.0877 9.80 <0.0001 0.7608 <0.0001 3 body weight 0.1162 13.34 <0.0001 0.6724 <0.0001 4 shank length 0.0366 3.84 0.0231 0.6478 <0.0001 5 chest circumference 0.0207 2.13 0.1220 0.6344 <0.0001 shank circumference 0.0005 0.05 0.9498 both sexes 1 chest circumference 0.1006 16.78 <0.0001 0.8994 <0.0001 2 wingspan 0.1890 34.83 <0.0001 0.7294 <0.0001 3 body length 0.1068 17.82 <0.0001 0.6515 <0.0001 4 body weight 0.0637 10.10 <0.0001 0.6100 <0.0001 5 shank length 0.0523 8.17 0.0004 0.5781 <0.0001 shank circumference 0.0005 0.08 0.9259 table 9. multivariate statistics and f approximations used in discrimination of the chicken populations. df = degrees of freedom; p = probability. group statistic value f value df den df p>f cocks wilks’ lambda 0.3912 8.68 12 174 <0.0001 pillai’s trace 0.6986 7.87 12 176 <0.0001 hotelling-lawley trace 1.3265 9.54 12 132.28 <0.0001 roy’s greatest root 1.1217 16.45 6 88 <0.0001 hens wilks’ lambda 0.6340 8.53 12 400 <0.0001 pillai’s trace 0.3916 8.16 12 402 <0.0001 hotelling-lawley trace 0.5367 8.91 12 308.04 <0.0001 roy’s greatest root 0.4459 14.94 6 201 <0.0001 both sexes wilks’ lambda 0.5778 15.51 12 590 <0.0001 pillai’s trace 0.4560 14.57 12 592 <0.0001 hotelling-lawley trace 0.6722 16.48 12 455.81 <0.0001 roy’s greatest root 0.5696 28.10 6 296 <0.0001 of hamer chickens, which might be related to their mothering ability. a previous study by dana et al (2010) which describes konso as a chicken ecotype took only two quantitative measurements (the body weight and shank length). in terms of body weight both cocks and hens of the current study were heavier than the konso chicken ecotype where the body weights of the konso cocks and hens were 1,411 g and 1,011 g respectively (dana et al, 2010). such phenotypic variations in body weight hint at the presence of genetic diversity that needs to be conserved and can also be used as a base in attaining genetic improvement through selection. therefore, in terms of body weight, the current chicken populations were different from the konso chicken ecotype. on the other hand, their shank length measurements were comparable. according to melesse and negesse (2011), the shank length is considered a good indicator of adaptation to lowland areas and skeletal development, which is related to the amount of meat a chicken 80 mustefa et al genetic resources (2021), 2 (4), 72–84 table 10. number and (percent) of observations classified into districts. group from district bena tsemay hamer male total cocks bena tsemay 19 (51%) 7 (19%) 11 (30%) 37 (100%) hamer 3 (11%) 21 (75%) 4 (14%) 28 (100%) male 8 (27%) 2 (7%) 20 (66%) 30 (100%) total 30 (32%) 30 (32%) 35 (36%) 95 (100%) error rate 49% 25% 34% 36% hens bena tsemay 43 (45%) 18 (19%) 34 (36%) 95 (100%) hamer 5 (11%) 33 (77%) 5 (11%) 43 (100%) male 24 (34%) 9 (13%) 37 (53%) 70 (100%) total 72 (35%) 60 (29%) 76 (36%) 208 (100%) error rate 55% 23% 47% 42% both sexes bena tsemay 65 (49%) 22 (17%) 45 (34%) 132 (100%) hamer 9 (13%) 52 (73%) 10 (14%) 71 (100%) male 31 (31%) 8 (8%) 61 (61%) 100 (100%) total 105 (35%) 82 (27%) 116 (38%) 303 (100%) error rate 51% 27% 39% 39% table 11. multivariate statistics, canonical coefficients of the quantitative variables, and class means outputs of the two canonical structures separately for each sex. can = canonical structure. cocks hens both sexes can 1 can 2 can 1 can 2 can 1 can 2 multivariate statistics canonical correlation 0.7271 0.4122 0.5553 0.2885 0.6024 0.3051 eigenvalue 1.1217 0.2047 0.4459 0.0908 0.5696 0.1026 proportion 0.8457 0.1543 0.8309 0.1691 0.8473 0.1527 cumulative 0.8457 1.0000 0.8309 1.0000 0.8473 1.0000 f value 8.68 3.60 8.53 3.65 15.51 6.08 p>f <0.0001 0.0052 <0.0001 0.0035 <0.0001 <0.0001 traits wingspan -0.3191 0.0338 -0.2780 0.1110 -0.3280 0.0350 body weight 0.0004 -0.0029 0.0019 -0.0031 0.0015 -0.0028 body length 0.1247 0.5403 0.0915 0.4719 0.0560 0.5070 chest circumference 0.1727 -0.0725 0.1483 0.0356 0.1828 0.0089 shank length 0.5074 -0.8638 0.3943 -0.5278 0.3388 -0.7575 shank circumference 0.4542 0.8747 -0.1134 0.0512 -0.0147 0.1737 class (district) mean bena tsemay 0.4234 0.5273 0.3164 0.2933 0.2980 0.3400 male 0.9488 -0.5150 0.3679 -0.3858 0.5594 -0.3871 hamer -1.5761 -0.1450 -1.298 -0.0200 -1.3419 -0.0869 can carry. this shows their high adaptability to the lowland areas of the pastoral and agropastoral community. comparable, higher and lower values of wingspan, body length, chest circumference, shank length and shank circumference measurements were reported in indigenous chickens in different parts of the country. however, surprisingly low values were also reported. for example, negassa et al (2014) reported 7.35–8.17 cm of wingspan and 22.6–24.2 cm of body length for chickens in southeastern ethiopia. similarly, halima et al (2007) reported 12.67–15.83 cm of wingspan and 0.53–0.93 cm of shank circumference for chickens in northern ethiopia. effect of sex cocks were bigger and heavier than hens, which follows rensch’s rule (rensch, 1950) where the males of an individual species are generally larger than the females. such differences between cocks and hens may be attributed to the differences in hormone secretion, genetic resources (2021), 2 (4), 72–84 ethiopian chicken genetic resources characterization 81 table 12. squared mahalanobis distance between districts; output of the multivariate analysis calculated using the quantitative measurements. *** indicates significance of the distance calculations at p<0.0001. group from district bena tsemay male hamer cocks bena tsemay 0 male 1.36** 0 hamer 4.45*** 6.51*** 0 hens bena tsemay 0 male 0.46** 0 hamer 2.70*** 2.91*** 0 both sexes bena tsemay 0 male 0.60*** 0 hamer 2.87*** 3.71*** 0 which leads to enlargement of muscle mass and skeletal development (baneh and hafezian, 2009). these results were in line with the reports of bekele et al (2015), getu et al (2014), negassa et al (2014), and melesse and negesse (2011), dana et al (2010) and halima et al (2007) on different indigenous chicken populations of ethiopia. similarly, sexual dimorphism was reported for most traits with males having higher values when compared to the females in other species of ethiopia, including goats (mustefa et al, 2019), cattle (mustefa et al (2020b)), donkeys (mustefa et al (2020a)) and sheep (hailu et al, 2020). qualitative characteristics observable qualitative characteristics, in addition to the quantitative measurements, have allowed us to characterize, identify and differentiate the studied chicken populations. dana et al (2010), bekele et al (2015), melesse and negesse (2011) reported normal feather morphology and distribution in the majority of indigenous chicken populations in ethiopia’s southern region that are comparable with the results of the current study. melesse (2000) described the nakedneck gene as one of the main genes responsible for heat tolerance of some ethiopian indigenous chicken populations by improving and enhancing heat dissipation due to the reduction in feather coverage. however, the current study also revealed the adaptation of indigenous chickens to the local pastoral and agropastoral lowland areas with a lower frequency of naked-neck chickens. this lower frequency of nakedneck chicken in the current study area specifically, as well as in the country in general, may also be a factor for their poor productivity (ajang et al, 1993). similarly, yunis and cahaner (1999) reported the probability of attaining higher egg and/or meat production in chicken with reduced feathering due to the saved feather protein. qualitative deviations were also observed between konso ecotype and the current chicken populations. firstly, the comb types, which vary greatly depending on the breed, are also a great indicator of a chicken’s health. bright fleshy combs indicate physical strength and good health. combs also help chickens regulate their body temperature – those with large combs are able to circulate blood faster through their combs, which helps to release body heat. this is why chickens adapted to warm environments have larger combs (like single combs and buttercup combs) while breeds from colder areas have smaller combs (like pea and rose combs) (vaughn, 2019). the majority of chickens from the current study area were single-combed while dana et al (2010) reported a higher frequency of pea-combed chickens in the konso chicken ecotype. dana et al (2010) also reported humid lowland to wet highland ecological zones as local areas of the konso chicken ecotype. the comb size of the studied chicken populations was found to be sex-dependent where hens of a given population had smaller combs than the cocks. this was supported by bell (2002) who reported the association of comb size with gonadal development and intensity of light. the earlobe colour of a chicken can determine the colour of the egg that it will lay. for example, if the chicken has a red earlobe, it will lay a brown-shelled egg and if it has a white earlobe, it will lay a white-shelled egg (bell, 2002). red earlobe colour was observed in the majority of chickens from the current study, while dana et al (2010) reported equal frequency of red and white earlobe colours in konso chicken ecotype. the observation of a higher proportion of hens with white earlobe colour than the cocks in each district was due to the sex-linked nature of the trait (luo et al, 2018). among the populations of the current study, a higher proportion of white earlobes was observed in hamer hens than the others, which might be due to their distinction from the others as earlobe colour is a breedspecific trait, though it could also be affected by the nutritional status of the chicken populations (melesse and negesse, 2011). the triangular body shape was dominantly found in the current study while the results of dana et al (2010) reported blocky body shape for the majority of the konso chicken ecotype. on the other hand, similar qualitative characteristics, like flat head shape, were observed on both konso chicken ecotypes (dana et al, 2010) and the current chicken populations. chicken populations, breeds and breeding groups can be identified by their plumage colours due to their decorative qualities. plumage colour is also a key trait during the interaction of chickens due to their welldeveloped visual perception of the world (makarova et al, 2019). in agreement with most ethiopian reports, several plumage colours were found in the current study area with a higher frequency of red-coloured cocks. on the other hand, the predominance of grey (faded white and black) colour observed in hens is in line with the reports of halima et al (2007). the diverse 82 mustefa et al genetic resources (2021), 2 (4), 72–84 plumage colours in the current study, which might be due to either genetic or environmental factors, showed the presence of diverse genetic resources, which can be an input in conservation and genetic improvement programmes for sustainable use. bekele et al (2015), getu et al (2014), halima et al (2007), dana et al (2010) reported yellow as the most widespread shank and skin colour in different indigenous chicken populations of ethiopia, which agrees with the current findings. according to hammond and harshaw (1941), a chicken’s shank and skin colour are influenced by breed, quantity of xanthophyll pigment in the diet, and quantity of a pigmentationsuppressing factor in the diet. bell (2002) stated that yellow shank colour is due to nutritional carotenoid colourants in the epidermis when the melanic pigment is absent. similarly, variable shades of black colour are the result of melanic pigment in the dermis and epidermis; if the black colourant is in the dermis and yellow in the epidermis, greenish shanks will appear. however, if both these pigments are completely absent, the shanks will be white (bell, 2002). commonly, red, yellow and black eye colours are due to the three pigments in the form of hemoglobin, carotenoids and melanin respectively. red eye colour is influenced by the degree of iridic vessels injection and hemoglobin content of the blood (nelson, 1947). according to nelson (1947), when chickens suffer from general anemia, a smaller amount of red colouration will be noted due to loss of blood volume. therefore, the dominant red eye colour observed in the current study is a confirmation that these chicken populations were free from such suffering. as per the same author, continual egg production can decrease the yellow factor in eye colour. therefore, the yellow eye colour chicken from bena tsemay district (27%) might indicate restrictions in egg productivity. multivariate analysis the use of more quantitative measurements during the discrimination analysis approaches the results towards reality. this is supported by the current results, where only one quantitative measurement in hens had low discriminatory power. in line with reports by mustefa et al (2020b) on raya cattle (61%) and hailu et al (2020) on tigray sheep (66%), the discriminant function analysis allowed the classification of an average 61% of the studied individuals into their respective locations. the observed highest classifications of individuals into their respective districts in hamer cocks and hens (75% and 77% respectively) showed their distinctness from the others due to the unique characteristics they possess. however, the lowest classifications (45% and 53%) were recorded in hens from bena tsemay and male district indicating strong similarities between them. the highly significant and longest pairwise mahalanobis distances between districts indicate the accuracy of the calculations and the distinctness of the populations from each other in a measurable group difference for the considered quantitative measurements. accordingly, the longest distances of hamer chickens from the other two chicken populations in this study showed its distinctness. in conclusion, the main contribution of this diversity study is the provision of information and data for breed differentiation, conservation and sustainable utilization of the chicken ecotypes in the districts, as well as the collation of information and data available on chicken ecotypes of ethiopia. accordingly, the chicken populations can be categorized phenotypically into two groups: the hamer group and the omo group (which includes the indigenous chicken populations from bena tsemay and male districts). however, such differences in phenotypic performances might be either due to genetic or environmental variations. therefore, we cannot conclude that the differences are solely due to genetic variations. thus, genetic characterization is recommended to understand their potential and the withinand among-population genetic diversity and population structures. data availability as baseline data for further research and development work, these data will be made available in the country’s focal institute for indigenous animal genetic resources, the ethiopian biodiversity institute (ebi) and the global domestic animal diversity information system (dadis) databases. acknowledgments the authors are highly grateful to the ethiopian biodiversity institute (ebi) for funding the work. our special appreciation also goes to the farmers for providing their animals for this work for free. we also take this opportunity to appreciate the animal science experts at zonal, district and kebele level for their endless help during the data collection. a special word also goes to our friend and work partner mr. tadesse hunduma for mapping the study area. author contributions all authors contributed to the study conception and design. material preparation and data collection were performed by amine mustefa, hizkel kenfo, and teklewold belayhun. data analysis and writing the first draft of the manuscript was performed by amine mustefa and all authors commented on previous versions of the manuscript. all authors read and approved the final manuscript. conflict of interest statement the authors declare no conflict of interest. genetic resources (2021), 2 (4), 72–84 ethiopian chicken genetic resources characterization 83 references ajang, o. a., prijono, s., and smith, w. k. 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(1999). the effects of nakedneck (na) and frizzle (f) genes on growth and meat yield of broilers, and their interactions with ambient temperatures and potential growth rate. poultry science 78, 1347–1352. doi: https://doi.org/10.1093/ ps/78.10.1347 https://doi.org/10.1007/s11250-020-02486-1 http://www.lrrd.org/lrrd31/6/amine31089.html https://doi.org/10.1017/s2078633614000319 https://doi.org/10.3382/ps.0260061 https://doi.org/10.3382/ps.0260061 https://www.sas.com/enus/software/stat.html https://www.sas.com/enus/software/stat.html https://doi.org/10.1007/s11250-017-1240-9 https://doi.org/10.1007/s11250-017-1240-9 https://www.communitychickens.com/chicken-comb-types-zbw1912ztil/ https://www.communitychickens.com/chicken-comb-types-zbw1912ztil/ https://www.communitychickens.com/chicken-comb-types-zbw1912ztil/ https://doi.org/10.1093/ps/78.10.1347 https://doi.org/10.1093/ps/78.10.1347 introduction materials and methods study areas study ecotypes data collection data analysis results quantitative measurements qualitative characteristics multivariate analysis for discrimination of chicken populations stepwise discriminant analysis discriminant analysis canonical discriminant analysis discussion quantitative measurements effect of sex qualitative characteristics multivariate analysis data availability acknowledgments author contributions conflict of interest statement original article genetic resources (2024), 5 (10), 107–116 doi: 10.46265/genresj.ofcr3000 https://www.genresj.org issn: 2708-3764 genetics solutions for improved chicken production in ghana richard osei-amponsah *,a, ricky aboagye poku a,b, ebenezer agyemang duah a,b, augustine naazie a, raphael ayizanga a, harrisson njamba c, wondmeneh esatu c, mulugetta y birhanu c and tadelle dessie c a department of animal science, school of agriculture, university of ghana, ghana b animal production directorate, ministry of food and agriculture, accra, ghana c international livestock research institute (ilri), addis ababa, ethiopia abstract: the tropical poultry genetics solutions project intervention in ghana, a collaboration between the international livestock research institute and the university of ghana, with support from ghana’s ministry of food and agriculture, seeks to test the adaptability, productivity and farmer preferences of tropically adapted improved dual-purpose chicken genotypes under different production systems. poultry farmers from two agroecological zones in five of the 16 regions of ghana were selected to participate in the project. these farmers were provided with selected tropically adapted chicken genotypes, namely kuroiler and hubbard, to manage under their production system, and data was collected on bird performance in terms of body weight, egg production, quality and overall acceptability by the farmers. the findings indicate a high farmer preference for, and significantly better (p ≤ 0.05) growth performance of the introduced chicken genotypes compared to local chickens. for instance, the introduced birds reached an average weight of 2kg within 18 weeks, compared to local chickens which required 30 weeks on average to reach 1.6kg under semi-intensive production management conditions. additionally, the egg production and carcass attributes of these selected genotypes were found to be comparable to those of the local chickens. based on these positive results, we recommend the multiplication and widespread adoption of kuroiler and hubbard chicken strains among smallholder farmers in ghana. by doing so, we anticipate improved chicken production, increased income generation, enhanced livelihoods and protein food security in the country. keywords: chicken breeds, dual-purpose, hubbard, kuroiler, sustainable development goals citation: osei-amponsah, r., poku, r. a., duah, e. a., naazie, a., ayizanga, r., njamba, h., esatu, w., birhanu, m. y., dessie, t. (2024). genetics solutions for improved chicken production in ghana. genetic resources 5 (10), 107–116. doi: 10.46265/genresj.ofcr3000. © copyright 2024 the authors. this is an open access article distributed under the terms of the creative commons attribution license (cc by 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. introduction poultry production in africa is mainly based on freerange indigenous chickens (dessie et al, 2011), which are well-valued in terms of their contribution to nutritious diets, household income and sociocultural attributes (aboe et al, 2006; dessie et al, 2011). indigenous chickens in africa are adaptive to the environment, survive on low-energy feeds, are often resistant to endemic diseases, and have been characterized to have good potential to be selected for meat and ∗corresponding author: richard osei-amponsah (rosei-amponsah@ug.edu.gh) eggs (ajayi, 2010; dessie et al, 2011). thus, african chicken resources constitute a reservoir of useful genes important for adaptation and meeting local breeding goals (osei-amponsah et al, 2013). such indigenous chickens are mostly maintained under traditional village production systems, typically characterized by low input and low egg and meat production (dessie et al, 2011; birhanu et al, 2023). in ghana, smallholder chicken production systems are predominantly based on unimproved genotypes maintained mostly in low-input extensive systems. despite their unique adaptive attributes, contribution to protein food security, low capital requirements and minimal production risks, their overall productivity remains relatively low. there is a need to select received: 05.06.2024 accepted: 23.09.2024 published online: 11.11.2024 https://www.genresj.org https://www.doi.org/10.46265/genresj.ofcr3000 https://www.genresj.org https://www.doi.org/10.46265/genresj.ofcr3000 mailto:rosei-amponsah@ug.edu.gh 108 osei-amponsah et al genetic resources (2024), 5 (10), 107–116 and improve indigenous chickens to take full advantage of their adaptive potential and genetic variability (oseiamponsah et al, 2013). genetic selection within the indigenous breed ideally relies on accurate data recording, including pedigree, and for the birds to be raised in a similar environment where the selection programmes have occurred. additionally, a large population with a substantial number of active and reserve sire and dam lines is needed to allow exhaustive exploitation of genetic variations for the meat yield trait (alemu et al, 2021). to overcome these challenges, the introduction of productive, yet tropically adapted chicken strains provides an opportunity to increase meat yield and egg number in developing countries (alemu et al, 2021). the african chicken genetic gains (acgg; https://af ricacgg.net/) project led by the international livestock research institute (ilri) was started in 2014 and implemented over five years in ethiopia, tanzania and nigeria. the programme’s vision was to increase smallholder chicken production and productivity growth as a pathway out of poverty in sub-saharan africa. to increase meat yield and egg number in a sustainable manner, selected chicken strains were made available to rural farmers in ethiopia, nigeria and tanzania, through the acgg project (alemu et al, 2021). sasso and kuroiler chickens were among the improved strains that had been tested in tanzania and were distributed on a large scale by the acgg project. these strains are suitable for backyard systems and can be used for meat and egg production, with minimal provision of commercial feed (dessie and getachew, 2016). following the success of the acgg project, dual-purpose tropically adapted chicken breeds such as hubbard, kuroiler, noiler and sasso are being promoted by ilri under the tropical poultry genetic solutions (tpgs) project (https://www.ilri.org/resea rch/projects/tpgs). the ghana component of tpgs is collaborating with local stakeholders to test the performance of the newly selected strains in terms of body weight, egg production and survivability in two different agroecological zones compared to the indigenous chicken in ghana. the superior performance of introduced strains such as kuroiler and hubbard over indigenous chickens will give smallholder farmers a good alternative for backyard poultry production. this should help improve income generation and livelihood of smallholder farmers in africa and contribute to the attainment of the un sustainable development goals (sdgs), particularly sdg 1 (no poverty), sdg 2 (zero hunger) and sdg 3 (good health and wellbeing). therefore, the objective of this research was to evaluate the performance of the tpgs-introduced chicken genotypes in terms of survivability, growth and egg production as well as their acceptance by stakeholders, particularly farmers. the findings of this study will serve as valuable input for chicken breeders, policymakers and other stakeholders in the development of selected more resilient chicken genotypes in africa. materials and methods project scope and farmer selection the ghana component of the tpgs project, managed by the animal science department of the university of ghana with support from the ministry of food and agriculture (mofa), supplied kuroiler and hubbard chickens to selected poultry farmers across two management systems – intensive and semi-intensive – in the forest and coastal savannah agroecological zones of ghana. the kuroiler is a tropically adapted dual-purpose chicken developed in india by kegg farms private limited. it is widely reported that this cross-bred chicken originated from crossing rhode island red, white leghorn, barred plymouth rock, and two indian local chicken breeds with some broiler blood infusion to obtain specific broiler characteristics (dessie and getachew, 2016). hens attain 2.5kg within 12 months, begin laying eggs at five to six months, and then lay 150–200 eggs during their 12–16-month egg-laying period, initially more than 20 eggs per month (dessie and getachew, 2016). males reach 4kg in 12 months and weigh at least 1kg at around three months. the breed thrives well on household waste, scraps and vegetation and, thus, does not compete with human food for grain or require any special feeding (dessie and getachew, 2016). hubbard has been a worldwide reference for broiler breeding stock, with the company supplying day-old grandparent and parent stock chicks all over the world. hubbard chickens are characterized by good growth, feed conversion ratio, excellent viability and good meat yield. the hubbard ja57 reach between 2.3 and 2.4kg on average at 20 weeks (https://www.hubbardbreeders.com/). the present project was undertaken in the greater accra, central, eastern, bono and ashanti regions of ghana (figure 1), the main areas of chicken production in the country. stakeholder engagement, particularly with the poultry farmers association, regional and district livestock officers of the mofa and the women in poultry value chain, enabled us to identify and agree with the targeted beneficiaries their needs and challenges in adopting improved technologies to enhance their production efficiency. two service providers located in the eastern and ashanti regions of ghana were contracted to hatch fertilized eggs and brood the chickens for a period of five weeks before they were distributed to the participating poultry farmers. the study sampled poultry farmers from five regions across two agroecological zones in ghana. these farmers were provided with kuroiler and hubbard chicken genotypes, which had been selected for improved growth and egg production performance, to raise under semi-intensive or intensive systems. in these systems, housing, feeding and veterinary care were provided for the chickens, with birds allowed to freely roam around the farm during the day in the semi-intensive system. six communities (villages/towns) were sampled from each of the selected regions. within each community, nine households (smallholder farmers) were selected genetic resources (2024), 5 (10), 107–116 chicken genetic improvement in ghana 109 as project beneficiaries. thus, 54 households per region and 270 farmers nationwide participated in the programme. three enumerators with minimum qualifications that met the standard selection criteria by ilri were chosen in each region to support the project in terms of data collection. the regional livestock officers of mofa in the participating regions were assigned as supervisors to oversee the activities of the enumerators. enumerators and their supervisors were trained on the data collection protocols, which were captured in real time using tablets installed with odk software (ictd, 2010). throughout the project, regular communication was established between enumerators and the project team to ensure speedy resolution of any challenges encountered in the field. egg incubation and mothering of chickens in total, 10,080 fertile kuroiler eggs were imported from uganda on 6 december 2021 and hatched by akro farms, akropong, eastern region, one of the selected private hatcheries whilst a second batch of 5,040 fertile eggs each of the hubbard ja57-i and ja57-ii strains were received from morocco on 9 april 2022 and hatched by topman farms, kumasi, ashanti region. the day-old chicks were raised intensively in mother units for up to five weeks. the feed provided was a commercially prepared chick mash with 3,080kcal/kg of energy and 21% crude protein. all prescribed prophylactic vaccines, including the 1st and 2nd newcastle and gumboro vaccinations, were provided. two hundred and seventy (270) beneficiary farmers received 40 birds of each of the three chicken strains at random, with no special preference for sex and size. data collection and analysis data on body weight, age at first egg (afe), egg number and weight were collected from participating farmers using odk tools. the first phase of tpgs ghana took place between december 2021 and march 2023. during this period, on average, poultry farms were visited monthly for sensitization and body weight measurements, starting when the birds were five weeks of age for approximately 8 visits in total. data entries were double-checked by supervisors before the final authorization to upload onto the server at ilri. a total of 180 eggs made up of 60 eggs each from kuroiler, local forest and local coastal savannah chicken ecotypes aged 34–36 weeks, were randomly sampled from farmers and kept at room temperature for three days before measuring egg quality parameters including egg weight, haugh unit, albumin height and yolk colour using an electronic egg analyzer (orka food technology llc, 2006) at the molecular genetics laboratory, department of animal science, university of ghana, legon. shell thickness was measured with an electronic vernier calliper by taking the average thicknesses of the large, central and narrow ends of individual eggs (melesse et al, 2010). the one-way analysis of variance (anova) procedure of genstat 12th edition (2009) was used for the data analysis. the effect of age and genotype of chicken were estimated based on the following model: yijk = µ+ ai + gj + ag(ij) + e ijk where yijk = body weight of chicken; µ = the population mean; ai = effect of age of chicken; gj = effect of genotype; ag(ij) = age and genotype interaction; eijk = residual or the random error term. the effect of management system and sex on growth performance of kuroiler chickens at week 17 was analyzed based on the following model: yijk = µ+ mi + sj +ms(ij) + e ijk where yijk = body weight of chicken; µ = the population mean; mi = effect of management system; sj = effect of sex; ms(ij) = management and sex interaction; eijk = residual or the random error term. the effect of genotype on egg quality traits of sampled eggs of kuroiler and two local chicken ecotypes from the forest and coastal savannah zones of ghana was analyzed using the following model: yij = µ + gi+eij where yij = egg quality parameter; µ = the population mean; gi = effect of chicken genotype; eij = residual or the random error term. where the anova indicated a significant genotype effect, the means were separated using tukey’s test at the 5% margin of error. tukey’s test was chosen for mean separation on account of its robustness, requiring a single value judging the significance of all mean differences and is thus quick and easy to use (steel and torrie, 1981). we also surveyed farmer trait preferences in the introduced chicken genotypes. farmers were asked to rank ten traits (fast growth, good survivability, good disease resistance, low feed intake, laying more eggs, laying big eggs, good mothering, plumage colour, tough meat and soft meat) indicating their preference for the introduced chicken genotypes compared to local chickens. in terms of hatchability, we used the twoproportion z-test to calculate the true difference in the hatching performance percentages between kuroiler and hubbard chicken eggs. results and discussion body weight and growth performance the growth rates of both the kuroiler and hubbard chicken genotypes are shown in figure 2. the growth rates of both the kuroiler and hubbard chickens were superior to those of the local chickens of ghana kept under semi-intensive management reported in an earlier study (osei-amponsah et al, 2012). this finding of superior growth performance and productivity of the introduced chicken genotypes compared to the local chickens aligns with previous studies that have reported on the benefits of using adopted breeds selected for improved production in smallholder systems (bamidele 110 osei-amponsah et al genetic resources (2024), 5 (10), 107–116 figure 1. map of ghana showing sampled regions. source: ghana statistical service, geographical information service (gis) section. et al, 2023). the significant reduction in the number of sampled chickens over time was largely due to the disposal (sale) of particularly male birds after 17 weeks to reduce the feeding costs of the farmer. the production system was found to influence the growth of chickens. on average, kuroiler chickens raised intensively weighed significantly (p < 0.05) more (2.0kg) than those kept under semi-intensive (1.8kg) management (table 1). the significant (p < 0.05) effect of the production management system on growth was expected because birds in confinement spend less energy moving around than those in the open. furthermore, husbandry practices, such as feeding, biosecurity and veterinary care, are expected to be better and easier in confinement. in a previous study on growth performance of chickens, osei-amponsah et al (2012) reported the average body weight (g) of female and male local ghanaian chickens at hatch, 20 and 28 weeks to be 25.8, 1,033.5 and 1,273.5g, respectively. in the male population, the average body weights at these ages were 26.6, 1,358.5 and 1,593.5g respectively indicating a clear sexual dimorphism. this growth performance in local chickens was however significantly (p ≤ 0.05) lower than the exotic sasso t44 raised under the same management condition which gave average body weights in females at the same ages as 38.2, 2355.0 and 2,635.0g with corresponding values of 37.6, 2,777.0 and 3,192.0g in males (osei-amponsah et al, 2012). the body weights recorded for kuroiler chickens in the current study were lower than those reported for the same breed and age on station in ethiopia (alemu et al, 2021; bedru, 2021). this emphasizes the importance of improved management practices for the growth and development of chickens. local chicken improvement programmes should incorporate supplementary feeding, veterinary care and housing to enhance productivity (osei-amponsah et al, 2012). under improved management, local ghanaian savannah and forest chicken ecotypes weighed between 1.2–1.7kg at seven months of age (osei-amponsah et al, 2012). this was lower than that recorded at four months for both kuroiler and hubbard chickens in the present study. the superior growth performance of kuroiler over local chickens is due to the genetic potential of the breed as a commercial strain (dessie and getachew, 2016), as it has been bred for superior growth through selection and strain crossing. this finding of considerable differences in live body weight among chicken strains is in line with those of previous studies (mulugeta et al, 2020; alemu et al, 2021; guni et al, 2021; kassa et al, 2021). sasso chickens have also been reported to be superior in growth compared to koekoek and sasso-rir crosses in ethiopia and were recommended for semi-scavenging production systems (fekede et al, 2021). there is thus a need for the selection and improvement of the growth potential of local chickens to take full advantage of their adaptive potential and genetic variability with respect to various environmental challenges (osei-amponsah et al, 2013). our findings indicated a significant (p ≤ 0.05) effect of management system on age at first egg in kuroiler chickens. kuroiler chickens raised under intensive management laid significantly earlier (22.3 weeks genetic resources (2024), 5 (10), 107–116 chicken genetic improvement in ghana 111 figure 2. growth performance of experimental chickens old) than those raised under semi-intensive management (25 weeks old). additionally, kuroiler chickens raised under intensive management grew significantly (p ≤ 0.05) faster than those under semi-intensive production systems (table 1). egg production performance the egg production performance of the kuroiler and the two hubbard lines was also superior to that of local ghanaian chickens. the age at first egg (afe) for the hubbard birds was 17.5 weeks compared to 28 weeks for the local chickens, and the expected egg number and egg weight (figure 3) of the hubbard chickens increased with age. we further compared the hatching performance of fertilized kuroiler and hubbard eggs under ghanaian conditions, based on the data received from the hatchery operator and shown in table 2. both genotypes recorded a relatively high percentage hatched on fertile (≥ 83%). egg hatchability is affected by egg physiology, egg size, environment (bird nutrition and handling of eggs), bird genotype, genetic diversity and their interactions. hence, the current results could not be solely associated with the genetic makeup of birds. the hatchability percentages of 83 and 85% of incubated kuroiler and hubbard eggs, respectively, and can be considered satisfactory, although a higher hatchability of 87.8% has been reported for indigenous chickens in the forest and savannah zones of ghana (osei-amponsah et al, 2014). comparison of hatching performance based on the proportion z-test indicated significantly (p < 0.001) higher fertility of hubbard chicken eggs compared to kuroiler eggs (table 2), which can be attributed to variations in egg storage and handling by the two hatchery operators. egg production performance of kuroiler and the two hubbard lines studied was superior to that of local chickens kept under similar management conditions in ghana (osei-amponsah et al, 2015). kuroiler birds raised intensively under on-station management in tanzania had an age at first egg of 22.7 weeks whilst 24.8 weeks was achieved for on-farm semi-scavenging management. in other studies, egg production traits of chickens selected for fast growth (sasso, bovans and koekock) were generally higher than those of local chickens, with variations observed across the genotypes (guni et al, 2021). the fact that chickens in the present study began laying from week 23 may imply an adaptation strategy for survival under semiintensive production conditions. egg production is very demanding of nutrients; therefore, under free range, it is likely that the bird must first build its reserves adequately before engaging in this nutrient-demanding activity and hence adapt to start later rather than earlier. the mean egg weight, albumin height and haugh unit of kuroiler chickens were all significantly (p ≤ 0.05) heavier than those of the indigenous birds; however, the shell thickness and yolk colour were not significantly different (table 3). the relatively higher egg weights obtained for kuroiler chickens compared to local chickens can be attributed to genetic differences. both albumin height (ah) and haugh unit were positively and significantly related to egg weight (moula et al, 2013; oseiamponsah et al, 2014; bekele et al, 2022). kuroiler eggs in this study were superior to the eggs from local chickens in terms of average albumin height and haugh unit, similar to their mean egg weight. previous findings indicate that local chicken eggs have relatively stronger shell and yolk ratios than sasso t44. however, the egg quality of local chickens is lower than that of sasso t44 chickens because of the negative correlation between yolk ratio and ah, the main determinant of egg quality (osei-amponsah et al, 2014). haugh unit 112 osei-amponsah et al genetic resources (2024), 5 (10), 107–116 table 1. range and mean body weight of kuroiler chickens (±se) by management system and sex at week 17. within-rows means followed by different superscripts are significantly (p ≤ 0.05) different. intensive semi-intensive cockerels (males) range (g) 1,000–3,940 700 – 3,920 mean (g) 2,231.65a±29.63 1,851.2b±28.33 n 536 530 pullets (females) range (g) 920–4,000 610–3,630 mean (g) 1,799.41a±18.35 1,696.55b±23.04 n 619 540 all range (g) 920–4,000 610–3,920 mean (g) 2,005.2a±18.12 1,806.79b±18.63 n 1,155 1,070 figure 3. variation of egg weight of hubbard chickens by age of bird under a semi-intensive system in ghana table 2. hatching performance of introduced chicken strains. *, the hatching performances of both hubbard lines were computed by the hatchery operator together. #, significant proportional differences (z statistics) are indicated by p ≤ 0.05. within rows, proportions followed by different superscripts are significantly different ( p ≤ 0.05). parameter chicken strains z statistic p-value # kuroiler hubbard* eggs received (n) 10,080 10,080 eggs set (n) 9,980 9,427 % egg set 99a 93b 20.54 < 0.001 fertile eggs (n) 8,700 8,671 % fertile 87b 92a -10.92 < 0.001 infertile (n) 1,280 750b % infertile 13a 8 11.08 < 0.001 % hatched on fertile 83b 85a -3.59 0.0003 % hatched on set 73b 78a -8.09 < 0.001 genetic resources (2024), 5 (10), 107–116 chicken genetic improvement in ghana 113 table 3. egg quality trait parameters of kuroiler and local chicken ecotypes of ghana. within-rows means followed by different superscripts are significantly (p ≤ 0.05) different. parameters n range kuroiler forest coastal savannah egg weight (g) 180 32.2–101.8 88.8a±9.42 58.70b±9.9 52.50c±10.3 albumin height (mm) 180 2.1–5.5 3.94a±0.68 3.54a±0.93 3.10b±0.52 shell thickness (mm) 180 0.24–0.48 0.39±0.05 0.38±0.04 0.38±0.04 yolk colour 180 1.0–11.0 5.80±2.01 6.27±2.26 6.43±2.79 haugh unit 180 43.3–97.6 77.30a±9.85 73.40a±9.05 66.40b±11.7 figure 4. distribution of farmers’ most preferred traits in chickens of eggs from the local chickens was lower than that of exotic chickens (osei-amponsah et al, 2014; kejela et al, 2019). the mean haugh unit of local chickens in ghana ranged from 64.97 to 67.34, lower than that of nakedneck and normal-feathered chickens in nigeria, which were 73.22 and 71.40, respectively (yakubu et al, 2008; osei-amponsah et al, 2014). farmer perception and acceptance of introduced chicken genotypes in terms of trait preferences, farmers were asked to rank ten traits (fast growth, good survivability, good disease resistance, low feed intake, laying more eggs, laying big eggs, good mothering, plumage colour, tough meat and soft meat). fast growth, good survivability, good disease resistance, low feed intake and increased egg number are the most preferred traits in this study (figure 4). the result of ‘fast growth’ and ‘increased egg number’ was expected. this is because, smallholder poultry farmers in sub-saharan africa mainly keep chickens for their meat and eggs hence these traits are of much importance to them (sonaiya and swan, 2004; ochieng et al, 2013; melesse, 2014). the ‘good survivability’ and ‘good disease resistance’ trait preferences are consistent with previous studies (faustin et al, 2010; terfa et al, 2019). diseases in all poultry enterprises can lead to huge economic loss hence farmers’ choice of ‘good survivability’ and ‘good disease resistance’ is not surprising (dar et al, 2018). beneficiary farmers provided positive accounts of experimental birds. they attested to the high performance of both kuroiler and hubbard in terms of their body weight, egg size and egg number compared to the local chicken. the socioeconomic gains and prestige these birds have brought to them in their communities were emphasized. almost all beneficiary farmers mentioned income generation through the sale of larger and more eggs as well as cockerels. the adaptation abilities of the introduced birds were evidenced by the relatively low mortality and their resilience to environmental and farmer management conditions. this led to nonbeneficiary poultry farmers expressing interest in the tpgs project and requesting inclusion in any future initiatives. the farmers showed a strong preference for the introduced breeds, as they were found to be more market oriented (bamidele et al, 2023). furthermore, the use of improved tropically adapted chicken breeds in smallholder flocks in sub-saharan africa led to increased production and productivity, generating more income while contributing to food security and social and ecological resilience (birhanu et al, 2023). as highlighted in the literature, livelihood enhancement programmes 114 osei-amponsah et al genetic resources (2024), 5 (10), 107–116 often provide beneficiaries with poultry birds and feed to contribute towards food security and income generation (singh et al, 2022). conclusion this study’s intervention of introducing improved, dualpurpose chicken breeds to smallholder farmers aligns with previous findings, demonstrating the potential to significantly enhance livelihoods through the adoption of better-suited poultry genetics. the fast-growing and productive nature of the introduced chicken strains, particularly in terms of egg production, suggests a high likelihood of acceptance among poultry farmers in ghana. although the introduced breeds are more effectively managed under intensive systems, they have also shown resilience and satisfactory performance in semi-intensive and extensive production systems. these findings, along with supportive evidence from other studies, underscore the potential of these dual-purpose chickens to support faster income generation for smallholder farmers under prevailing production conditions in ghana. the introduction of adapted, dual-purpose chicken genotypes, such as the kuroiler and hubbard chickens has shown promising results in enhancing productivity and marketoriented performance. this study thus provides valuable insights for stakeholders to make informed decisions on local chicken breed diversification, conservation and improved production through efficient management, breeding and nutrition practices. the government and relevant stakeholders should work to ensure that tropically adapted chicken strains, selected for improved production, are made widely accessible to smallholder farmers. this can significantly boost poultry production and income generation across rural farming communities. future initiatives should include comprehensive economic assessments, particularly cost-benefit analyses, to evaluate the long-term profitability and sustainability of the introduced chicken breeds. such data will guide strategic genetic improvement programmes and investments in the poultry sector. to maximize the benefits of the new chicken strains, farmers should be trained in efficient management practices, including breeding, feeding and health care. this will boost productivity and profitability of the introduced poultry under varied production systems. the ministry of food and agriculture should develop supportive frameworks that promote the distribution and management of the introduced chicken lines as well as financial and technical support, alongside market linkages, to enhance the success of tpgs and similar interventions. authors contribution richard osei-amponsah: conceptualization, supervision, data analysis, preparation of initial draft, review and approval of final draft. ricky aboagye poku: data collection, review and approval of final draft. ebenezer agyemang duah: data collection, data analysis, review and approval of the final draft. augustine naazie: supervision, data analysis, review and approval of the final draft. raphael ayizanga: data collection, data analysis, review and approval of final draft. harrisson njamba: data collection, data analysis, review and approval of final draft. wondmeneh esatu: sourcing of fertilized eggs, review and approval of final draft. mulugetta y. birhanu: sourcing of fertilized eggs, review and approval of the final draft. tadelle dessie: conceptualization, fund acquisition, review and approval of the final draft. acknowledgements the authors are grateful to the tropical poultry genetics solution (tpgs) project of ilri and the university of ghana for permission to use the project data for this study. the tropical poultry genetics solution (tpgs) project of ilri was funded by the bill and melinda gates foundation. conflict of interest statement the authors declare no conflicts of interest. data availability the authors affirm that all data necessary to confirm the conclusions of the article are presented within the article, figures and tables. raw data analyzed for this article are available from the 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(2008). productivity and egg quality characteristics of free range nakedneck and normal feathered nigerian indigenous chickens. int. j. poult. sci 7(6), 579–585. https://doi.org/10.1111/agec.12502 https://doi.org/10.1111/agec.12502 introduction materials and methods project scope and farmer selection egg incubation and mothering of chickens data collection and analysis results and discussion body weight and growth performance egg production performance farmer perception and acceptance of introduced chicken genotypes conclusion authors contribution acknowledgements conflict of interest statement data availability original article genetic resources (2024), 5 (10), 117–125 doi: 10.46265/genresj.ihxu5248 https://www.genresj.org issn: 2708-3764 the eurisco-eva information system, an innovative approach to the data management of multi-site crop evaluation data suman kumar *,a, filippo guzzon b, sandra goritschnig b and stephan weise a a leibniz institute of plant genetics and crop plant research (ipk) gatersleben, corrensstr. 3, 06466, seeland, germany b european cooperative programme for plant genetic resources (ecpgr), c/o alliance of bioversity international and ciat, via di san domenico 1, rome, 00153, italy abstract: this paper introduces eurisco-eva, an extension of the european search catalogue for plant genetic resources (eurisco) hosted at and maintained by the leibniz institute of plant genetics and crop plant research (ipk) gatersleben on behalf of the european cooperative programme for plant genetic resources (ecpgr). this information system facilitates standardized data collection, sharing and analysis for plant genetic resources for food and agriculture (pgrfa) characterization and evaluation. in the framework of the european evaluation network (eva), public–private partnerships aiming at the evaluation of crop accessions conserved in european genebanks, eurisco-eva provides a standardized data repository for multi-site evaluations of different crops. through centralizing metadata maintenance, eurisco-eva ensures uniformity in trait definitions, experimental designs and passport data, promoting the efficient exchange of observed phenotypic data. eurisco-eva currently stores more than half a million phenotypic data points for 4,845 pgrfa accessions from 6 genera and 17 species, collected through 382 phenotypic experiments conducted at 115 experimental locations across 33 countries, involving 89 project partners. this platform offers a user-friendly web interface, empowering its users with features such as map-based filtering of trial locations, statistical overviews and customizable reports. eurisco-eva’s robust administrative functionalities, coupled with standardization efforts, enhance data quality and harmonization, providing a robust and scalable system for storage of and access to crop evaluation data that could be further enhanced by adding analysis modules. eurisco-eva also formed the basis for the data management of two research projects (agent and increase) under the european union horizon 2020 funding programme, providing the background organization of complex datasets used to address future challenges in european agriculture. keywords: crop evaluation, genebank, information system, plant genetic resources, metadata citation: kumar, s., guzzon, f., goritschnig, s., weise, s. (2024). the eurisco-eva information system, an innovative approach to the data management of multi-site crop evaluation data. genetic resources 5 (10), 117–125. doi: 10.46265/genresj.ihxu5248. © copyright 2024 the authors. this is an open access article distributed under the terms of the creative commons attribution license (cc by 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. introduction in the coming decades, a growing population, climate change and the need to protect ecosystems will create new challenges for agriculture and global food security. to address these challenges, sustainable farming and increased crop production are required. developing crop varieties with resilient traits like disease resistance, ∗corresponding author: suman kumar (kumar@ipk-gatersleben.de) drought and heat tolerance will be crucial to achieving these goals (mccouch et al, 2013; pixley et al, 2023). this requires access to a diverse pool of plant genetic resources for food and agriculture (pgrfa) conserved ex situ by genebanks to identify and incorporate valuable traits into new crop varieties (sanchez et al, 2023; king et al, 2024). the accessibility of these pgrfa accessions and their related passport, characterization and evaluation data, is strictly linked with the existence and updating of information systems. this involves gathering data for accesreceived: 02.07.2024 accepted: 04.11.2024 published online: 13.11.2024 https://www.genresj.org https://www.doi.org/10.46265/genresj.ihxu5248 https://www.genresj.org https://www.doi.org/10.46265/genresj.ihxu5248 mailto:kumar@ipk-gatersleben.de 118 kumar et al genetic resources (2024), 5 (10), 117–125 sions from many germplasm collections and projects into centralized sources, facilitating a smooth flow of germplasm material and data among institutions. therefore, developing an information system that collects data from various sources in a standardized format and creates searchable datasets on genetic resources is key to pgrfa access and sustainable use (khoury et al, 2010; guzzon and ardenghi, 2018; ecpgr, genres bridge project consortium, erfp & euforgen, 2021). a standardized phenotypic data platform is essential for interoperability, enabling integration and comparison of data from diverse sources, allowing also for the feeding and operation of international information systems on plant genetic resources (pgr) (weise et al, 2020). a standardized information system promotes reproducibility and validation of research findings, fostering transparency and collaboration among researchers. standardization streamlines data integration and analysis processes, improving efficiency and reducing duplication of effort and resources, allowing the implementation of the fair (findable, accessible, interoperable, reusable) data principles (wilkinson et al, 2016; papoutsoglou et al, 2023). data standardization ensures also data quality and integrity by defining clear guidelines for collection, storage and validation. in this framework, miappe (minimum information about a plant phenotyping experiment) provides a community data standard for the plant phenotyping domain (krajewski et al, 2015; papoutsoglou et al, 2020). to exploit the genetic wealth of pgr conserved ex situ in genebanks, multi-site pre-breeding characterization and evaluation of pgrfa are fundamental to inform and speed up the complex process of crossing, selection and testing of plant material needed to produce a new elite cultivar and preparing material ready to be incorporated into crop breeding programmes (cockel et al, 2022). in europe, the european evaluation network (eva) for pgrfa (http://www.ecpgr.org/eva/ ), coordinated by the european cooperative programme for plant genetic resources (ecpgr), is an international initiative aimed at increasing the use of crop genetic diversity as well as the diversity of stakeholders in plant breeding. in joint efforts with partners from both public and private sectors, eva is producing standardized evaluation data for various crop cultivars and landraces found in european genebanks. this data includes both phenotypic characteristics and genotypic information, which can be used to identify suitable breeding materials and genetic markers for relevant traits. eva operates through specific networks tailored to different crops, including cereals and vegetables. initially established as five different crop networks (carrot, lettuce, maize, pepper, and wheat and barley), ecpgr launched a new eva network on legumes in 2024, which covers seven different crop groups: chickpea, common bean, faba bean, lentil, lupin, pea and orphan legumes, thus vastly expanding the project partnership. eva provides an opportunity to promote the sustainable use of pgrfa to facilitate the adaptation of european agriculture to climate change and to contribute towards achieving related sustainable development goals. in this paper we describe the eurisco-eva information system, which was developed as an extension of the european search catalogue for plant genetic resources (eurisco; see weise et al (2017); kotni et al (2023)) and is a service provided by ecpgr to the pgrfa user community. the system provides partners with a central data repository and allows the collection of standardized phenotypic data in the framework of eva. it features filter and display options and can facilitate the publication of datasets after the project embargo through integration with eurisco. eurisco-eva served as a blueprint for data management infrastructures developed in other european projects like agent (https://agent-project.eu /) and increase (https://www.pulsesincrease.eu/). by adopting a common model and protocols, these projects standardize and thereby facilitate the exchange of data and information among different databases and systems. this interoperability enhances collaboration and coordination among various stakeholders involved in pgrfa conservation and breeding efforts across europe and beyond. the eurisco-eva information system database content the eurisco-eva database currently stores the data of five crop networks: carrot, lettuce, pepper, maize, and wheat and barley. the wheat and barley network accounts for three different crops, barley (hordeum vulgare l.), durum wheat (triticum turgidum l. subsp. durum (desf.) husn.) and common wheat (triticum aestivum l.). in the lettuce network, data on wild prickly lettuce (lactuca serriola l.) are stored together with data on cultivated lettuce (lactuca sativa l.). in the pepper network, five species are considered: capsicum annuum l., c. baccatum l., c. chacoense hunz., c. chinense jacq., c. frutescens l. at the time of writing, these five networks thus cover a total of eight crops accounting for a total of 4,845 accessions and 282 phenotypic traits with data. the 89 network partners work in 33 countries, carrying out characterization and evaluation activities in 115 experiment locations. as of june 2024, more than 500,000 total phenotypic data points have been collected in 382 phenotypic trials and this number is continuously growing. table 1 provides an overview of the data of the different crop networks and figure 1 presents a summary of the data points, phenotypic trials, traits and evaluated accessions by country as of june 2024. in addition to phenotypic evaluation data, euriscoeva stores relevant metadata such as accession passport data, trait and method definitions, information on phenotypic trials, network partners and genotyping experiments. a new record for any of the metadata can be created or the existing record can be updated as needed. a new partner can join an existing network; however, they will only have access to the network’s genetic resources (2024), 5 (10), 117–125 119 table 1. summary overview of the data available on eurisco-eva of the current five eva networks (as of 11 june 2024). countries of operation are those where trials are performed, experiment locations refer to locations within countries. all networks carrot lettuce maize pepper wheat & barley crops 8 1 2 1 1 3 accessions evaluated 4,845 67 291 861 181 3,445 partner institutes 89 14 12 18 15 47 countries of operation 33 8 8 9 13 25 experiment locations 115 14 6 30 10 58 phenotyping experiments evaluated 382 27 13 63 15 264 traits evaluated 282 138 21 51 26 46 phenotypic data points 510,097 88,199 10,217 90,359 19,327 301,995 figure 1. summary of evaluation data and metadata in eurisco-eva for all crop networks disaggregated by evaluation countries. a) total number of evaluation data points obtained by country, b) total number of phenotypic trials evaluated by country, c) total number of traits evaluated by country, and d) total number of accessions evaluated by country (data as of 11 june 2024). eurisco-eva: innovative multi-site crop data management 120 kumar et al genetic resources (2024), 5 (10), 117–125 data created on or after their joining date. the eva accessions’ passport data follow the multi-crop passport descriptors standard (alercia et al, 2015) along with some eva-specific identifiers, e.g. material type (original accession, single-seed descent line, cross or check), eva id, male and female parent for crosses, and parent dois, where applicable. these project-specific parameters allow categorization of the accessions in the network and can be adjusted centrally by the eva coordinator. each trait is defined by a unique trait acronym, its trait name, a detailed trait method description, a crop ontology term, trait group, measurement unit and allowed scores. the allowed trait scores are of two types, metric and rating scores, and define the range of allowed values or the allowed scoring entries, respectively. traits can be further grouped into different categories, such as morphological, agronomic, quality or (a-)biotic stress traits, to facilitate searches and filtering. the trial definition broadly consists of the trial location (ideally by gps coordinates), experimental and field design as well as meteorological and soil conditions, which are important parameters when comparing data from multiple locations. the evaluation data are stored as trait scores that were observed for the accessions under defined trial conditions. the system can also host additional data like accession images and links to genotypic data repositories. for every partner involved in the project, there is information on the location of the organization and the trials as well as contact information of the responsible persons. technical requirements, features and technology the eurisco-eva infrastructure was developed as an extension of the european search catalogue for plant genetic resources (eurisco), operated by the leibniz institute of plant genetics and crop plant research (ipk) gatersleben, germany, on behalf of and under the supervision of ecpgr, providing additional necessary functionality beyond the existing eurisco infrastructure in the framework of the abovementioned eva network. the eurisco-eva information system is also maintained on behalf of ecpgr and has been developed by ipk. it has been available online to the eva network partners since 2022 and its management is handled by the ecpgr eva coordinator. the primary access point to eurisco-eva is its web interface (https://eva.ipk-gatersleben.de/), developed using the oracle application express (apex) technology, version 21. partners can use their login credentials to access their network and view their network’s data collected on or after their joining date. there are two entry points: a general homepage and a main page for each crop network. after logging in, users first access the common homepage, which introduces the eva project, offers user manuals, provides downloadable templates (see supplemental data 1 and 2) for recording or uploading data, and includes general information about the data and metadata stored in the system. every crop network has its homepage with the same type of information, accessible to the network partners only. the network homepage has four tabs (figure 2). the first tab ‘trial locations’ shows a map giving users an overview of the geographic diversity of the experimental trial locations. the data on the map can be filtered and searched using one or more available filters, such as crop, year, experiment group, organization, country or trial id. the second tab ‘all data’ provides on the left-hand side a statistical overview of available data for the crop network and the total data of all crop networks (figure 2). moreover, it has several cards that act as hyperlinks to generate reports on accession passport and phenotypic data, partner information, specifics of the phenotypic traits as well as phenotyping and genotyping experiments. the third tab ‘available metadata’ provides numerical information on relevant available metadata, including passport data, trial details, trait definition and network partner details, already grouped by important parameters such as accession material type or trait group. the hyperlinks in the statistical reports open a filtered report corresponding to the selected parameter. the fourth tab ‘available observed data’ displays phenotypic data already grouped by crop or species, predefined experiment group or year as well as country of origin of the accessions and institutes maintaining the material. the user interface of this application is designed to offer multiple methods for retrieving and filtering data to suit different needs and preferences. below is a detailed overview of these methods and how they work: 1. default search and filter options: the default search feature allows users to quickly find information by entering keywords or criteria into a search bar. for example, if a user is looking for data on ‘genotype a,’ they can simply type this term into the search bar, and the system will display all relevant records containing the string ‘genotype a.’ additionally, users can apply basic filters from a predefined set of options such as date ranges or data categories to narrow down the search results. 2. one-click filters: for more common searches, the interface provides one-click filters. by clicking on them, users can instantly retrieve the data without having to apply a filter manually. for example, to retrieve phenotypic data specifically for wheat, simply click on ‘wheat’ in the one-click filter menu labelled ‘grouped by crop’. 3. advanced searches with interconnected dropdown filters: the advanced search functionality provides a more detailed and precise approach to data retrieval through a series of interconnected drop-down menus, where the selection made in one filter dynamically updates the options available in the following. it provides only those options in the drop-down menus that will lead to actual records in the search results. for instance, if ‘trial 2024’ is selected in one drop-down menu, the subsequent trait options will be limited to those associated with ‘trial 2024.’ this dynamic interacgenetic resources (2024), 5 (10), 117–125 121 figure 2. eurisco-eva crop homepage for the eva wheat and barley network showing overview statistics of the network on the left and the shortcut cards leading to reports for metadata and phenotypic data. additional tabs provide access to pre-filtered data and metadata reports tabs (data as of 25 september 2024). tion ensures that users are presented with only relevant choices, facilitating a more refined and accurate search. 4. phenotypic data reports: the interface also includes several types of phenotypic data reports to cater to different reporting needs: • default report: provides a general overview of the data, with each individual data point listed. • overview report: groups data by trait and trial, offering a broader perspective on the data collected. • customizable pivot report: users can create customized pivot reports by selecting a crop and up to five specific traits, along with optional parameters. the report displays the selected traits side-by-side, allowing for a comparative view. 5. report download options: all reports generated through the interface can be easily downloaded in excel, csv, html or pdf format. this functionality allows users to further analyze or share their data outside the application. this combination of search methods and reporting tools provides users with a robust mechanism for data retrieval and analysis, ensuring they can access and manipulate the information in ways that best suit their needs. the chart functionality within the web interface enhances user engagement by offering a flexible and interactive experience (figure 3). users can customize charts by selecting data through interconnected dropdown menus, ensuring precision in data selection. moreover, the chart feature provides access to detailed pages, offering in-depth insights into the viewed data, such as displaying the distribution of data, the frequency of observed values across all (or selected) experiments or comparisons of data collected over different timepoints. beyond its customizable nature, the chart functionality serves as a gateway to further analysis via the download of associated data as described above. users can explore detailed reports presenting the total data points per experiment, providing a comprehensive overview of the dataset. additionally, the chart facilitates the visualization of statistical measures, including mean, variance, median and mode. this multifaceted approach not only empowers users to create personalized visualizations but also supports comprehensive data exploration and analysis through detailed information pages and statistical insights, as can be seen in figure 3. data standardization and upload in eva, a big effort was put into the standardization of data collection. the standardization process involves the central maintenance of metadata, i.e. trait definitions, experimental design and passport data. the partners collecting phenotypic data reference these metadata, and the uniform methods and scales of the collected observed phenotypic data make the data easy to understand, analyze and exchange among providers and users. generic data collection templates that include all important information for collecting multilocation trial data were developed in order to ease the upload of the data. to reduce the load on background programmes for reading and validating data, and eurisco-eva: innovative multi-site crop data management 122 kumar et al genetic resources (2024), 5 (10), 117–125 figure 3. examples of data visualization in the eurisco-eva user interface. a) comparison between distribution of two trait scores using box plot, b) histogram showing the frequency of scores of a specific trait. to make the processing faster, a data collection template consisting of two excel files was created and is available in the documents section of the eva website (see https://www.ecpgr.org/eva/documen ts-and-links/evaluation-protocols-and-templates and the current version v1 included as supplemental data 1 and 2). one data collection template (supplemental data 2) was designed for collecting and updating the observed evaluation data with minimal trial details and is used by the data providers. the second collection template (supplemental data 1) is used for creating and updating metadata, including trial details, trait definitions, accession passport data and partner details. this template is centrally completed by the eva coordinator with input from partners, making the overall data collection simpler, faster and less prone to error. the eurisco-eva data templates aim to fulfil the miappe standard in the best possible way while keeping them simple for use by diverse stakeholders. moreover, most of the accessions that are part of eva are already documented in eurisco. to keep the passport data in the two systems consistent, a process was implemented that automatically synchronizes the passport data from eurisco to the eurisco-eva information system. the overall phenotypic data upload and management consists of three parts (figure 4): 1) data file upload: the website features a streamlined four-step file uploader, allowing users to easily upload the phenotypic data using the aforementioned generic data collection template, 2) data processing: the background import programme initiates upon file upload, reads data from the file, validates the information, and subsequently writes it into the designated database tables, 3) data presentation: data is retrieved from these tables and is utilized on the front end to generate reports and charts on the web application (figures 2 and 3). users are only able to upload data for experiments that they are responsible for. if errors are detected during data upload and validation, a message is logged and shared with the user, with a description of the error and an explanation of how to fix it in the data template. most errors are with formatting or values that are outside the allowed range and the error log enables users to easily identify and fix the issues. once the processing is successfully finished, an email with the processing log is sent to the user, confirming the successful upload. apart from the evaluation data, up to five images per accession can be uploaded to illustrate its appearance and specific characteristics. the images are associated with the accession and thus enrich the passport data available for accessions in eurisco-eva. the image uploader allows the user to map the images also to a certain trait or trial, where applicable, which makes the images searchable. moreover, users can upload additional files to their experiment that contain further trial information. since the data in these files are not written into the database, they can include various file formats providing for example graphical representation of field layouts or initial statistical analyses. these files genetic resources (2024), 5 (10), 117–125 123 figure 4. eurisco-eva dataflow and capabilities overview. are included in the trial detail report and are available for download by all users. database implementation the eurisco-eva information system is based on an oracle relational database management system version 19c. the foundational database schema encompasses 49 tables (see supplemental data 3), while the business logic was implemented using pl/sql, primarily focused on ensuring data quality, enhancing performance, enabling user-specific download capabilities, facilitating reporting tasks, automating data manipulations and uploads, and executing scheduled removal of unnecessary data. the eurisco-eva information system provides a set of functionalities for administrators to maintain the website content. the website is highly scalable to new crop networks and is currently being expanded to host the data of the eva legumes network, which started operations in 2024, as well as a demo network for the interested public. eva’s data model is trial-based, and designed for collaborative, multi-environment evaluations, incorporating data from both public and private partners. unlike accession-based models like eurisco, which focus on cataloguing genetic resources, eva emphasizes ongoing evaluation data collected continuously throughout active trials. moreover, eva standardizes trait definitions and measurement scales across all experiments, ensuring that data from one partner is fully compatible and usable by all other partners. this consistency significantly improves data comparability and usability, making it highly valuable for advanced data analysis, including comparative studies, large-scale data mining and breeding decision support. the combination of continuous data collection and standardized traits enables eva to deliver more actionable, high-quality insights for breeding and research compared to existing solutions. compared with other information systems developed for managing data on pgrfa characterization and evaluation such as germinate (shaw et al, 2017) or grassroots (bian et al, 2017), eurisco-eva is not an open-source project software that can be easily installed and applied to new projects. instead, eurisco-eva is operated by ecpgr as a long-term service to the pgrfa community and is very closely linked to eurisco as the central european pgr information system. outlook since its inception, eurisco-eva has facilitated data curation and management and enabled the analysis of complex datasets by the existing eva networks, which have produced several publications (goritschnig et al, 2023; tripodi et al, 2023; balconi et al, 2024). although eurisco-eva has been built as a platform with restricted access to the data, its use of the same background system as eurisco can easily facilitate the incorporation of phenotypic data in the public database after the end of the data embargo periods. ensuring the public availability of the generated evaluation data is one of the core values of the eva networks and will be provided through eurisco. however, some discussions are still ongoing about how to ensure that useful data is available to the public (e.g. all raw data vs. experiment means, considering also the quality of data from individual trials). the eurisco-eva information system has a wide range of features for a beginner as well as an advanced user among eva network partners. a beginner user, who may find manual searches and filters challenging, can easily open reports by applying a pre-defined filter eurisco-eva: innovative multi-site crop data management 124 kumar et al genetic resources (2024), 5 (10), 117–125 with just one click. a more advanced user can apply searches and filters with several drop-down selection lists to customize their reports. the data collection uploader is self-explanatory, highly intuitive and easy to debug. besides common features allowing users to view, filter, visualize, email and download data, the web application provides additional features like pivot reporting and data visualization, which take user input to dynamically create custom pivot reports or highly intuitive charts. it is a highly scalable system in which the administrator can easily configure a new network with the existing import programme and template for partners. as mentioned, the eva legumes network has recently been added. in the future, the error management of data processing could be made more robust and user-friendly. also, an immediate value could be added to the euriscoeva information system by integrating a data analysis module into it. moreover, an open-access demo network will be configured so that new potential partners can have the feel of eurisco-eva’s user interface, background programmes, easy-to-use reports and data visualization features, without accessing restricted data. the eurisco-eva information system is an ongoing initiative that integrates data on pgrfa evaluations from multiple partners and locations. considering that crop characterization and evaluation data are often scattered in various data sources and publications and lack standardization (ćwiek kupczyńsk et al, 2016), the euriscoeva information system provides a user-friendly and versatile environment that enhances data interoperability as well as standardization (by considering uniform traits and methods) of phenotypic data in the framework of the evaluation activities of the eva initiative. conclusions in this paper, we described the development and implementation of eurisco-eva, an information system for pgrfa, which supports the management of metadata and experimental phenotypic data for eva, with the possibility to provide fair public access to data after an embargo through its interoperability with eurisco. eurisco-eva is being maintained by ecpgr and upgrading with additional elements will be possible in the future. eurisco-eva provides a gateway for important evaluation data describing genetic resources, adding value to genebank collections and enabling users across the globe to access phenotypic data for genebank accessions through eurisco. acknowledgements the authors wish to thank all partners of the eva networks for their feedback on earlier versions of the database and suggestions for improvements and new functionalities. this work was supported by the german federal ministry of food and agriculture through grant genres 2019-2 to ecpgr for the implementation of the eva networks. the authors are grateful to the two reviewers for their useful comments and suggestions on an earlier version of this manuscript. supplemental data supplemental data 1: metadata creation templates supplemental data 2: phenotypic data collection templates for users supplemental data 3: foundational database scheme of eurisco-eva authors contribution suman kumar was responsible for the initial conception and design. suman kumar also wrote the initial draft of the manuscript, with filippo guzzon contributing by enhancing and refining the content. sandra goritschnig and stephan weise reviewed and edited the manuscript, providing critical input to ensure its relevance. all authors read and approved the final manuscript. conflict of interest statement the authors declare that they have no conflicts of interest. references alercia, a., diulgheroff, s., and mackay, m. 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(2016). the fair guiding principles for scientific data management and stewardship. sci data 3. doi: https://doi.org/10.1038/sdata.2016.18 https://doi.org/10.17660/actahortic.2023.1384.8 https://doi.org/10.17660/actahortic.2023.1384.8 https://doi.org/10.1007/s10531-017-1485-7 https://doi.org/10.1007/s10531-017-1485-7 https://doi.org/10.1007/s10722-010-9534-z https://doi.org/10.1007/s10722-010-9534-z https://doi.org/10.1111/gcb.17440 https://doi.org/10.1111/gcb.17440 https://doi.org/10.1093/nar/gkac852 http://doi.org/10.1093/jxb/erv271 https://doi.org/10.1038/499023a https://doi.org/10.1038/s41597-023-02364-z https://doi.org/10.1038/s41597-023-02364-z https://doi.org/10.1111/nph.16544 https://doi.org/10.1016/j.molp.2023.09.003 https://doi.org/10.1016/j.molp.2023.09.003 https://doi.org/10.1073/pnas.2205780119 https://doi.org/10.1073/pnas.2205780119 https://doi.org/10.2135/cropsci2016.09.0814 https://doi.org/10.2135/cropsci2016.09.0814 https://doi.org/10.3389/fpls.2023.1252777 https://doi.org/10.3389/fpls.2023.1252777 https://doi.org/10.3390/plants9081050 https://doi.org/10.3390/plants9081050 https://doi.org/10.1093/nar/gkw755 https://doi.org/10.1093/nar/gkw755 https://doi.org/10.1038/sdata.2016.18 introduction the eurisco-eva information system database content technical requirements, features and technology data standardization and upload database implementation outlook conclusions acknowledgements supplemental data authors contribution conflict of interest statement supplemental data for: protonotariou, s. v., thanopoulos, r., katsileros, a., bebeli, p. j., mandala, i. (2023). evaluating agromorphological traits of greek wheat landraces and exploring their potential for bread and pasta making based on seed physical properties. genetic resources 4 (8), 37–53. doi: 10.46265/ genresj.hfwz5263. contents supplemental figure 1: the frequency percentage for the four qualitative morphological traits observed in the 15 wheat accessions studied. supplemental figure 2: simple linear regression model to describe the relationship between seed firmness (sf) and thousand kernel weight (tkw) of wheat seeds. supplemental table 1: summary table of friedman test results and medians of qualitative variables; plant growth habit; color of ear; ear shape in profile and awn color, for 15 triticum spp accessions. supplemental table 2: analysis of variance for quantitative agromorphological traits (anova tables). supplemental table 3: spearman rank correlations between each pair of variables (qualitative: plant growth habit, color of ear, ear shape in profile and awn color versus all the variables). supplemental table 4: mean values of image analysis-shape factor measurements of 15 greek wheat accessions. supplemental table 5: pearson correlation test between quantitative parameters. https://doi.org/10.46265/genresj.hfwz5263 supplemental figure 1 the frequency percentage for the four qualitative morphological traits observed in the 15 wheat accessions studied. supplemental figure 2 r-squared = 70.58 percent r-squared (adjusted for d.f.) = 68.35 percent standard error of est. = 0.7451 mean absolute error = 0.5401 the equation of the fitted model is tkw (g) = 2.0574 + 0.0326*sf (n) since the p-value in the anova table is less than 0.05, there is a statistically significant relationship between tkw (g) and sf (n) at the 95.0% confidence level. y = 0.0326x + 2.0574 r² = 0.7058 0 1 2 3 4 5 6 7 8 9 0 50 100 150 200 250 s f ( n ) tkw (g) supplemental table 1 summary table of friedman test results and medians of qualitative variables; plant growth habit; colour of ear; ear shape in profile and awn colour, for 15 triticum spp accessions. accession code plant growth habit colour of ear ear shape in profile awn color w1 3,0 2,0 5,0 1,0 w2 1,0 2,0 5,0 2,0 w3 2,0 2,0 1,4 1,0 w4 1,0 1,0 3,0 1,0 w5 8,0 1,0 5,0 1,0 w6 3,0 1,0 5,0 1,0 w7 2,0 1,2 5,0 4,0 w8 7,0 1,0 5,0 1,0 w9 2,0 1,0 5,0 4,0 w10 2,0 1,8 5,0 1,8 w11 3,0 1,0 4,3 1,0 w12 6,0 1,9 3,3 3,3 w13 3,0 1,0 5,0 4,0 w14 4,0 1,4 5,0 1,5 w15 5,0 1,0 5,0 1,0 friedman χ² 27,6 36,2 34 39,5 p 0,016 < .001 0,002 < .001 where w1=yekora-t. aestivum, cultivar; w2= elisavet-t. aestivum, cultivar; w3= elpida-t. durum, cultivar; w4= mexicali 81-t. durum, cultivar; w5= zoulitsa-t. aestivum, landrace; w6= ntopio-t. aestivum, landrace; w7= mavragani-t. durum, unknown status from skyros; w8= grilos-t. aestivum, landrace; w9= leventis-t. polonicum, landrace; w10= kopaida-t. dicoccum schrank, landrace; w11=asprositi-t. aestivum, landrace from kalavrita; w12= asprositi-t. durum, landrace from kozani; w13= mavragani-t. durum, unknown status from lemnos; w14= aspratherit. durum landrace and w15= kokkinositaro-t. aestivum, landrace. supplemental table 2 analysis of variance for quantitative agromorphological traits (anova tables). all f-ratios are based on the residual mean square error. since p-value is less than 0.05, the factor has a statistically significant effect on the variable at the 95.0% confidence level (bold). source sum of squares df a:variety 3963 14 b:block 14,68 2 residual 101 28 total (corrected) 4079 44 source sum of squares df a:variety 136,9 14 b:block 1,758 2 residual 35,75 28 total (corrected) 174,4 44 source sum of squares df a:variety 30870 14 b:block 254,9 2 residual 2580 28 total (corrected) 33710 44 source sum of squares df a:variety 185,6 14 b:block 1,707 2 residual 19,12 28 total (corrected) 206,4 44 0,683 0,854 1,25 0,302 13,26 19,4 0,000 main effects mean square f-ratio p-value ear length excluding awns 92,13 127,5 1,38 0,267 2205 23,9 0,000 main effects mean square f-ratio p-value 1,277 plant length 0,879 0,69 0,511 9,781 7,66 0,000 main effects mean square f-ratio p-value total number of tillers 3,607 7,339 2,03 0,150 283,1 78,50 0,000 main effects time of ear emergence mean square f-ratio p-value plant weight sum of squares 10420 91,66 1894 12400 number of spikelet (first tiller) sum of squares 177,9 4,485 35,98 218,4 sum of squares 26040 181,3 1111 27330 ear length including awns sum of squares 663,4 18,29 231,2 913total (corrected) 44 residual 28 8,26 b:block 2 9,15 1,11 0,344 a:variety 14 47,4 5,74 0,000 mean square f-ratio p-value main effects source df total (corrected) 44 90,6 2,28 0,121 residual 28 39,7 b:block 2 a:variety 14 1860 46,9 0,000 main effects source df mean square f-ratio p-value total (corrected) 44 residual 28 1,29 stem length (base to ear base) b:block 2 2,24 1,74 0,193 a:variety 14 12,7 9,89 0,000 main effects source df mean square f-ratio p-value total (corrected) 44 residual 28 67,7 11 0,000 b:block 2 45,8 0,68 0,516 df mean square a:variety 14 744 f-ratio p-value main effects source supplemental table 3 spearman rank correlations between each pair of variables (qualitative: plant growth habit, colour of ear, ear shape in profile and awn colour versus all the variables). variables plant growth habit εar color shape in profile awn color area -0,161 -0,423 -0,278 0,313 aspect -0,255 0,350 -0,059 0,442 den./inten. (mean) 0,244 0,007 0,008 -0,279 diameter (max) -0,355 -0,080 -0,057 0,498 diameter (min) -0,063 -0,482 -0,113 0,358 diameter (mean) -0,138 -0,392 -0,137 0,385 perimeter -0,289 -0,132 -0,262 0,531 roundness 0,040 0,153 -0,230 0,292 size (length) -0,323 -0,122 -0,036 0,509 size (width) 0,018 -0,513 -0,121 0,403 firmness -0,161 -0,093 -0,282 0,367 weight -0,143 -0,200 -0,371 0,367 volume -0,170 -0,261 -0,318 0,364 tap volume -0,174 -0,261 -0,331 0,364 bulk density -0,048 0,102 -0,236 -0,124 tap density -0,154 0,267 -0,175 0,113 ci -0,253 -0,045 0,113 0,324 time of ear emergence 0,453 -0,434 0,486 0,066 weight/plant 0,525 -0,337 0,000 0,094 total number of tillers 0,623 -0,004 0,113 -0,139 number of spikelet in the first 0,126 0,230 0,435 0,407 plant length 0,538 -0,528 0,153 -0,074 ear length excluding awns -0,247 0,078 -0,367 -0,701 ear length including awns 0,412 -0,380 0,375 0,269 stem length (base to ear base) 0,529 -0,536 0,177 -0,119 l* 0,040 0,334 -0,572 -0,143 a* 0,321 -0,100 0,283 -0,144 b* 0,027 0,196 -0,393 0,045 wi 0,148 0,202 -0,596 -0,237 plant growth habit -0,278 0,088 -0,309 ear colour -0,042 0,191 ear shape in profile 0,168 ci: car index, l* indicates lightness, a* indicates hue on a green (−) to red (+) axis, b* indicates hue on a blue (−) to yellow (+) axis, and wi: white index. values in bold denote significance level at p<0.05. supplemental table 4 mean values of image analysis-shape factor measurements of 15 greek wheat accessions. for each trait, different letters following the means indicate significant differences at 95 % level, using tukey's honestly significant difference (hsd) method w1, yekora 17,24 abc 2,10 cd 142,68 g 6,77 b 3,11 bc 4,55 bc 17,56 b 1,44 a 6,81 b 3,30 bc w2, elisavet 13,85 a 1,89 a 136,19 def 5,91 a 2,97 b 4,17 a 15,71 a 1,43 a 5,96 a 3,13 ab w3, elpida 18,24 d 2,47 cd 127,41 cdef 7,94 ef 3,11 gh 4,73 cd 20,58 ghi 1,92 de 8,01 f 3,33 bc w4, mexicali 19,91 abc 2,07 cd 133,56 cde 7,70 e 3,56 fgh 5,06 def 20,11 fgh 1,63 abc 7,73 e 3,79 def w5, zoulitsa 16,21 abc 2,09 cd 129,45 bc 6,75 b 3,04 b 4,50 abc 18,27 bcd 1,67 abc 6,82 b 3,34 bc w6, ntopio 17,48 ab 1,98 b 134,35 cdef 6,81 b 3,25 cd 4,61 bc 18,16 bc 1,51 abc 6,85 b 3,41 bcd w7, mavragani skyros 21,44 bcd 2,27 e 129,69 bc 8,15 fg 3,47 efg 5,17 ef 21,00 hi 1,67 abc 8,20 fg 3,73 cdef w8, grilos 18,39 abc 2,08 cd 130,69 bc 7,26 cd 3,34 de 4,77 cd 19,30 def 1,63 abc 7,30 cd 3,62 cdef w9, leventis 26,08 cd 2,37 f 127,74 ab 9,26 h 3,67 h 5,66 g 23,62 j 1,72 bcd 9,30 h 3,85 ef w10, kopaida 14,90 e 2,92 g 123,95 a 7,94 ef 2,53 a 4,25 ab 19,64 efg 2,10 e 8,04 f 2,72 a w11,asprositi kalavrita 17,34 a 1,86 a 137,28 ef 6,55 b 3,33 de 4,59 bc 17,92 bc 1,48 ab 6,60 b 3,49 bcde w12, asprositi kozani 17,73 abc 2,10 cd 138,92 fg 7,16 c 3,28 cd 4,61 bc 19,34 def 1,73 bcd 7,18 c 3,50 bcde w13, mavragani lemnos 23,35 abc 2,14 d 130,73 bc 8,31 g 3,72 h 5,41 fg 21,49 i 1,60 abc 8,35 g 3,94 f w14, aspratheri 16,79 abc 2,07 cd 131,70 bcd 7,28 cd 3,46 efg 4,83 cde 18,90 cde 1,74 cd 7,32 cd 3,65 cdef w15, kokkinositaro 19,17 ab 2,04 bc 138,77 fg 7,43 d 3,39 def 4,81 cde 19,25 def 1,55 abc 7,47 de 3,58 cdef perimeter (length of the seed outline) roundness size length (feret diameter along major axis) size width (feret diameter along minor axis) area of seed aspect (ratio between major and minor axis) density/ intensity. mean (average optical density) diameter max (length of longest line joining two points of outline ) diameter min (length of shortest line joining two points) diameter mean (average length of diameters ) w1 and w2 t. aestivum, cultivar; w3 and w4 t. durum, cultivar; w5, w6, w8, w11 and w15 = t. aestivum, landrace; w7 and w13= t. durum, unknown status; w9= t. polonicum, landrace; w10= t. dicoccum schrank, landrace; w12 and w14= t. durum, landrace supplemental table 5 pearson correlation test between quantitative parameters. values in red denote p-values below 0.05 indicating statistically significant non-zero correlations at the 95.0% confidence level. variables area aspect den./inten. (mean) diameter (max) diameter (min) diameter (mean) perimeter roundness size (length) size (width) sf tkw volume volume tap bulk density tap density ci time of ear emergence plant weight total number of tillers number of spikelet in first tiller plant length ear ength excluding awns ear length including stem length l* a* aspect 0,086 0,761 den./inten. (mean) -0,217 -0,716 0,437 0,003 diameter (max) 0,822 0,611 -0,598 0,000 0,016 0,019 diameter (min) 0,799 -0,437 0,129 0,431 0,000 0,103 0,647 0,109 diameter (mean) 0,976 0,037 -0,247 0,807 0,864 0,000 0,895 0,376 0,000 0,000 perimeter 0,878 0,496 -0,555 0,980 0,536 0,863 0,000 0,060 0,032 0,000 0,040 0,000 roundness -0,010 0,894 -0,762 0,538 -0,370 -0,001 0,469 0,972 0,000 0,001 0,039 0,175 0,998 0,078 size (length) 0,812 0,627 -0,612 1,000 0,413 0,796 0,978 0,551 0,000 0,013 0,015 0,000 0,126 0,000 0,000 0,033 size (width) 0,801 -0,415 0,083 0,445 0,991 0,868 0,557 -0,334 0,427 0,000 0,124 0,768 0,097 0,000 0,000 0,031 0,225 0,113 sf 0,711 0,075 -0,073 0,626 0,593 0,711 0,687 0,127 0,615 0,569 0,003 0,791 0,796 0,013 0,020 0,003 0,005 0,651 0,015 0,027 tkw 0,871 -0,051 -0,120 0,690 0,828 0,887 0,786 0,031 0,677 0,829 0,840 0,000 0,856 0,671 0,005 0,000 0,000 0,001 0,913 0,006 0,000 0,000 volume 0,938 0,096 -0,265 0,811 0,785 0,939 0,886 0,119 0,801 0,791 0,791 0,978 0,000 0,733 0,340 0,000 0,001 0,000 0,000 0,673 0,000 0,000 0,000 0,000 volume tap 0,936 0,086 -0,244 0,801 0,777 0,931 0,878 0,104 0,790 0,781 0,806 0,982 0,998 0,000 0,761 0,381 0,000 0,001 0,000 0,000 0,711 0,001 0,001 0,000 0,000 0,000 bulk density 0,097 -0,720 0,585 -0,275 0,524 0,160 -0,151 -0,508 -0,293 0,496 0,462 0,473 0,283 0,311 0,731 0,003 0,022 0,321 0,045 0,570 0,592 0,053 0,290 0,060 0,083 0,075 0,308 0,259 tap density 0,219 -0,641 0,458 -0,128 0,608 0,291 -0,009 -0,431 -0,146 0,579 0,500 0,574 0,401 0,418 0,969 0,433 0,010 0,086 0,649 0,016 0,293 0,974 0,109 0,604 0,024 0,058 0,025 0,138 0,121 0,000 ci 0,507 0,457 -0,629 0,676 0,277 0,537 0,634 0,418 0,677 0,277 0,223 0,446 0,546 0,496 -0,271 -0,053 0,054 0,087 0,012 0,006 0,317 0,039 0,011 0,121 0,006 0,319 0,425 0,096 0,035 0,060 0,328 0,851 time of ear emergence -0,184 0,225 -0,369 -0,009 -0,334 -0,201 -0,018 0,299 0,001 -0,317 -0,067 -0,266 -0,183 -0,154 -0,408 -0,435 -0,064 0,512 0,420 0,177 0,976 0,224 0,473 0,949 0,278 0,997 0,249 0,813 0,337 0,514 0,584 0,132 0,105 0,822 plant weight 0,140 -0,012 -0,013 0,159 0,169 0,143 0,237 0,222 0,151 0,163 0,626 0,390 0,311 0,340 0,408 0,418 -0,001 0,404 0,618 0,967 0,963 0,571 0,546 0,612 0,396 0,426 0,590 0,563 0,013 0,150 0,260 0,215 0,132 0,121 0,998 0,135 total number of tillers -0,451 0,240 -0,090 -0,181 -0,476 -0,458 -0,189 0,406 -0,175 -0,447 -0,099 -0,320 -0,341 -0,314 -0,133 -0,208 -0,296 0,584 0,570 0,092 0,389 0,751 0,519 0,073 0,086 0,500 0,133 0,534 0,095 0,725 0,244 0,214 0,255 0,637 0,456 0,284 0,022 0,026 number of spikelet in the first -0,235 -0,012 0,244 -0,158 -0,179 -0,238 -0,197 0,052 -0,162 -0,240 0,314 -0,077 -0,151 -0,122 0,256 0,229 -0,134 0,256 0,644 0,392 0,399 0,965 0,382 0,575 0,524 0,392 0,483 0,854 0,564 0,389 0,254 0,785 0,590 0,664 0,357 0,412 0,634 0,358 0,010 0,148 plant length 0,033 -0,082 -0,055 0,055 0,117 0,059 0,112 0,142 0,052 0,102 0,353 0,212 0,169 0,197 0,231 0,232 -0,034 0,674 0,796 0,608 0,375 0,907 0,771 0,845 0,846 0,678 0,835 0,692 0,613 0,854 0,716 0,197 0,448 0,548 0,481 0,407 0,406 0,904 0,006 0,000 0,016 0,168 ear length excluding awns -0,219 -0,059 0,199 -0,216 -0,164 -0,211 -0,251 -0,120 -0,214 -0,181 -0,129 -0,092 -0,145 -0,141 0,198 0,153 -0,227 0,409 0,242 0,344 0,207 0,456 0,433 0,836 0,477 0,440 0,560 0,451 0,367 0,671 0,444 0,519 0,648 0,744 0,605 0,616 0,478 0,587 0,415 0,131 0,385 0,209 0,459 0,088 ear length including awns 0,354 0,098 -0,005 0,404 0,300 0,357 0,399 0,160 0,397 0,293 0,422 0,430 0,432 0,437 0,113 0,176 0,260 0,267 0,526 0,267 0,325 0,643 0,042 0,195 0,729 0,987 0,135 0,277 0,192 0,141 0,568 0,143 0,289 0,117 0,109 0,108 0,103 0,690 0,531 0,349 0,337 0,044 0,337 0,238 0,010 0,883 stem length (base to ear base) 0,018 -0,092 -0,102 0,030 0,106 0,045 0,094 0,134 0,028 0,093 0,288 0,153 0,123 0,150 0,161 0,157 -0,043 0,710 0,759 0,611 0,330 0,988 0,499 0,523 0,949 0,745 0,717 0,915 0,708 0,875 0,740 0,634 0,920 0,741 0,297 0,587 0,662 0,594 0,568 0,577 0,880 0,003 0,001 0,016 0,230 0,000 0,058 0,045 l* 0,053 -0,404 0,514 -0,172 0,328 0,073 -0,071 -0,248 -0,183 0,329 0,170 0,337 0,214 0,233 0,649 0,651 -0,215 -0,630 -0,005 -0,285 -0,099 -0,243 0,290 -0,202 -0,266 0,850 0,136 0,050 0,539 0,233 0,795 0,803 0,373 0,514 0,231 0,545 0,220 0,444 0,403 0,000 0,009 0,442 0,012 0,985 0,303 0,726 0,383 0,294 0,471 0,338 a* -0,550 -0,344 0,005 -0,564 -0,236 -0,439 -0,593 -0,215 -0,563 -0,247 -0,317 -0,456 -0,516 -0,498 0,100 0,076 -0,174 0,376 0,085 0,211 0,411 0,191 -0,098 -0,108 0,218 -0,353 0,034 0,209 0,985 0,028 0,397 0,102 0,020 0,441 0,029 0,374 0,249 0,088 0,049 0,059 0,724 0,787 0,535 0,168 0,763 0,450 0,128 0,495 0,728 0,703 0,434 0,198 wi 0,082 -0,301 0,479 -0,114 0,257 0,063 -0,012 -0,180 -0,123 0,266 0,160 0,343 0,240 0,260 0,565 0,565 -0,202 -0,600 -0,009 -0,240 -0,169 -0,233 0,300 -0,159 -0,265 0,974 -0,501 0,771 0,275 0,071 0,687 0,355 0,822 0,968 0,522 0,663 0,338 0,569 0,210 0,389 0,349 0,028 0,028 0,472 0,018 0,974 0,389 0,548 0,403 0,278 0,571 0,340 0,000 0,057 sf: seed firmness, tkw: thousand kernel weight, ci: car index, l* indicates lightness, a* indicates hue on a green (−) to red (+) axis, and wi: white index. values in bold denote significance level at p<0.05. short communication genetic resources (2023), 4 (8), 64–70 doi: 10.46265/genresj.ibnu2035 https://www.genresj.org issn: 2708-3764 management practice of the sheko cattle breed in ethiopia: a review melkam aleme *,a and gezahegn mengistu b a ethiopian institute of agricultural research, teppi agricultural research center, teppi, ethiopia b ethiopian institute of agricultural research, holeta agricultural research center, addis ababa, ethiopia abstract: the great diversity of agroecological conditions and production systems present in ethiopia contributes to the country’s large livestock population and makes it suitable for various forms of livestock production. the ethiopian livestock sector provides significant economic and social benefits at household levels and to the national economy. ethiopia has 32 recognized indigenous cattle breeds, of which the sheko is a known trypano-tolerant breed found in the southwest of the country. however, in recent times, the sheko population has been declining mainly due to interbreeding with local zebu cattle and to a shift in the production system. this paper aims to review and illustrate the current status and management practices of the sheko cattle breed towards conserving and improving the breed, and the production system. the feed resources in the sheko home area are natural pasture and crop residues, with limited utilization of cultivated improved forage. husbandry practices such as feeding, watering, housing and veterinary services are priority areas where improvements are needed. enhancing the conservation and improvement of the breed would greatly benefit from the active improvement of various stakeholders, including governmental policymakers and non-governmental organizations. therefore giving special attention to enhancing the management systems in the breed’s home area is crucial. this involves the direct engagement of research centres, extension workers; and higher learning institutions in proximity to the area all aimed at the conservation and improvement of the sheko cattle breed. keywords: conservation, ethiopia, husbandry practice, livestock, trypanosomiasis citation: aleme, m., mengistu, g. (2023). management practice of the sheko cattle breed in ethiopia: a review. genetic resources 4 (8), 64–70. doi: 10.46265/genresj.ibnu2035. © copyright 2023 the authors. this is an open access article distributed under the terms of the creative commons attribution license (cc by 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. introduction ethiopia is a forefront country in africa with an important livestock sector. the great diversity of agroecological conditions and production systems found in ethiopia contributes to the country’s large livestock population and makes it suitable for various forms of livestock production. the country is home to 32 recognized cattle breeds with an estimated population of 65 million heads (dagris, 2007; dessie, 2012; csa, 2020). although livestock farming in ethiopia is subsistence-oriented, the sector provides significant economic and social contributions at household levels and to the national economy (tolera et al, 2012; desta et al, 2011). for instance, the livestock sector in the ∗corresponding author: melkam aleme (melekamaleme@gmail.com) country serves as a source of food, income, employment opportunities, draught power and savings. the sector contributed 20% of the total gross domestic product (gdp), 40% of agricultural gdp, and 20% of national foreign exchange earnings (world bank, 2007). the occurrence of serious diseases also limits cattle production through increased mortality rates and their effects on fertility, growth and production. the major disease affecting cattle in the area is trypanosomiasis, locally called ‘gendi’, which is caused by flagellate protozoa belonging to the genus trypanosoma and transmitted by tsetse fly (glossina spp). the disease appears at all times of the year but it reaches its peak point after the rainy season (may–october). both direct and indirect loss is brought on by the disease. lack of equipment and transport for the field services, a weak tsetse and trypanosomiasis control unit, and a shortage of adequately trained workforce are some received: 17.07.2023 accepted: 15.11.2023 published online: 19.12.2023 https://www.genresj.org https://www.dx.doi.org/10.46265/genresj.ibnu2035 https://www.genresj.org https://www.dx.doi.org/10.46265/genresj.ibnu2035 mailto:melekamaleme@gmail.com genetic resources (2023), 4 (8), 64–70 sheko cattle breed in ethiopia 65 of the problems associated with cattle health in the area (dagris, 2007). the direct loss is primarily caused by disease-related costs, death, morbidity and infertility in infected cattle. according to seyoum et al (2013), trypanosomiasisrelated mortality, morbidity and control expenses result in a yearly direct loss of us$200 million. more substantial than the direct loss is the indirect loss, which includes the lack of use of cattle and animal power for agricultural production in productive land in tsetse-infested areas. food security and the reduction of poverty are both significantly hampered by trypanosomiasis (kitila et al, 2017). blackleg and other diseases including babesiosis and pasteurellosis are also present in the area. there is heavy tick infestation, especially in the lowland areas of southwest ethiopia (dawit, 1992). the sheko cattle breed is known for its trypano-tolerance, which makes it very suitable for areas affected by the disease but populations are facing conservation challenges. this paper aims to review and illustrate the current status and management practices of sheko cattle towards conserving and improving the breed and its production system. location and characteristics of sheko cattle breed indigenous cattle breeds have a multipurpose function and are distributed across the country’s diverse topographic and climatic conditions. sheko is one of the taurine-known cattle breeds (humpless) found in the southwestern part of ethiopia, particularly in the benchsheko, sheka and keffa zones of the southern nations nationalities and peoples’ (snnp) region and adjacent to the sudanese border (hanotte et al, 2000) (figure 1). sheko cattle are also called ‘mizan’ or ‘goda’ by the local community. phenotypically, the breed is characterized by short-horned and/or hornless heads, a small body size, humplesseness, particularly in females, and a diverse range of coat colours such as red, red-brown, patchy red, white, and black (taye et al, 2009; bayou et al, 2014). the breed is known for its hardiness, trypanotolerance (ability to survive in trypanosomiasis endemic areas), good mothering ability, better feed conversion efficiency, fast growth rate and comparable milk production to other indigenous cattle breeds in ethiopia (taye et al, 2009; stein et al, 2011; desta et al, 2012). farmers in the mid-altitude agroecological zone keep the sheko cattle breed primarily for draught power followed by milk and income whereas farmers in the lowland agroecological zones often keep the breed for milk and as a source of income (bayou et al, 2014). however, the breed is endangered, estimated at around 4,000 heads due to gradual interbreeding with local zebu, castration of the male at an early age, shift of production systems and shrinkage of grazing land (takele, 2005). studies done by hanotte et al (2000) indicated that approximately 90% of the inspected sheko bulls had their particular taurine allele almost 10% replaced by the zebuoriginated indicine allele, showing an alarming amount of crossbreeding zebu. conservation and maintenance of pure-breed individuals are essential to the long-term survival of any species to withstand the changing environmental conditions and ensure their sustainable use in the future. conservation of animal genetic resources is undertaken to maintain their diversity, fostering contributions to food and agricultural production, enhancing productivity and safeguarding ecological resources and cultural values for current and future generations. according to mekuriaw and kebede (2015), genetic conservation can be carried out using different methods. in situ conservation refers to the conservation of livestock through continued use by livestock keepers in the production system in which the livestock evolved or are normally found and bred. it involves the production of animals in their original production environment either on-farm or communitybased (figure 2). feeds and feeding feed is the main input of livestock production and largely determines its profitability since it accounts for 65–75% of the total cost of livestock operation (walli et al, 2012; makkar, 2018; matope et al, 2020). in addition, feeding is the foundation of livestock systems as it directly or indirectly affects the entire livestock sector, including animal productivity, health and welfare, and the environment (garg et al, 2014). feed sourcing and feeding are at the very interface where the ‘positive’ and ‘negative’ effects of livestock production are negotiated (blummel et al, 2018). feed resources in the home area of the sheko cattle breed are natural pasture, crop residues, and to a small extent the cultivation and utilization of cultivated improved forage. for instance, during cropping season (early june to late august), tinned maize (zea mays l.) and the green stovers remaining after the green cob is harvested are used for animal feeding (bayou et al, 2014). crop residues, such as teff or barley straw are also major feeds. improved forage like elephant grass (pennisetum purpureum) to some extent, sesbania (sesbania sesban), cassava (manihot esculenta), taro (colocasia esculenta), sweet potato (ipomoea batatas), leaf and stem parts of enset (ensete ventricosum), steudner’s dragon tree (dracaena steudneri), banana (musa sapientum), sugarcane (saccharum officinarum) and homemade conventional feeds are common and well known. no reports are available regarding the production of roughage, such as hay for feeding livestock during the dry season. straw treatments to produce feed include cutting and chopping long stalks and utilizing molasses and urea to enhance digestibility (takele and workneh, 2011; hailu, 2020). there is a general lack of feed availability for livestock production in the area due to the use of grazing lands for crop production. feed shortage is rampant when most of the farmlands are covered with food crops during the wet season, and grasses are depleted 66 aleme and mengistu genetic resources (2023), 4 (8), 64–70 figure 1. map of the distribution areas of sheko cattle breed figure 2. sheko cattle breed maintained at the teppi agricultural research center genetic resources (2023), 4 (8), 64–70 sheko cattle breed in ethiopia 67 during the dry season (bayou et al, 2014). the lack of improved feed conservation knowledge, shortage of land to plant forage, lack of adequate extension service to inform farmers on improved forage technology and unavailability of forage seed or planting materials are the most common limiting factors in the production and productivity of sheko cattle breed (desta et al, 2011). feed resources are declining due to the expansion of crop fields in the face of fast human population growth. consequently, sheko cattle keepers increasingly resort to tethered feeding. therefore, the average cattle herd size is declining, which also discourages the maintenance of the sheko cattle breed (desta et al, 2011; mengistu et al, 2017). there is a notable lack of awareness and poor feed resource management, utilization, preservation, treatment and development (hailu, 2020). feeding troughs are made of wood clay dishes, and synthetic materials such as plastic bath and plastic pot are used (desta et al, 2011). the challenge lies in creating linkages between innovation sources, such as research centres and agricultural extension services, and the expansion of technology as well as coordination with budgetary administrations. accordingly, addressing the issues related to the quantity and quality of feeds is essential (moa, 2013). water resources and watering systems within the region where the sheko cattle are grown, agriculture has actively harnessed water resources, predominantly drawing from natural resources such as springs and ponds. however, watering frequency is an additional limiting factor for sheko cattle rearing (bunke, 2019). the irrigation system adds further challenges, particularly if access, water quality, timing and frequency of watering are limiting factors (hailu, 2020). according to mengistu et al (2017), water sources for cattle in the area are rivers, springs and ponds. the majority of the people give water to their cattle twice a day during the dry season, others offer water to their cattle once a day and some of them water their animals three times a day by tracking the animals to the watering point. the majority of producers report there is no water shortage in the area, but for the remaining 27%, water is scarce during the dry season. the distance to a water hole is another factor that limits the rearing of cattle. about 86.4% of producers travelled 1–4 km to reach drinking water for their animals (mengistu et al, 2017). some communities need more accessible watering points and natural mineral licks to fully utilize the genetic potential of the sheko cattle breed (desta et al, 2011). housing system the majority of cattle keepers in mejenger (81.2%) and 47% in bench-sheko zones keep their animals in separately constructed houses made of wood with a grass-covered roof (mengistu et al, 2017). at night, cattle are housed not far from the family house to protect them from cold, rain, predators and theft (alemu, 1990). feeder cattle are well protected from adverse weather conditions, predator attacks and theft. to keep the stall warm, the walls are covered with leaves and grasses. enough space is provided in the stall to reduce competition for feed among stall-fed cattle (takele and habtamu, 2009). calves are kept separate from their dams, either within an annex connected to most houses or independently situated in and around the family house, typically within a horse shelter. often the roof of a cattle house is made of grasses (mengistu et al, 2017; hailu, 2020). husbandry practices husbandry practices are carried out with family individuals and sometimes with neighbours. to utilize manure for trim generation, farmers frequently clean cattle faeces and urine and bolster refusals from the barn and the compound (takele and habtamu, 2009). tools such as nose rings are used to tame aggressive sheko bulls and oxen in the area. traditionally, farmers use ear rings for the same purpose and this practice needs to be closely examined for its effectiveness (lund, 2002). breaking and training animals at a younger age and alternating herding with tethering can soften the aggressive behaviour of the breed. larger herd sizes increase the likelihood of selection and maintenance of breeding studs in the villages. male cattle are usually castrated at an early age. such males are kept in the herd for a long time adorned with decorations and are a source of pride to their owners. another reason for the observed high number of castrates in household herds is that the number of castrated males is an indicator of social status ranking (terefe et al, 2012). marketing practices the breed has the potential for high marketability in large parts of south-western ethiopia, which are challenged with medium to high tsetse and trypanosomiasis presence. recent government initiatives, aimed at relocating smallholder farmers from densely populated highlands into underutilized fertile and sparsely populated valleys in south-western ethiopia, have created a demand for adapted breeding cattle with the sheko breed emerging as the optimal choice. the dairy and draught qualities of the breed may be worth investigating even for other agroecologies. the agriculture extension services and the national research systems need to support this effort until market interests gain momentum. in light of the threat of extinction, implementing an incentive system is essential for the sheko breed. owners who successfully rear a sheko calf could be rewarded, promoting efficiency as proposed in some studies and encouraging breed preservation efforts by discouraging crossbreeding (zander et al, 2009). 68 aleme and mengistu genetic resources (2023), 4 (8), 64–70 additionally, the creation of a breeders’ society for sheko cattle will ensure farmers’ involvement in identifying elite animals, creating market opportunities, recruiting herd registration and taking the lead in conservation activities (hegde, 2005). targeted promotion outside the existing market niche should focus on the special merits of the breed, such as its trypano-tolerance and dairy quality potential. the breed management plan should also be widely communicated to relevant stakeholders using appropriate media as part of marketing strategies (terefe et al, 2012). production system the livestock production system dominating in sheko district and its surrounding areas is a mixed crop–livestock farming system. permanent crops like coffee and bananas are widely produced. coffee is the main cash crop. smaller areas of land are also used for cereal production like maize and sorghum. fruits such as avocado and mango are also highly produced. cattle are important as a source of diverse foods such as milk, butter and meat, and also a source of income through sales of live animals and animal products like leather (fasil, 2004). the margin for increasing forage production from pasture is limited. free grazing is the main animal management system in the area, but a few farmers provide supplementary feed at home. most of the farmers keep their animals outdoors during the night near their homestead. some farmers have enclosures made for their animals, whereas others keep animals together within the family house in a partially enclosed area (moa, 1984). sheko cattle are said to be better milk producers, reproduce faster than other breeds of cattle in tsetse-infested zones, and have good grazing and browsing ability during critical periods of forage scarcity (workeneh, 2001). the average production and reproduction performances of the sheko breed are presented in table 1. the most important characteristics of sheko cattle include tolerance to disease especially to trypanosomes, long lactation length, good milking potential, good adaptation to heat stress, good traction power and table 1. production and reproduction performances of the sheko cattle breed (takele, 2005) parameter and measure values age at first mating (years) 3 age at first calving (years) 4 calving interval (years) 1.5 gestation length (days) 276.8 body weight at calving (kg) 20 body weight at weaning (kg) 102 daily milk yield (litre) 2.3 lactation milk yield per calving (litre) 698.3 lactation length (months) 9.9 females life span calving (in number) 8 adaptation to internal parasitic and tick infestations. other positive traits attributed to sheko cattle include good mothering ability, less selective feeding behaviour, attractive coat colour appropriate for the local environment, tolerance for biting flies, shorter calving interval and better adaptation to the terrain of the area. on the other hand, sheko are known for their aggressive behaviour and their relatively high feed requirement of the farm households (elias, 2008). constraints of production the overall trend indicates a decline in the population of sheko cattle in its production region. the reason for this decline could be identified with sheko cattle’s energetic and aggressive behaviour, which decreases their acceptance by the local community (alemu, 2002). the lower male-to-female ratio in the sheko population might expose the animals to inbreeding and crossbreeding with bulls of other breeds. this situation is exacerbated by little consideration given to maintaining the endangered breed (alemu, 2002). due to their aggressive behaviour, sheko cattle are difficult to harness and control by old persons, women, children and disabled persons. in addition, the early castration of bulls exercised by some farmers to control their aggressive behaviour seriously limits pure breeding of sheko cattle (takele, 2005). the most important constraints affecting sheko cattle production are their sparse distribution, absence of conservation efforts, declining interest of the community in sheko cattle due to their aggressive behaviour, inbreeding and hornlessness, which makes them difficult to restrain using rope (elias, 2008; bayou et al, 2014). given that the home area of sheko cattle is renowned for coffee plantations, nearly all available land except for the swampy areas and hill-tops, is utilized for cultivation. the potential for increased forage production from pasture is severely limited and cattle rely entirely on natural pasture. consequently, natural pastures are overgrazed causing the proliferation of undesirable plant species. seasonal feed shortage is common during the dry season. proper management, along with the allocation of grazing area and conservation of feed, is rarely practised. all stocks are grazed together with no attempt to provide special treatment for different classes of the stock (moa, 1984). conclusion and way forward in ethiopia’s rural areas, livestock is the most longlasting feature of the way of life. sheko cattle are among the three categories of ethiopian breeds, which include small east africa zebu and large east africa zebu. characterized by short horns and lack of hump, sheko cattle are predominantly located in the benchmaji area, which was formerly part of the bench-sheko zone. additionally, they can be found to some extent in the sheka and kefa zones of south-western ethiopia. genetic resources (2023), 4 (8), 64–70 sheko cattle breed in ethiopia 69 sheko cattle are raised within mixed farming systems and are sustained through natural pasture and crop residues. the breed’s most important traits include resistance to trypanosomiasis, better milk yield compared with other local breeds, drought power and better feed conversion efficiency. safeguarding the breed is vital to preserving its distinctive characteristics. to counteract the loss of the breed’s genetic diversity, a diverse array of conservation and management practices needs to be in place. these include establishing in situ breeding stations and breed studs in its breeding tract, promoting niche markets and improving husbandry practices. crucial measures involve addressing production challenges like feed shortages during the dry season, ensuring proper housing and feeding management, and allowing the breed to thrive in its natural habitat. the creation of incentives can encourage owners to keep breeding stock under good management, contributing to the breed’s preservation. consequently, the establishment of a sheko breed conservation fund may be necessary. conflict of interest the authors declare no conflict of interest. author contributions melkam aleme provided contributions to the current versions of the article through collection, review, writing and interpretation, while gezahegn mengistu provided a review of the work before its final submission and overall assistance. references alemu, r. 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(2023). is the ecosystem services concept relevant to capture the multiple benefits from farming systems using livestock biodiversity? a framework proposal. genetic resources 4 (8), 15–28. doi: 10.46265/genresj.mrbt4299. © copyright 2023 the authors. this is an open access article distributed under the terms of the creative commons attribution license (cc by 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. introduction livestock biodiversity has been threatened since the second half of the 20th century, as many local breeds ∗corresponding author: anne lauvie (anne.lauvie@inrae.fr) have been replaced by a few specialized breeds and hybrids, which, among other traits, have been intensively selected to increase production, and have become mainstream breeds (audiot, 1995; fao, 2015). however, local breeds are both a resource for and a product of livestock farming. globally, fao (2015) reported received: 22.03.2023 accepted: 15.06.2023 published online: 06.09.2023 https://www.genresj.org https://www.dx.doi.org/10.46265/genresj.mrbt4299 https://www.genresj.org https://www.dx.doi.org/10.46265/genresj.mrbt4299 mailto:anne.lauvie@inrae.fr 16 lauvie et al genetic resources (2023), 4 (8), 15–28 a total of 8,774 breeds from 38 domesticated animal species, resulting from long-term livestock farming in diverse territories with different goals. the first challenge for livestock biodiversity faced by stakeholders and the scientific community, is its conservation, mainly focused on breed conservation and withinbreed genetic variability management (hall and bradley, 1995; ajmone-marsan and consortium globaldiv, 2010). beyond this conservation challenge, adding value to local breeds has been underlined as a key process for farm animal biodiversity, in particular through the production of quality food products (verrier et al, 2005; mathias et al, 2010; ligda and casabianca, 2013). various authors in the field of local breeds management have stressed that the supply of animal products is associated with other benefits: provision of a high diversity of food and other goods, services related to landscape and environmental management, uses related to cultural and heritage dimensions (audiot, 1995; alexandre et al, 2002; gandini and villa, 2003; rege and gibson, 2003; verrier et al, 2005; berland et al, 2006; fontaine et al, 2008; naves et al, 2011; leroy et al, 2018; hall, 2019). these material and immaterial benefits have been used as one of the arguments to underline the importance of local breeds’ conservation (fao, 2015). few authors use ecosystem service approaches to analyze the material and immaterial benefits provided by the raising of local breeds (hoffmann et al, 2014). however, from being a simple metaphor to raise public awareness (norgaard, 2010; barnaud et al, 2011), since the millennium ecosystem assessment (2005), the ecosystem services concept is being increasingly used (kull et al, 2015; droste et al, 2018). a prolific literature highlights the diversity of ecosystem services produced or used by agricultural ecosystems (zhang et al, 2007) and how it is linked to human practices (lescourret et al, 2015). up to now, when the ecosystem services approach was applied to livestock biodiversity, it was mainly at a global scale, through the generic inventory of ecosystem services (leroy et al, 2018), or to focus on specific dimensions of them, like the cultural and heritage values of breed diversity in the alpine area (marsoner et al, 2018). hall (2019) hypothesized that the underuse of this framework by stakeholders and the scientific community is partly due to a lack of recognition of livestock biodiversity by the ecosystem services community, and suggested it would be useful and beneficial that linkages be strengthened among several scientific communities. however, velado-alonso et al (2021) highlighted the interest in considering both cultural and ecological dimensions of the relationships between livestock breeds and ecosystem services. moreover, as underlined by beudou et al (2017), the most commonly used ecosystem services approaches are quantitative and, as a consequence, neglect the social dimension of livestock farming systems, while qualitative approaches are relevant to comprehend the complex processes and interrelations underlying the production of ecosystem services (barnaud et al, 2018). indeed, we can wonder if a qualitative ecosystem service approach could help better understand the dynamics underlying the multiple benefits obtained from farming systems using livestock biodiversity. the purpose of the present paper is to propose a qualitative approach to ecosystem services provision by different farming systems that exploit livestock diversity. considering that the ecosystem services notion applies at the ecosystem scale, we do not look here at ecosystem services provided by livestock biodiversity but ecosystem services provided by farming systems using livestock biodiversity (martin-collado et al, 2019). in this paper, we present a framework for the analysis of ecosystem services provided by farming systems using livestock biodiversity focusing on the processes at play and the interactions supporting them. then, we discuss to what extent the notion of ecosystem services is appropriate to deal with the multiple benefits obtained from farming systems using livestock biodiversity. materials and methods our work is based on empirical research on nine ruminant breeds in six different regions. all the case studies are located in france, including in french overseas territories, reunion island (indian ocean) and guadeloupe island (french west indies). the locations were chosen to represent diverse (i) biogeographical and climatic conditions, (ii) species, (iii) main uses and (iv) population sizes (figure 1). we analyzed data and empirical knowledge on those breeds and the associated livestock farming systems, taken from previous or ongoing studies (table 1). all the breeds concerned are local except one, the montbéliarde breed, which is the second most important dairy cattle breed in france. for this breed, we repurposed work carried out in its cradle of origin (located in the east of france, in the bourgogne franche comté region), which allowed us to have a wide variety of situations on the fourth criterion mentioned above (gaillard et al, 2018). we developed an inductive approach (woo et al, 2017) based on several working meetings that brought together researchers from different animal sciences, and social and economic sciences (the authors of the present paper). we share a research position that relies on the importance of a qualitative approach, tackling empirical issues to build realistic conceptualizations. we also share a vision of ecosystem services as social constructs: barnaud et al (2018) illustrated that in such a vision, “an open landscape does not ‘naturally’ or ‘intrinsically’ provide a cultural ecosystem service but someone, in a given geographical, cultural, and historical context, attributes a specific patrimonial or aesthetic value to such landscape.” indeed, even for the services that result from ecological dynamics, the way humans qualify them as services, recognizing them as such, has a social dimension. consequently, our aim is not to describe ‘objective’ ecosystem services and genetic resources (2023), 4 (8), 15–28 ecosystem services concept applied to local breeds 17 figure 1. the six regions and the nine breeds analyzed in the present study (source of the population size estimates: inra (2014)). table 1. references associated with the described case studies. case study species references creole cattle and goat alexandre et al (2002); gautier and naves (2011); naves et al (2011); boval et al (2012); gourdine et al (2021) bretonne pie noir cattle couix et al (2016); lauvie et al (2017) pëı goat fontaine et al (2008) montbéliarde cattle gaillard et al (2018) räıole, caussenarde des garrigues, rouge du roussillon sheep lauvie et al (2017); nozieres-petit and lauvie (2018) corse sheep lauvie et al (2017); perucho et al (2020) quantify or assess them (and such a vision is not adapted to do this kind of assessment). our aim is rather to take into account services as social constructs: such a vision is adapted to a qualitative and comprehensive approach of complex processes in agroecosystems. first, we organized three online workshops in 2016, during which we shared our knowledge of the case studies (table 1 and figure 1) by presenting (i) their main characteristics, and (ii) the ecosystem services produced by the systems that used the breeds concerned. all the researchers were invited to the online workshops and the attendance ranged from 11 to 12, according to the individual availability for each workshop. the ecosystem services were identified from a researcher’s point of view, resulting in an interpretation of what could constitute a service, and to whom. we organized two 2-day workshops in 2017 during which a transverse analysis resulted in a first analytical grid. eleven researchers participated in the first and nine in the second workshop. in these workshops, we raised the following questions: i) who were the beneficiaries or potential beneficiaries of the ecosystem services identified, ii) who provided the services and iii) the role of the breed in the process of providing 18 lauvie et al genetic resources (2023), 4 (8), 15–28 the service, thanks to available data and expertise. this analysis raised several theoretical and methodological questions reported in the result section. we organized four online workshops in 2017 and 2018 to complete the iterative development of the framework, identifying loops between knowledge of the case studies, the questions we wanted to ask and the notions and concepts used to tackle these questions in the literature. this iterative collective back and forth between the case studies and the literature provided the background necessary to discuss to what extent the concept of ecosystem services (and associated concepts) was useful to deal with the multiple benefits of systems using livestock biodiversity. the process is summarized in figure 2. results services provided by farming systems using livestock biodiversity: our proposed framework our transversal approach enabled us to develop a framework to analyze the provision of ecosystem services in farming systems using livestock biodiversity, focusing on the main elements to be taken into account and their interactions. we developed this framework in the form of a list of items to be considered in studies of ecosystem services produced by farming systems using livestock biodiversity. the main themes of the framework are summed up in figure 3. we formulated all the items as questions possibly applicable to other situations. the data from our empirical studies did not necessarily enable us to answer all the questions with the same level of detail, but the questions were considered key to understanding the processes for at least one case, even if empirical data to answer them were not available for all the cases. considering ecosystem services as a social construct implies the analysis of each ecosystem service identified, including its temporal dynamics, with these questions: • how is the service formulated or qualified? the formulation or qualification may be plural, depending on from whose point of view the service is considered. it needs to consider dynamics over time and space. • what processes underlie service provision? – who is concerned? – who are the beneficiaries and/or the people who help provide the service? – who identifies the service as such? – is the service produced intentionally or not (and by whom)? – what are the interactions between the people concerned? – what is the role of the breed in the process? – is the effect of the breed linked with biological characteristics and the abilities of the animals of the breed (direct or indirect effects)? if so, which abilities are considered to be involved in the provision of services by the different people concerned (several points of view are possible)? – is the effect of the breed linked with other attributes of the breed that are not directly linked to biological characteristics (e.g. local heritage or image)? if so, which attributes are considered to be at play by the different people involved in service provision (several points of view are possible)? – can the breed be considered a marker of the search for innovation/alternatives in farming systems? – does the breed play a catalytic role in relation to collective action to provide a service? – what are the interactions with other services? – do other services result from this service? i.e. does the fact that this service is produced enable the provision of other services? (e.g. cascades or bundles of services) – are there any variations in the generic formulation of the service? – do other services – synergies or trade-offs – interact with it? • to what extent is the service taken into account in the management of the breed? in the following sections, for the different items in the framework, we present each item and provide details on: (i) how the transverse analysis of our case studies enabled the identification of the item (ii) how creating linkages between our cases and the literature helped build the framework and provided insights into the usefulness of the notion of ecosystem services. we explore which elements from the literature helped us in the analysis of the case studies. the elements presented in those paragraphs result from linkages between empirical data (from case studies) and theoretical contributions (from the literature and the questions raised by our cross-cutting analysis). dynamics of production of a diverse range of services the first step was to identify each service. we first established that systems that use livestock biodiversity are involved in the production of a wide range of ecosystem services. table 2 summarizes this diversity by giving examples from our case studies. our aim here was to illustrate the diversity and put it in perspective with a classification frame proposed in ecosystem services literature, but not to make an exhaustive, generic or ‘objective’ inventory. indeed, the temporal (and spatial) dynamics of ecosystem services provision are important in the cases we studied. genetic resources (2023), 4 (8), 15–28 ecosystem services concept applied to local breeds 19 figure 2. process used from the six case studies analysis to the building of the framework figure 3. main themes of the framework 20 lauvie et al genetic resources (2023), 4 (8), 15–28 table 2. the diversity of services identified in the case studies. services identified examples given in the case studies food products (main products of the systems studied, diversity of meat and dairy products mainly subject to market valuation but can also be subject to non-market valuation) without geographical indication, sold through direct sales or through intermediaries, like the meat and dairy products from the bretonne pie noir protected designation of origin (pdo) products like comté cheese made from the milk of cows belonging to the montbéliarde or simmental breeds and the brocciu whey cheese in corsica, which can only be made from corsican sheep and goat milk inedible products wool from the räıole breed skins of goats in guadeloupe manure used to fertilize the soil (e.g. by caussenarde des garrigues, raiole and rouge du roussillon or creole cattle in guadeloupe) maintenance of some specificities of the farming systems the sheep breeds raiole, rouge du roussillon and caussenarde des garrigues are associated with pastoral systems in the region concerned, and are reputed for their ability to adapt to those systems services linked to the management of habitats or the preservation of associated landscapes wildfire prevention (particularly in the mediterranean region) birds nesting open areas (recorded in typical guadeloupean savannahs grazed by tethered creole cattle (zoom-guadeloupe, 2012)) use of animals from the systems for religious rites in réunion and guadeloupe islands, goats are commonly used for hindu sacrifices. a diversity of phenotypes are sought. pëı and créole goats contribute to this diversity, more often in guadeloupe than in la réunion, along with other goats from various breeds and crossbred animals. educational use in several cases, farmers have allowed farm visits by schoolchildren contribution to heritage and cultures this dimension can be recognized through the associated landscape, e.g. the pastoral landscapes in the causses and cevennes area, which are designated unesco world heritage sites local breed animals, with their specific phenotypic attributes (e.g. colour, horns) also contribute to the aesthetics of the landscape and its original identity, and thus serve as an image vector for agro-tourism through an informal pathway, they may simply be part of the cultural patrimony, like the creole society in reunion island or guadeloupe. contribution of farming systems to the global dynamics of the territories (by helping maintain an agricultural activity and/or to the image of the territory concerned). a cow from the bretonne pie noir breed was named ‘star cow’ at the 2017 paris agricultural fair, and the pays de redon where the cow originated benefited from this event being reported in the press. genetic resources (2023), 4 (8), 15–28 ecosystem services concept applied to local breeds 21 for example, the use of créole cattle in guadeloupe in oxen drawing (bœufs tirants) contests, has played a role in conserving the breed, thanks to the success of this cultural event. however, the increasing success has led to a modification in the rules to include categories of larger animals, shorten the distance to be covered, and no longer place the yoke directly on the horns. these changes have increased the inclusion of exotic breeds with greater muscle development (limousin, charolais, blonde d’aquitaine) than that of the local breed. the cultural service is thus developing, but the link between the service and the local breed is dwindling. a second example of temporal and spatial dynamics of ecosystem services is the montbéliarde cattle breed in the comté cheese territory (a protected designation of origin (pdo) cheese). the milk is collected and processed by small-scale processors (called fruitières in french) distributed throughout the territory. this organization and the related livestock farming systems are associated with the production of a specific landscape, which can be considered a service. however, we identified changes in farm structure with an increase in the size of the herds. this trend is due to the increasing demand for comté cheese (and a readiness to make the most of the attractive price of the milk for comté), the pleasure involved in raising highyielding dairy cows, as well as the desire to reorganize labour. the tendency to graze the herd on land close to the farm homestead rather than on more distant pastures (more typical of the jura landscape), as well as to increase the size and homogeneity of the plant covers used in pastures, has also led to changes in the landscape (gaillard et al, 2018). we compared data gathered from the case studies with data in the literature, which provided benchmarks for the identification of ecosystem services. the wellknown categories suggested by the millennium ecosystem assessment (2005) or the common international classification of ecosystem services (cices) (hainesyoung and potschin, 2018) make it possible to distinguish provisioning, regulation and maintenance, cultural, and support services. identification of services can be facilitated by using such a classification. we used the provisioning, support and regulation and cultural categories to describe the services in our cases, which helped ensure we covered a wide range of services. however, as we will see hereafter, the services we identified were not always easy to classify into a single category. moreover, the view of ecosystem services as social constructs (barnaud et al, 2018) underlines the fact that they are not intrinsic properties of ecosystems, and that the classification of specific services provided at a given time by an ecosystem may not still be relevant when a dynamic approach is used. how can services be qualified and classified? after the first step of identification of services, the first item of the framework is: how can the services be formulated or qualified? our analysis of case studies raised the question of how to qualify (and classify) the services. by using the word qualify we mean describe and attribute one or several qualities to a service by naming it/them. the main products of systems using the breeds concerned are food products. however, systems that produce traditional food products, can be considered to provide both a provisioning service and a cultural service. indeed, the food products concerned often have a cultural dimension, which can be recognized, for instance, through a pdo. similarly, a pasture system that has shaped a typical landscape can be considered both as a regulation and a cultural service, like the causses and the cévennes, where the pastoral landscapes are unesco designated world heritage sites. many services produced by farming systems using local breeds have a cultural dimension. ovaska and soini (2016) noted the overlapping of ecosystem services categories and for instance, d́ıaz et al (2018), underlined the importance of recognizing “the central and pervasive role that culture plays” in the production of nature’s contributions to humans, as well as the importance of applying a context-specific perspective. increasing research in this field has enabled the development of multiple approaches that might be complementary (peterson et al, 2018) to qualify and classify ecosystem services. following barnaud et al (2018), we consider ecosystem services as “subjective perceptions, socially situated and constructed”. the points of view of the people involved in the situation studied should be taken into account when qualifying and classifying ecosystem services. people concerned by the production of services the first question regarding the second item of the framework, is: who is concerned? our analysis of the case studies confirmed the diversity of actors involved in the systems using the breeds concerned and in the processes linked to the provision of ecosystem services. for instance, the range of livestock keepers involved in the use of the bretonne pie noir cattle breed includes both hobby breeders and professional breeders, and both dairy and meat herds (couix et al, 2016). other stakeholders include researchers, consumers, environmental management associations and restaurant owners. interactions among actors influence the provision of ecosystem services by systems using the breed. in several ecosystem services that we identified, the same actors, particularly farmers, can be both beneficiaries and providers. for instance, some farmers who raise bretonne pie noir cattle use areas of ecological 22 lauvie et al genetic resources (2023), 4 (8), 15–28 interest, like wetlands, as feed resources for their cattle. the farmers thus participate in the production of the management service of those areas through grazing, and at the same time, profit from the vegetation in the areas to feed their herds. the ecosystem functions are translated into services when they are used, consumed or enjoyed by humans (fisher et al, 2009). this makes ecosystem services beneficiary-dependent (different individuals or collectives benefit from different services (d́ıaz et al., 2018)). in the framework they proposed for the analysis of social interdependencies underlying ecosystem services dynamics, barnaud et al (2018) identified two other categories of actors together with beneficiaries: providers, and possibly intermediaries between beneficiaries and providers. to grasp the complexity of the actors involved, we suggest adding a step before qualifying actors as a beneficiary, provider or intermediary: identifying what we call the ‘people concerned’. the aim of this step is to ensure different levels of concern are included. the people concerned can then be qualified as beneficiaries, producers, intermediaries or, in some cases, may belong to more than one category. identifying the people concerned is a step in the process of tracing actors who play a direct role in the processes underlying the provision of services and helps grasp the multiple perceptions, values and practices associated with such services (dendoncker et al, 2018; teixeira et al, 2018). some of the ecosystem services produced at the scale of the human-driven farming system are produced intentionally, e.g.edible and inedible goods. however, this is not necessarily the case for all ecosystem services. the provision of cultural services, for instance, may sometimes be considered involuntary, as they are inherited from a long history of co-evolution of the breeds, their environment and the human practices involved in the farming system. it is not always easy to determine whether a service is provided intentionally or not, especially when several dimensions of ecosystem services production are interconnected. however, the distinction proposed by aznar et al (2007) may be useful to deal with the general question of whether or not ecosystem services are produced intentionally. based on the economy of services, this author defines provisioned services as services provided by agriculture which lead a farmer to maintain or change support and/or contribute technical or human capacities. these provisioned services are supplied by the farmer intentionally and are co-built by the farmer and the beneficiaries/applicant. aznar et al (2007) distinguished them from service externalities that are supplied without the intention of the supplier and from service functions which refer to services supplied to humans by nature. the role of the breed in service production: not only biological characteristics are at play the second question regarding the second item in the framework, is: what is the role of the breed? the provision of ecosystem services may be directly linked to specific abilities of the animals of the breeds involved. in creole cattle, for instance, a signature of selection has been identified in the genomic region that can be linked to the strength and the shape of the horns, directly inherited from their use as draught animals (naves et al, 2011). resistance to specific sanitary problems, such as internal parasites in creole goats, or to ticks and associated infectious diseases in creole cattle, also help provide some ecosystem services (naves et al, 2011). the integrated management of these diseases enabled by the use of resistant animals reduces the need for treatment, in turn reducing the quantity of chemical residues in edible products, but also in animal dung, which may be useful in agroecological or organic production systems. however, the animals’ specific abilities are not sufficient to describe all the characteristics of the breeds that can play a role in the provision of ecosystem services. the raiole, caussenarde des garrigues and rouge du roussillon breeds, for instance, show that breeds also play a role in gathering together a group of farmers who exchange breeding animals, technical knowledge or projects to add value to products (nozieres-petit and lauvie, 2018). the appropriate scale to identify the provision of ecosystem services is the ecosystem (or the farming system as far as farming activity is concerned); as a consequence, the breed is one of the elements in the system that can contribute to their provision (martincollado et al, 2019). relevant concepts in the literature to better identify the role of the breed are not specifically related to the notion of ecosystem services. the most relevant concepts to identify the role of the animals’ specific abilities are two concepts from animal sciences: the concept of animal abilities and the concept of animal performances. those concepts are used to describe specific biological characteristics of animals that are useful in livestock farming (directly linked with food production, like milk yield, or indirectly linked with food production, like the walking ability of animals, which is useful in pastoral systems). a global term, often used for local breeds, covering their functional abilities, and not only their productive feature, is the hardiness of animals. hardiness covers a wide range of abilities which depend on the situation and the point of view, as described by hubert (2011). being hardy means being not very demanding and therefore enabling the herd to survive even in harsh conditions (poussard et al, 2016). the hardiness of a breed is difficult to measure and even to define precisely, as it covers a set of different animal functions interacting with the environment. moreover, these genetic resources (2023), 4 (8), 15–28 ecosystem services concept applied to local breeds 23 functions are more difficult to evaluate than productive traits (e.g. milk yield, protein content, daily gain, etc.). but such abilities are highly valued in nonintensive farming systems and can be directly linked with the provision of ecosystem services (naves et al, 2011; marshall et al, 2016). they generally result from long-term breeding and from natural selection of the animals in specific environments, and are now being increasingly elucidated thanks to recent genomics technologies (amills et al, 2017). for example, a complex trait such as the adaptation to climatic stress or the ability to walk is very useful in pastoral management, and some specific physical attributes useful in such conditions are present in local breeds (for example, short hair, dark hooves or a hump in cattle) (naves et al, 2011; flori et al, 2012). however, these concepts from animal sciences are not sufficient to describe other dimensions that are not biological but that may also be involved in the provision of ecosystem services, for instance, the ability to trigger collective action. as a consequence, we propose to use the term ‘breed attributes’ to cover characteristics of breeds that are both biotechnical (abilities and performances) and sociotechnical. in this definition, both the ‘ability to exploit native vegetation in pastoral systems’ and the ‘ability to trigger a collective action’ could be considered as complementary breed attributes and combined in underlying processes to produce a set of ecosystem services. relations between services the third question regarding the second item of the framework is: ‘what are the interactions with other services?’ our case studies also underline the fact that diverse ecosystem services are interrelated. for instance, in the case of räıole, caussenarde des garrigues and rouge du roussillon breeds, we identified interactions among ecosystem services: some services result from others, like the contribution to wildfire prevention that results from the animals’ capacity to graze local resources. some services are divisions of others: ‘participate in education’ is a subset of the service ‘the social role of livestock farming’. several ecosystem services are produced jointly in similar farming systems (nozierespetit and lauvie, 2018). for instance, farms using the corsican sheep breed produce milk, processed into cheeses and whey cheese (on farm or in industrial dairy plants); and they also help maintain the pastured vegetal resource, thus indirectly helping shape the landscape and preventing wildfires. creole cattle raised for meat production and manure provision are tethered in natural savannahs, sustaining small-scale family farms, while shaping the typical hilly landscapes of guadeloupe (mornes). direct provisional services can be reinvested through the loop of cultural service since they enhance gastronomy, with dishes like goat curry in guadeloupe, or can provide raw material for the production of musical instruments. the use of goats in hindu sacrificial rites in guadeloupe or in reunion island illustrates a case of service (with a cultural and religious dimension) that over time has enhanced the development of the entire goat meat sector (service provision). ‘bundles of services’ is a useful notion in the literature to jointly consider a diverse range of services produced in similar farming systems, and it can help tackle synergies and trade-offs between services (cord et al, 2017; dumont et al, 2019). links between services and management of the breeds the last item in the framework is: what are the links with the management of the breed? we found examples in the case studies in which the intentional production of a new service engendered changes in the collective management of the breed. for instance, for the räıole breed, the farmers developed collective marketing of the wool, and during the sale of rams organized by the breeders’ association, they provided information to farmers about the wool quality of rams sold, so that they could consider it in their choice of a breeding animal (lauvie et al, 2017). biodiversity and ecosystem services are often closely linked. for instance, mace et al (2012) underlined how biodiversity generally plays a key role in ecosystem services provision, as a regulator of underpinning ecosystem processes, as a final ecosystem service, and as a good subject for valuation. this link between services and management of the breed feeds the question of the links between services and biodiversity dynamics. indeed, collective management of the breed is an important lever of livestock biodiversity dynamics: through the collective choice of selection criteria that influence the direction of breed management, but also through collective promotion actions that can have an impact on the number of animals, for instance. discussion in this paper, we used several case studies to explore the question of the multiple benefits of farming systems that use livestock biodiversity and developed a framework to address the processes underling the ecosystems services provided by them. one of our aims was to discuss the extent to which the notion of ecosystem services is useful to deal with these multiple benefits. services are indeed increasingly taken into account in livestock farming research (rodŕıguez-ortega et al, 2014; alexandre et al, 2014; ryschawy et al, 2017; dumont et al, 2019). steger et al (2018) argue that the diversity of definitions and approaches in ecosystem services research has prevented it from being structured by a single discipline, and maintained it as a boundary object. choosing the ecosystem services notion as an entry point of our analysis did not provide us with a turnkey approach, but rather gave us room to include different visions (and notions) of animal scientists, social scientists 24 lauvie et al genetic resources (2023), 4 (8), 15–28 and economists in our analysis. some of the concepts associated with ecosystem services in the literature appeared to be relevant to our empirical findings in case studies (e.g. the bundle of services) or fed our reflection on the processes underlying the provision of services (e.g. the beneficiaries). however, we also considered other concepts, not related to the ecosystem services literature, which were useful in our analysis, like the concept of animal abilities used in animal sciences. choosing the notion of ecosystem services as an entry point also favoured a systemic analysis. indeed, such a view is central to our communities as the farming system is a key notion, particularly for the livestock farming systems community (dedieu et al, 2008). the meeting point of systemic views in several scientific communities is the agroecosystem. however, the view on agroecosystem might favour complementary foci depending on the communities, which could enrich each other. to go further, it would indeed be interesting to broaden our already multidisciplinary vision by including the points of view of ecological scientists in our framework. other notions or theoretical frameworks could also be adapted to tackle the question of the multiple benefits obtained by farming systems which use domestic animal biodiversity and are complementary to the ecosystem services approach. the notion of multifunctionality, for instance, is equally constructed and situated (barnaud and couix, 2020), and makes it possible to consider the different functions of farming activities. huang et al (2015) reported the proximity of the scientific communities that use multifunctionality and ecosystem services concepts when dealing with agriculture. however, the entry point of the approach is agricultural activity whereas the entry point of the ecosystem services notion is the ecosystem. huang et al (2015) pointed out that the two approaches would benefit from being integrated, and barnaud and couix (2020) associated them by using an ecosystem services lens to answer a multifunctionality question. the framework proposed by d́ıaz et al. (2018) is another relevant framework for our purpose. d́ıaz et al. argued that the “nature’s contribution to people” (ncp), the core concept of this framework, is a concept which makes it possible to go beyond some of the limits of the ecosystem services concept, in particular to respond to criticisms regarding the lack of social and humanities sciences involved. this framework indeed emphasizes the importance of the cultural context in understanding ncp, which our case studies highlight. d́ıaz and her colleagues’ proposal led to a broad debate on the real novelty of the concept compared to ecosystem services (braat, 2018; faith, 2018; peterson et al, 2018). without going further in this debate, we note that the use of the word ‘nature’ (instead of ‘ecosystem’) as the subject of the contribution, and the people only as the beneficiaries, can question the importance of human action in the production of several of these services. as noted by peterson et al (2018): “a focus on ‘nature,’ therefore de-emphasizes the ecosystems that are home to and provide the necessities of life to most of the world’s population”. in our case, the services are clearly co-produced by humans and animals in farming systems and talking about them as ncp may seem counterproductive. the framework we propose in this paper is the first step in identifying and understanding the services provided by farming systems using livestock biodiversity. this framework could however be put to the test and enriched by applying it to other cases. steps to enrich it could be to include the points of view, values and practices of the different stakeholders involved. answers to the questions proposed in the framework can consequently include a diversity of points of view. the interest of the framework is not to provide an irrevocable single answer to each question, but rather contribute to better understanding the processes underlying services production, in their complexity. however, this framework cannot be used for a quantitative assessment of the dynamics of livestock biodiversity or the values of services. the framework can contribute to better understanding the link between livestock biodiversity dynamics and the provision of services in the farming systems concerned. indeed, from a perspective of livestock biodiversity management and conservation, one of the scientific challenges is to understand the issues underlying the management dynamics of each breed, the corresponding management objectives, and their translation into practices, at both individual and collective levels. this will be a key step in understanding whether or not the provision of services production is at stake for the people concerned. in our framework, we gathered important items to be taken into consideration to analyze the dynamics of services produced as social constructs and to understand the underlying processes. such an approach implies including a diversity of biotechnical and sociotechnical dimensions, and accounting for their interactions and their dynamics in multiple services over time. the notion of ‘breed attributes’ actually helps explain how livestock biodiversity contributes to the provision of ecosystem services. we propose this notion to describe not only the biological but also the sociotechnical characteristics of breeds. our aim is not to objectify the role of a given breed in providing a given ecosystem service (martincollado et al, 2019). as ecosystem services are seen as constructs and breeds are seen as dynamics (and not categories with static properties), our aim is rather to question whether accounting for the wide range of productions (including the different services) of farming systems using local breeds would enable a better understanding of the overall dynamics of livestock biodiversity conservation and management . to conclude, if the ecosystem services notion can help include different visions and develop a multidisciplinary approach, it is not the only notion 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(2007). ecosystem services and dis-services to agriculture. ecological economics 64(2), 253–260. doi: https://doi.org/10. 1016/j.ecolecon.2007.02.024 zoom-guadeloupe (2012). bois jolan. url: http://zoom-guadeloupe.fr/component/content/ article?d4dad6935f632ac35975e3001dc7bbe8= a9597d0cde95aa2b7dedeee2f2dd7eca&showall=1& id=42. https://doi.org/10.1016/j.ecolecon.2007.02.024 https://doi.org/10.1016/j.ecolecon.2007.02.024 http://zoom-guadeloupe.fr/component/content/article?d4dad6935f632ac35975e3001dc7bbe8=a9597d0cde95aa2b7dedeee2f2dd7eca&showall=1&id=42 http://zoom-guadeloupe.fr/component/content/article?d4dad6935f632ac35975e3001dc7bbe8=a9597d0cde95aa2b7dedeee2f2dd7eca&showall=1&id=42 http://zoom-guadeloupe.fr/component/content/article?d4dad6935f632ac35975e3001dc7bbe8=a9597d0cde95aa2b7dedeee2f2dd7eca&showall=1&id=42 http://zoom-guadeloupe.fr/component/content/article?d4dad6935f632ac35975e3001dc7bbe8=a9597d0cde95aa2b7dedeee2f2dd7eca&showall=1&id=42 introduction materials and methods results services provided by farming systems using livestock biodiversity: our proposed framework dynamics of production of a diverse range of services how can services be qualified and classified? people concerned by the production of services the role of the breed in service production: not only biological characteristics are at play relations between services links between services and management of the breeds discussion acknowledgments conflict of interest statement author contributions supplemental data for: therkildsen, m., vestergaard, m., kargo, m., keto, l., ertbjerg, p., thorkelsson, g., gudjónsdóttir, m., kjetså, m., honkatukia, m., egelandsdal, b., svartedal, n., røe, m., fikse, w. f., karlsson, a. h., hessle, a. (2023). carcass characteristics of nordic native cattle breeds. genetic resources 4 (7), 1–19. doi: 10.46265/genresj.lwup7415. table of contents native and reference breeds description .................................................................................................... 2 danish native breeds .................................................................................................................................. 2 finnish native breeds ................................................................................................................................. 2 icelandic native breed ................................................................................................................................ 3 norwegian native breeds ........................................................................................................................... 3 swedish native breeds ............................................................................................................................... 4 reference breeds ....................................................................................................................................... 5 references ................................................................................................................................................... 5 supplemental table 1 .................................................................................................................................... 7 supplemental table 2 .................................................................................................................................... 8 supplemental table 3 .................................................................................................................................... 9 supplemental table 4 .................................................................................................................................... 9 supplemental table 5 .................................................................................................................................. 10 supplemental table 6 .................................................................................................................................. 11 https://doi.org/10.46265/genresj.lwup7415 native and reference breeds description danish native breeds jysk kvæg jysk kvæg was firstly recorded in the pedigree herd book in 1881 and were up to 1949 used both for milk and meat production (sørensen and hunnicke nielsen, 2017). in 1949, the breed merged into one herd book for all danish black and white cattle (sdm) together with the breeds of black and white cattle originating from holland, and since then the breed became nearly extinct with only a few purebred animals remaining. the breed has nowadays become part of a conservation-breeding program. in 2016, the original population included 630 females above the age of one year. the phenotype is described with a height of 134 cm and a live weight of 550 kg for adult females and a height of 145 cm and live weight of 1,000 kg for adult males (sørensen and hunnicke nielsen, 2017). cows calving age is approximately 31 months. the 305-day milk yield is 6,000 kg with 3.9% fat and 3.3% protein (sørensen and hunnicke nielsen, 2017). rdm-1970 rdm anno 1970 was first pedigree-recorded in 1878 and was widely used in the danish dairy production in the 20th century. however, from 1970, genetics from american brown swiss and canadian red were introduced to counteract the severe inbreeding within the population. then the pure breeding of the original rdm-1970 was given up in the mainstream red breed in denmark. today only, a small population exists of the pure rdm-1970. in 2016, there were 224 females above the age of one year (sørensen and hunnicke nielsen, 2017). the phenotype is described with a height of 134 cm and a live weight of 550 kg for adult females and a height of 150 cm and a live weight of 1,050 kg for adult males, with a dressing percentage of 53.9% and europ conformation class of 5.8 for bull calves (sørensen and hunnicke nielsen, 2017). the 305-day milk yield is 6,700 kg with 4.8% fat and 3.7% protein (sørensen and hunnicke nielsen, 2017). finnish native breeds länsisuomenkarja (western finncattle) this polled beige-brown cattle breed was the dominant finncattle in the beginning of the 20th century. länsisuomenkarja association was established in 1906 (luke, 2015a). the breed has been kept for dairy purpose and it is informally characterized as one of the most productive native dairy breeds. the number of breeding females is 1,160 (luke, 2022). on average, female height is 133 cm and the live weight 556 kg. the height of the bulls is on average 140 cm and live weight 625-820 kg (felius, 1995). the annual energy corrected milk yield (ecm) of the breed is 7,660 kg (nokka, 2021). itäsuomenkarja (eastern finncattle) the reddish-brown cattle is known for a broad white line on its back. the pattern is also known as colorsided in other breeds. itäsuomenkarja, as the name implies, is traced to the eastern part of finland, where it was prevailing. it is the oldest of the finncattle breeds as the breed association was established in 1898. pedigree registration began afterwards, in 1914 (felius, 1995, luke, 2015b). it suffered, however, severe losses during the second world war. as a results of war, a huge evacuation operation was executed to relocate more than four hundreds of thousands finnish with their cows and bulls to other parts of the country inside the new borders. the breed lost both its geographical area and a significant part of its population. the number of breeding females was 1,556 in 2021 (luke, 2022). the breed has been kept for milk and meat production. the height of the cows is 118 cm and live weight is 440 kg. bulls are on average 135 cm and their live weight is 600 kg (felius, 1995). the annual ecm of itäsuomenkarja is 4,550 kg (nokka, 2021). pohjoissuomenkarja (northern finncattle) the polled white cattle, the rarest of finncattle breeds, is often characterized as the sister breed of swedish fjällko and norwegian sidet trønderfe og nordlandsfe (stn). the herdbook was established in 1905. the breed was kept in large herds in north, finnish lapland before the second world war. a massive rescuing operation was carried out in the autumn of 1944 when the cattle was evacuated mainly to sweden. after the war, only portion of the cattle came back with their owner and crossbreeding with länsisuomenkarja and commercial breeds nearly caused disappearance of the pure breed before its rescue in 1980’s. as there were no purebred bulls left in 1980s, swedish fjällko bulls were used to save the remains of pohjoissuomenkarja. it is bred for milk and meat (juvani, 2014; luke 2015c). the number of breeding females is 842 (luke, 2022). the height of the cows is on average 110-115 cm and their live weight is 300400 kg. the height of the bulls is on average 128 cm and live weight 450-650 kg (felius, 1995). the annual ecm of pohjoissuomenkarja is 5,650 kg per year (nokka, 2021). icelandic native breed íslenska kúakynið the native icelandic cattle has been almost completely isolated from other breeds for over 1100 years, or since the settlement of the island, which makes it genetically distinct from other cattle breeds (gautason et al, 2020). the breed is most closely related to the northern nordic indigenous breeds, the finncattle breeds länsisuomenkarja, itäsuomenkarja, pohjoissuomenkarja and the swedish fjällko (gautason et al, 2020). the population size is approximately 81,000 animals, whereof around 26,000 dairy cows (statistics iceland, 2022). the breed is still the commercial cattle breed of the country and is mostly used for dairy production. meat production is considered a side production and the growth potential is relatively low. the average height and weight are 125 cm and 470 kg of females and 150 cm and 800-1,000 kg for bulls. the average annual milk yield is 6,300 kg (the icelandic association of cattle farmers, 2022). norwegian native breeds sidet trønderfe og nordlandsfe (stn) the breed has its background in the central and northern areas of norway. it is mainly a dairy breed, adapted to the conditions in valley and mountain settlements in norway. in 2020 the number of breeding females was 1,806 (sæther et al, 2021) where 56% were suckler cows and 44% dairy cows (holene and sæther, 2021). the live weight of cows is about 430 kg and of bulls 600 kg. the annual milk yield is 4,000 kg (sæther et al, 2021; fao, 2022). telemarkfe telemarkfe is the oldest cattle breed in norway. it was established in 1856 and it was thought to be especially adapted to the mountains. it was originally a dairy breed. in 2020, the number of breeding females was 485 (sæther et al, 2021) where 59% were suckler cows and 41% dairy cows (holene and sæther, 2021). it is small, with a live weight of cows ranging from 350-500 kg and an annual milk yield of 4,000 kg (sæther et al, 2021; fao, 2022). dølafe dølafe is originally from the southeastern part of norway. these areas were good grasslands but were still not close enough to cities for easy milk distribution, and so the breed was often used as a dual-purpose breed. in 2020, the number of breeding females was 305 (sæther et al, 2021) where 56% were suckler cows and 44% dairy cows (holene and sæther, 2021). in the early 1900s, it was considered one of the heaviest breeds in the country with a live weight of 350 kg. today the breed live weight is 500 kg with an annual milk yield of 3,000 kg (sæther et al, 2021). østlandsk rødkolle østlandsk rødkolle is a red polled cattle breed and it was the dominant breed in south-east norway until second world war. after this, the breed was heavily crossed with norwegian red cattle and the breeds were considered the same breed from 1961. in 2020, the number of breeding females was 473 (sæther et al, 2021) where 90% were suckler cows and 10% dairy cows (holene and sæther, 2021). the live weight is 490 kg (johansson, 1953). the annual milk yield is 4,000 kg (sæther et al, 2021). vestlandsk raudkolle vestlandsk raudkolle is a red polled cattle breed, originating from the south-west part of norway. it is considered as a dual-purpose breed. in 1947, vestlandsk raudkolle and vestlandsk fjordfe were merged to one breed: sørog vestlandsfe. however, in 1990s in the early period of the conservation work, both the original vestlandsk raukolle and vestlandsk fjordfe were found, and it was decided to separate the breeds again. in 2020, the number of breeding females was 233 (sæther et al, 2021) where 72% were suckler cows and 28% dairy cows (holene and sæther, 2021). the cow live weight is between 350 and 500 kg and its annual milk yield is 4,000 kg (sæther et al, 2021). vestlandsk fjordfe vestlandsk fjordfe originates from the western part of norway where the landscape is dominated by fjords, steep hills and nutrient-poor grasslands. the cow is thus small and hardy. vestlandsk fjordfe has a big range in colour and size, and it was both polled and horned. in 2020 the number of breeding females was 1,018 (sæther et al, 2021) where 74% were suckler cows and 26% dairy cows (holene and sæther, 2021). today the live weight is 400-500 kg and the annual milk yield 4,000 kg (sæther et al, 2021). swedish native breeds fjällko this polled cattle type has probably been kept in northern sweden since the viking age (hallander, 1989; upadhyay et al, 2019) and it was named as a specific breed in the early 1800s. a pathological selection towards predominantly white animals a century ago narrowed the breeding base drastically. there is about 2,600 cattle of fjällko breed at present (svensk fjällrasavel, 2022). the fjällko is a dairy cow with a milk protein composition giving rise to a high cheese yield (hallander, 1989). the yearly milk yield of today is approximately 6,000 kg ecm for the 575 milk-recorded cows (växa sverige, 2021). the live weight of adult females is 400-450 kg and the height is 120-130 cm (hallander, 1989). rödkulla this red polled cattle from central sweden became officially a breed in 1912. the breeding programmes for fjällko and rödkulla were then merged for a long period and the rödkulla got a unique breed code as late as in 2004. due to very low number of cattle in the start of the conservation work, some similar-type norwegian and finnish sires were used (hallander, 1989; sveriges rödkulleförening, 2021). in 2020, there were 1,856 females and 733 males. originally, the rödkulla was a dairy breed with a yearly milk yield up to 5,500 kg ecm but is today more often kept as a suckler cow. the live weight of cows is 400-650 kg (sveriges rödkulleförening, 2021). väneko the väneko is named after the hundreds of väne (meaning grazing) in south-western sweden, where a group of animals since the 1850’s until the conservation work started in 1992 had largely not been crossed with other breeds (föreningen allmogekon, 2021). in 2019, there were 176 females and 25 males (fao, 2022). the väneko is horned with a great variation in color, often black or reddish-sided. the live weight is 500-600 kg for cows and 700-800 kg for bulls (föreningen allmogekon, 2021). bohuskulla the bohuskulla is a southern variant of the fjällko, of which a small remnant was found in south-western sweden in 1990’s and named from the county. the polled bohuskulla has the same variation in drawing and color as the fjällko. in 2019, there were 90 females and 24 males (fao, 2022). the live weight of cows is 400-500 kg, with slightly heavier bulls (föreningen allmogekon, 2021). ringamålako the reddish and horned ringamålako got its name from the hamlet ringamåla in south-eastern sweden, where in 1993 a herd was found that for over 40 years largely had kept up with its own bulls and not crossed with other breeds. two groups of cattle from rögnaröd and loshult in the same region have later been merged with the breed (föreningen allmogekon, 2021). in 2019, there was 118 females and 38 males (fao, 2022). the live weight for cows is 400-500 kg, while bulls are heavier (föreningen allmogekon, 2021). reference breeds two beef breeds, charolais and hereford, and one dairy breed, holstein, was included as reference breeds in denmark, sweden, finland, and norway. in addition, red dairy cattle were used as further reference breeds in norway and sweden. as there is only one cattle breed of any importance in iceland, no reference breed was included from iceland. charolais charolais was developed in the middle-east france in the 19th century based on local french breeds and some import of british shorthorn, but since the middle of the late 19th century the breed has been developed through selective breeding, and the breed is today the most used beef breed in france. in the 1960s a huge export of charolais cattle and semen took place, and the breed also settled as one of the largest beef breeds in numbers in the nordic countries. charolais is denoted as a late maturing beef breed meaning it has capacity for fast growing in intensive rearing systems. the live weight for cows is 750 kg, while live weight of adult bulls is from 1,150 kg (fao, 2022). hereford hereford is a british breed developed in herefordshire around year 1800. the breeding goal of the hereford cattle have gone through different directions since then. at the beginning, it was a quite heavy beef cattle type. however, in the 20th century it became important that they developed fast under semi-extensive environments. this created the small and quite fat type of hereford cattle. since the 1970s the breeding goal has again been larger and less fat cattle, and this is also the present type of hereford in the nordic countries. however, hereford is still regarded as an early maturing beef breed. the live weight for cows is 675 kg, while the live weight of adult bulls is 1,075 kg (fao, 2022). holstein holstein dairy cattle is the numerically largest dairy breed in the western world. in europe, the breed is based on the gene pool of national black and white cattle, which were substituted with the gene pool from us holstein through semen import, which became available in the 1960s where freezing of semen became possible. today, nearly all genes in the nordic holstein populations can be traced back to us holstein, which originally was imported from holland as a dual-purpose breed and selected towards today’s dairy type over more than 100 years. the holstein of today are tall animals with mature cow weight of approximately 700 kg (fao, 2022) and an annual milk yield of 11,000 kg of ecm in the nordic countries (ryk, 2022; växa sverige, 2021). red dairy cattle the norsk rødt fe (nrf) is the main commercial dairy breed in norway and the svensk röd och vit boskap (srb) is the second biggest commercial dairy breed in sweden. they are composite breeds heavily based on import of scottish ayrshire, starting in late 1800s. the imported breeds were crossed with the local native scandinavian breeds, and after the second world war nrf and srb were spread through the whole norway and sweden, respectively, with artificial insemination, and quickly outcompeted the other breeds with higher milk and meat yield. both breeds are considered as dual purpose, although milk yield is weighed higher in the breeding programs than the carcass traits. annual milk yield is slightly above 8,000 kg for nrf and slightly above of 10,000 kg of ecm for srb (sæther et al, 2021; växa sverige, 2021). references fao. (2022). domestic animal diversity information system (dad-is). http://www.fao.org/dad-is/browse-by-country-andspecies/en/ (accessed 23.07.2022) http://www.fao.org/dad-is/browse-by-country-and-species/en/ http://www.fao.org/dad-is/browse-by-country-and-species/en/ felius, m. (1995). cattle breeds – an encyclopedia. doetinchem, netherlands: misset uitgeverij. 799 p. föreningen allmogekon. (2021). plan för avel med nötkreatur av raserna väneko, ringamålako och bohuskulla (allmogekor) 2015 – 2020. https://allmogekon.se/. (accessed 8.02.2022) gautason, e., schönherz, a. a., sahana, g., guldbrandtsen, b. (2020). relationship of icelandic cattle with northern and western european cattle breeds, admixture and population structure, acta agriculturae scandinavica, section a — animal science, 69:1-2, 25-38, doi:10.1080/09064702.2019.1699951 hallander, h. (1989). svenska lantraser. bokförlaget blå ankan ab, veberöd. isbn 91-87956-00-4. 600 pp. holene, a.c., sæther, n. (2021). flere ammekyr av bevaringsverdige storferaser engasjerer. nibio pop, 7(20). https://hdl.handle.net/11250/2761544 (accessed 12.08.2022) johansson, i. (1953). husdjursraserna, grøndal & søn, oslo. 523 p. juvani, j. 2014. pohjoissuomenkarjan kantakirja-analyysi. thesis. oulu university of applied sciences. 78 p. luke 2015a. eläingeenivarat, länsisuomenkarja. https://portal.mtt.fi/portal/page/portal/www/tietopaketit/elaingeenivarat/sailytysohjelmat/nauta/lansisuomenkarja (accessed 02.08.2022) luke 2015b. eläingeenivarat, itäsuomenkarja. https://portal.mtt.fi/portal/page/portal/www/tietopaketit/elaingeenivarat/sailytysohjelmat/nauta/itasuomenkarja (accessed 02.08.2022) luke 2015c. eläingeenivarat, pohjoissuomenkarja. https://portal.mtt.fi/portal/page/portal/www/tietopaketit/elaingeenivarat/sailytysohjelmat/nauta/pohjoissuomenkarja (accessed 02.08.2022) luke 2022. luke’s statistics. https://www.luke.fi/en/statistics (accessed 02.08.2022) nokka, s. (2021). lypsykarjan tuotosseurannan tulokset 2020. https://www.proagria.fi/sites/default/files/attachment/lypsykarjan_tuotosseurannan_tulokset_2020.pdf (accessed 12.08.2022) ryk. (2022). aktuelle tal i ydelsenkontrollen. https://www.ryk-fonden.dk/aktuelle-tal-i-ydelseskontrollen (accessed 08.07.2022) statistics iceland. (2022). stat.icel. https://px.hagstofa.is/pxis/pxweb/is/atvinnuvegir/atvinnuvegir__landbunadur__landframleidsla/lan10201.px (accessed 20.02.2022) svensk fjällrasavel. (2022). avelsplan för svensk fjällras. https://fjallko.se/avel/avelsarbetet/avelsplan (accessed 24.07.2022) sveriges rödkulleförening. (2021). rödkullan. https://rodkullan.se/ (accessed 08.02.2022) sæther, n., holene, a.c., fjellstad, k.b., frøiland, c. (2021). nøkkeltall 2020 fra norsk genressurssenter. nibio rapport 7(107). 130 s. nibio, ås. sørensen, l. h., hunnicke nielsen, v. (2017) danske husdyrgenetiske ressourcer. dca – nationalt center for fødevarer og jordbrug. dca rapport nr. 100. 63 p. the icelandic association of cattle farmers. (2022). landssamband kúabænda. 2022. https://naut.is/gagnlegarupplysingar-2/ (accessed 13.01.2022) upadhyay, m., eriksson, s., mikko, s., strandberg, e., stålhammar, h., groenen, m. a. m., crooijmans, r. p. m. a., andersson, g., johansson, a. m. (2019). genomic relatedness and diversity of swedish native cattle breeds. genetics selection evolution 51:56 doi:10.1186/s12711-019-0496-0. växa sverige. (2021). cattle statistics 2020. https://www.vxa.se/fakta/styrning-och-rutiner/mer-om-mjolk/statistik/ (accessed 05.02.2022) https://allmogekon.se/ file://uni.au.dk/users/au223081/animalske%20f%c3%b8devarer/nordic%20meat%20network/publication/030222/holene,%20a.c.,%20s%c3%a6ther,%20n.%20(2021).%20flere%20ammekyr%20av%20bevaringsverdige%20storferaser%20engasjerer.%20nibio%20pop,%207(20).%20available%20at%20 file://uni.au.dk/users/au223081/animalske%20f%c3%b8devarer/nordic%20meat%20network/publication/030222/holene,%20a.c.,%20s%c3%a6ther,%20n.%20(2021).%20flere%20ammekyr%20av%20bevaringsverdige%20storferaser%20engasjerer.%20nibio%20pop,%207(20).%20available%20at%20 https://hdl.handle.net/11250/2761544 https://portal.mtt.fi/portal/page/portal/www/tietopaketit/elaingeenivarat/sailytysohjelmat/nauta/lansisuomenkarja https://portal.mtt.fi/portal/page/portal/www/tietopaketit/elaingeenivarat/sailytysohjelmat/nauta/itasuomenkarja https://portal.mtt.fi/portal/page/portal/www/tietopaketit/elaingeenivarat/sailytysohjelmat/nauta/pohjoissuomenkarja https://www.luke.fi/en/statistics https://www.proagria.fi/sites/default/files/attachment/lypsykarjan_tuotosseurannan_tulokset_2020.pdf https://www.ryk-fonden.dk/aktuelle-tal-i-ydelseskontrollen https://px.hagstofa.is/pxis/pxweb/is/atvinnuvegir/atvinnuvegir__landbunadur__landframleidsla/lan10201.px https://fjallko.se/avel/avelsarbetet/avelsplan https://rodkullan.se/ https://naut.is/gagnlegar-upplysingar-2/ https://naut.is/gagnlegar-upplysingar-2/ https://doi.org/10.1186/s12711-019-0496-0 https://www.vxa.se/fakta/styrning-och-rutiner/mer-om-mjolk/statistik/ supplemental table 1 reference cattle in denmark, finland, norway, and sweden – breeds, categories, numbers, and age (mean and standard deviation (sd)) country denmark finland norway sweden # of animals age mo. sd mo. # of animals age mo. sd mo. # of animals age mo. sd mo. # of animals age mo. sd mo. holstein young bull < 12 mo 681,159 10.0 0.79 1,824 9.23 2.59 1,893 7.0 1.56 82,380 9.1 1.17 bull > 12 mo 226,313 14.4 3.85 338,033 20.5 3.65 14,821 19.3 4.23 404,588 19.8 7.44 steers 10,173 27.1 6.39 63 22.5 5.06 30 23.5 2.87 90,081 26.9 5.29 heifersa 120,930 23.2 6.60 53,256 22.0 7.64 1,118 20.2 4.20 61,485 28.5 11.81 young cow < 48 moa 216,672 33.7 5.33 38,855 34.2 5.15 7,137 36.1 7.46 82,669 34.8 4.76 cow > 48 mo 727,690 65.5 17.96 160,510 68.3 20.19 5,519 64.1 15.20 333,786 67.4 20.06 red dairy cattle young bull < 12 mo 88,399 7.3 1.67 27,359 9.2 1.22 bull > 12 mo 790,444 19.8 4.16 231,608 20.2 8.10 steers 12,082 23.0 4.73 80,208 27.4 5.55 heifersa 89,471 19.8 4.57 41,612 28.3 9.53 young cow < 48 moa 340,177 36.5 7.63 52,248 35.1 4.571 cow > 48 mo 393,122 68.1 18.13 234,081 68.2 20.35 charolais young bull < 12 mo 295 11.0 1.00 140 9.2 2.78 1,585 7.9 1.87 1,243 10.7 1.49 bull > 12 mo 1,452 20.7 17.59 22,526 21.7 9.28 32,529 19.8 9.20 39,319 19.6 12.98 steers 5 27.1 9.09 89 23,2 5.60 1,365 24.1 7.15 heifersa 773 19.8 7.12 12,427 18.8 6.66 8,745 18.3 4.70 21,844 23.4 8.33 young cow < 48 moa 883 34.2 5.15 940 33.7 5.07 6,965 35.2 8.87 2,958 34.1 4.66 cow > 48 mo 5,397 96.6 39.96 6,404 93.5 36.22 9,054 90.0 33.98 18,213 93.5 40.73 hereford young bull < 12 mo 131 10.9 1.21 113 10.2 1.54 3,907 7.7 2.22 485 9.5 2.46 bull > 12 mo 1,488 23.8 20.17 32,529 22.9 9.50 29,325 20.4 8.61 28,349 23.1 18.39 steers 40 23.6 8.96 3 36.4 5.41 490 23.7 5.87 2,931 25.9 5.21 heifersa 488 22.4 9.41 14,465 19.5 6.76 8,110 18.7 4.85 13,359 25.7 9.86 young cow < 48 moa 2,316 34.2 5.32 1,255 33.3 5.00 6,743 35.7 9.87 2,502 33.9 4.65 cow > 48 mo 12,883 89.9 36.44 9,007 95.9 36.83 11,542 93.4 35.06 13,271 89.3 35.64 a in norway a heifer is a female between 12 and 24 mo. and a young cow is between 24 and 48 mo., whereas calving instead of age separate the two categories in the other countries. 8 supplemental table 2 danish native cattle – breeds, categories, numbers, and age (mean and standard deviation (sd)) breed categories # of animals age mo. sd mo. rdm young bull < 12 mo 39 10.6 0.88 bull > 12 mo 137 23.6 17.49 steers 17 28.7 5.96 heifers 23 29.9 9.30 young cow < 48 mo 47 34.1 4.81 cow > 48 mo 183 77.3 30.33 jysk kvæg young bull < 12 mo 47 10.1 0.81 bull > 12 mo 127 22.7 11.41 steers 28 30.5 7.25 heifers 39 33.1 12.79 young cow < 48 mo 57 35.6 5.91 cow > 48 mo 322 89.4 35.08 9 supplemental table 3 finnish native cattle – breed, categories, numbers, and age (mean and standard deviation (sd)) breed categories # of animals age mo. sd mo. länsisuomenkarja young bull < 12 mo 392 8.0 2.87 bull > 12 mo 5,589 21.9 5.92 steers 26 23.0 5.96 heifers 1,636 25.0 11.20 young cow < 48 mo 1,087 33.3 5.03 cow > 48 mo 3,007 72.2 25.26 itäsuomenkarja young bull < 12 mo 225 8.7 2.36 bull > 12 mo 1,685 23.2 9.93 steers 28 24.5 10.18 heifers 398 28.2 13.57 young cow < 48 mo 252 33.5 5.53 cow > 48 mo 785 78.5 30.42 pohjoissuome young bull < 12 mo 785 8.5 2.46 bull > 12 mo 2,150 21.9 8.81 steers 85 45.7 36.56 heifers 776 24.8 12.36 young cow < 48 mo 380 32.8 5.38 cow > 48 mo 1,144 75.4 27.87 supplemental table 4 icelandic native cattle – breed, categories, numbers, and age (mean and standard deviation (sd)) breed categories # of animals age mo sd mo. íslenska kúakynið young bull < 12 mo 368 9.6 3.00 bull > 12 mo 28,417 25.5 4.20 steers 0 heifers 3,688 25.7 7.80 young cow < 48 mo 8,147 37.5 6.60 cow > 48 mo 17,610 74.7 24.00 10 supplemental table 5 norwegian native cattle – breed, categories, numbers, and age (mean and standard deviation (sd)) breed categories # of animals age mo. sd mo. stn young bull < 12 mo 2,282 7.1 2.14 bull > 12 mo 3,034 19.8 8.26 steers 371 22.2 6.77 heifers 731 18.5 5.45 young cow < 48 mo 1,567 35.5 7.97 cow > 48 mo 2,096 78.8 26.34 telemarkfe young bull < 12 mo 484 7.5 2.23 bull > 12 mo 547 18.7 9.68 steers 42 18.6 3.57 heifers 141 18.5 5.43 young cow < 48 mo 292 36.4 7.98 cow > 48 mo 470 80.4 30.25 dølafe young bull < 12 mo 214 7.3 2.06 bull > 12 mo 337 20.5 7.47 steers 31 18.7 5.43 heifers 40 19.3 4.70 young cow < 48 mo 76 36.0 8.08 cow > 48 mo 185 88.2 30.46 østlandsk rødkolle young bull < 12 mo 419 7.4 1.55 bull > 12 mo 482 19,5 7.33 steers 6 21.0 3.80 heifers 71 18.5 5.43 young cow < 48 mo 122 35.0 8.03 cow > 48 mo 212 87.4 38.0 vestlandsk raudkolle young bull < 12 mo 205 7.0 2.09 bull > 12 mo 253 19.3 6.70 steers 15 22.7 4.94 heifers 43 17.6 5.30 young cow < 48 mo 103 34.1 7.51 cow > 48 mo 161 74.8 25.5 vestlandsk fjordfe young bull < 12 mo 757 7.9 5.53 bull > 12 mo 1,179 21.0 8.73 steers 188 21.7 3.14 heifers 295 18.4 5.46 young cow < 48 mo 440 36.2 9.24 cow > 48 mo 702 82.3 31.56 11 supplemental table 6 swedish native cattle – breed, categories, numbers, and age (mean and standard deviation (sd)) breed categories # of animals age mo. sd mo. fjällko young bull < 12 mo 238 7.7 2.46 bull > 12 mo 772 25.7 11.69 steers 256 29.0 8.89 heifers 164 28.6 10.54 young cow < 48 mo 184 33.5 5.74 cow > 48 mo 701 88.0 40.78 rödkulla young bull < 12 mo 138 8.3 2.12 bull > 12 mo 660 24.9 12.52 steers 178 26.9 5.99 heifers 197 27.0 9.23 young cow < 48 mo 76 33.9 4.92 cow > 48 mo 475 101.2 42.35 väneko young bull < 12 mo 6 8.8 0.44 bull > 12 mo 24 29.6 8.63 steers 7 40.3 23.05 heifers 11 32.4 15.26 young cow < 48 mo 6 38.2 1.85 cow > 48 mo 29 92.1 32.34 bohuskulla young bull < 12 mo bull > 12 mo 11 21.3 5.60 steers 5 27.9 2.39 heifers 4 36.4 5.45 young cow < 48 mo cow > 48 mo 2 76.2 31.46 ringamålako young bull < 12 mo 5 10. 8 0.90 bull > 12 mo 32 26.1 12.87 steers heifers 6 22.3 10.58 young cow < 48 mo cow > 48 mo 5 102.5 35.99 native and reference breeds description danish native breeds finnish native breeds icelandic native breed norwegian native breeds swedish native breeds reference breeds references supplemental table 1 supplemental table 2 supplemental table 3 supplemental table 4 supplemental table 5 supplemental table 6 short communication genetic resources (2024), 5 (9), 36–44 doi: 10.46265/genresj.wqzs1824 https://www.genresj.org issn: 2708-3764 a public mid-density genotyping platform for cultivated blueberry (vaccinium spp.) dongyan zhao a, manoj sapkota a, jeff glaubitz b, nahla bassil c, molla f mengistd, massimo iorizzod, kasia heller-uszynska e, marcelo mollinari f, craig t beil a and moira j sheehan *,a a breeding insight, cornell university, ithaca, 14853, ny, usa b institute of biotechnology, cornell university, ithaca, 14853, ny, usa c national clonal germplasm repository, usdaars, or, 97333, corvallis, usa d plants for human health institute, north carolina state university, nc, 28081, kannapolis, usa e diversity arrays technology, act 2617, bruce, australia f north carolina state university, campus box 7609, nc, raleigh, 27695, usa abstract: small public breeding programmes have many barriers to adopting technology, particularly creating and using genetic marker panels for genomic-based decisions in selection. here we report the creation of a dartag panel of 3,000 loci distributed across the tetraploid genome of blueberry (vaccinium corymbosum) for use in molecular breeding and genomic prediction. the creation of this marker panel brings cost-effective and rapid genotyping capabilities to public and private breeding programmes. the open access provided by this platform will allow genetic data sets generated on the marker panel to be compared and joined across projects, institutions and countries. this genotyping resource has the power to make routine genotyping a reality for any breeder of blueberry. keywords: vaccinium spp., amplicon-sequencing, plant breeding, dartag genotyping, microhaplotype citation: zhao, d., sapkota, m., glaubitz, j., bassil, n., mengist, m. f., iorizzo, m., heller-uszynska, k., mollinari, m., beil, c. t., sheehan, m. j. (2024). a public mid-density genotyping platform for cultivated blueberry (vaccinium spp.). genetic resources 5 (9), 36–44. doi: 10.46265/genresj.wqzs1824. © copyright 2024 the authors. this is an open access article distributed under the terms of the creative commons attribution license (cc by 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. introduction molecular techniques have been employed for nearly four decades to enhance and speed the breeding efforts for major staple food crops like tomato, maize and barley (tanksley (1983); helentjaris et al (1985); feuerstein et al (1990) and reviewed in hasan et al (2021)). over time, molecular biology techniques have been paired with high-quality phenotypic data to perform genomewide association studies (gwas), genomic selection and prediction, further fuelling breeding for quantitative or complex traits (eathington et al, 2007; heffner et al, 2009; lorenzana and bernardo, 2009). while these achievements are significant, many crop species grown for human consumption are still unable to apply ∗corresponding author: moira j sheehan (moirasheehan@cornell.edu) these techniques in breeding efforts. many breeders would like to adopt molecular breeding tools and techniques, but sometimes doing so is hampered by large barriers-to-entry challenges. the range of barriers, and how surmountable they are, varies from species to species and is impacted by species-specific challenges in logistics, technical know-how, biology and the growing environment. blueberries (vaccinium spp) are native to north america and are a relatively recent crop, having been cultivated only since 1916 (ushbc, 2021). the united states (us) is the largest global producer of blueberries (faostat, 2021). in 2022, the us produced over 282 million kilograms (622 million pounds) of cultivated blueberries and harvested 35.2 million kilograms (77.6 million pounds) of wild blueberries, which amounted to a total crop value of usd1.04 received: 14.02.2024 accepted: 02.04.2024 published online: 11.04.2024 https://www.genresj.org https://www.doi.org/10.46265/genresj.wqzs1824 https://www.genresj.org https://www.doi.org/10.46265/genresj.wqzs1824 mailto:moirasheehan@cornell.edu genetic resources (2024), 5 (9), 36–44 a fast and robust genotyping platform for blueberry breeding 37 billion (nass, 2023). blueberries are considered a ‘superfruit’ for human nutrition due to their high levels of essential nutrients, fibre and antioxidants. because of their nutritional value, blueberries are produced for a wide range of markets including fresh eating (and upick), frozen whole berries, frozen juice, powders and dried leaves for herbal tea. cultivated blueberries are categorized by growing region and chilling requirements. in northern states, most varieties are northern highbush types (nhb; v. corymbosum) that only flower after about 800–1,000 hours of exposure to temperatures between 0◦c–7◦c (32◦f–45◦f) (hancock, 2009). the southern highbush types (shb) are complex hybrids between v. corymbosum with the evergreen species v. darrowii native to florida. southern highbush varieties have reduced chilling requirements (200–300 hours) and enhanced adaptation to southern climates and soils (hancock, 2009). the half-high blueberry (hhb) is derived from crosses between nhb with v. angustifolium, a wild northern species. half-high blueberry is preferred for commercial environments that require varieties with enhanced hardiness. unlike nhb, shb and hhb which are tetraploid types, the fourth cultivated type, rabbiteye (re; v. virgatum), is hexaploid. rabbiteye blueberry, known for its high vigour and heat tolerance, is native to the southeastern us (edger et al, 2022). although most of the breeding efforts are focused on these four cultivated types, some pre-breeding work has included parents from wild species (also known as lowbush blueberry) of the cyanococcus section of the subgenus vaccinium. blueberry breeding is a long and tedious process (gallardo et al, 2018). traditional breeding approaches can take 9 to 20 years from crossing and testing to the release of new cultivars (gallardo et al, 2018). some of the breeding challenges are that cultivated blueberries are perennials, outcrossing, highly heterozygous and autotetraploid, where random chromosome pairing during meiosis predominates (qu and hancock, 1995; qu et al, 1998; lyrene et al, 2003). traditional biallelic snp marker systems designed for inbred or diploid species often fall short when applied to heterozygous and polyploid species due to their inability to identify multiallelic dosages accurately. a more sophisticated genotyping system is needed to address the unique challenges posed by blueberry’s autotetraploid nature, yet the investment cost and reliance upon skilled bioinformatics support for each genotyping run make this a high-risk endeavour for breeders. the first and most tractable place to build capacity and tools for molecular breeding is to create a rapid genotyping pipeline that fits within both the breeding and selection cycles and can deliver on the breeder’s objectives (hawkins and yu, 2018; mejiaguerra et al, 2021) . as stated here, a pipeline refers to a complete workflow starting with a genetic marker platform, vendors for services and bioinformatic tools to transform returned raw data into a usable format for breeders. there are several factors to consider when choosing a genetic marker platform: cost per data point, vendor services, turnaround times and what genetic analyses can be done with the resulting data. for blueberry, we hypothesized that a targeted-amplicon sequenced-based approach would be the most beneficial for breeders. unlike genotyping-by-sequencing (gbs), targeted amplicon-based genotyping technologies such as dartag (diversity array technology dart), flex-seq (rapid genomics), and capture-seq (lgc genomics) have low missing data rates and query the same loci in all samples across genotyping projects, allowing new data to be easily appended to existing data (darrier et al, 2019; telfer et al, 2019; wang et al, 2020). the amount of data returned is in the tens of thousands or less, rather than the millions of reads from gbs, simplifying downstream bioinformatics processing (darrier et al, 2019; milner et al, 2019). this in turn speeds up the analysis time for marker-assisted selection (mas), introgression tracking, linkage mapping, gwas and genomic prediction (darrier et al, 2019). here, we report the creation of a mid-density dartag panel of 3,000 marker loci distributed across the blueberry genome for use in molecular breeding and genomic prediction. dartag is a hybridization/amplicon-based targeted genotyping platform developed by dart (blyton et al (2023); https:// www.diversityarrays.com/services/targeted-genotying/) available to the public. materials and methods germplasm selection and whole-genome sequencing of a blueberry diversity panel a total of 31 cultivated blueberry accessions focused on elite north american breeding lines were selected for skim sequencing. this panel consisted of 12 nhb, 10 shb, 2 nhb x shb hybrids, 5 re, and 1 re x shb accessions (supplemental table 1, entries marked with asterisks). two biological replicates of each sample in the discovery panel were processed, where the sequencing libraries (average insert dna size of 300bp) were prepared using either illumina nextera wgs library prep at the genomics facility of cornell institute of biotechnology or nebnext ultra dna library prep kit at novogene. whole-genome sequencing was done using illumina novaseq 6000 at novogene (https:// en.novogene.com). snp discovery and selection of 3k marker loci for building dartag genotyping panel raw fastq sequences were processed by removing residual adapter sequences and low-quality bases using trimmomatic (leading:10 trailing:10 slidingwindow:4:15 minlen:30) (bolger et al, 2014). cleaned reads were then aligned to the haploid set (i.e., the first set out of the four homologous chromosomes) of the blueberry reference genome as described by colle et al (2019) using bwa-mem (li, 2013). structural variants (snps and indels) were called using the dnaseq https://www.novogene.com https://en.novogene.com https://www.diversityarrays.com/services/targeted-genotying/ 38 zhao et al genetic resources (2024), 5 (9), 36–44 pipeline developed by sentieon (https://www.sentieon. com). a total of 600k snps were discovered in the diver-sity panel. a high-confidence set of 10k snps (figure 1) was then identified using the following criteria: (1) not located within 5bp from an indel, (2) qual > 30, (3) minimum and maximum read depths of 20 and 1,500, respectively, (4) at each heterozygous site, at least one read supporting the reference allele and two reads supporting the alternative allele, (5) no missing genotype per snp position, (6) with a minor allele frequency greater than 0.25, (7) not located in transposable elements or within 1kb of chromosome termini and (8) even genomic distribution and mostly located in genic regions. the 10k snps were submitted for qc to dart (diversity arrays technology pty ltd, www.diversityarrays.com), from which a 3k snp set was selected. additionally, a few experimentally validated snps were also force-included in the panel. custom oligo probes were then synthesized, and genotyping was done at dart. a total of 1,445 and 1,555 marker loci (supplemental file 1) were designed to produce amplicons from the plus and minus strands based on the reference genome, respectively (colle et al, 2019). based on the ‘draper’ reference genome and gene assembly v1.0 from colle et al (2019), 97% (2,924) reside in genic regions, with only 3% (76) residing in non-genic regions (supplemental file 1). among the 3,000 loci selected, each chromosome harbors between 219 loci on chr07.1 to 296 loci on chr02 with an average of 250 loci per chromosome. in addition, there is a positive correlation (r2=0.70) between the number of genes on a chromosome and the number of targeted loci on that chromosome, indicating that chromosomes with more genes have better marker coverage (supplemental file 1). the dartag genotyping technology produces multi-allelic data as 54bp and 81bp amplicons (referred to as microhaplotypes in this study) encompassing the 3k target snp sites, therefore, we refer to these target sequences as marker loci. selection of samples for validating the dartag panel and genotyping results the dartag genotyping assay consists of four steps based on principles described in krishnakumar et al (2008) and implemented as described in zhao et al (2023). briefly, the pool of 3,000 blueberry oligos, each targeting one genetic variant plus adjacent flanking sequence, is hybridized to denatured gdna in step 1, followed by snp/indel copying into dartag molecules by dna polymerase in step 2. after ligation into circular molecules also in step 2, and nuclease treatment to remove uncircularized molecules in step 3, dartag products are subsequently amplified i n s tep 4 with the simultaneous addition of sample unique barcodes used downstream for demultiplexing. the products of dartag assay, after purification a nd quantification, are sequenced on ngs platforms (e.g. novaseq 6000, illumina) with a depth of around 200x, demultiplexed and the genetic variants are detected using the dart proprietary analytical pipeline. the blueberry 3k marker panel was tested using a set of 375 samples, including: (1) a diverse set of cultivated blueberries (n = 171), (2) a ‘draper’ x ‘jewel’ (dxj) f1 population (n = 175), (3) wild vaccinium species and other interspecific hybrids (vaccinium subgenus) (n = 24), and (4) a small number of cultivated cranberry varieties (n = 5) (oxycoccus subgenus) (supplemental table 1). the raw genotyping data included fastq and the missing allele discovery count (madc) file (supplemental file 2). the madc file was first filtered at the microhaplotype level. a microhaplotype was retained if it was present in at least 10 samples and each sample had at least 2 reads detected. first, samples with ≥ 95% missing data were removed. then, filtering of marker loci was based on ≥ 10 samples with each having ≥ 10 reads for each marker locus per sample. all snps, including both target and off-target snps were extracted from all remaining marker loci for downstream analyses. principal component analysis was conducted using read count data from all samples using addpca function in polyrad (clark et al, 2019) and plotted using ggplot2. genetic map construction the dxj f1 population was derived from a ‘draper’ x ‘jewel’ cross. ‘draper’ is a nhb variety released by michigan state university in 2004, whereas ‘jewel’ is a shb variety released by the university of florida in 1999. the true parental plants that were used to make the dxj cross are no longer available, so we genotyped five draper accessions and five jewel accessions from across several public programmes. genotype dosage calls for each snp in the dxj population were determined with updog software (gerard et al, 2018). a pca was performed in polyrad and identified 1 4 d xj progenies that do not appear to be true f1s (supplemental figure 1a). before mapping, these 14 individuals were removed leaving 161 dxj f1 progeny and the most similar parents to the true parents, which were not available, ‘draper 2004.001 s10-42’ and ‘jewel 2157.001 g04-01’ were identified a s p roxy p arents. ( supplemental figure 1b). of the 8,955 snps detected, 4,918 were noninformative in the dxj f1 population and were removed from further mapping on the ‘true’ 161 f1s in the dxj population. the average missing data for this population was 15% (range 6–26%) (supplemental figure 2). marker loci with > 5% missing rate (n = 840), and that did not fit expected mendelian segregation (n = 1,203) were also removed from further analysis leaving 1,994 markers available for map construction. to construct the f1 population genetic map mappoly2 was used (https:// github.com/mmollina/mappoly2; mollinari and garcia (2019); mollinari et al (2020)). a recombination fraction matrix was calculated and used to cluster the markers into linkage groups. screening snps based on recombination frequency via the rf filter function eliminated additional snps (n = 497). for each linkage https://www.sentieon.com www.diversityarrays.com https://github.com/mmollina/mappoly2 https://www.diversityarrays.com/ genetic resources (2024), 5 (9), 36–44 39 raw snps keep snps that: 1. are > 5bp away from an indel. 2. have map qual > 30 3. have read depths between 20 and 1500. 4. have ≥1 read supporting the reference allele and ≥2 reads supporting the alternative allele. 5. have no missing genotypes at snp position. 6. have >0.25 minor allele frequency. 7. do not reside in transposable elements. 8. do not reside within 1 kb of chromosome termini. breeding diversity panel filter 600k snps thin snps to achieve even genomic distribution and select snps in genic regions. filtered snps 10k snps dart performs in-silico performance & qc testing to remove snps likely to have problems in pooled oligo sets. thinned to genic regions 3k  breeding insight selects the set of 3k from snps passing darts qc testing and dart orders the oligos. genomic distribution & qc open panel to public thin poolqc figure 1. filters and criteria applied to produce the 3k dartag marker loci panel from the wgs of the blueberry diversity panel. note that the 3k marker loci selection contains the 3k target snps discovered from the snp discovery using a diversity panel of 31 blueberry lines. abbreviations: k is thousands. group, genomic order (physical position) of the markers was used to perform phasing and generate the genetic map. the construction of the genetic map involved initially creating individual maps for each parent, which were then integrated into a comprehensive hmm model using the merge single parents maps function, resulting in a consolidated map. additional unmapped markers were incorporated using the augment phased map function, which adds markers with redundant map information. the final f1 genetic map was constructed with 1,301 unique (1,487 total) markers (supplemental figure 3b). lastly, the haplotypes of the f1 individuals were reconstructed by employing genotypic conditional probabilities through the calc homoprob function (example shown in supplemental figure 3c). results validation of the 3k blueberry dartag panel and genotyping results to assess the quality and completeness of data, a validation set of 375 samples was genotyped using the 3k dartag panel to (1) assess diversity among cultivated blueberries, (2) construct a genetic linkage map, and (3) evaluate its usefulness across species and subgenera. dart generates genotyping results in several formats, among which the madc format (missing allele discovery count) provides all the microhaplotypes (54–81bp) discovered based on amplicons for the 3k marker loci. these microhaplotypes contain target snps per assay design as well as off-target snps, which are present in flanking amplicon sequences. to better distinguish these microhaplotypes, those matching the reference and alternative alleles at the target snp site and containing no other variant nucleotide are denoted as ref and alt microhaplotypes, respectively. additional haplotypes that contain off-target snps in variant nucleotides in the flanking sequences are denoted as refmatch (when target snp matches ref) and altmatch (target snp matches alt) with consecutive numbering for uniqueness (figure 2). the madc report (supplemental file 2) was filtered at the microhaplotype level by requiring at least 5% of total samples, each having a minimum of 2 reads to retain a refmatch or altmatch. out of 16,340 refmatch and altmatch, 8,370 were filtered out due to high missing data and 7,970 remained. panel effectiveness in extant accessions the marker loci detection rate was determined at both sample and marker levels, respectively. all 375 samples contained data from ≥ 25% marker loci, therefore, no samples were removed. about 95% (n = 355) of total samples have data from ≥ 75% marker loci, indicating the high detection efficiency of the marker panel. at the marker level, data presence ranged from 5% to 100% in samples. it is worth noting that 1,722 (57%) marker loci were detected in ≥ 95% of samples and 299 (10%) marker loci were detected in all the samples surveyed, representing the most conserved marker loci in the blueberry genome and its related species. a total of 101 marker loci with data in < 5% of total samples were excluded for downstream analyses. the average missing data for each cultivated blueberry type was as follows: 19% for nhb (range 8–24%), 18% for shb (range 13–26%), 20% for hhb (range 18–21%), and 21% for re (range 17–24%) (supplemental figure 2). wild species from the vaccinium subgenus had a missing data rate ranging from 18–56% (supplemental figure 2), whereas the five cranberry samples (oxycoccus subgenus) exhibited the highest missing rates ranging from 54–73%. marker loci that worked across subgenera a fast and robust genotyping platform for blueberry breeding 40 zhao et al genetic resources (2024), 5 (9), 36–44 figure 2. example of dartag sequencing reads from blueberry locus chr6.1 000263165. each sequence is a microhaplotype detected in breeding material tested on the panel. the dartag assay was designed to detect the target snp and distinguish the reference allele from the alternative allele. additional variant positions (yellow fill) distinguish the individual microhaplotypes. physpos refers to the physical nucleotide position within the sequencing read from left to right. newly discovered haplotypes are named with incrementing left-padded numbers with a prefix of ‘refmatch’ or ‘altmatch’ depending on which allele they match the ref or alt nucleotide at the target snp, respectively. are likely linked to conserved regions of the blueberry and cranberry genomes. creation of a linkage map a bi-parental population of ‘draper’ (nhb) and ‘jewel’ (shb) (dxj) was genotyped to test if the 3k dartag panel can be used to generate a linkage map. the population was created by michigan blueberry grower marketing and clones of the parents, ‘draper’ and ‘jewel’, were distributed widely to researchers and growers nationwide. the true parents of the dxj population were not available to be genotyped so we genotyped five different samples of both ‘draper’ and ‘jewel’. genetic evidence supported that ‘draper 2004.001 s10-42’ and ‘jewel 2157.001 g04-01’ were close proxies for the true parents (see materials and methods; supplemental figure 1). the final dxj f1 linkage map consisted of 12 linkage groups with 1,301 markers and a total length of 1,368.6cm (average density of 0.96 markers/cm) from 161 progeny (figure 3; supplementary figure 3). linkage group length ranged from 90.50cm to 148.30cm, with an average of 114.05cm. markers were well distributed throughout the 12 linkage groups. supplemental file 3 contains linkage groups with marker order, positions in cm, and parental phasing. additionally, haplotypes (indicating recombination events) for all individuals in f1 population were reconstructed (supplemental figure 3c) based on the genotypic conditional probabilities. discussion and conclusion the blueberry dartag panel is now publicly available and open for any researcher or breeder to order through dart (https://www.diversityarrays.com). the panel was designed on the legacy technology to produce 54bp reads but worked equally well with the current technology (81bp reads) with the caveat that some residual adapter sequences may be included (readthrough of the entire fragment into the adapter). raw data in fastq can be requested as can the missing allele discovery file (madc) that indicates the read depth of each microhaplotype in each sample. the high detection rate and repeatability make this panel suitable for genetic map construction, marker-assisted selection, whole-genome association mapping, reconstruction of recombination patterns, allele dosage estimation and parental confirmation in north american cultivated nhb, shb, re, and hhb, with some limited application in other vaccinium species. the efficacy of the panel on breeding materials outside of north america has not been tested at this time. the dartag assay can be processed from blueberry gdna or leaf tissue to genotyping data extraction in a 3–4-week turnaround time. the dart genotyping data report comprises allele dose calls and raw data with custom report formats available upon request. one benefit that dartag has over fixed array platforms is the ability to update and improve the marker panel as required over time. the panel is a pool of 3,000 oligos, one per locus, which is used to generate the sequencing libraries from the assayed material. because the pool is created from individual oligo stocks, the removal of suboptimal loci or the addition of new loci can be easily done by creating a new pool. to determine which loci should be considered for removal, extensive genotyping (> 10,000 samples) is underway to identify and remove those loci that consistently underperform or fail. independently, as new significant qtl markers and/or markers specific to other germplasm are detected, they can be targeted for inclusion in the original pool in the next version(s) of the panel. dart offers re-pooling services once per year at low or no cost, but more frequent requests could result in labour surcharges being applied (andrzej kilian, personal communication). researchers interested https://www.diversityarrays.com/ genetic resources (2024), 5 (9), 36–44 41 figure 3. genetic map of the dxj bi-parental f1 population. a) marker distribution across 12 linkage groups of the blueberry genome. scale bar is shown in cm. b) relationship plots of genetic distance (cm) to physical distance (mb) for each of the 12 linkage groups. a fast and robust genotyping platform for blueberry breeding 42 zhao et al genetic resources (2024), 5 (9), 36–44 in initiating projects with dart are encouraged to contact dart directly for consultation. another benefit of genotyping using the blueberry 3k dartag panel is the ability to detect and catalogue all microhaplotypes into a fixed allele database, which will improve combining data sets across genotyping projects (manuscript in preparation). if after testing on thousands of samples, there are too few markers for gwas for a given trait of interest, additional dartag panels can be made to complement this panel, or larger platforms like the flex-seq 22k panel (flex-seq panel code: fs 1903) from rapid genomics could be utilized (nahla bassil, personal communication). another option is to add the required loci to the existing panel up to the technical limit of 7k, which is a more cost-effective option for the routine genotyping service with scalability. we chose to create a panel of 3,000 marker loci due to cost and technical reasons, but smaller complementary panels can be made at lower up-front and downstream usage costs. the practical upper limit for the maximum number of probes on a dartag panel is 7,000 loci, though the optimal maximum may differ by species and genome complexity, and read depth required to sufficiently c all g enotypes ( andrzej kilian dart, personal communication). the blueberry breeding community could decide to create a complementary 3k panel to result in more detailed genotypic data, however, this would nearly double the cost of genotyping per sample. data availability statement the fastq files from the whole-genome skim sequencing for the 31 blueberry accessions used for identifying the candidate snp variants are housed in the ncbi short read archive under the bioproject id prjna1020150. the targeted regions used to create the 3k dartag markers are available on dryad (prepublication url: https://datadryad.org/stash/share/ue w2rmvu2bbxtkm0sbmukp6vjvfshe72um9mavakqj a; doi: 10.5061/dryad.j6q573nnc). the code and data for the construction of the f1 map in mappoly2 are available in our github repository for those interested in reproducing our analysis (https://github.com/breedi ng-insight/blueberry dartag panel paper#blueberry d artag panel paper). supplemental data supplemental table 1. accessions used in the construction and testing of the blueberry 3k dartag panel supplemental figure 1. principle component analy-sis (pca) plots of the ‘draper’ x ‘jewel’ f1 population supplemental figure 2. missing data rates for different grouped subsets of genetic material supplemental figure 3. blueberry genetic map construction for the f1 population supplemental file 1. genomic information of the blueberry 3k dartag marker panel supplemental file 2. madc report for the 375 samples used to validate the 3k dartag panel supplemental file 3. linkage group with their marker order, positions in cm, and parental phasing information where p1 represents ‘draper’ and p2 represents ‘jewel’. acknowledgements breeding insight is acknowledged for project design, marker development, curation and data processing. diversity arrays technology created the oligo array, provided sequencing services and contributed to the manuscript. we thank chad finn, nahla bassil, ebrahiem babiker, massimo iorizzo and mark ehlenfeldt for providing germplasm. we also thank alexandra casa and sharon mitchell for careful reading and revision of the manuscript. breeding insight was funded for this work through cooperative agreements between usda-ars and cornell (project numbers: 8062-21000-043-004-a, 8062-21000-052-002-a, and 8062-21000-052-003-a). marcelo mollinari was funded by a usda nifaawarded afri grant (project number: 2022-6701336269). author contributions dz, nb, and mjs contributed to experimental design and planning. dz, nb and mjs selected the diversity panel for wgs. nb collected and prepared all plant materials used in the study. dz performed all the wgs analyses, snp database creation, filtering pipelines, and quality control analyses to create the 3k panel. khu managed the panel creation at diversity arrays technology. dz, ms and mm executed the data analyses and genetic mapping. mfg and mi assisted with ‘draper’ and ‘jewel’ parental identification. dz, ms and mjs wrote the initial draft of the manuscript. cb managed experiments and 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(2023). a public mid-density genotyping platform for alfalfa (medicago sativa l.). genet resourc j 4(8), 55–63. doi: https://doi.org/10. 46265/genresj.emor6509 https://doi.org/10.1534/g3.119.400620 https://doi.org/10.1534/g3.119.400620 https://downloads.usda.library.cornell.edu/usda-esmis/files/zs25x846c/zk51wx21m/k356bk214/ncit0523.pdf https://downloads.usda.library.cornell.edu/usda-esmis/files/zs25x846c/zk51wx21m/k356bk214/ncit0523.pdf https://downloads.usda.library.cornell.edu/usda-esmis/files/zs25x846c/zk51wx21m/k356bk214/ncit0523.pdf https://doi.org/10.2307/2446540 https://doi.org/10.1007/bf00220946 https://doi.org/10.1007/bf02680255 https://doi.org/10.1007/bf02680255 https://doi.org/10.1371/journal.pone.0222640 https://doi.org/10.1371/journal.pone.0222640 https://blueberry.org/about-blueberries/history-of-blueberries/ https://blueberry.org/about-blueberries/history-of-blueberries/ https://doi.org/10.1038/s41598-020-73321-8 https://doi.org/10.1038/s41598-020-73321-8 https://doi.org/10.46265/genresj.emor6509 https://doi.org/10.46265/genresj.emor6509 introduction materials and methods germplasm selection and whole-genome sequencing of a blueberry diversity panel snp discovery and selection of 3k marker loci for building dartag genotyping panel selection of samples for validating the dartag panel and genotyping results genetic map construction results validation of the 3k blueberry dartag panel and genotyping results panel effectiveness in extant accessions creation of a linkage map discussion and conclusion data availability statement supplemental data acknowledgements author contributions conflict of interest statement original article genetic resources (2024), 5 (9), 1–12 doi: 10.46265/genresj.nlvc6810 https://www.genresj.org issn: 2708-3764 leaf trichome diversity, acylsugar concentration, and their relationships to leaf area in solanum galapagense ilan henzler a and hamid khazaei *,a,b a world vegetable center, shanhua, tainan, 74151, taiwan b current address: natural resources institute finland (luke), helsinki, finland abstract: glandular trichomes are physical and chemical barriers used by some tomato wild relatives to confer resistance against insect pests and diseases transmitted by them. solanum galapagense has been identified a s o ne o f t he potential sources of insect pest resistance. the present study aimed to examine the trichome diversity and acylsugar concentration of 26 accessions of s. galapagense along with one cultivated tomato (s. lycopersicum) and one cherry tomato (s. l. cerasiforme) cultivar. the results revealed large phenotypic variation among s. galapagense accessions for all studied traits. the s. galapagense accessions had significantly higher t richome types iv density on the adaxial and abaxial surfaces of the leaf and greater acylsugar concentration but a smaller leaflet area than the cultivated tomato. the selected cherry tomato line represents greater trichome type iv density and acylsugar concentration than other groups. the acylsugar concentration was positively associated with trichome type iv but negatively associated with trichome type v on both leaf surfaces. dna markers revealed the presence of two previously identified whitefly-resistance alleles in s. galapagense accessions. this study will support breeding programmes aiming to improve insect pest resistance in tomato cultivars using crop wild relatives. keywords: acylsugar, crop wild relatives, leaflet area, s. galapagense, tomato, trichomes citation: henzler, i., khazaei, h. (2024). leaf trichome diversity, acylsugar concentration, and their relationships to leaf area in solanum galapagense. genetic resources 5 (9), 1–12. doi: 10.46265/genresj.nlvc6810. © copyright 2024 the authors. this is an open access article distributed under the terms of the creative commons attribution license (cc by 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. introduction cultivated tomato (solanum lycopersicum) is the most valuable vegetable crop by fruit weight globally, generating revenues of us$70 billion from 187 million tonnes of fresh fruit in 2020 (fao, 2022). improving fruit quality and yield through domestication in this crop, has led to the loss of important plant defence characteristics (paudel et al, 2019), with tomato cultivation now heavily relying on pesticides to control biotic stresses (dari et al, 2016). the chemical treatments are not only costly, but they also harm the environment (damalas and eleftherohorinos, 2011). developing resistant tomato cultivars could reduce the reliance on pesticides and their associated burden. ∗corresponding author: hamid khazaei (hamid.khazaei@gmail.com) tomato wild relatives are important sources of genetic diversity and are commonly used as reliable sources of resistance genes against biotic and abiotic stress (ebert and schafleitner, 2015; khazaei and madduri, 2022). sources of resistance to insect pests have been identified in some tomato wild species, including s. galapagense, s. habrochaites, s. pennellii, s. cheesmaniae, and s. pimpinellifolium (kennedy, 2003; schilmiller et al, 2012; leckie et al, 2016; rakha et al, 2017b; vosman et al, 2018). among them, s. galapagense has been identified as one of the most promising sources of insect pest resistance (firdaus et al, 2012; lucatti et al, 2013). it has been the focus of most tomato breeding programmes aiming to improve biotic and abiotic stress resistance due to its close relationship to cultivated tomatoes (vendemiatti et al, 2022). the s. galapagense species originates from the galápagos islands, an archipelago 1,000km west of ecuador, where received: 04.10.2023 accepted: 21.12.2023 published online: 16.01.2024 https://www.genresj.org https://www.dx.doi.org/10.46265/genresj.nlvc6810 https://www.genresj.org https://www.dx.doi.org/10.46265/genresj.nlvc6810 mailto:hamid.khazaei@gmail.com 2 henzler and khazaei genetic resources (2024), 5 (9), 1–12 it formed a diverse range of phenotypes due to the islands’ unique ecosystem (darwin et al, 2003). while genetic studies revealed a narrow genetic diversity within the s. galapagense germplasm (darwin, 2009; pailles et al, 2017), it presents distinct morphological characteristics. these include yellow-green foliage, orange fruit at maturity, small seed size and highly divided leaves (darwin et al, 2003; fenstemaker et al, 2022). plants have developed a variety of defence mechanisms to counter biotic and abiotic stress conditions (levin, 1973; oksanen, 2018). one of these is the presence of fine outgrowths, called trichomes, on the surface of flowers, fruits, stems and leaves as physical and chemical lines of defence. numerous studies have been conducted on the nature of these epidermal outgrowths, including function, quantification and effectiveness (glas et al, 2012; vendemiatti et al, 2022). seven types of trichomes are characterized on plants, four are termed glandular due to the cells at their tip which can store and secrete metabolites (luckwill, 1943). the presence of glandular trichome types iv and vi has been associated with higher insect pest resistance (lucatti et al, 2013; firdaus et al, 2013; rakha et al, 2017b; zhang et al, 2020). these types of glandular trichomes deter insects through the release of secondary metabolites such as acylsugars, which cause behaviour changes and reduced survival in the arthropods that land on them (antonious et al, 2005; bleeker et al, 2011, 2012; dias et al, 2016). in addition to acylsugars, other trichome metabolites such as terpenoids, methylketones and flavonoids play a key role in plant defence mechanisms (huchelmann et al, 2017). two major genomic regions conferring whitefly resistance (wf-1 and wf-2), largely based on glandular trichomes type iv, have been identified in s. galapagense (accession id pri95004; (firdaus et al, 2013; vosman et al, 2019)). most likely, they regulate the formation of glandular trichome type iv on the leaf epidermis and subsequently control the accumulation of acylsugar on trichome type iv. trichome diversity and density, and their relationship with insect pest resistance have been investigated in tomato wild relatives, including s. galapagense (firdaus et al, 2012; lucatti et al, 2013; rakha et al, 2017b). an aspect deserving further attention is to harness the genetic diversity of morphological and biochemical characteristics of large germplasm of s. galapagense accessions and their relationships with leaf area. so, this study aims to uncover differences in trichome diversity, leaf characteristics and acylsugar concentration in this species. this is supported by the analysis of dna markers associated with insect pest resistance phenotypes (firdaus et al, 2013). materials and methods plant material this study was conducted on 26 accessions of s. galapagense, one accession of cherry tomato (s. lycopersicum var. cerasiforme, abbreviated as s. l. cerasiforme), and one cultivated tomato (s. lycopersicum). detailed information on accession number, origin and habitat at collection sites are presented in table 1. all s. galapagense accessions originated from the galápagos islands, ecuador (figure 1). more than a third were collected on isla isabela, the largest island. this study is the first to screen accessions vi037867, vi037869, vi045262, vi057457, vi063173, vi063178, vi063179, vi063181, vi063182 and vi063183 for insect-pest resistancerelated traits. the cultivated tomato is a breeding line from the world vegetable center (worldveg) carrying multiple tomato yellow leaf curl virus resistance genes (ty-1/3 and ty-2). the sm131 cherry tomato is a selection from accession vi063893 due to its high density of trichome type iv (unpublished data). all accessions were acquired from the worldveg genebank. seed treatment tomato seeds acquired from the worldveg genebank were treated with hydrogen chloride for 15 minutes and washed under running water. they were then treated with trisodium phosphate for one hour, washed under running water and dried in an incubator room at 60% for two days. growing conditions experiments were conducted in a glasshouse at the worldveg in shanhua, taiwan. seeds were sown in a nursery on 25 february 2022, and after two weeks, were transplanted into 8-inch pots filled with cultivable soil collected from tomato fields. the pots were arranged in a randomized complete block design with four replicates. plants were watered once a day in the morning and fertilized with a blend of 15–15–15 (n–p–k) at week four after transplanting. relative humidity was about 80±15%. the temperature was set to 28±3◦c during the day and to 22±2◦c during the night. measurements leaflet area the leaflet area was measured ten weeks after sowing using the third leaf from the apex. it was measured using a li-3100 leaf area meter (li-cor inc., lincoln, ne, usa). the same leaflet was also used for subsequent trichome and acylsugar measurements. trichome analysis analysis of leaf trichomes was conducted eight and ten weeks after sowing using a stereo microscope (leica® m-series stereo microscopes, ernst leitz wetzlar, gmbh, germany). leaf samples were collected at the third node from the apex using sterile forceps. the density of glandular trichome types i, iv, v and vi were measured from four randomly chosen leaflets within 1mm2. g enetic resources (2024),5 (9),1–12 d iversity of trichom es and acylsugar in solanum galapagense 3 table 1. species, accession number, origin and habitat at collection sites of solanum species used in this study. more information about accessions can be found at https://genebank.worldveg.org/ (accessions with ‘vi’ code) and http://www.ars-grin.gov/ (accessions with ‘pi’ code). passport data was obtained for accessions with ‘la’ code from the tgrc, http://tgrc.ucdavis.edu/. *, this accession was first classified as s. cheesmaniae and later reclassified as s. galapagense. species accession no. origin elevation (m) other name(s) habitat/phenotype s. galapagense vi007099 bartolome, galápagos islands 15 la0317, pi231257 lava flow, amongst basalt rock, very arid, coastal arid zone vi037239 isabela, galápagos islands 40 la0436 sandy, near lava outcrop vi037241 pinta, galápagos islands 150 la0526, sal254 west side abingdon island vi037339 isabela, galápagos islands 5 la1401, pi365897 among rocks in large beach with magma and lava flows at each end, collected few metres above tide line vi037340 isabela, galápagos islands 200 la1408, pi379039 ridge of cape berkeley volcano vi037867 floreana, galápagos islands la1136, tl01054 garnder near floreana islet vi037868 rabida, galápagos islands 10 la1137, tl01055 on cinder ash vi037869 santiago, galápagos islands 650 la1141, tl01056 interior walls of crater, purple fruit colour (fenstemaker et al, 2022) vi045262 santiago, galápagos islands selection from la1141, tl01572 vi057400 fernandina, galápagos islands la483, 6201a, sal241 vi057408 isabela, galápagos islands selection from la1401 vi057457 galápagos islands la3909 vi063173 bartolome, galápagos islands 15 la0317 lava flow, amongst basalt rock, very arid vi063174 isabela, galápagos islands 30 la0438, sal192 rocky basalt outcropping in first hills to w. (7km) from villamil, 1km from coast vi063175 isabela, galápagos islands 20 la0480a, sal238 along coast in bay facing cowley islet not far from shore, in broken terrain without shade vi063176 santa cruz, galápagos islands la0528, sal256 academy bay vi063177 fernandina, galápagos islands 580 la0530, sal258 inside crater at edge vi063178 galápagos islands 5 vi063179 santiago, galápagos islands 6 la0747 in lava formation near shore vi063180 santiago, galápagos islands 3 la0748 in lava formation vi063181 isabela, galápagos islands 4 la0929 growing in lava, roots in sand, above sea level continued on next page 4 h enzler and k hazaei g enetic resources (2024),5 (9),1–12 table 1 continued species accession no. origin elevation (m) other name(s) habitat/phenotype vi063182 isabela, galápagos islands 4 la0930 growing in lava, roots in sand, same as la 0929 vi063183 bartolome, galápagos islands la1044 vi063184 isabela, galápagos islands 400 la1452 on the trail from punta ecuador to crater rim mid-elevation in longer of two lava flows vi063185 isabela, galápagos islands 100 la1627 volcanic cone above darwin’s salt lake likely tagus cove vi063187* santiago, galápagos islands 10 la1411, pi379040 on soft bright red rock formation, margin of beach s. l. cerasiforme vi063893 fernandina, galápagos islands sm131 s. lycopersicum cln3682c breeding line avto1424 pedigree: cln3682f1-10-3-4-27-3-16 genetic resources (2024), 5 (9), 1–12 diversity of trichomes and acylsugar in solanum galapagense 5 the number of trichomes was counted from four different microscopic fields at 5x magnification and converted to the number per mm2 using a standard scale. the identification of trichome types on the leaf surface followed a schematic drawn by luckwill (1943). after measuring trichomes on the adaxial surface, the leaflets were flipped to measure the trichomes on the abaxial surface. acylsugar concentration analysis of acylsugar content was conducted at eight and ten weeks after sowing. polyethylene vials were used to collect four 3±1cm lateral leaflets from each plant at the third node from the apex. samples were dried in an incubator at 29◦c for three days before washing them with 3ml methanol. of this suspension, 100µl was added to 100µl 6m ammonium hydroxide in 96 well elisa plates with two biological replicates, following a protocol developed by martha mutschler (savory, 2004). the samples were incubated overnight and left to dry under the hood for three days before adding 200µl pgo reagent to each well and placing it on an orbital shaker. after three hours, absorbance values at 490nm were measured using biotek’s uquant (agilent technologies inc., santa clara, ca, usa) and converted into acylsugar concentration using a sucrose standard curve. dna extraction and dna marker assay genomic dna was extracted from 10-week-old plants using the ctab method (doyle and doyle, 1990). the wf-1 and wf-2 detailed marker sequences presented in firdaus et al (2013) were used for genotyping the studied germplasm for the presence/absence of corresponding bands. the term ‘wf ’ stands for whitefly and represents markers previously identified for whitefly resistance. these markers are located in tomato chromosomes 2 and 9, respectively. purified dna samples were digested with restriction enzymes ddei and hpych4iv for wf-1 and wf-2 markers, respectively. digested samples were amplified along with markerspecific primers using pcr reactions as described by mahfouze and mahfouze (2019). the pcr-amplified samples were run on a 5% acrylamide gel for 30 minutes at 100v and stained using an etbr-out stain. the gel was scanned in a bio-1000f scanner, and the amplified bands visualized using microtek mibio fluo software (both from microtek international, inc., hsinchu, taiwan). statistical analysis the r statistical package (r core team, 2021) was used for data analysis. correlation analysis was performed to determine the relationships between morphological measurements. the dataset was subjected to a oneway analysis of variance (anova), and the sem (standard error of means) was calculated. principal component analysis (pca) was employed to illustrate relationships between accessions and leaf morphological measurements. the online mapping tool at maps.co was used to plot the coordinates of accessions in figure 1 (https://maps.co/). the geographic coordinates of s. galapagense accessions were obtained from the tomato genetics resource center (c.m. rick tgrc, https:// tgrc.ucdavis.edu/) and the worldveg (https:// genebank.worldveg.org/#/) genebank databases. results trichome densities varied significantly a m ong studied germplasm (table 2 and supplemental table 1). trichome type iv density ranged from 6.3 to 13.5 for the abaxial and 0.7 to 10.9 for the adaxial surface of s. galapagense accessions, while the cultivated tomato (cln3682c) had none on either surface. accessions vi057408, vi063174, vi063177, vi063185 and vi057400 had the greatest number of trichome type iv on both surfaces. the cherry tomato (vi063893) had 22% greater (on both surfaces) trichome type iv compared to the average values of s. galapagense accessions. within s. galapagense accessions, trichome type vi varied from 0.4 to 2.7 on the abaxial and 0.3 to 2.8 on the adaxial side. for the abaxial surface, this was 30% and 14% lower than cultivated and cherry tomato cultivars, respectively. for the adaxial surface, it was 94% lower and 30% higher than cultivated and cherry tomatoes, respectively (table 2). most studied accessions had fewer trichomes on the adaxial than on the abaxial side, with 13% less for type iv and 40% less for type vi. comparing 8and 10-week trichome phenotyping at the abaxial surface, s. galapagense trichome densities remained stable with a 3% increase for type iv, and a 12% decrease in type vi. acylsugar concentration varied significantly (p < 0.001) among s. galapagense accessions, ranging from 5.43 to 58.03 µmol/g. the cultivated tomato cultivar (cln3682c) showed a very low acylsugar concentration of 0.94µmol/g and the cherry tomato (vi063893) showed a moderate level of 21.02µmol/g (table 2). on average, 10-week-old s. galapagense accessions had 45% greater acylsugar concentrations than 8-week-old plants. accessions vi063181, vi037869 and vi045262 had the highest concentration of acylsugar, all above 50µmol/g. the leaflet area varied significantly (p < 0.001) among s. galapagense accessions, ranging from 2.43 to 9.15cm2. leaflet area was significantly greater for cultivated tomato, with 14.36cm2 than for all s. galapagense accessions. on average, cherry tomato leaflets were 10% larger than s. galapagense accessions (table 2). correlations between trichome types and acylsugar concentration are presented in table 3. acylsugar concentration was positively associated with trichome type iv and negatively associated with trichome type v. the negative correlation between acylsugar and trichome type vi was only significant at the 10-weekold plant stage. in addition, leaflet area was negatively correlated with trichome iv density of abaxial surface 6 henzler and khazaei genetic resources (2024), 5 (9), 1–12 figure 1. geographical distribution of s. galapagense accessions in the galápagos islands. (p < 0.001, figure 2a) and acylsugar concentration (p = 0.078, figure 2b). the pca plot (figure 3) showed three clusters based on acylsugar, trichome type and leaf area measurements. the first g roup c onsisted o f s . g alapagense accessions and the cherry tomato genotype (vi063893), which was correlated with high acylsugar concentration as well as high levels of trichome type iv on the adaxial surface. the cultivated tomato (cln3682c) was separated from s. galapagense group mainly due to its greater leaflet area and high levels of trichome type v. one of the s. galapagense accessions, vi007099, was grouped closer to the cultivated tomato mainly due to its larger leaflet area (its leaf morphology was similar to cultivated tomatoes) compared to other accessions from the galápagos islands. the dna marker results are presented in supplemental table 3. all accessions of s. galapagense showed both resistance bands at 46 and 40bp for the wf-2 marker (indicated by ‘++’) while both s. lycopersicum cultivars (cln3682c and vi063893) showed the susceptible band at 87bp (indicated by ‘-‘). the wf-1 marker showed a resistance band at 139bp (indicated by ‘+’). both s. lycopersicum cultivars showed the susceptible band at 157bp (indicated by ‘-‘). discussion in this study, we screened a large germplasm collection of s. galapagense accessions revealing a considerable phenotypic variation in glandular trichome density and acylsugar concentration. our results indicated that more than 50% of s. galapagense accessions had acylsugar concentration more than 30µmol/g – on average, 34 times greater than the studied s. lycopersicum (cln3682c) genotype. however, not all s. galapagense had high concentrations and trichomes types iv and vi density. there was a 12-fold difference between the greatest and lowest s. galapagense accessions for acylsugar concentration. however, only a 2-fold variation was observed for trichome type iv density in this species. our results also showed that tomato plants with smaller leaves had higher densities of trichome type iv. the selection of tomato plants with higher densities of glandular trichomes iv and vi has shown to be an effective criterion for obtaining superior resistance to insect pests. this has been widely proven in resistance to spider mites (tetranychus urticae, andrade et al (2017); rakha et al (2017a); de souza-marinke et al (2022)), whitefly (bemisia tabaci, lucatti et al (2013); rakha et al (2017b)), thrips (escobar-bravo et al, 2017), and cabbage looper caterpillar (mymko and avilasakar, 2019). these studies generally attribute insectpest resistance to the excretion of acylsugars by the tip of glandular trichome type iv, thereby acting as a biopesticide. several studies have reported genetic diversity for trichome density and acylsugar concentration in tomato (lucatti et al, 2013; baier et al, 2015; rakha et al, 2017b). these have allowed tomato breeding programmes globally to exploit the available diversity and improve insect-pest resistance cultivars. we screened a relatively larger number of s. galapagense accessions (26) compared to previous studies, which genetic resources (2024), 5 (9), 1–12 7 table 2. mean ± sd (standard deviation) for trichome iv and vi measurements (abaxial and adaxial surfaces) at ten weeks after sowing and acylsugar concentration and leaflet area on 26 solanum galapagense accessions along with one cherry tomato and one cultivated tomato genotype. sem, standard error of means. trichome types per mm2 – abaxial trichome types per mm2 – adaxial acylsugar (µmol/g) leaflet area (cm2) species accession no. iv vi iv vi s. galapagense vi063181 8.0±5.7 1.6±1.3 7.1±1.6 2.6±0.9 58.02±8.32 8.01±1.84 vi037869 10.5±1.6 0.6±0.4 9.1±1.8 0.2±0.5 57.68±15.42 6.12±2.67 vi045262 11.3±1.2 1.0±0.4 8.7±1.3 0.4±0.1 54.02±11.27 5.95±1.56 vi057408 13.5±1.3 1.0±0.5 9.1±0.7 2.8±0.7 46.64±12.57 3.83±0.65 vi063184 10.1±1.1 1.1±0.7 7.5±1.8 1.4±0.3 45.56±4.43 5.92±0.95 vi063187 9.2±1.3 1.0±0.8 8.6±1.4 0.5±0.2 44.25±9.63 3.81±1.45 vi063185 12.0±0.5 1.5±0.3 9.0±3.0 1.1±0.9 43.49±5.71 5.15±0.45 vi057400 11.8±0.9 0.9±0.6 10.8±0.8 0.3±0.3 43.07±4.51 4.40±0.18 vi063177 12.1±1.0 1.1±0.5 9.7±2.5 0.4±0.1 41.75±11.28 5.15±1.46 vi037241 10.1±1.4 0.3±0.1 6.0±1.4 0.5±0.2 34.00±0.01 2.88±0.17 vi037339 7.7±0.6 0.7±1.5 9.3±2.5 0.3±0.0 32.40±0.01 6.89±0.0 vi063182 11.1±1.9 0.8±0.3 8.5±1.0 0.6±0.3 32.39±6.25 6.15±1.78 vi057457 10.0±2.7 2.5±1.5 9.0±1.2 0.3±0.0 31.87±6.97 2.65±1.01 vi063174 12.4±0.9 1.0±0.5 10.8±1.5 1.2±0.6 30.58±8.12 6.81±3.33 vi063179 6.2±5.5 2.0±1.8 4.3±0 1.3±0.0 25.20±1.86 6.59±0.83 vi037340 11.3±1.3 1.1±0.9 8.5±1.5 0.8±0.6 24.65±2.52 7.07±1.03 vi037239 9.7±0.7 1.2±0.7 9.5±1.1 1.1±0.2 24.60±0.01 6.60±1.2 vi063175 12.0±1.4 2.6±1.6 7.5±1.1 1.6±1.4 22.53±5.67 4.60±1.62 vi037868 10.6±2.3 1.8±0.4 9.5±1.6 1.4±0.5 22.08±5.05 5.40±1.43 vi063183 10.3±1.5 2.4±1.4 7.2±1.7 0.8±0.1 22.07±1.52 2.43±1.66 vi037867 9.0±0.4 1.5±0 7.7±1.8 0.7±0.5 18.46±1.81 5.17±0.38 vi063176 9.3±5.3 0.9±0.9 7.7±5.4 0.5±0.5 16.06±4.36 4.37±1.86 vi063173 11.6±0.5 2.4±1.6 9.4±2.8 1.6±1.2 15.29±4.70 5.46±1.92 vi063180 10.3±1.8 1.4±0.5 8.1±2.5 0.3±0.3 15.23±2.15 7.07±1.77 vi063178 10.0±1.3 1.6±1.5 8.7±2.7 1.1±0.3 14.8±3.61 5.32±0.98 vi007099 6.6±2.9 1.6±0.5 0.6±0.8 1.8±0.5 5.43±5.88 9.15±5.5 range (s. galapagense) 6.2–13.5 0.3–2.6 0.6–10.9 0.2–2.8 5.43–58.02 2.43–9.15 mean (s. galapagense) 10.3±1.7 1.4±0.6 8.2±2.0 1.0±0.7 31.6±14.9 5.50±1.59 s. l. cerasiforme vi063893 12.6±1.5 1.6±0.4 10.0±2.2 0.7±0.3 21.02±15.93 6.10±0.94 s. lycopersicum cln3682c 0.0 2.0±0.8 0.0 4.6±2.3 0.94±0.23 14.36±3.64 sem 1.7 0.2 1.6 0.2 16.95 1.3 table 3. correlations between trichome types and acylsugar (as) concentration at 8-week (n = 26) and 10-week (n = 28) intervals on abaxial surface. *, p < 0.05; **, p < 0.01. data for 8-week measurements is presented in supplemental table 2. trichome type as (8-week-old) as (10-week-old) type i -0.140 -0.238 type iv 0.473* 0.410* type v -0.540** -0.459* type vi -0.347 -0.471* diversity of trichomes and acylsugar in solanum galapagense 8 henzler and khazaei genetic resources (2024), 5 (9), 1–12 figure 2. relationships between trichome type iv (a) and acylsugar concentration (b) with leaflet area at 10-week-old seedlings (n = 28). the red triangle and red square represent cherry and cultivated tomato cultivars, respectively. figure 3. the biplot illustrates the principal components (pc) analysis for the 26 accessions of solanum galapagense and two s. lycopersicum lines with measurements as vectors. vectors that are close together are correlated in terms of the leaf measurements. ad, adaxial surface; ab, abaxial surface. i, iv, v, and vi refer to trichome types. genetic resources (2024), 5 (9), 1–12 9 only characterized 10 (rakha et al, 2017b) or 11 accessions (lucatti et al, 2013). our study revealed a wider variation for trichome type iv, however, smaller values for acylsugar concentration compared to a similar study by rakha et al (2017b). the cherry tomato genotype (vi063893) was previously characterized by high trichome type iv density (unpublished data). our results confirm that its trichome type iv density was higher than 96% of studied s. galapagense accessions (table 2). this cherry tomato genotype may be used as a source of insect resistance in cherry and cultivated tomato germplasm. a moderate positive correlation (p < 0.05) was observed between acylsugar concentration and trichome type iv at two different sampling times. previous studies also reported a similar trend (lucatti et al, 2013; rakha et al, 2017b). a possible explanation for this could be the poor phenotyping of trichomes under the microscope, as counting the number of trichomes is an inherently delicate task. this difficulty highlights the need for high throughput methods to measure trichomes. another explanation for the lack of strong correlation between trichome iv density and acylsugar could be that acylsugar production is not solely linked to trichome density but also their metabolic activity, whereby the same trichome types in different accessions produce varying levels of acylsugar (zhang et al, 2008; bergau et al, 2015). following this reasoning, isolated trichomes could be tested for metabolic activity through gc-ms as described for l. hirsutum by fridman et al (2005). a negative correlation was observed between trichome type iv and leaflet area, a trend that has been reported in other plant species, including s. berthaultii hawkes (pelletier, 1990) and silver birch (betula pendula roth) (lihavainen et al, 2017). mymko and avilasakar (2019) reported that unexpanded leaves had greater trichome density and resistance than expanded (larger) leaves at different growth stages of tomatoes. in our study, leaflet area was one of the main drivers in allocating tomato species into three different groups (figure 3). accession vi007099 had the greatest leaflet area and lowest trichome density and acylsugar concentration among s. galapagense and presented leaf morphology between wild and cultivated tomatoes. on the other hand, accession vi063181 had the second-largest leaflet area among s. galapagense accessions but also the greatest acylsugar concentration among all accessions. this controversy was also evident by the weak correlation between leaflet area and acylsugar concentration (figure 2). these results suggest that acylsugar concentration may not be derived by leaf size and trichome type iv in s. galapagense germplasm. no clear pattern between leaf trichome measurements and accessions’ geographical origin in the galápagos islands was observed. the only accession from pinta island, vi037241, was separated from other s. galapagense. pinta island is the northernmost of the main islands and has a semi-arid ecosystem (hunter and gibbs, 2014). the main driver for the deviation of other s. galapagense from the core cluster (figure 3) was leaflet area (vi063181, vi063179, vi037339 and vi007099) and higher trichome type vi density (vi063175 and vi063183). the three accessions with the highest trichome type iv density were all found among volcanic rock with vi057408 and vi06374 found within 1km of the sea on isla isabela and vi063177 inside a volcanic crater on isla fernandina. an explanation for this correlation could be the presence of silicon in volcanic soil as it has been shown to boost plants’ resistance to pests by accumulating magnesium at the base of trichomes (ma, 2004). some s. galapagense accessions originating from the galápagos islands have been exposed to dry and saline growing conditions (pailles et al, 2020) and high insect pressure (peck, 2008), thus may represent a generous source of alleles that can be explored to improve biotic and abiotic stress. as this species can easily hybridize with cultivated tomatoes (rick, 1961), they have been used as donors for stress tolerance genes, which could be transferred into commercial varieties by introgression breeding (zamir, 2001). for example, vi037339 (la1401) and vi007099 accessions have already been utilized as donors of high trichome iv density into modern cultivated tomato cultivars through interspecific crosses (andrade et al, 2017; rakha et al, 2017b; dasilva et al, 2019; vendemiatti et al, 2022). however, in this study, these accessions were not among those with the highest trichome type iv density and acylsugar concentration. this could be due to the genotype-by-environment interactions as the experiments were carried out under different growing conditions. from the analysis of dna markers, we could see that most s. galapagense accessions were homozygous for wf-1 and wf-2 but neither s. lycopersicum cultivars. this suggests that the morpho-chemical measurements in this study were linked to the genetic background of s. galapagense accessions. however, the cherry tomato genotype vi063893 (sm131), which showed high levels of trichome type iv and acylsugar, did not amplify either band of the dna markers. this was not surprising as those dna markers were developed from an interspecific population derived from s. galapagense (firdaus et al, 2013). a potential reason why the cultivated tomato accession (cln3682c) did not show the resistance markers is that the related genes may have been lost during domestication. we conclude that wf-1 and wf2 may be more suitable to be used in genetic materials derived only from s. galapagense. the other possibility is that the source(s) of resistance in vi063893 is nonallelic to wf-1 and wf-2. in conclusion, our study focused on screening a large s. galapagense germplasm, supporting breeding programmes aiming to improve insect-pest resistance in tomatoes using crop wild relatives. the ultimate goal is to develop tomato cultivars with insect-pest resistancerelated traits that help farmers reduce pesticide use diversity of trichomes and acylsugar in solanum galapagense 10 henzler and khazaei genetic resources (2024), 5 (9), 1–12 and produce a high-quality and chemical-free tomato crop. the glandular trichome density and chemistry are highly affected by the genotype by environment interactions (wang et al, 2021). this needs to be considered when selecting these traits under field conditions. supplemental data supplemental table 1. mean ± sd (standard deviation) for trichomes i and v measurements (abaxial and adaxial surfaces) supplemental table 2. trichome density and acylsugar concentration at 8-week-old seedlings of s. galapagense supplemental table 3. dna marker assay for b. tabaci whitefly resistance alleles wf-1 and wf-2. n, missing data data availability the data that support this study will be shared upon reasonable request to the corresponding author. conflicts of interest the authors declare no conflicts of interest author contributions ih and hk did the conceptualization, data curation and formal analysis, led the methodology, and created the original draft of the manuscript. acknowledgement we would like to thank yun-che hsu (grace) and jean lin for their kind assistance during the experiments. we also thank dr roland schafleitner (flagship leader, vegetable diversity & improvement) and dr maarten van zonneveld (genebank manager) for their valuable suggestions during the experiments. in addition, the first author would like to thank the national cheng kung university (ncku), taiwan for its support. funding financial support was provided by long-term strategic donors to the world vegetable center: taiwan, uk aid from the uk government, the united states agency for international development (usaid), the australian centre for international agricultural research (aciar), germany, thailand, philippines, korea, and japan. references andrade, m. c., silva, a. a. d., neiva, i. p., oliveira, i. r. c., de castro, e. m., francis, d. m., and maluf, w. r. 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(2024). a case study on lentil to demonstrate the value of using historic data stored in genebanks to guide the selection of resources for research and development projects. genetic resources 5 (10), 139–153. doi: 10.46265/genresj.hlsn8777. © copyright 2024 the authors. this is an open access article distributed under the terms of the creative commons attribution license (cc by 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. introduction sustainable agriculture and global food security depend on the availability of well-described plant genetic resources. however, differences in the methods used to evaluate and describe germplasm held in genebanks in different countries, and in the accessibility of data, are a major obstacle, leading to the underutilization ∗corresponding author: nadiia vus (vus.nadezhda@gmail.com) of these resources and negatively impacting future prospects (egan et al, 2022). a better understanding of the stability and potential of measured traits can be achieved by considering data from different experiments, thereby improving the prospects of using genetic resources in novel breeding programmes (cristobal et al, 2014). consequently, genetic resource collections can become more utilized through enhanced cooperation and sharing not only seeds but also the accumulated knowledge gained over many years of resource regeneration and/or research (guerra-garćıa et al, 2021). received: 15.03.2024 accepted: 21.11.2024 published online: 16.12.2024 https://www.genresj.org https://www.doi.org/10.46265/genresj.hlsn8777 https://www.genresj.org https://www.doi.org/10.46265/genresj.hlsn8777 mailto:vus.nadezhda@gmail.com 140 vus et al genetic resources (2024), 5 (10), 139–153 lentil (lens culinaris medik.) is one of the most important pulse crops and is an essential component of a balanced diet. historically, the primary producers of lentils have been asian countries and canada. in europe, lentils account for only 4% of the total area devoted to pulses. eu countries produce significantly fewer lentils than required to meet domestic consumption needs (terresunivia, 2021). france and spain are the main producers and consumers of lentils within the european union. the area harvested for lentils has doubled between 2010 and 2018 (kaale et al, 2023). the expansion of lentil production in europe depends on the development of novel, advanced, high-yielding and nutritionally improved varieties (rajpal et al, 2023). the initial step in this process is a comprehensive review and analysis of the available genetic resources in genebanks to identify suitable sources for selecting and crossing in order to breed elite lines. the creation of special medium-core and mini-core collections allowed for more detailed study of traits, systematization of accumulated material and information, and generally better management of research (bisht et al, 1998; d́ıez et al, 2018). however, starting this process from scratch is long and costly. it is, therefore, more expedient to utilize the material and information available in genebanks to accelerate the preliminary phase. some genebanks, such as the indian national gene bank (icar), which currently hosts 2,324 lentil accessions, have initiated this process. this genebank has characterized the entire collection for dozens of agromorphological traits to identify the most suitable accessions for use in breeding programmes (tripathi et al, 2022). the lentil collection of the national plant genetic resources centre of ukraine (ncpgru) in kharkiv was established in 1993 and currently comprises 1,140 accessions, which were collected through interbank exchanges, independent expeditions and collaboration with breeders (kobyzeva et al, 2011; kir’yan et al, 2014; vus et al, 2020c). all genotypes were subjected to a comprehensive study over three years, during which their main phenotypic, morphological and agronomic characteristics were outlined. based on the collected data, a basic collection, an educational collection and a reference basic set were created and registered (bezuhla and kobyzeva, 2021). accessions that successfully completed the research cycle and exhibited specific traits were included into a specialized trait-oriented database. this database served as the source of information for the first exchange of lentil seed material between french researchers and ncpgru, which took place in 2019. this exchange facilitated evaluating the performance of selected samples under different climatic conditions. this also enabled the assessment of the potential of using additional data from the donor genebank for a more comprehensive analysis of the genotypes. developing methods for comparing accessions despite the differences in cultivation and research techniques, as well as the soil and climatic characteristics of the regions, was particularly significant. information on accessions from the ncpgru database collected during the period from 1993 to 2016 was processed for a preliminary evaluation and recommendation of genotype characteristics and a preliminary forecast of their use in breeding programmes. the results of field studies conducted in ukraine (2019) and france (2021) were used to assess the accuracy of the provided forecasts, clarify the outcomes of the research, and refine the methodological approaches employed in such studies. the aim of this research was to analyze data from the ncpgru traits database to identify the most promising sources for breeding. the results were evaluated through trials conducted in two ecogeographical locations. the methods used took into account the specificity of each trial in terms of experimental design and choice of standards. materials and methods plant material a total of 37 lentil (lens culinaris medik.) accessions from ncpgru were included in this study, 29 of them belong to the subsp. microsperma (100-seed weight < 4.5g), while the rest belongs to subsp. macrosperma (100-seed weight > 4.5g). the accessions under study were from 19 countries (table 1). transferring of lentil accessions was accompanied by smta agreement, according to the terms of the international treaty on plant genetic resources for food and agriculture (fao, 2009). for the list of 37 lentil accessions, which were transferred to the genebank of france (inrae, dijon), a preliminary evaluation was carried out according to the characteristic database of the genebank of ukraine. preliminary recommendations for their use were provided, together with a comparative assessment of their resistance to stress factors and potential productivity in comparison with standard accessions, based on phenotypic data during historic regenerations (1993-2016). in order to verify the applicability of the given prediction based on preliminary data from the genebank of ukraine, two studies were conducted in ukraine (2019) and in france (2021). figure 1 illustrates the complete data set, showing the accessions and the years in which they were evaluated. field trials the field trials in ukraine were carried out at the plant production institute of named after v. ya. yuriev, kharkiv, ukraine (ppi naas) at the laboratory of genetic resources of grain legumes elitne village, kharkiv district, kharkiv region (49º59’31”n, 36º26’55”e; 95m above sea level). a 4-year crop rotation was applied with winter wheat being systematically grown as the preceding crop for lentil. the experiments were conducted according to the methodical recgenetic resources (2024), 5 (10), 139–153 141 table 1. list of lentil accessions used in this study. the accession id has been provided here to facilitate reference to the accessions. genebank id, accession name, country of origin and subspecies information are provided according to the national plant genetic resources centre of ukraine (ncpgru) database. the accessions were grouped in four clusters, for more details on clustering see figure 4. accession id genebank id accession name country of origin subspecies cluster 1 ud0600006 cf 17-5 morocco microsperma 3 2 ud0600007 mel m 5 france microsperma 1 3 ud0600028 ethiopia microsperma 3 4 ud0600036 krasnohrads’ka 49 ukraine macrosperma 2 5 ud0600052 stepova 244 ukraine microsperma 4 6 ud0600065 giza 9 egypt microsperma 1 7 ud0600084 anicia france microsperma 4 8 ud0600086 mel c 4 morocco microsperma 4 9 ud0600090 dieu printemp foncé france microsperma 1 10 ud0600091 du puy france microsperma 4 11 ud0600092 spatz albinzée france microsperma 1 12 ud0600095 ge ic-p hungary microsperma 1 13 ud0600103 france macrosperma 1 14 ud0600112 krasnohrads’ka 250 ukraine macrosperma 2 15 ud0600119 france macrosperma 1 16 ud0600131 gornostepnaia armenia macrosperma 1 17 ud0600145 code 35 morocco microsperma 4 18 ud0600163 france macrosperma 2 19 ud0600203 flip 86-38l syria microsperma 3 20 ud0600248 syria macrosperma 1 21 ud0600347 ill 481 lebanon microsperma 3 22 ud0600403 nadejda bulgaria microsperma 4 23 ud0600423 cdc sunrise canada microsperma 1 24 ud0600437 israel microsperma 4 25 ud0600443 iran microsperma 4 26 ud0600444 ethiopia microsperma 1 27 ud0600468 france macrosperma 2 28 ud0600490 afghanistan microsperma 1 29 ud0600496 mexico microsperma 2 30 ud0600521 china microsperma 1 31 ud0600530 slovyanka russia microsperma 2 32 ud0600550 bulgaria microsperma 4 33 ud0600563 ethiopia microsperma 4 34 ud0600614 precoz brazil microsperma 1 35 ud0600638 eston canada microsperma 4 36 ud0600686 france microsperma 2 37 ud0601021 beluga israel microsperma 1 ommendations for studying the genetic resources of grain legumes (kobyzeva et al, 2016). the area of each experimental plot was 1m2, and the sowing design was 20cm × 10cm, 6 rows of 10 plants each, or 60 plants per plot. the seeds were hand sown. weeds were removed manually. in accordance with the methodology of genetic resources studies, and considering that no replicates were included, a block of standards sown every 20 plots was systematically added for the correct evaluation of the accessions in the field. for lentil evaluation in ncpgru, the three historically used standard accessions were included in each standard block, namely stepova 244 (ud0600052), krasnohrads’ka 49 (ud0600036) and krasnohrads’ka 250 (ud0600112). field trials in france were conducted at the epoisses experimental unit (u2e), managed by inrae, in bretenière, france (05◦05’57”n, 47º14’11”e; 210m above sea level). agroecological management practices were applied. weeds were removed manually. seeds were sown mechanically and he experimental plot consisted of three rows of 1m each with 20 seeds per row, or 60 plants per plot. three replications per accession with anicia (ud0600084) and beluga (ud0601021) were used as standard accessions. using historic genebank data for material selection 142 vus et al genetic resources (2024), 5 (10), 139–153 figure 1. upset plot showing the years in which each lentil accession considered in this study was sown in the field for seed multiplication and phenotyping, including historical data (1993–2016 in ukraine) and field trials conducted in 2019 in ukraine and 2021 in france. for example, phenotyping data for ud0600006 are available for 1993, 1995, 1996, 1997 and 2019. plant phenotyping traits were characterized according to the lentil crop ontology (agrawal, 2016). phenological and agronomic traits were assessed on each accession. phenological traits included days to 50% of flowering and pod filling period. agronomic or yield component traits included seed yield per area (kg/m2), seed yield per plant (g), number of seeds per plant, height of the lowest pod (cm), plant height (cm) and weight of 100 seeds (g). to allow genotype comparison in a representative way, each individual yield was normalized compared to the median of the standard accessions (cy, %) according to the formula below: cy = ya/ym*100, (1) where cy is the relative productivity (%), ya is the accession’s yield (kg/m2), and ym is the median of standards’ yield (kg/m2). statistical analysis statistical data processing and visualisation were carried out using r software version 4.2.2 (r core team, 2023) and in particular the following packages: tidyverse and rlang (henry and wickham, 2023), openxlsx (schauberger and walker, 2022), ggplot2 (wickham, 2016), cluster (maechler et al, 2022), metan (olivoto and lúcio, 2020) and factoshiny (vaissie et al, 2023). results ncpgru manages the ex situ conservation of 1,140 lentil accessions. each of these accessions has undergone three years of mandatory field and laboratory research since 1993. some have been grown in additional years to produce new seed lots and collect phenotyping data. all the information collected is stored in a database designed for this specific use. this study illustrates the use of historic genebank information on a subcollection of 37 lentil accessions from 19 countries. these accessions were requested by and made available to a research team in france in 2019. following seed distribution, these accessions were evaluated in the field in ukraine in 2019 and in france in 2021. the main objective was to show how access to the phenotyping genetic resources (2024), 5 (10), 139–153 143 data collected between 1993 and 2016 can provide valuable information on the potential of the accessions to be used for research and breeding, including outside ukraine. comparison of weather conditions in trials in ukraine (1993–2019) and france (2021) the sowing dates of lentil accessions in kharkiv, ukraine varied each year depending on weather conditions. in general, sowing took place between the end of march and the beginning of may. plants matured between the end of july and the beginning of august and the average length of the vegetation period was 84 days average daily temperatures during the lentil growing season in ukraine in the years of our observations (1993–2016) ranged from 15.6 to 21.3◦c. the average sum of temperatures during the lentil period of vegetation was 1,661.3◦c, and the average sum of precipitation during the same period was 176.8mm (figure 2). weather conditions in 2019, when the research trial was conducted in ukraine, were characterized by high precipitation at the beginning of the growing season and intermittent showers during flowering and maturity. the end of the growing season featured a prolonged period of drought and high temperatures, which led to rapid desiccation of the plants and had a negative impact on seed filling. in bretenière, france (bourgogne-franche-comté region), in 2021, sowing took place on 25 february and germination was observed on 10 march. the plants reached maturity at the beginning of july, with an average vegetation period of almost 119 days. the weather conditions in the region during this year were atypical, exhibiting a greater degree of dryness and heat than is typical for a multi-year period. the weather conditions that accompanied the study in france were characterized by a lower temperature (average sum 1,528.1◦) and a significantly higher amount of rainfall (258.1mm) during the growing season, compared to most of the trials conducted in ukraine (figure 2). analysis of the lentil yield data collected in kharkiv, ukraine in the period ranging from 1993 to 2016 and 2019 showed a rather high variability due to a wide range of weather conditions over the study years (figure 3). the years with high productivity were 2006 and 2015, while the years with the lowest seed production were 1996 and 2014. this allowed a comprehensive assessment of the stability of the performance of the lentil accessions. analysis of ukrainian genebank data the database of the ncpgru lentil collection includes results from multiple years of research in the field and in the laboratory. the analysis of variance for 37 lentil accessions from the genebank database in this study, which provided insights into the genetic diversity and variability, is presented in table 2. considering ten agronomic traits with a significant coefficient of variation (ranging from 11.7 to 75.92%), it was found that the duration of the growing season (11.7%) and the number of days to flowering (15.87%) were the most stable traits. conversely, seed yield per plot exhibited the highest coefficient of variation (75.92%). the generalization of the long-term data (1993–2016) of the genebank of ukraine was carried out using principal component analysis (pca) (figure 4). pca of agronomic traits (seed yield per plant (p1), weight of 100 seeds (p2), seed yield per area (p3), number of seeds per plant (p6), plant height (m1), height of the lowest pod (m2), and relative yield to standards (cy)) and weather parameters including sum of effective temperatures (and sum of precipitations by different phenophases) revealed that the first two components accounted for over 50% of the total variance. a strong correlation was observed between yield components and weather conditions (both total and relative yield, weight and number of seeds, height of attachment of the lower pod). for the second axis, the most significant indicator was the duration of the growing season and the sum of temperatures experienced during this period. the qualitative factor map (figure 4b) illustrates that dimension 1 opposes individuals located on the right of the graph, characterized by strongly positive coordinates on the axis to individuals characterized by strongly negative coordinates on the axis (left of the graph). this group, characterized by positive coordinates on the axis, showed high values for variables like seed yield per plant (p1), seed yield per area (p3), relative yield to standards (cy), number of seeds per plant (p6), plant height (m1), height of the lowest pod (m2), days from germination to full maturity (f5), sum of effective temperatures during the vegetation period (s t c), days to 50% of flowering (f8) and days to 50% of flowering (f2). the variables are ranked from the strongest to weakest. a k-means cluster analysis was performed to identify groups of lentil accessions based on the similarity of their agronomic characteristics. the 37 accessions were grouped into four clusters with no direct relationship between cluster separation and the geographical origins of the accessions (table 1, figure 4). cluster 1 and cluster 4 were the largest with 15 and 10 total accessions, respectively. the average indicators for each cluster are summarized in table 3. cluster 4 is the most promising for breeding. the accessions have a high mean yield (0.156kg/m2) and a high mean percentage yield compared to the standards (103.45%) and all belong to the subspecies microsperma. three of them are from france. the complete characteristics of the lentil accessions from this cluster, according to the most important agronomic traits, are given in table 4. in addition, cluster 2, which combines seven accessions of the macrosperma type, is important for further breeding processes. cluster 2 includes two standard accessions from ukraine and five landraces – three from france, one from mexico and one from using historic genebank data for material selection 144 vus et al genetic resources (2024), 5 (10), 139–153 figure 2. sum of temperature (◦c) and sum of precipitation (mm) during the lentil vegetative period in ukraine (1993–2016 and 2019) and france (2021). the red dot refers to the year when the accessions provided by the national plant genetic resources centre of ukraine (ncpgru) were evaluated in the field in france. the green dot refers to the year when the maximum number of accessions evaluated in france were considered for seed multiplication and phenotyping in ukraine. the dashed lines correspond to the average sum of temperature and sum of precipitation: blue for kharkiv, ukraine (1993–2016) and red for bretenière, france (2011–2021). table 2. analysis of variance for 37 lentil accessions from the national plant genetic resources centre of ukraine (ncpgru) database (1993–2016). sd, standard deviation; cv, coefficient of variation; ***, statistically significant at p < 0.001. traits min–max mean sd cv, % source of variation genotype year plant height (m1), cm 0–80 37.54 13.76 36.66 4.617*** 11.491*** height of the lowest pod (m2), cm 0–26 14.43 6.11 42.35 3.496*** 8.842*** number of seeds per plant (p6) 0–329 93.3 69.08 74.04 5.116*** 6.319*** weight of 100 seeds (p2), g 0–87.6 39.45 19.92 50.50 12.412*** 6.156*** seed yield per plant (p1), g 0–9.5 2.96 1.96 66.24 3.496*** 8.842*** seed yield per area (p3), kg/m2 0–0.43 0.137 0.104 0.076 4.978*** 16.621*** relative productivity (cy), % 0–457.45 145.44 110.42 75.92 4.978*** 16.621*** days from germination to 50% flowering (f2) 25–56 42.6 6.76 15.87 14.04*** 29.13*** days from 50% flowering to full maturity (f8) 13–66 41.38 8.49 20.51 2.479*** 18.948*** days from germination to full maturity (f5) 61–108 83.98 9.83 11.70 7.231*** 26.837*** table 3. agronomic traits characterizing the four clusters of lentils (1993–2016) (means for cluster). cluster plant height, cm height of the lowest pod, cm number of seeds per plant weight of 100 seeds, g yield of seeds per plant, g yield per plot, kg/m 2 relative yield to standards, % 1 33.39 14.91 70.63 3.4 1.85 0.080 59.63 2 42.91 19.56 59.86 5.9 2.71 0.110 85.9 3 25.44 8.95 23.13 3.2 0.88 0.024 23.69 4 39.66 15.17 115.41 2.9 3.15 0.156 103.45 genetic resources (2024), 5 (10), 139–153 145 figure 3. boxplots showing seed yield (kg/m2) of lentil accessions per research year in kharkiv, ukraine. the red line represents the median of the yield of three standard accessions, stepova 244 (ud0600052), krasnohrads’ka 49 (ud0600036) and krasnohrads’ka 250 (ud0600112), regularly sown and phenotyped in each year. table 4. agronomical traits of ten microsperma lentil accessions composing cluster 4 (national centre for plant genetic resources of ukraine (ncpgru), 1993–2016). accession id plant height, cm height of the lowest pod, cm number of seeds per plant weight of 100 seeds, g yield of seeds per plant, g yield per plot, kg/m 2 relative yield to standards, % ud0600052 44.5 15 187 2.6 3.9 0.196 97.94 ud0600084 32.2 13.3 182.7 2.7 3.75 0.161 144.72 ud0600086 34.5 14.5 113.5 2.6 2.95 0.168 104.1 ud0600091 45.7 13.7 55.9 2.5 2.9 0.149 97.13 ud0600145 36.3 20 88 3.9 3.6 0.163 85.65 ud0600403 34.8 12.4 75 3.4 2.4 0.156 107.76 ud0600437 40.6 18.4 109 2.7 2.6 0.090 78.9 ud0600443 34.3 10.25 122 3 3 0.152 92.25 ud0600550 52.1 18.6 98 2.5 2.55 0.108 109.54 ud0600638 41.6 15.5 123.00 3.1 3.8 0.217 116.5 using historic genebank data for material selection 146 vus et al genetic resources (2024), 5 (10), 139–153 figure 4. principal component analysis of phenological, agronomic and meteorological parameters. a, variables factor map; b, qualitative factor map. phenology traits include: f2, days to 50% of flowering; f5, days from germination to full maturity; f8, days from 50% of flowering to full maturity. agronomic traits include: p1, seed yield per plant, g; p2, weight of 100 seeds, g; p3, seed yield per area, kg/m2; p6, number of seeds per plant; m1, plant height, cm; m2, height of the lowest pod, cm; cy, relative yield to standards. weather parameters: s t c, sum of effective temperatures during the vegetation period; s p c, sum of precipitation during the vegetation period; s t g, sum of temperature during the pod filling period; s p g, sum of precipitation during the pod filling period; s t v, sum of temperature during the period from germination to the beginning of flowering, s p v, sum of precipitation during the period from germination to the beginning of flowering. figure 5. cluster plot highlighting four groups of lentil accessions based on seven agronomical traits (1993–2016). genetic resources (2024), 5 (10), 139–153 147 russia.table 5 shows the complete characteristics of the lentil accessions from this cluster. comparative analysis of ukrainian genebank data, and data from field experiments in ukraine (2019) and france (2021) a comparison of the duration of the phenophases in france and ukraine, conducted in conjunction with the long-term observations of the genebank, showed that despite the significantly earlier sowing date in france, the growing season was not significantly longer, with an average of 118.72 days in france versus 105.24 days in ukraine (2019). the experimental year in ukraine (2019) was characterized by particularly unfavourable conditions for lentil cultivation, with a much shorter growing season than usual (63.94 days). the results of the field experiments in ukraine (2019) and france (2021) are presented in table 6. we found that the examined set of accessions (37 genotypes) showed a significant decrease in productivity, in both ukraine (2019) and france (2021) compared to the characteristic indicators of the genebank for the evaluation period 1993–2016 (table 2), most likely due to weather conditions. the average seed yield per plot in france was almost half that in ukraine (0.042kg/m2 and 0.089kg/m2respectively), but this reflected the typical conditions of the year of the study and the design of the experiment, as can be seen from the fact that the relative yield to the standard is almost the same in both experiments (95.45% and 94.57%). the most stable trait in both experiments (ukraine and france), as in the analysis of long-term data of ncpgru, was the duration of the growing season. the most variable features were those representing yield components (table 6). the biplot analysis of the genotype×environment interaction and the ranking of genotypes revealed a notable discrepancy in environmental conditions between the long-term observations conducted by ncpgru (1993–2016) and the experimental years in ukraine (2019) and france (2021). this enabled a comprehensive evaluation of the selected accessions, with ud0600638 eston (canada) and ud0600052 stepova 244 (ukraine) identified as the most closely aligned with the ‘ideal’ genotype (figure 5). the most informative environment, as expected, turned out to be the conditional environment of genebank (information from the database of ncpgru). the ranking of genotypes in relation to the ’ideal’ genotype should be based on two criteria: average efficiency and stability across environments. the ideal genotype, represented by the point in the centre of the concentric circles, represents ‘absolute stability’ in the positive direction and has a vector length equal to the longest vectors of genotypes on the positive side of the highest average seed yield per plot. consequently, genotypes that are closer to the ideal genotype are more desirable. by comparison with the ideal genotype, six accessions were selected, located in the first three rounds of the concentric circles in the graph (figure 5): ud0600638, ud0600052, ud0600145, ud0600403, ud0600084, ud0600086. significant differences in environmental conditions were analyzed by biplot analysis based on the indicator of relative yield to the standard (standard accessions were removed from the ranking). this approach gave a much larger range of variation in all three environments (figure 6). as a result, eight accessions were selected within the circles around the ideal genotype. five of these accessions belong to cluster 4 and one to cluster 2 (table 7). discussion the evaluation of genetic resources and the identification of the most promising parental lines is crucial in the development of new varieties (sivaraj et al, 2022). the exchange of seed material and information on accessions and their characteristics is not always optimal due to the locations of genebanks in a wide range of climatic zones. it is known that environmental conditions have a significant influence on the yield of lentil genotypes (khatun et al, 2022). the study of the lentil gene pool has a long history in ukraine (bezuhla and kobyzeva, 2021), while it is a relatively new endeavour in france. it was therefore decided, upon seed material request, to accompany seeds with related phenotyping data to increase their utility use efficiency. the initial phase of the study involved gathering historic data on these accessions from ncpgru, collected during previous regeneration cycles. it became evident that significant discrepancies in weather patterns and regional variations in agricultural technology and applied methods preclude a straightforward comparison of lentil productivity. consequently, a comparison of the relative yield against the median of the standards included in field trials was conducted, accompanied by an assessment based on a set of characteristics. the principal components analysis of the ncpgru data revealed that pc1 was influenced by yield indicators, while pc2 was affected by weather conditions. the combination of temperature and precipitation exerted the most significant influence on the generative phase of lentil development, thereby determining the duration of the growing season in general (maphosa et al, 2023). the characteristics of yield per plot (direct and relative to standards), the height of pods attachment, and the weight and number of seeds per plant exhibited the strongest correlation. this finding aligned with literature data (hussain et al, 2022; ilyas et al, 2024). accessions situated in the lower right quadrant demonstrated a greater contribution in terms of yield, while those in the upper right quadrant exhibited a greater contribution in terms of the length of the growing season. the results of the anova indicated that the duration of phenophases, yield, plant height and the attachment of the lower pod bean exhibited a greater degree of using historic genebank data for material selection 148 vus et al genetic resources (2024), 5 (10), 139–153 table 5. agronomic traits of seven macrosperma lentil accessions composing cluster 2 (national centre for plant genetic resources of ukraine (ncpgru), 1993–2016). accession id plant height, cm height of the lowest pod, cm number of seeds per plant weight of 100 seeds, g yield of seeds per plant, g yield per plot, kg/m 2 relative yield to standards, % ud0600036 48.4 22.9 62.5 6.7 4.9 0.188 104.93 ud0600112 44 18.7 60.5 6.9 4.05 0.160 100 ud0600163 40 18 36 6.9 2.05 0.077 67.44 ud0600468 38.8 19.1 47.5 6.4 1.85 0.073 81.27 ud0600496 40 19.9 39.5 5.2 1.45 0.734 72.7 ud0600530 46.3 19.7 98.5 4.5 2.25 0.106 111.34 ud0600686 42.9 18.6 74.5 4.6 2.4 0.089 63.64 table 6. comparison of the main traits of lentils in experimental years in ukraine (2019) and france (2021). sd, standard deviation; cv, coefficient of variation; ***, statistically significant at p < 0.001. ukraine france traits min–max mean sd cv, % min–max mean sd cv, % plant height, cm 25–39 31.75 3.68 11.59 10–35 21.3 6.12 28.73 height of the lowest pod, cm 8–23 15.86 3.26 20.55 5–23 12.58 3.97 31.56 number of seeds per plant 9–117 48.61 30.69 63.14 1.3–128.77 49.9 27.85 55.81 weight of 100 seeds, g 2.13–6.6 3.77 1.37 36.45 0–2.65 1.64 0.34 20.73 yield of seeds per plant, g 0.3–3 1.54 0.76 49.35 0–2.6 0.83 0.49 59.04 yield per plot, kg/m2 0.016–0.181 0.089 0.05 52.99 0–0.117 0.042 26.22 61.90 relative yield, % 16.7–192.55 94.57 50.18 53.06 0–266.7 95.45 59.81 62.66 days from germination to 50% flowering 13–44 39.47 5.22 13.23 68–90 83.88 3.45 4.11 days from 50% flowering to full maturity 19–64 24.47 7.25 29.63 25–44 34.84 3.60 10.33 days from germination to full maturity 63–77 63.94 2.32 3.63 110–128 118.72 4.25 3.58 table 7. characteristics of the most promising lentil accessions revealed across trials conducted in ukraine and france.*, accessions were not evaluated in france. accession id seed yield per plot, kg/m 2 relative yield per plot from median of standards (cy), % cluster genebank (median of years) ukraine (2019) france (2021) genebank (median of years) ukraine (2019) france (2021) ud0600006* 0.021 0.140 13.03 148.94 3 ud0600065* 0.082 0.136 42.71 144.68 1 ud0600086 0.197 0.099 0.034 102.87 105.32 76.72 4 ud0600145 0.168 0.158 0.044 71.04 168.09 99.29 4 ud0600437 0.092 0.088 0.047 64.17 93.62 106.75 4 ud0600530 0.102 0.110 0.047 105.66 117.02 106.16 2 ud0600550 0.096 0.167 0.031 177.66 78.63 69.92 4 ud0600638 0.252 0.181 0.030 111.43 192.50 68.38 4 variation in response to the prevailing conditions of the year (lázaro et al, 2001). in contrast, seed size and time to 50% flowering were largely influenced by genotype (bhartiya et al, 2015). cluster analysis allows the grouping of a large number of genotypes based on a list of characteristics and the selection of a group of accessions that meet the requirements of a specific breeding programme (ahamed et al, 2014; vus et al, 2020a; zayed et al, 2022; amit et al, 2023). the clustering of the 37 accessions from this study allowed the identification of a cluster of lines (cluster 4; table 3) as the most promising for breeding with high and stable productivity. all accessions of this cluster were microsperma type and had a higher yield than accessions of macrosperma type, consistent with results from mohammed et al (2019) and preiti et al (2024) who suggested that this is caused by earliness of this type of lentils. cluster 4 included wellknown accessions such as ud0600638 eston (canada), the most popular canadian variety. however, this one is also susceptible to ascochyta blight (sari et al, 2018). this cluster also contained accession ud0600052 stepova 244 (ukraine) – a reference for yield stability in ukraine (bezuhla and kobyzeva, 2021), which has been genetic resources (2024), 5 (10), 139–153 149 figure 6. biplot analysis of genotype-environment interaction by yield of seeds per plot. ranking genotypes relative to the ‘ideal’ genotype (centre of the concentric circles). green dots are environments and blue dots are genotypes. used in many studies aiming at understanding drought tolerance (vus et al, 2020b) or long-term seed storage behaviour (zadorozhna et al, 2015). the bulgarian variety nadejda (ud0600403) is largely used in research by bulgarian scientists. its resistance to four of the seven pathotypes of ascochyta blight known in the country has been established (stanoeva and koleva, 2017), but also the high sensitivity to changes in weather conditions was noted (milev, 1999; tonev et al, 1999). this was also confirmed in our research: in france, the variety yielded significantly lower than the standard (13.52%), while in ukraine it performed well (171.28%). large-seeded lentil genotypes in cluster 2 (table 4) included two ukrainian reference accessions (ud0600036 and ud0600112) and ud0600530 slovyanka, which has been identified as a source of the highest first pod (kobyzeva et al, 2012). this feature is an important character for mechanization in lentil, as it shows high variability depending on growth conditions and can cause significant yield losses (gaad et al, 2018; kuzbakova et al, 2022). accession ud0600444 is part of a set of lentils with high seed nutritional value (vus et al., 2020a). it should be noted that some genotypes in this cluster, which have important breeding traits such as high weight of 100 seeds (ud0600163) or high number of seeds per plant (ud0600686), had a high susceptibility to environmental changes and a low yield compared to the standard (27.42 and 32.6%, respectively), consistently with results from (mohammed et al, 2019). the ranking of lentil accessions under different environmental conditions – using the genebank’s median data as one environment point, along with data from field trials conducted in ukraine (2019) and france (2021) – was carried out using biplot analysis based on the feature ‘seed yield per plot’. it was noted that the years of research trials in ukraine and france were significantly different from the long-term observations of ncpgru. in france, a low variability of lentil accessions was observed, compared to ukraine. according to the relationship to the ‘ideal’ genotype – represented by the centre of circles in the pca – an ideal genotype should have both high mean performance and high stability across environments. therefore, genotypes closer to the ideal are considered more desirable (gedif and yigzaw, 2014). six lentil accessions located within the first three circles from the centre were selected as more promising for the three environments (yan and tinker, 2006; khan et al, 2021). significantly, two of them were standards: ud0600052 (in ukraine) and ud0600084 (in france), with a consistently high yield in the study regions. all of these accessions were also identified earlier as promising using historic genebank data for material selection 150 vus et al genetic resources (2024), 5 (10), 139–153 figure 7. biplot analysis of genotype-environment interaction by relative yield (without standards accessions). ranking genotypes relative to the ‘ideal’ genotype (centre of the concentric circles). green dots are environments and blue dots are genotypes. by results of multi-year observations in ncpgru (cluster 4). ‘stable’ genotypes are desirable only when they have high mean performances. it should also be noted that accessions ud0600095 and ud0600423 showed the highest level of productivity in the conditions of the year of research in france. it has been confirmed (bezuhla and kobyzeva, 2021) that ud0600423 cdc sunrise was determined as a source of two valuable features: high protein content and low cooking time. conclusions the present study demonstrated how the valuable information stored in genebank databases is essential for supporting research and development, using an underutilised crop – lentil – as a key example. the performance of lentil accessions under different annual and agroclimatic conditions in ukraine (kharkiv region) allowed the identification of promising accessions that could be confirmed in parallel trials conducted in ukraine and in france (bourgogne-franche-comté region). accessions that consistently outperformed the standards are recommended for use in breeding to create new lentil varieties. authors contributions nadiia vus: conceptualization, data curation, analyses conduction, investigation, methodology, resources, provision of study materials, validation, verification of the overall result replications, visualization, prepared the figures and tables, writing (original draft, review and editing); olha bezuhla: supervision, data curation, resources, provision of study materials, validation; hervé houtin: resources, validation, provision of study materials, verification of the overall result replications; florence naudé: resources, validation, provision of study materials, verification of the overall result replications; antonina vasylenko: analysis conduction the analyses, methodology, validation, verification of the overall result replications, writing (review and editing); anthony klein: resources, validation, verification of the overall result replications, provision of study materials; oleh leonov: data curation, analysis conduction, validation, verification of the overall result replications; nadim tayeh: supervision, conceptualization, data curation, provision of study materials, formal analysis, investigation, resources, validation, visualization, writing (review and editing), acquisition of financial support. all authors read and approved the final manuscript. genetic resources (2024), 5 (10), 139–153 151 conflict of interest statement the authors confirmed that no conflict of interest exists. acknowledgments this research work was supported by the casdar project résilens (french ministry of agriculture, no c2018-08). references agrawal, s. k. 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(2022). genetic diversity and principal component analysis (pca) of faba bean landraces based on yield traits and protein sds-page. journal of global agriculture and ecology 13(4), 1–16. doi: http://dx.doi.org/10. 56557/jogae/2022/v13i47506 using historic genebank data for material selection https://doi.org/10.18699/vj20.617 https://doi.org/10.18699/vj20.617 https://doi.org/10.6001/zemesukiomokslai.v27i2.4337 https://doi.org/10.6001/zemesukiomokslai.v27i2.4337 https://doi.org/10.30835/2413-7510.2020.206962 https://doi.org/10.30835/2413-7510.2020.206962 http://dx.doi.org/10.4141/p05-169 http://genres.com.ua/ua/arxv-vidan/2015-vipusk-16/xranenie-semyan-goroxa-nuta-i-checheviczyi-v-kontroliruemyix-usloviyax/ http://genres.com.ua/ua/arxv-vidan/2015-vipusk-16/xranenie-semyan-goroxa-nuta-i-checheviczyi-v-kontroliruemyix-usloviyax/ http://genres.com.ua/ua/arxv-vidan/2015-vipusk-16/xranenie-semyan-goroxa-nuta-i-checheviczyi-v-kontroliruemyix-usloviyax/ http://dx.doi.org/10.56557/jogae/2022/v13i47506 http://dx.doi.org/10.56557/jogae/2022/v13i47506 introduction materials and methods plant material field trials plant phenotyping statistical analysis results comparison of weather conditions in trials in ukraine (1993–2019) and france (2021) analysis of ukrainian genebank data comparative analysis of ukrainian genebank data, and data from field experiments in ukraine (2019) and france (2021) discussion conclusions authors contributions conflict of interest statement acknowledgments original article genetic resources (2023), 4 (8), 71–90 doi: 10.46265/genresj.dvkv8430 https://www.genresj.org issn: 2708-3764 status and prospects of plant genetic resource conservation in yemen maeen ali aljarmouzi *,a, khalil m alsharjabi a and luigi guarino b a agricultural research and extension authority (area), dhamar, republic of yemen b global crop diversity trust, platz der vereinten nationen 7, 53113, bonn, germany abstract: this study provides a comprehensive examination of the conservation status of plant genetic resources in yemen, with a particular focus on the national genebank, natural sites and field genebanks. employing a historical and descriptive approach, the study sheds light on the inception of field inventories and collection surveys, the roles and contributions of various projects and institutions since the late 1980s, and the quantities of seed samples collected from different crop varieties and species. additionally, the study highlights the challenges faced, especially during the last decade following the eruption of war in early 2015. specifically, it emphasizes the detrimental impact of the war and sanctions, resulting in the loss of conserved seed samples, and the damage and sabotage of field genebanks. in contrast, there is a need to ensure the enhancement of functioning seed systems and agriculture production even during times of conflict to reduce the impact of food insecurity. to conclude, the study puts forth several proposals, with a strong emphasis on expanding conservation efforts beyond natural sites, enhancing the capabilities for seed conservation in genebanks, and building genetic resources capacity. keywords: agricultural research, conservation, genebank, field genebanks, natural sites, yemen citation: aljarmouzi, m. a., alsharjabi, k. m., guarino, l. (2023). status and prospects of plant genetic resource conservation in yemen. genetic resources 4 (8), 71–90. doi: 10.46265/genresj.dvkv8430. © copyright 2023 the authors. this is an open access article distributed under the terms of the creative commons attribution license (cc by 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. introduction for thousands of years, farmers in yemen have inherited and managed their local seeds. in particular, the sorghum crop shows a remarkable level of diversity, serving as a clear testament to their commitment. they have cultivated, conserved, exchanged and managed the seeds of agricultural crops since the early stages of history when dams, irrigation canals and highland terraces were developed across different regions during the eras of ancient yemeni kingdoms and states dating back to between the tenth and fifth centuries bce (alafif cultural foundation, 2003). however, the systematic conservation, characterization and use of plant genetic resources in a modern scientific manner can be traced to the establishment of agricultural research work in yemen during the midto late-1940s. the first agricultural research station was established in the el-kod district of abyan governorate, which ∗corresponding author: maeen ali aljarmouzi (maeen669@gmail.com) later evolved into the fully-fledged el-kod agricultural research center (earc) in 1955. the first systematic activities related to the conservation and use of plant genetic resources in yemen took place in this centre in the early 1970s. since then, various yemeni government bodies, including the ministry of agriculture and irrigation (mai), the ministry of water and environment (mwe), as well as institutions like the agricultural research and extension authority (area) and the environment protection authority (epa), have made diverse contributions to the conservation and sustainable use of genetic resources. these bodies have been actively involved in supporting the implementation of numerous national programmes, projects and activities in collaboration with local, regional and international organizations. in recent decades, there has been an increasing interest in plant genetic resources due to their recognition as a national asset and a sovereign resource for any country worldwide. these resources play a vital role in achieving food security, sustainable development, resilience, better livelihoods and higher income. yemen, received: 09.07.2023 accepted: 03.11.2023 published online: 20.12.2023 https://www.genresj.org https://www.dx.doi.org/10.46265/genresj.dvkv8430 https://www.genresj.org https://www.dx.doi.org/10.46265/genresj.dvkv8430 mailto:maeen669@gmail.com genetic resources (2023), 4 (8), 71–90 plant genetic resource conservation in yemen 72 characterized by diverse climates and a rich and distinct plant diversity, unfortunately, has not made optimal use of its traditional plant genetic resources. on the contrary, these resources have been diminishing and lost due to various factors, including human interventions such as urban expansion, road construction at the expense of agricultural lands, and the expansion of qat (catha edulis) cultivation, an important cash crop in yemeni social life but with negative effects on human nutrition and health, as well as the introduction of new crops. the deterioration and loss of plant genetic resources can in addition be attributed to factors such as the absence of legislation, weak institutional frameworks, limited national programmes and inadequate material and human capacities to leverage modern technologies for conservation and sustainable use. since 2015, the ongoing war and conflict have also significantly impacted yemen’s plant genetic resources, resulting in severe economic and social consequences. the collapse of state institutions, the paralysis of public service agencies and facilities, the internal and external displacement of millions of citizens, currency devaluation, limited availability of goods and services, rising prices, declining investment and the loss of income sources for many yemeni families all exert immense pressure on natural resources, including the country’s biodiversity in all its forms. in recent years, amid worsening conditions and the effects of the war and recurring food crises, official authorities and decision-makers, prompted by cases of tampering with plant genetic resources, have called for more attention to be given to the country’s biodiversity. the need for implementing relevant research and raising awareness about these issues has become increasingly urgent. the present study was undertaken in response to these calls. the request made by international bodies and organizations such as the commission on genetic resources for food and agriculture (cgrfa) and the international treaty on plant genetic resources for food and agriculture (itpgrfa), among others, for member states to contribute to monitoring and assessing the state of plant genetic resources, served as further justification for conducting this study. materials and methods to assess the status of plant genetic resource conservation in yemen, the authors employed a historical descriptive approach. this involved gathering and reviewing national and international documents, reports, studies and surveys related to plant genetic resources. the authors also utilized the available electronic and paper information systems, including databases and records, of the genebank of the national genetic resources center (ngrc) within the agricultural research and extension authority (area) in dhamar, yemen. furthermore, various institutions contacted during the study provided additional information that was incorporated into the research. additionally, the authors relied on their personal knowledge as co-witnesses, drawing from their extensive experience in research and agricultural technologies, as well as their involvement in various activities related to plant genetic resources. they also considered events and developments they have witnessed or been exposed to up until the time of preparing this study. it is worth noting that the authors followed the methodology established by the commission on genetic resources for food and agriculture of the food and agriculture organization of the un (fao), as well as the approved indicators, to guide the monitoring of the implementation of the second global plan of action for plant genetic resources for food and agriculture (second gpa). a total of 55 indicators from this guide were used in the study to assess the state of conservation of plant genetic resources in yemen from 2012 to 2022 (fao, 2019). the assessment was conducted based on the themes and activities outlined in the second gpa, as well as the medium-term plan of area and the ngrc’s plan. the execution of this study encountered numerous difficulties and obstacles, with the most significant ones being: • limited availability of data: the study faced challenges due to the scarcity of available data. the existing information was dispersed across multiple sources and exhibited a diversity of forms and formats, making it challenging to gather comprehensive and standardized data. • low response rate from contacted agencies: despite efforts to collect relevant and available data, the study encountered a low response rate from most of the local agencies contacted. this lack of cooperation hindered the acquisition of crucial information necessary for the research. • impact of war: the study was conducted amidst the ongoing war, which created adverse conditions and posed additional challenges. coordinating and communicating with relevant entities and individuals became difficult due to the disrupted infrastructure and limited resources. obtaining the requested information became a daunting task in such a context. these difficulties and obstacles significantly influenced the implementation of the study, impacting the comprehensiveness and accuracy of the collected data. nonetheless, the authors made their best efforts to overcome these challenges and ensure the reliability of the findings within the existing constraints. 73 aljarmouzi et al genetic resources (2023), 4 (8), 71–90 table 1. studies surveying natural vegetation in the different yemeni regions and numbers of species identified. study title no. of species source survey and classification of medicinal and aromatic plants in sana’a and dhamar governorates 858 ngrc (2022) survey and classification of the forest-pastoral vegetation cover in the al-jawf governorates 93 rnrrc (2022) inventory of the natural plant species in important areas in the arabian peninsula: bani omar, taiz governorate, yemen 135 al-khulaidi et al (2000) survey of the natural flora of some districts in ibb, taiz, al-mahweet and sana’a governorates 1,603 area (2015) studying the existing natural resources and the climate change dimension in taiz governorate using remote sensing and geographic information systems techniques (second phase) 389 area (2014) surveying, collecting and classifying the vegetation cover in bura’a 240 area (2014) survey of the natural plants in al-makha district 167 ngrc (2013) survey of the natural plants in ibb governorate 316 al-khulaidi (2013) surveying and collecting plant germplasm of pasture and forest, medicinal and aromatic plants, and the unexploited plants in the highlands region of ba’adan and al-sher’ar districts, ibb governorate 219 ngrc (2011) survey and evaluation of the deterioration of the vegetative cover in the upper stream area of wadi rasyan 332 mufarreh et al (2011) surveying and collecting unexploited plant germplasm of pastures, forests, and medicinal and aromatic plants in the highland region of sana’a, al-mahweet amran hajjah governorates 1,329 area (2013) surveying and collecting unexploited plant germplasm of pastures, forests, and medicinal and aromatic plants in mareb governorate 294 ngrc (2009) surveying and collecting unexploited plant germplasm of pastures, forests, and medicinal and aromatic plants in 14 districts of the coastal plain of hadramout and al-mahrah governorates 172 ngrc (2008) inventory and evaluation of the pastural genetic resources in al-kusumah district of raymah governorate 289 mufarreh (2004) inventory and evaluation of the pastural genetic resources in belad al-ta’am district of raymah governorate 311 mufarreh (2003) survey of the natural vegetative cover of madinat al-sharq – wadi rama’a 78 al-khulaidi (1994) genetic resources (2023), 4 (8), 71–90 plant genetic resource conservation in yemen 74 results and discussion state of in situ conservation and on-farm management surveys and inventories of plant genetic resources understanding crop diversity, including its distribution and changes over time, is a crucial prerequisite for the development and implementation of effective and efficient conservation strategies. various studies have been conducted to survey and inventory plant diversity across different regions in yemen by agencies such as the agricultural research and extension authority (area), colleges of agriculture and sciences in yemeni universities, and institutions like the social fund for development (sfd). al-khulaidi (2013) reported the identification of 2,838 plant species, as part of the yemeni flora, of which 2,602 are native, 129 are cultivated and 107 are introduced. among these species, 608 were found to be endemic or semi-endemic (457 are endemic, among which 307 on socotra island alone), confined to yemen or the arabian peninsula. the diverse topography of yemen’s plains, mountains, plateaus, valleys and deserts, with varying altitudes, has contributed to the emergence of this unique plant diversity in the country. the uniqueness of such diversity extends to the varied crops grown in the country, which includes both tropical and temperate crops. additionally, there is a diversity within species, with multiple crop varieties adapted to different environments or agroecological zones in the country. for instance, sorghum is characterized by a great diversity of types that mature over a range of durations (after three, four, five or even six months). table 1 presents the results of some recent studies and surveys, which involved counting and classifying plant species, compiling lists with their scientific and local names, providing photographs, and mapping their distribution in specific areas. these studies also identified the uses of the plant species, such as food, fodder, medicine and other purposes. additionally, they focused on identifying threatened, rare, endemic and semi-endemic plants, some of which are introduced plants. on-farm management and improvement of plant genetic resources for food and agriculture plant diversity plays a vital role in food security and adaptation to climate change. therefore, it is crucial to focus on on-farm management and improvement of local crop varieties, including neglected and underutilized crops. by doing so, the resilience and adaptability of the cropping systems can be enhanced, enabling better withstanding of biotic and abiotic stresses and eventually better production and food security for the farmers’ households. ngrc has undertaken various activities to support the management and improvement of genetic resources on farm through multiple projects (table 2). these initiatives primarily involved the characterization, evaluation and dissemination of local varieties suitable for different agroclimatic zones. the germplasm collected by various projects has been safety duplicated at the national genebank. the aim was to increase genetic diversity and broaden the range of options available to farmers for selecting varieties that are well-adapted and tolerant to biotic and abiotic stresses prevalent in their specific local conditions. providing seeds to farmers and researchers in case of disasters to restore cropping systems ngrc is ideally positioned to play an important role in preserving plant genetic resources, especially in the face of natural disasters (such as droughts and floods) and man-made disasters (like conflicts and wars). ngrc’s mandate includes providing high-quality seeds adapted to farmers’ environmental conditions in disaster-affected areas, in coordination with relevant national authorities and organizations like regional agricultural research stations, the general improved seed multiplication corporation (gismc), the public corporation for grain development and production (pcgdp), and provincial mai offices. however, due to limited material and human resources, ngrc’s efforts are constrained, particularly in the current challenging conditions within the country. in 2016, the regional station for agricultural research in the western coastal plain (al-kadan, sardoud, tihama region) was severely damaged by airstrikes. the seed stores in the station were significantly affected, leading to spoilage and loss of different varieties that had been collected and preserved by the station’s researchers over many years (figure 1). nonetheless, ngrc has managed to provide services to farmers and researchers in conflict and war-prone zones in some instances. this has been achieved through quality testing of locally distributed seeds and by providing seeds of important local strains to research stations in the respective agricultural regions. for example, ngrc collaborated with other organizations and international agencies operating in yemen to provide seeds of cereal, legumes and vegetable crops to affected farmers as part of humanitarian relief activities. organizations such as fao, the red cross, acted and others have contributed to seed distribution efforts. however, the distribution mechanism faced drawbacks, including poor coordination with official agencies, poor seed quality and sometimes unsuitable seeds for the targeted agricultural climatic zones. these issues created a negative impression among farmers and decisionmakers regarding the effectiveness and safety of the distribution mechanism. in response, measures were established to mitigate these negative effects. these measures included conducting seed quality tests in laboratories, seed screening and purification by gismc, and ensuring direct coordination with government authorities and institutions in the targeted areas. 75 aljarm ouziet al g enetic resources (2023),4 (8),71–90 table 2. projects implemented by the national genetic resources center (ngrc) that have contributed to enhancing the on-farm conservation of genetic resources in yemen. area, agricultural research and extension authority; bsf, the benefit-sharing fund; fao, food and agriculture organization of the un; gef, global environment facility; icarda, international center for agricultural research in the dry areas; ifad, international fund for agricultural development; mai, ministry of agriculture and irrigation; wb, world bank. project name funding agency implementing agencies implementation period implementation site activity participation of the rural community in raising crops (barley and lentils) in the mountain terraces (ceccarelli, 2002) icarda area/farmers’ groups in the selected sites, agriculture office 1998–2010 kuhlan affar, hajjah governorate evaluation of strains of barley and lentil on farmers’ fields. an economic and social study for the farmers of the selected villages; study and analysis of gender (the role of the farm household, male and females). the rainfed agriculture and livestock project (yralp, 2005) wb area/grc-sana’a university, gcism 2006–2013 sana’a, al-mahweet, hajjah, al-hodeidah and lahj governorates farmer-based seeds improvement and management system. evaluation of several local genotypes of sorghum, millet and cowpea the participatory rural development of the raimah governorate (ifad, 2010) ifad area and dhamar mai office 2001–2007 raimah governorate evaluation and dissemination of adapted cereal and legume cultivars irrigation improvement project (iip, 2009) wb area, agricultural services corporation, tehama development authority and nasser’s faculty of agriculture sciences (lahj) 2005–2007 al-hodeidah, lahj, and abyan governorates evaluation and dissemination of adapted cultivars of cereals, cotton and sesame the participatory rural development of al-mahrah governorate (ifad, 2011) ifad area, mai office 2002–2006 al-mahrah governorate evaluation and dissemination of adapted maize and sesame cultivars the participatory rural development of dhamar governorate (ifad, 2012) ifad area, mai office 2006–2010 dhamar governorate evaluation and dissemination of adapted cereal and legume cultivars improvement of the medicinal, and aromatic plants and underutilized crops (gotor and cherfas, 2012) fao ngrc, mai offices 2009 sana’a, dhamar and lahj governorates cultivation of neglected and underutilized crops on farmers’ fields continued on next page g enetic resources (2023),4 (8),71–90 plant genetic resource conservation in yem en 76 table 2 continued project name funding agency implementing agencies implementation period implementation site activity enhancing food security in the arab countries (yemen) (communication team icarda, 2018) icarda area and dhamar mai office 2012–2019 dhamar dissemination of wheat, lentils and peas varieties on farmers’ fields agricultural biodiversity and adaptation to climatic changes (yaccap, 2010) gef area, sana’a college of agriculture, and the gcism 2014 sana’a, taiz, ibb and al-mahweet governorates evaluation of local sorghum strains on farmers’ fields participatory conservation and sustainable use of landraces to improve farmer’s livelihood and their resilience in adapting to climate changes in yemen (fao, 2023) bsf area and mai offices 2019–2023 dhamar and hadramout governorates evaluation and characterization of local strains of sorghum, wheat, barley, lentils and peas on farmers’ fields 77 aljarmouzi et al genetic resources (2023), 4 (8), 71–90 unfortunately, there have been instances where poorquality seeds have been distributed. in april 2021, mai destroyed a large quantity of rotten seeds that were provided by fao for distribution to farmers in al-hodeidah governorate. the spoiled quantity was estimated to be more than 240 tonnes. these seeds, intended as donated assistance to yemeni farmers, were infected with fungi and deemed unsuitable for cultivation. this incident highlights the importance of strict quality control measures and coordination among relevant authorities and organizations to ensure the effective and safe distribution of seeds in yemen in line with the recommendation of the international plant protection convention (ippc) on “ensuring safe provision of seed during humanitarian assistance disbursement” (ippc, 2021). changes and trends in on-farm genetic resources conservation yemen is known as one of the main countries that are growing qat. qat or khat or khata (catha edulis) is a mild stimulant plant consumed by most yemenis in a daily afternoon session by chewing its fresh leaves (zahran et al, 2019). the expansion of qat cultivation at the expense of the main cereals and legumes (sorghum, millet, wheat, barley, lentil and pea) poses a significant threat to crop diversity in yemen. the yearly agricultural statistics book indicates a decline in the cultivated area of cereal crops over the years, while qat cultivation has increased (gdsid/mai, 2022). however, the actual increase in qat cultivation may be even greater than what is reflected in the official figures (figure 2). due to the war and resulting consequences, including the institutional divide and ineffectiveness, no agricultural census has been conducted for the past two decades. the urban population expansion on agricultural lands is another factor threatening crop diversity. figure 3 shows the trend in population growth over the past 70 years. a study conducted by the renewable natural resources research center (rnrrc) in 2021 on the urban expansion in dhamar governorate revealed a dramatic increase over the past 50 years. reports from field surveys conducted by ngrc and rnrrc highlight the expansion of urban centres and population at the expense of agricultural lands (figure 4). this phenomenon is considered one of the most critical factors endangering agricultural biodiversity. the disappearance of certain plant species and fruit varieties (quince, walnuts, pear, peach, apricot, fig and grapes) from areas like sana’a governorate, which used to be known for cultivating them, further emphasizes the negative impact of urban expansion on agricultural biodiversity. parks and orchards (for example of walnuts, peach and grapes) have been replaced by commercial and residential areas, resulting in a significant decrease in green coverage. on the other hand, there has been an expansion in the cultivation of some vegetable crops (tomato, onion, potato, pepper, carrot, cucumber, zucchini, cabbage), including varieties introduced from abroad. although the cultivated area of vegetable crops fluctuates, the number of greenhouses in different regions of yemen has increased. strawberry cultivation has also grown in response to high demand from fresh juice shops in major cities. the cultivation of fruit crops, particularly almonds, has expanded due to relatively low water requirements and the profitability of production. the almond cultivation area has increased between 2011 and 2020, with significant expansion in sana’a governorate, particularly in the districts of al-haima and bani matar. coffee cultivation has also expanded due to increasing demand for yemeni coffee locally and internationally. the cultivation area has increased over the past few years much more than the estimated figures made in the agricultural census book for 2020, which indicated an increase of 2,000 hectares in the coffee area between 2016 (33,900ha) and 2020 (35,900ha). also, there has been a focus on improving agricultural processes and post-harvest operations to meet desired quality standards. high government authorities and mai have shown increased interest in the coffee sector, establishing the national center for coffee research and supporting farmers’ associations. a decree was issued in 2022 banning the import of both processed and unprocessed coffee from abroad to encourage domestic cultivation and increase the income of coffee farmers. overall, while there is an expansion in certain crops, the threats to crop diversity posed by the expansion of qat cultivation, urbanization and population activities remain significant challenges in yemen. status of ex situ conservation of plant genetic resources (genebanks) the ngrc in yemen operates under the umbrella of area and has its headquarters outside dhamar city, about 100km from the yemeni capital sana’a. in addition, there are two other genetic resource centres working under the faculties of agriculture sciences of sana’a and aden universities and some field genebanks as detailed in table 5. supporting the targeted collection of plant genetic resources the primary drivers for implementing targeted collecting of genetic resources were the risk of loss of on-farm diversity, opportunities for use, and the need to address deficiencies in ex situ conservation. bawazir (2004) conducted a study on cereal diversity in southern yemen and emphasized the importance of surveying and collecting genetic resources of cereal crops and their wild relatives across different agroclimatic zones in yemen. the study aimed to document these resources and utilize them in breeding and genetic improvement programmes. the study’s findings revealed that sorghum varieties in these areas exhibited genetic variation within each environmental zone, as evidenced by differences in genetic resources (2023), 4 (8), 71–90 plant genetic resource conservation in yemen 78 figure 1. examples of the effects of the air raids of the war coalition against yemen on the buildings, equipment and seed stores of the tehama regional agricultural research station in the western coastal plain, al-kadan, sardoud, tihama zone, al-hodeidah governorate (a and c), including damage to the preserved seed samples of various crops (b). figure 2. expansion of qat cultivated area from 1990 to 2020. source: cso (2020) phenotypic characteristics, anatomical features and resistance to environmental stress conditions. the researcher pointed out that the varieties of sorghum grown in coastal areas showed early maturity (100–120 days), while the varieties grown in the highland areas matured late (160–180 days). according to the study, sorghum varieties in the highland areas were tallest (300cm) while in the coastal areas plant height ranged from 250–300cm. figure 3. trend of population growth in yemen over the past 70 years. source: macrotrends (2023) the study also demonstrated that local wheat varieties possessed drought tolerance. this may be accounted for by their morphological and anatomical traits, such as the smaller number of seminal roots and the small size of seminal root vessels. most of the local varieties of wheat grown in the southern, mediumaltitude regions of yemen belong to triticum aestivum. in some areas, there may be a mixture of t. aestivum and t. durum. the local varieties of millet grown in the southern yemeni regions belong to pennisetum glaucum (pearl 79 aljarmouzi et al genetic resources (2023), 4 (8), 71–90 figure 4. urban sprawl on fertile agricultural lands in dhamar city (km2) (1973–2021) millet), pennisetum setaceum, pennisetum rigidum (little millet) and eleusine coracana (finger millet). misibli is the local name for pearl millet throughout yemen, kanab is the local name for finger millet, and heba is the local name for small (short) millet (and an early-maturing type) on socotra island. the efforts made in collecting and conserving plant genetic resources in yemen can be divided into three phases: 1) first phase (1970s and 1980s): during this period, international organizations collaborated with researchers from the el-kod research station in abyan governorate and the southern highlands regional agricultural research station (shrar) in osaiferah, taiz governorate to collect hundreds of samples. notable collecting missions during this period in the different yemeni regions are presented in table 3. unfortunately, all the samples collected during this period were lost due to inadequate storage conditions, except for those preserved outside yemen by supporting organizations, such as fao, the international plant genetic resources insitute (ipgri), the united states agency for international development (usaid), the international maize and wheat improvement center (cimmyt) and others (al-mua’alem et al, 1993). 2) second phase (1990s): during this period, a specialized unit for plant genetic resources was established under rnrrc of area in dhamar. over 2,000 plant samples were collected during this phase. notably, a joint team from area and the international center for crop research in the semi-arid and arid tropical areas (icrisat) conducted significant collecting trips. they collected 685 seed samples, mostly of sorghum and millets, from al-dhalea, radfan, yafe’a, abyan, lahj and tehama. details on this collection trip and other trips are presented in table 3. despite having a refrigeration room for cold storage (-18°c), frequent power shortages and blackouts led to the loss of many collected samples, despite their data being recorded in ngrc’s records. 3) third phase (2000s to present): this stage witnessed the establishment of ngrc in 2002, an increase in specialized staff and the establishment of various departments within the centre. fao stated that ngrc in dhamar held 3,281 samples in its genebank (fao, 2009). the number of samples collected from different regions of the country has increased to over 6,500 accessions at present. notable collecting operations, covering most yemeni governorates, conducted by ngrc during this phase are displayed in table 3. the collecting trips conducted by ngrc between 1989 and 2013 to collect seeds from natural and cultivated plants, targeting all geographical regions in yemen are shown in figure 5. most of the collection activities previously reviewed were carried out with support and funding within bilateral or multilateral cooperation or joint work projects between yemen and several partners over the previous decades, and copies of samples of genetic resources that were collected found their way abroad and have been preserved by institutions outside yemen. early reports indicate that over 8,000 samples were deposited in various genebanks abroad (al-ghouri et al, 1996). the second national report on the state of plant genetic resources for food and agriculture stated that international organizations held 8,619 samples from yemen (fao, 2009). currently, data on the genesys platform show 8,958 samples from yemen conserved in 19 genebanks, covering 44 plant species, with a focus on grain crops (genesys, 2021). data from fao wiews and the svalbard platform also align closely with these figures (table 4). expanding diversity in genebanks it is crucial to expand ex situ conservation efforts for neglected crops, crop wild relatives and forages to facilitate research and crop improvement. the genebank of ngrc currently conserves seeds from 45 different crops. these crops encompass cereals such as sorghum, maize, millet, wheat and barley; legumes including lentils, beans, kidney beans, cowpeas, peas and fenugreek; vegetables like onions, tomatoes, chilli peppers, cucumbers, zucchini, mallow, radishes, eggplant and okra; oil-producing crops like sesame and peanuts; less commonly utilized crops, such as black seed (nigella sativa), henna (lawsonia inermis), coriander (coriandrum sativum), caladium, arugula (eruca sativa) and mustard (brassica juncea l.). additionally, field genebanks situated in various regions hold a diverse range of crops, including date palms, mangoes, citrus fruits, almonds, grapes, bananas and papayas, along with forage and forest crops. there are eight field genebanks in various regions – the northern, central and southern highlands, the tihama coastal plains region, the southern coast region (alkud, abyan) and the eastern plateau region (marib and sayun). more details are provided in table 5. it is important to note that the acquisition of new species and samples to expand the genetic resources reserve at ngrc was temporarily halted between 2014 and 2018 due to the prevailing conditions of the war and the resultant socioeconomic deterioration and institutional ineffectiveness in the country. g enetic resources (2023),4 (8),71–90 plant genetic resource conservation in yem en 80 table 3. details of the main phases of plant genetic resource collection and conservation in yemen main genus/species no. of accessions location(s) year project/programme reference phase 1: 1970s–1980s sorghum, millet and sudan grass 4,500 northern yemeni governorates 1975–1977 the american sorghum improvement assistance project hakimi and ya’ni (2008); jaradat (1997) wheat, barley and some legumes 490 1978–1979 the german technical cooperation agency (gtz) field crops (sorghum, millet, wheat, barley and some legumes) 783 1980–1981 the international board for plant genetic resources (ibpgr) 30 crops of human and animal food 351 southern and eastern yemeni governorates 1969–1989 ibpgr hakimi and ya’ni (2008); guarino (1989) phase 2: 1990s 70 plant species 617 tehama, central and south highlands, and east regions 1990–1999 area programme jaradat (1997) sorghum and millets 685 al-dhalea, radfan, yafe’a, abyan, lahj, and tehama. 1992 sorghum and millet improvement project sorghum and millet 294 saada, taiz, sana’a, al-dhalea, ibb and socotra 1992 sorghum and millet improvement project amer and al-dahmashi (1997) continued on next page 81 aljarm ouziet al g enetic resources (2023),4 (8),71–90 table 3 continued main genus/species no. of accessions location(s) year project/programme reference phase 3: 2000–present time sorghum bicolor, pennisetum americanum, zea mays, vigna spp., cajanus cajan, phaseolus vulgaris, trigonella foenum-graecum, sesamum indicum, raphanus spp. corchorus olitorius, abelmoschus esculentus, eruca sativa, cyamopsis tetragonoloba, cucumis spp, nicotiana spp., capsicum annuum, gossypium spp. 629 sana’a, hajjah, al-mahweet, al-hudiedah and lahj 2007 the rainfed agricultural project ngrc (2008) 36 species of human and animal food 390 western and eastern coastal plain 2008 area research programme ngrc (2009) sorghum bicolor, zea mays, pennisetum americanum, triticum aestivum, hordeum vulgare, lens culinaris, trigonella foenum-graecum, vicia faba, pisum sativum, vigna spp., lablab vulgaris, phaseolus vulgaris, trifolium spp., abelmoschus esculentus, corchorus olitorius, capsicum annuum, sesamum indicum, brassica napus, linum usitatissimum, coffea arabica 330 sana’a, amran, ibb, dhamar, hajjah, al-mahweet and al-hodeidah 2009 area research programme ngrc (2010) fruit crops (vitis vinifera, prunus amygdalus, prunus persica, cydonia oblonga, ficus carica, malus sylvestris, morus alba, pyrus spp., olea spp., ceratonia siliqua, mangifera indica, citrus spp., manilkara achras (mill), musa spp., psidium guajava l., carica papaya l., passiflora edulis sims, annona spp., opuntia ficus-indica, phoenix dactylifera) 150 sana’a, amran, al-mahweet and hajjah 2009 area research programme ngrc (2010) honeydew pumpkin (cucurbita spp.) 70 belad alrous district, sana’a governorate 2010 area research programme ngrc (2011) sorghum bicolor (30), pennisetum americanum (2) zea mays (14), triticum durum (1), hordeum vulgare (2), faba bean (1), vigna spp. (14), sesamum indicum (1), trigonella foenum-graecum (1), phaseolus spp. (1), cajanus cajan (5) 72 al-salafiya directorate and bilad al-ta’am in raymah governorate 2012 area research programme ngrc (2013) continued on next page g enetic resources (2023),4 (8),71–90 plant genetic resource conservation in yem en 82 table 3 continued main genus/species no. of accessions location(s) year project/programme reference sorghum bicolor (33), zea mays (7), triticum durum (26), hordeum vulgare (16), lens culenaris (11), vigna spp. (11), pisum sativum (6), trigonella foenum-graecum (9), brassica spp. (1), linum usitatissimum (3), allium spp. (1), cucumis sativum (1), phaseolus spp. (1) 126 sabah district in al-bayda governorate 2012 area research programme ngrc (2013) sorghum bicolor (33), zea mays (7), triticum durum (32), hordeum vulgare (20), lens culenaris (4), vigna spp. (3), pisum sativum (8), trigonella toeniccum (1) 108 ans, jahran and al-hada’a in dhamar governorate 2012 area research programme ngrc (2013) cereals, vegetables and fruits 282 sana’a, al mahwit, ibb and taiz governorates 2014 the agricultural biodiversity and climate adaptation project ngrc (2013) the project team collected a total of 368 landraces (228 from dhamar and 140 from hadramout and almahrah governorates) and more than 20 species: sorghum, maize, wheat, barley, millet, cowpea, pea, bean, fava bean, lentils, fenugreek (trigonella, spp.), sesame, lablab purpureus, mustard (brassica spp.), flax (linum spp.), pepper (capsicum annum), eleusine spp., pennisetum spp., roselle (hibiscus sabdariffa), plectranthus spp., black caraway (nigella sativa l.), fennel (foeniculum vulgare) 383 several districts in the governorates of dhamar, hadramout and al-mahra governorates 2019 the conservation and sustainable use of local landraces project bsf ngrc (2022) 83 aljarmouzi et al genetic resources (2023), 4 (8), 71–90 efforts have also been made to conserve genetic resources of threatened and endemic species ex situ in various research stations located in different agricultural regions. notably, a specialized team from the el-kod research station in abyan governorate collected the socotra wild pomegranate (punica protopunica balf. f.) in 1989 and 1990. this threatened species, which is endemic to the island, has been confirmed by international organizations such as the international union for conservation of nature (iucn) to be one of the species at risk of overexploitation without natural regeneration. the collected seeds were grown, and the trees were nurtured at the el-kod research farm (bazara’a, 2000). regeneration and multiplication of genebank seed samples even under optimal storage conditions, it is necessary to periodically regenerate and multiply seed accessions due to the decline in viability over time and the limited quantity of preserved seeds resulting from their distribution to users such as researchers and farmers. hence, ngrc carries out annual processes to regenerate seed accessions that are at risk of viability loss and to multiply accessions with limited quantities. the most recent regeneration and multiplication initiative was conducted by ngrc in 2014. due to the prevailing war conditions, regeneration operations were suspended from the beginning of 2015 until 2020, resulting in no activity in this regard during that period. subsequently, with the initiation of the ‘participatory conservation and sustainable use of local varieties’ project, funded by the benefit sharing fund (bsf) of the itpgrfa, approximately 200 plant accessions (mainly cereal crops) were regenerated in the governorates of dhamar and hadramout (seiyun) (fao, 2023). table 6 shows the numbers of seed samples that have been regenerated until 2014 and those still requiring regeneration, as reported by ngrc in dhamar. conservation changes and trends in genebanks the study period witnessed several notable positive changes and trends in the field of genebank conservation, including: • increase and expansion of plant samples: the number of seed accessions in the ngrc genebank increased from 3,281 in 2006 to approximately 6,500 accessions in 2021. similar growth was observed in the genetic resources center (grc) of sana’a university, with the number of accessions rising from 1,528 to over 3,000 (table 5). • improved energy security: noteworthy progress was made in securing electrical energy for cold storage in the ngrc genebank in dhamar. solar energy systems were introduced in 2016, funded by the agricultural and fisheries production encouragement fund (afpef). additionally, the public corporation for grain development and production (pcgdp) provided a solar energy system for the grc at the faculty of agriculture, sana’a university, in 2019. • in 2022, with support from the crop trust and the itpgrfa, the ngrc genebank in dhamar was provided with several solar energy batteries to improve the electricity supply for cooling the longterm seed storage. • safety duplication: copies of seed accessions from cereal and leguminous crops were sent to icarda in 2013 and subsequently stored in the svalbard seed vault (table 4). however, there were also negative changes and trends observed in germplasm conservation in genebanks. negative impact on field genebanks: the conservation of plant genetic resources in field genebanks managed by research stations across the various regions was significantly affected by the war and its associated socioeconomic and institutional consequences. the lack of operational budgets hindered essential agricultural activities such as ploughing, levelling, weeding, pruning, grafting, fertilizing and irrigation. fuel shortage and high prices further exacerbated the challenges faced by research stations in adequately maintaining the field genebanks. in certain field genebanks in the northern (al-errah) and central highlands (dhamar), some fruit trees were cut down by residents for firewood during periods of gas shortage and high prices resulting from the war and siege (figure 6). complete destruction of field genebanks occurred in the southern highlands agricultural research station (osaiferah, taiz) for mango, guava and coffee varieties, as well as in the southern coast agricultural research station in elkod, abyan governorate. similarly, the field genebank for pomegranate at the college of agriculture and veterinary medicine, university of dhamar, experienced the same fate, with all preserved trees being cut down due to war and siege conditions. limited implementation of biotechnologies: the use of biotechnologies, such as tissue culture, for the conservation of plant genetic resources has not been possible due to a lack of resources, including electrical energy sources, laboratory materials and equipment. thus, no duplicates of the accessions maintained in field genebanks had been maintained in tissue cultures and thus, as safety duplicates. limited multiplication and regeneration of seed samples: except for a limited number of seed accessions regenerated and multiplied through funded projects such as the ‘participatory conservation and sustainable use of landraces’ (2019–2023) (ngrc, 2022) and activities supported by the general corporation for grain production and development, the war’s economic effects halted vital regeneration programmes and seed multiplication activities. improper storage conditions, insufficient seed quantities and difficulties in providing seeds to farmers and researchers have led to a decline in seed viability. hundreds of samples are currently at risk of spoilage and loss, requiring regeneration and multiplication. genetic resources (2023), 4 (8), 71–90 plant genetic resource conservation in yemen 84 figure 5. areas of plant genetic resources collection in various parts of yemen (1989–2019). source: ngrc (2021), ngrc database (2021) and fao (2023) table 4. number of accessions of some types of yemeni plant genetic resources preserved outside yemen (sgsv, 2023). icrisat, the international crops research institute for the semi-arid tropics; cimmyt, the international maize and wheat improvement center; icarda, the international center for agricultural research in the dry areas; iita, international institute of tropical agriculture; us npgs, united states national plant germplasm system. *, includes the following genera: capsicum, nigella, citrullus, linum, coriandrum, medicago, teramnus and ricinus. crop icrisat icarda iita cimmyt us npgs others svalbard sorghum 2,144 3,376 45 5,565 maize 2 1 3 wheat 13 33 12 115 173 barley 66 25 55 146 millet 289 49 338 cowpea 25 12 37 lentil 38 48 86 bean 3 3 peas 50 50 others* 2 7 33 88 130 total 2,435 127 25 35 3,446 463 6,531 85 aljarmouzi et al genetic resources (2023), 4 (8), 71–90 table 5. changes in the number of species and accessions of genetic material in seed and field genebanks in yemen (2006–2020). source: fao (2009) and area (2021). centre/reserve institution site no. of species no. of accessions 2006 2020 2006 2020 national genetic resource center (ngrc) agricultural research and extension authority dhamar 56 47 3,281 6,300 genetic resource center college of agriculture, sana’a university sana’a 38 54 1,528 > 3,000 genebank nasser’s college of agricultural sciences, university of aden lahj 9 0 136 0 field genebank of date palm and lemon the valley and the desert agricultural research station, seiyun seiyun 1 1 67 48 field genebank of coffee, guava and mango the southern highlands agricultural research station osaifr, taiz 16 0 36 0 field genebank of pomegranate the college of agriculture and veterinary sciences, dhamar university dhamar 1 0 22 0 field genebank of mango and date palm tihama plateau agricultural research station al-kadan, surdud, alhudeidah 5 2 64 47 field genebank of apricot, grapes, almond, pomegranate, fig and olive the northern highlands agricultural research station al-errah, sana’a 11 6 218 78 field genebank of apple, olive and almond. the central highlands agricultural research station dhamar – 65 55 144 field genebank of banana, papaya, mango and date palm the southern coast agricultural research station al kodabyan 65 67 230 > 400 field genebank date palm, coconut and jasminum sambac the eastern coast agricultural research station mukalla, hadramout 15 24 68 > 200 total 217 266 5,705 10,217 figure 6. remains of the field genebank of mango, coffee and guava crops in the field genebank of the osaifera experimental farm, taiz genetic resources (2023), 4 (8), 71–90 plant genetic resource conservation in yemen 86 table 6. number of seed accessions in ngrc in dhamar that have been regenerated until 2014 and those that need regeneration. source: data were collected during the genetic resources inventory of ngrc in 2014 (ngrc, 2022). crop scientific name no. of accessions no. of regenerated accessions no. of accessions needing regeneration cereals sorghum sorghum bicolor 2,436 679 1,757 millet pennisetum glaucum 589 225 364 maize zea mays 505 108 397 barley hordeum vulgare 362 164 198 wheat triticum aestivum 351 229 122 finger millet eleusine coracana 36 0 36 al-tahf eragrostis spp. 11 0 11 legumes cowpea vigna unguiculata 451 88 363 lentils lens culinaris 180 78 102 fenugreek trigonella foenum-graecum 141 141 0 beans vicia faba 100 40 60 kidney beans phaseolus vulgaris 90 90 0 peas pisum sativum 80 80 0 lablab lablab purpureus 45 0 45 vegetables okra abelmoschus esculentus 95 0 95 radish raphanus sativus 47 33 14 eggplant solanum melongena 42 0 42 tomatoes solanum lycopersicum 41 0 41 carrot daucus carota 19 0 19 cumin cuminum cyminum 20 0 20 arugula eruca sativa 10 7 3 onion allium cepa 10 0 10 leek allium porrum 3 0 3 chilli capsicum spp. 40 0 40 cucumber cucumis sativus 8 0 8 pumpkin cucurbita spp. 6 0 6 melon cucumis melo 60 0 60 watermelon citrullus lanatus 10 0 10 calabash/squash cucurbita maxima 53 0 53 mallow corchorus olitorius 37 0 37 oil and cash crops sesame sesamum indicum 88 71 17 peanut arachis hypogaea 9 0 9 underutilized crops coriander coriandrum sativum 50 24 26 black seed nigella sativa 49 23 26 gaps and challenges facing the conservation of genetic resources in yemen this list of gaps and challenges in the conservation of plant genetic resources in yemen, in addition to the recommendations and conclusions, is based on government documents, including the first, second and third country reports prepared as contributions to the fao reports on the state of the world’s plant genetic resources for food and agriculture. area, as a national public agricultural research institution in yemen, has asked the national focal point of the itpgrfa and cgrfa to prepare a working paper to be presented at the planned national conference on genetic resources but postponed it to an undefined future time after preparatory efforts of more than a year. such a paper was prepared and reviewed by the steering committee and included a set of gaps, conclusions and recommendations. moreover, the recently prepared documents on a road map of the agricultural research programmes and projects (area, 2022), prepared 87 aljarmouzi et al genetic resources (2023), 4 (8), 71–90 by a specialist team, discussed thoroughly with the area management, and endorsed by higher agricultural authorities, are compatible with what is stated in this article. however, the content of this paper and other developments will serve as a background primary paper for any national symposium or conference when the time comes after the resumption of peace in the country. based on the above-reported situation, the most significant points can be summarized as follows. in situ conservation gaps and challenges • limited knowledge about rich areas/regions of plant genetic diversity and regions at risk of extinction and loss of diversity • inadequate methodologies for monitoring and evaluating genetic diversity, including geographical and plant species gaps • lack of a policy for restoring cropping systems after disasters and wars • absence of targeted collecting, propagation and reintroduction of rare and threatened species to their natural habitats • insufficient sustainable use of economically promising plant species • absence of in situ conservation and management of crop wild relatives and wild food plants • weak technical capabilities, including a shortage of specialized staff in farm systems management, biodiversity, environmental systems management, plant taxonomy, remote sensing, control and monitoring, and evaluation systems. additionally, laboratories in agricultural research, environmental protection and university science colleges lack certain devices and equipment. • lack of support and funding for managing natural sites, and inadequate funding for scientific research activities • lack of coordination between relevant authorities, particularly between area and epa, which serve as the national focal points for the itpgrfa and cbd, respectively. ex situ conservation gaps and challenges • weak integration between on-farm/in situ and ex situ conservation • absence of a plan for regeneration and multiplication under normal and emergency circumstances. • lack of a specialized supportive unit for communication, publication and coordination of genetic resources • genetic collection operations are not comprehensive and do not cover most geographical areas, genera or plant species • weak human, technical and institutional capabilities in ngrc • weakness in the basic infrastructure for managing plant genetic information • weak participation of local partners and government institutions in collecting, preserving and using plant genetic resources • lack of annual budgets to cover the expenses of collecting and preserving plant genetic resources • weak cooperation and participation in regional and international initiatives in the field of conservation and use. conclusion and recommendations based on the preceding results and discussion, the plant genetic resources system in yemen faces numerous problems and gaps, which have been exacerbated by the ongoing war since 2015. however, it is possible to identify a set of needs to develop conservation processes for both in situ and ex situ plant genetic resources in the country. in this regard, the most important recommendations can be summarized as follows: on farm/in situ • assess the impact of threats and pressures on crop diversity based on scientific foundations • identify areas of diversity and threats and develop an action plan for their conservation • conduct targeted collecting of threatened and rare species • reintroduce threatened or rare species and varieties into yemen through a coordinated effort • conduct inventory and survey operations for crop wild relatives, in coordination with ex situ activities • promote in situ conservation and management of crop wild relatives and wild food plants • develop a national seed policy that includes provisions for seed distribution mechanisms during and after disasters and wars • enhance technical expertise in plant taxonomy, agriculture and environmental systems management, and monitoring and evaluation • provide adequate funding and financial support for programmes and projects focused on managing natural sites and on-farm conservation • develop effective mechanisms to enhance cooperation and coordination among various relevant agencies. ex situ (genebanks) • improve storage conditions in genebanks by providing electricity, preservation equipment and materials, and conducting seed quality tests • establish a centrally organized seed health system by providing resources and expertise • enhance the national genetic resources information system, documentation and data exchange, and encourage the use of available information and accumulated knowledge • allocate operational budgets to cover the expenses of genebanks • implement seed regeneration and multiplication programmes for accessions at risk of loss • develop the capabilities of genebank personnel for routine genebank operations including information management systems genetic resources (2023), 4 (8), 71–90 plant genetic resource conservation in yemen 88 • provide ngrc with young male and female professionals and enhance their scientific and technical capacities in various crucial areas such as laboratory conservation using biotechnologies, genetic data and information management, plant taxonomy and characterization (particularly wild relatives), among others. it is also important to foster ngrc to assume coordinating responsibilities across the different areas and activities. • develop an awareness-raising strategy and plan highlighting the importance of the country’s genetic resources and biodiversity • establish and strengthen communication, cooperation and coordination among all relevant local, regional and international organizations involved in genetic resources and genebanks • conduct inventory and survey operations for crop wild relatives, in coordination with on-farm or in situ activities. acknowledgments we extend our sincere thanks and appreciation to dr muhammad al-khasha’a, director general of the agricultural research station on the southern coast, al-kod, abyan governorate, mr amjad bgigo from the eastern coast agricultural research station, mukalla, hadramout, mr ali abdul-mughni al-maqtari from the tihama plain agricultural research station, and dr saeed saif, director general of the agricultural research station in the southern highlands, taiz governorate. we would also like to express our gratitude to dr muhammad hizam al-mashreqi, dr fouad al-qadasi and dr nadir al-absi, from rnrrc, and thanks to all ngrc employees and the leadership of area for their support and cooperation in providing the necessary data, information and images for this study. furthermore, we express our thanks and gratitude to the benefit sharing fund of the itpgrfa for their support to the ngrc committee during the period 2019–2023, and in disseminating the project’s outputs, including this study. author contributions maeen ali al-jarmouzi prepared the study proposal, collected, organized, analyzed the data, wrote and improved the manuscript’s drafts. khalil m. alsharjabi contributed to reviewing and improving the study proposal, collecting the data, writing the manuscript, editing and improving the manuscript’s drafts at different stages. luigi guarino provided support through guidance, reviewing and improving the manuscript draft, enhancing the translation of the manuscript and providing important references. conflict of interest the authors declare no known conflicts of interest or any financial or personal relationships influencing the work or materials appearing in the article. references al-ghouri, m., sailan, a. a., bahumaish, a., al-muallem, a. b., bazara’a, m., and al-khulaidi, a. a. 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(2019). qat farms in yemen: ecology, dangerous impacts and future promise. pages 107. doi: https: //doi.org/10.1016/j.ejbas.2013.09.002 http://www.nordgen.org/sgsv http://documents.worldbank.org/curated/en/721331468334834796/environmental-and-social-impact-assessment http://documents.worldbank.org/curated/en/721331468334834796/environmental-and-social-impact-assessment http://documents.worldbank.org/curated/en/721331468334834796/environmental-and-social-impact-assessment http://documents.worldbank.org/curated/en/438861468334798669/republic-of-yemen-rainfed-agriculture-and-livestock-project http://documents.worldbank.org/curated/en/438861468334798669/republic-of-yemen-rainfed-agriculture-and-livestock-project http://documents.worldbank.org/curated/en/438861468334798669/republic-of-yemen-rainfed-agriculture-and-livestock-project https://doi.org/10.1016/j.ejbas.2013.09.002 https://doi.org/10.1016/j.ejbas.2013.09.002 introduction materials and methods results and discussion state of in situ conservation and on-farm management surveys and inventories of plant genetic resources on-farm management and improvement of plant genetic resources for food and agriculture providing seeds to farmers and researchers in case of disasters to restore cropping systems changes and trends in on-farm genetic resources conservation status of ex situ conservation of plant genetic resources (genebanks) supporting the targeted collection of plant genetic resources expanding diversity in genebanks regeneration and multiplication of genebank seed samples conservation changes and trends in genebanks gaps and challenges facing the conservation of genetic resources in yemen conclusion and recommendations acknowledgments author contributions conflict of interest original article genetic resources (2024), 5 (9), 72–82 doi: 10.46265/genresj.weta7514 https://www.genresj.org issn: 2708-3764 morphological and molecular characterization of ‘saragolla’ wheats (triticum turgidum subsp. durum from abruzzo, italy) agata rascio *,a, vanessa de simone a, lorenzo goglia b, silvana paone a, maria pellegrino a and giuseppe sorrentino b a council for agricultural research and economics, research centre for cereal and industrial crops s.s, 673 km 25, 200 71122, foggia, italy b institute for sustainable plant protection italian national research council (ipsp-cnr), piazzale enrico fermi, 1, 80055, portici (na), italy abstract: a morphological and genetic characterization of autochthonous ‘saragolla’ wheats, currently cultivated in abruzzo region (italy), was carried out. using 15 simple sequence repeat (ssr) markers and 24 upov morphological traits we compared: (a) 13 ‘saragolla’ genotypes with traits of the italicum/apulicum botanical varieties (saragolla (sar.) italicum), (b) 26 ‘saragolla’ genotypes with traits of leucurum/affine botanical varieties (sar. leucurum), (c) 8 breeding varieties (pure lines), and (d) 5 italian autochthonous wheats and 1 turanicum line (old wheats). one hundred twenty-six (126) alleles were identified. the number of alleles per locus spanned from 4 to 15 and the number of alleles per genotype varied between 12 and 21. values of gene diversity (nei) across the 53 genotypes was 0.17. the groups of sar. leucurum and sar. italicum genotypes were morphologically distinguishable from the groups of old wheats and pure lines. likewise, the analysis of molecular data using the discriminant analysis revealed that genotypes with the sar. italicum phenotype displayed distinct genetic differences from sar. leucurum, pure lines and old wheats. these results make sar. italicum genotypes distinguishable and eligible as a conservation variety. ward’s clustering analysis of the 53-genotype pool showed that the ‘saragolla’ landrace is a valuable repository of genetic diversity. keywords: abruzzo region, ’saragolla’ landrace, durum wheat diversity, genetic characterization citation: rascio, a., de simone, v., goglia, l., paone, s., pellegrino, m., sorrentino, g. (2024). morphological and molecular characterization of ‘saragolla’ wheats (triticum turgidum subsp. durum from abruzzo, italy). genetic resources 5 (9), 72–82. doi: 10.46265/genresj.weta7514. © copyright 2024 the authors. this is an open access article distributed under the terms of the creative commons attribution license (cc by 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. introduction traditional crops are generally viewed by consumers as more natural and healthier options (rascio et al, 2015, 2016). their reintroduction into cultivation and reinforcing their value chain can play a vital role in bolstering conservation efforts and elevating the value of their cultivation environment. this, in turn, can substantially increase the economic benefits for rural communities (pallante et al, 2016). before the onset of ∗corresponding author: agata rascio (email:agata.rascio@crea.gov.it) the breeding era in italy, initiated by strampelli in the early twentieth century (scarascia-mugnozza, 2005), cultivated wheat primarily consisted of autochthonous varieties. although there is not a worldwide consensus on this definition, these wheats are often referred to as old/ancient wheats, or landraces (negri et al, 2009). autochthonous wheats are regarded as valuable sources of alleles for breeding programmes (terzi et al, 2005). indeed, over time, environmental conditions and, to a certain extent, purposeful farmer selection have resulted in the cultivation of plants exhibiting high adaptability and superior performance within their respective cultivation regions (zeven, 1998). for received: 18.10.2023 accepted: 10.04.2024 published online: 14.05.2024 https://www.genresj.org https://www.doi.org/10.46265/genresj.weta7514 https://www.genresj.org https://www.doi.org/10.46265/genresj.weta7514 mailto:e\protect \discretionary {\char \hyphenchar \font }{}{}mail:agata.rascio@crea.gov.it genetic resources (2024), 5 (9), 72–82 characterization of italian autochthonous ‘saragolla’ wheats 73 these reasons, the eu (eu, 2009) defines landraces or varieties which are naturally adapted to local and regional conditions as conservation varieties (spataro and negri, 2013). they are frequently composed of mixed genotypes (populations) and exhibit a high degree of genetic diversity (zeven, 1998), in contrast to modern breeding varieties (cultivars or pure lines). following the ratification of the international treaty on plant genetic resources for food and agriculture by the un food and agriculture organization (fao, 2009) in 2001, most italian regions implemented laws aimed at protecting and promoting indigenous crops. they also provided funding for projects dedicated to studying the conservation varieties’ distinctive characteristics. their registration involves a formal process where these varieties are officially recognized, documented, and often included in seed catalogues or databases. once registered, conservation varieties are often conserved in community seedbanks or similar facilities where they can be accessed by farmers. enhancing access to these varieties is important to support agricultural biodiversity, improve resilience in the face of environmental challenges, and contribute to the long-term sustainability of food production systems. some countries, by officially recognizing and documenting these varieties, provide legal protection to conservation varieties to prevent unauthorized use or commercial exploitation (eu, 2009). this protection is designed to encourage the continued conservation and sustainable use of these valuable genetic resources. in southern italy, the cultivation of a wheat type known as ‘saragollo forte’ which is particularly wellsuited for pasta production, has been extensively documented in various commercial agreements dating back to the seventeenth century (fiore, 2013). as introduced by old botanists (draghetti, 1927; de cillis, 1927), the use of the plural noun ‘saragolle,’ highlights the presence of multiple forms of ‘saragolla’ wheat, all falling under the saragolla (sar.) leucurum botanical variety, which was one of the 22 botanical varieties of triticum turgidum documented at the start of the 1900s (percival, 1921). in 2004, the ‘produttori sementi bologna’ company registered a variety also named ‘saragolla’. this variety resulted from crosses between the ‘iride’ cultivar and the ‘0114’ elite line. therefore, this ‘saragolla’ pure line is an enhanced variety and not a local one. today, a significant number of farmers in central and southern italy are cultivating the old ‘saragolla’ wheat, either for personal consumption or to establish short food supply chains of autochthonous wheat. the resurgence of interest in ‘saragolla’ can be attributed to its adaptability to low-fertility soils and its suitability for cultivation with minimal input methods, making it especially attractive for agriculturally marginal regions. moreover, this revived interest in ‘saragolla’ has been magnified by online sources (eccellenze d’abruzzo, 2024), which suggest that it can be an italian alternative to khorasan wheat, marketed under the name ‘kamut’ (piergiovanni, 2013). kamut is a registered variety belonging to the tetraploid species triticum turanicum jacubz, initially described by percival (1921) as t. orientale. this hypothesis is reinforced by the morphological resemblance between the elongated seeds of ‘saragolla’ cultivated in the abruzzo region and the seeds of ‘kamut’ t. orientale percival. considering the complexity of the situation, in the years 2018–2020, the local authorities of the italian region abruzzo funded the sarab project, ‘characterization of local saragolla durum wheat populations’, to characterize the ‘saragolla’ wheat currently cultivated. through intensive cataloguing based on morphology, the project focused on the main species and botanical varieties of ‘saragolla’ cultivated in 11 different sites within the abruzzo region (rascio et al, 2021). the results revealed a heterogeneous botanical composition both within and among these sites (rascio et al, 2022). nine botanical varieties of durum wheat were observed, the majority belonging to the italicum or apulicum botanical varieties, primarily differing in glume pigmentation intensity. there was also a smaller number of genotypes falling into the leucurum or affine botanical varities (referred to here as saragolla (sar.) leucurum), which exhibited variations in seed pigmentation intensity. the main goals of this study were to conduct genetic and morphological characterizations of representative plants belonging to the most prevalent botanical varieties collected in 11 farms that participated in the sarab project. additionally, these varieties were compared with a collection of both pure lines (modern) and traditional italian (old) wheat varieties. the results here shown indicate that, in contrast to the sar. leucurum, the italicum genotypes shared a close genetic similarity among themselves. they are widespread in the abruzzo region and genetically distinct from the groups of modern and old genotypes examined in this study, hence they are eligible for registration as a conservation variety. the study also explores the degree of diversity of the four groups of genotypes. material and methods plant material eleven samples of ‘saragolla’ wheats from 11 locations in abruzzo (shown in supplemental table 1) were grown and characterized at the council for agricultural research and economics research centre for cereal and industrial crops (crea-ci) in foggia (rascio et al, 2022). about 1,000 individual plants of these heterogeneous wheats were morphologically examined and 434 durum wheat plants were found. a total of 39 representative spikes of this subset, selected to be indicative of the prevailing botanical varieties italicum/apulicum and leucurum/affine were used for further genetic and morphological characterization in this study. for comparison, eight modern varieties/durum cultivars and six samples of old autochthonous durum wheat samples 74 rascio et al genetic resources (2024), 5 (9), 72–82 belonging to the crea-ci working collection were also included (supplemental table 2). they comprised four genotype groups: 1) sar. italicum: 13 ‘head to row’ of ‘saragolla’ genotypes exhibiting at least four out of five traits of the italicum/apulicum botanical varieties 2) sar. leucurum: 22 ‘head to row’ of ‘saragolla’ genotypes from abruzzo and four from puglia displaying at least four out of five traits of the leucurum/affine botanical varieties 3) modern pure lines : eight seed samples of durum wheat cultivars: ‘ciccio’, ‘cappelli’, ‘capeiti’, ‘colosseo’, ‘duilio’, ‘simeto’, ‘svevo’ and ‘saragolla’ 4) old wheats: six seed samples of autochthonous durum wheats, primarily sourced from sicily (fiore et al, 2019) and belonging to the crea-ci working collection. these include: ‘realforte’, ‘russello’, ‘sammartinara’, ‘scorsonera’, ‘vallelunga pubescent’, and the t. turanicum pure line (pi166959), selected at crea-ci. phenotypic assessment in 2021, a total of 53 rows, each 1m in length and spaced 30cm apart, were sown according to the usual agronomic practices (rascio et al, 2016). throughout the growth stage, each row was carefully examined to ensure its purity, and one plant was selected for dna extraction and morphological characterization. the assessment was performed on 24 traits with value scales employed for evaluation in part adhering to the guidelines outlined by the international union for the protection of new varieties of plants (upov, 2012) (table 1). molecular marker analysis for each genotype, the extraction of dna was performed according to the protocol used by marone et al (2009). twenty-eight microsatellite single sequence repeat (ssr) markers were selected based on published map data (marone et al, 2009, 2012), according to the following criteria: locus-specific amplification, high level of polymorphism, and good genome coverage (one marker per chromosome arm). the sequences of the ssr are available in the graingenes database (http://wheat.pw.usda.gov). the pcr reactions were performed in 25µl volume in applied biosystems 2720 thermal cyclers. the reaction mixture contained 60ng of template dna, 0.2mm of dntps, 1x buffer (10mm tris–hcl—ph 8.3, 50mm kcl, 1.5mm mgcl2), 0.4µm labelled reverse primer (fam or hex or ned or tet), 0.4µm unlabelled forward primer and 0.2u of taq dna polymerase (5u/µl) (kapa). thermal cycling conditions were as follows: 94◦c for 3min, followed by 45 cycles of 94◦c for 30s, the specific annealing temperature (ta) for each primer for 30s, 72◦c for 30s, with a final extension at 72◦c for 2min. the amplification products were analyzed by means of capillary electrophoresis (abi3130), multiplexing different fluorescent dyes. electropherograms were analyzed with genemapper version 4.0. the internal molecular weight standard was 500-rox (life technologies). statistical analysis genotypic characterization was performed with 15 ssr markers which gave a clear electrophoretic pattern. to this aim, the genotypic data were transformed into a binomial matrix as present (1) or absent (0) for each marker and this matrix was used to construct ward’s dendrogram tree to assess genetic diversity. nei’s gene diversity, percentage of polymorphic loci and shannon’s information index were determined using the popgen 1.31 software (yeh et al, 1999). for assessing marker polymorphism and informativeness, the average polymorphic information content (pic) was calculated using the following formula introduced by anderson et al (1993): pic = 1-σ(pi)2 where pi is the number of polymorphic loci/all the number loci. the distances among the four groups of genotypes (sar. leucurum, sar. italicum, pure lines and old wheats) were examined by multivariate discriminant analysis and cluster analysis, using the statistica (statsoft inc.) software. results morphological characterization as shown in figure 1 and in supplemental table 3, the sar. italicum/apulicum-like genotypes have rather compact, hairy glumes, lightly pigmented spikes, long red or brown-red awns, yellow-amber and elongated grains. the sar. leucurum/affine genotypes have white and glabrous glumes, elongated, compact spikes, and white or red seeds. the mean values of each morphological and phenological trait for genotypes belonging to the four groups (sar. leucurum, sar. italicum, modern cultivars or old wheats) show that the sar. italicum/apulicum genotypes registered the highest values for glume hairiness and 1,000 seed weight (table 2). in contrast, the sar. leucurum/affine genotypes exhibited the highest values for the shape of the lower glume beak. modern varieties displayed smaller height and earlier heading dates, a result of the extensive breeding efforts they underwent. results of stepwise discriminant analysis performed using the four groups of genotypes as classification categories and visualized through the biplot of canonical variables (figure 2) showed that the model had a high discriminatory power (lambda wilks: 0,0075245; approx. f (48,90) = 7,8312; p < 0,0000), with two discriminant functions that accounted for 92.9% of the explained variance (table 3). based on the absolute values of standardized coefficients of the canonical variables (table 3) the main traits that horizontally contributed to the 4-group separation were the glume hairiness, which was absent in sar. leucurum and the upper neck glaucosity, which genetic resources (2024), 5 (9), 72–82 characterization of italian autochthonous ‘saragolla’ wheats 75 table 1. the 24 traits used for the morphological characterization of wheat genotypes, and the value scale employed for evaluation (upov, 2012). trait measure units/score assessment scale 1 1,000 seed weight g 2 curvature of lower glume beak 1–7 1 = absent, 7 = strong 3 lower glume length of beak 1–9 1 = very short, 7 = long 4 lower glume hairiness 1–9 1 = absent, 9 = very present 5 straw: pith in cross section 1–7 1 = thin, 7 = thick 6 grain shape 3–7 3 = slightly, 7 = strongly elongated 7 grain: length of brush hair 3–7 3 = short, 7 = long 8 grain weight/plant g 9 awn colour 1–5 1 = white, 3 = medium dark, 5 = very dark 10 spike colour 1–5 1 = white, 3 = medium dark, 5 = very dark 11 awn tip/ear length ratio 1–3 1 = low ratio, 3 = high ratio 12 spike length cm 13 spike shape in profile 1–5 1 = tapering, 2 = parallel sided, 3 = slightly clavate, 4 = strongly clavate, 5 = fusiform 14 ear glaucosity 1–9 1 = absent, 9 = very strong 15 awn divergence 1–2 1 = adherent, 2 = divergent 16 spike density 3–7 3 = lax, 5 = medium, 7 = dense 17 plant height cm 18 growth habit 1–9 1 = erect, 9 = prostrate (score 9) 19 recurved flag leaves 1–9 1 = absent, 9 = very high 20 heading date from april 1st days 21 flag leaf: glaucosity of sheath 1–9 1 = absent, 9 = very strong 22 flag leaf lower side glaucosity 1–9 1 = absent, 9 = very strong 23 upper node hairiness 1–9 1 = absent, 9 = very strong 24 upper neck glaucosity 1–9 1 = absent, 9 = very strong figure 1. comparison of distinguishing characteristics of glume (a), and spike and seed (b) of saragolla botanical types sar. italicum and sar. apulicum: hairy with red glumes and red awns; sar. leucurum and sar. affine: glabrous with white glumes and white awns. 76 rascio et al genetic resources (2024), 5 (9), 72–82 table 2. mean values of morphological traits of the four groups of durum wheat genotypes. the qualitative traits, for which no specific unit of measurement is provided, were assessed using the value scale established by the international union for the protection of new varieties of plants (upov, 2012). trait sar. leucurum sar . italicum modern cultivars old wheats shape of lower glume beak mean 6,9 2,0 2,7 2,1 sd 0,6 1,3 2,0 1,6 glume length of beak mean 4,0 4,9 4,3 3,6 sd 1,8 1,6 3,0 1,9 glume hairiness mean 1,3 8,2 1,3 1,0 sd 1,5 1,7 0,8 0,0 straw: pith in cross section mean 5,5 6,7 6,7 6,4 sd 1,9 0,6 0,8 1,0 ear length (cm) mean 9,2 9,4 8,5 8,4 sd 1,2 1,0 2,3 2,2 grain shape mean 4,6 6,8 3,7 3,6 sd 1,7 0,6 1,0 1,5 grain: length of hair mean 3,3 4,1 3,1 3,0 sd 0,9 1,3 0,2 0,0 awn colour mean 1,8 2,3 2,2 2,6 sd 1,1 0,9 1,6 1,4 glume colour mean 1,7 2,0 1,5 2,0 sd 0,8 0,9 0,5 1,5 awn tip/ear length ratio mean 1,6 2,7 1,7 1,6 sd 0,7 0,6 0,8 0,8 ear shape mean 2,0 2,2 3,0 2,0 sd 0,9 1,2 1,3 0,8 awn compactness mean 1,4 1,5 1,5 1,9 sd 0,6 0,8 0,5 1,5 ear density mean 5,2 4,7 4,7 6,1 sd 1,9 1,4 1,5 1,1 plant height (cm) mean 108,7 120,0 79,2 101,4 sd 16,2 7,9 17,7 21,7 grain weight/plant (g) mean 19,7 15,2 19,0 15,3 sd 6,3 4,2 1,9 3,4 1,000 seed weight (g) mean 46,5 73,3 48,7 46,4 sd 8,5 3,6 5,3 7,0 growth habit mean 3,7 4,4 2,3 4,4 sd 1,4 1,0 2,1 1,0 % recurved flag leaves mean 6,1 6,7 2,7 5,0 sd 1,6 0,8 1,5 2,0 heading date mean 28,3 29,4 18,1 20,7 (days from april 1st) sd 4,1 1,6 6,6 6,3 flag leaf glaucosity mean 5,1 5,8 4,7 4,4 sd 1,5 1,3 2,0 2,2 flag leaf lower glaucosity mean 3,4 3,0 3,7 3,3 sd 1,4 0,0 1,0 1,4 upper node hairiness mean 3,3 3,0 2,3 3,0 sd 1,3 0,0 1,0 1,2 upper neck glaucosity mean 5,1 3,3 5,0 4,4 sd 1,5 0,8 1,3 1,5 spike glaucosity mean 4,8 6,7 5,7 5,3 sd 1,4 1,1 1,0 1,4 genetic resources (2024), 5 (9), 72–82 characterization of italian autochthonous ‘saragolla’ wheats 77 figure 2. the biplot of canonical variables obtained using a stepwise discriminant analysis based on 24 upov morphological descriptors and four groups of genotypes. the percentages of explained variance by the two roots are shown. was lacking in sar. italicum (table 2). the differences in heading date along with the upper neck green glaucosity degree (table 2) mainly contributed to the vertical separation of sar. leucurum and italicum from the pool of new and old genotypes. the mahalanobis distances between groups were all highly significant (table 4), except between pure lines and old wheats; sar. leucurum and sar. italicum were the two most distant and hence morphologically different groups. ssr patterns the molecular analysis utilized a set of 15 ssr markers out of initially 28 tested, each characterized by a distinct electrophoretic pattern for all genotypes. in total, 126 alleles were identified, with the number of alleles per locus spanning from 4 to 15. the polymorphic information content (pic) values of the ssr markers ranged from 0.52 (for gwm60) to 0.91 (for wmc606 and gwm459), resulting in an average pic value of 0.77 per locus (table 5). excluding from the analysis the alleles that occurred at a low frequency (p < 0.05), the number of alleles per genotype varied between 12 and 21, with the most frequent value being 16 (table 6). this occurrence was four times higher than what was observed in a study where 104 ethiopian durum wheat genotypes, representing 13 populations, three regions, and four altitudinal classes, were analyzed using 14 ssr markers (dagnaw et al, 2023). diversity the diversity analysis for all cultivars based on ssr markers (table 7) yielded low mean values (0.28±0.20) of shannon’s index. the values (0.176±0.5) of nei’s gene diversity were lower than the minimum observed in 40 winter wheat genotypes (petrović et al, 2017) coming from european countries (croatia, austria, france, italy, and russia) and lower than that (0.56) resulting for 124 ethiopian genotypes (dagnaw et al, 2023). the among-groups comparison indicated that old wheats and sar. leucurum exhibited the highest percentage of polymorphic loci, followed by pure lines and old wheats. the measurement of gene diversity, estimated by both nei’s gene diversity and shannon’s information index, yielded similar values for sar. leucurum, breeding lines, and old wheats, and the lowest values for sar. italicum. in the case of sar. leucurum, sar. italicum, old wheats and pure lines, the highest average number of amplified alleles per locus was observed in 1b (long arm), 6b (short arm) and 6b (long arm), respectively, with average values of 9.0, 6.5, 2.5 and 2.3, respectively. it’s worth noting that old wheats and modern pure lines exhibited the highest percentage of polymorphisms detected by ssr markers in the b genome (table 8), likely originating from a species, or several species closely related to aegilops speltoides tausch, a cross-pollinating species; while the sar. leucurum and sar. italicum sets had the highest percentage of polymorphisms in the a genome, which can be traced back to diploids like t. urartu thumanjan ex gandilyan (wang et al, 2007). a similar clustering pattern was observed when analyzing both the morphological traits (figure 2) and molecular marker profiles (figure 3) of all 53 genotypes. this analysis employed a hierarchical grouping method, without missing data in the dataset. the resulting dendrogram revealed four major clusters (figure 3). cluster 1a comprised all 13 sar. italicum genotypes, 9 out of 22 sar. leucurum genotypes of abruzzo and 1 sar. leucurum genotype from puglia. the second major cluster, 1b, could be further subdivided into two subclusters: 1b1 and 1b2. the 1b1 cluster included two subgroups: the first subgroup contained four closely related breeding lines (‘colosseo’, ‘simeto’, ‘ciccio’ and ‘capeiti’), two sar. leucurum genotypes, and the old wheat ‘vallelunga pubescent’; the second subgroup was larger, consisting of modern varieties (‘duilio’, ‘realforte’, ‘svevo’ and the ‘saragolla’ pure line), some old wheats (‘russello’, ‘scorsonera’, ‘sammartinara’, ‘cappelli’ and the t. turanicum line), along with eight sar. leucurum genotypes from puglia or abruzzo. cluster 1b2 included six strongly related sar. leucurum genotypes: one was from puglia and five from abruzzo. discussion the morphological and genetic characterization of autochthonous wheats serves the dual purpose of safeguarding the economic interests of farmers and increasing consumers’ trust in the origin and quality of food products entering the market (terzi et al, 2005). a recent morphological analysis conducted by the sarab project on wheat crops in 11 farms across 78 rascio et al genetic resources (2024), 5 (9), 72–82 table 3. values of the standardized coefficients for the canonical variables included in the discriminant functions, obtained using the four groups of genotypes (sar. leucurum, sar. italicum, pure lines and old wheats) as classification categories. root 1 root 2 glume hairiness -0,99 -0,26 heading date (days from 1/4) 0,05 -1,39 upper neck glaucosity 0,73 -0,98 growth habit -0,48 0,64 grain shape -0,06 -0,50 1,000 seed weight 0,03 0,44 spike glaucosity -0,43 -0,05 spike shape -0,27 -0,08 glume colour 0,18 -0,57 awn colour -0,40 0,38 plant height 0,48 -0,17 flag leaf lower side glaucosity 0,19 -0,49 grain weight/plant 0,09 -0,36 straw: pith in cross section -0,23 0,20 lower glume: length of beak -0,36 -0,29 spike density 0,21 0,31 eigenvalue 12.4 3.4 explained cumulative variance (%) 72.6 92.9 table 4. pairwise square mahalanobis distances (plain text) and probability values (italics) for the contrasts between the four groups of genotypes. sar. leucurum sar. italicum pure lines old wheats sar. leucurum 0,0000 0,00036 0,00003 sar. italicum 69,48 0,00000 0,00000 pure lines 23,76 78,44 0,01962ns old wheats 27,19 68,77 20,95 table 5. list of ssr markers used for molecular analysis, number of alleles and polymorphic information content (pic) obtained for each marker in the 39 saragolla wheat lines. a, a genome; b, b genome; l, long arm; s, short arm. marker chromosome no. of alleles pic 1 gwm311 2a(l) 11 0,83 2 gwm1042 3a(l) 6 0,55 3 gwm299 3b(l) 7 0,74 4 barc45 3a(s) 4 0,77 5 gwm495 4b(s) 8 0,82 6 gwm1093 4a(s) 14 0,83 7 gwm1084 4b(l) 8 0,75 8 gwm865 5a(l) 8 0,80 9 gwm154 5a(s) 9 0,73 10 gwm499 5b(l) 11 0,80 11 gwm1017 6a(l) 6 0,80 12 gwm459 6a(s) 12 0,91 13 gwm193 6b(l) 4 0,76 14 gwm60 7a(s) 7 0,52 15 wmc606 7b(s) 11 0,91 genetic resources (2024), 5 (9), 72–82 characterization of italian autochthonous ‘saragolla’ wheats 79 table 6. average number of ssr alleles per genotype. only alleles occurring with a frequency higher than 11 (p < 0.05) are included. the codes from s1 to s11 indicate the cultivation sites (see supplemental table 1) of saragolla wheats from abruzzo and s12 indicates the cultivation site in puglia. the extra letters and numbers differentiate the genetically characterized plants within each site. old wheats and pure lines are described in supplemental table 2. group genotypes average allele no. group genotypes average allele no. sar. leucurum s1f3 13 sar. italicum s1h3 16 s2e 13 s2p2a 15 s2f 14 s3p27f 20 s3p27a 16 s4p23b 14 s3p3b 15 s5p49b 21 s3p3i 16 s6p6a 16 s4p4b 16 s7p43c 16 s4p4a 16 s8p8d 16 s4z 15 s8p9a 16 s5p5c 16 s10p50c 15 s5p31d 16 s11p11a 17 s6p21d 15 s7a 16 s6p41c 15 s7c 14 s8a1 14 old wheats ‘sammartinara’ 15 s8r1 14 ‘realforte’ 12 s8 15 ‘scorsonera’ 17 s9p22d 15 ‘russello’ 16 s9p9d 23 ‘vallelunga pubescent’ 13 s9p22a 15 ‘turanicum’ 17 s10p32a 14 modern pure lines ‘capeiti’ 14 s11p24a 15 ‘ciccio’ 15 s12p12a 14 ‘simeto’ 15 s12p54b 16 ‘colosseo’ 15 s12p35a 14 ‘duilio’ 16 s12p54a 14 ‘saragolla’ 15 s1e4 15 ‘cappelli’ 13 ‘svevo’ 16 table 7. genetic diversity indices over 15 ssr loci for all 53 italian genotypes tested in the study, as well as for the four groups categorized by ‘saragolla’ botanical variety or control group (means±sd). pic, polymorphic information content. shannon’s information index percentage of polymorphic loci nei’s gene diversity pic sar. leucurum 0.19±0.19 71.4 0.10±0.13 0.902±0.08 sar. italicum 0.08±0.18 22.2 0.05±0.13 0.097±0.08 old wheats 0.17±0.22 39.7 0.10±0.14 0.929±0.10 modern pure lines 0.19±0.19 61.1 0.11±0.12 0.764±0.11 all genotypes 0.28±0.20 97.63 0.17±0.15 0.673±0.35 table 8. percentage of polymorphism detected by ssr in a and b genomes of four groups of genotypes ‘saragolla’ leucurum ‘saragolla’ italicum old italian wheats modern pure lines genome a 54,1 54,5 46,2 48,5 genome b 45,9 45,5 53,8 51,5 80 rascio et al genetic resources (2024), 5 (9), 72–82 figure 3. ssr-based genetic distances among the 53-genotype pool, through ward’s clustering. the percentage of the prevalent type of genotypes included in each cluster is indicated. characters: red = sar. leucurum/affine genotypes from abruzzo; blue = sar. leucurum from puglia; black = sar. italicum/apulicum genotypes from abruzzo; violet = modern and old varieties. codes are as in table 6. the abruzzo region revealed significant morphological diversity within the cultivated ‘saragolla’ variety (rascio et al, 2022). this diversity poses a challenge in accurately defining their distinct traits. the morphotypes that are both quantitatively and widely spread, across most of the 11 wheat farms included in the sarab project, belong to the sar. italicum/apulicum or the sar. leucurum/affine botanical varieties (rascio et al, 2022). the genetic characterization described here aimed to validate whether the observed morphological similarity among the genotypes from abruzzo corresponds to genetic similarity and to develop a tool to differentiate them. the results presented here confirm the efficacy of ssr markers to characterize wheat genotypes (wang et al, 2007; dagnaw et al, 2023). in fact, the 15 ssr primers used in this experiment showed detectable polymorphisms in all the 53 genotypes and their mean polymorphic information content (pic = 0.77) makes their use very informative. ‘saragolla’ genotypes, belonging to the italicum/apulicum botanical varieties can be eligible as conservation varieties. these genotypes are widely cultivated across the abruzzo region, and they also exhibit a noteworthy genetic similarity, as indicated by the low values of nei’s gene diversity and shannon’s information index. additionally, sar. italicum genotypes display both a distinct phenotype and genotype in comparison to sar. leucurum, older wheats and pure lines. it is worth noting that ward’s clustering analysis of the 53-genotype pool revealed significant genetic diversity between sar. italicum and sar. leucurum genotypes. out of the 26 sar. leucurum genotypes examined, only 9 displayed a significant genetic resemblance to sar. italicum. six were categorized within the broader groups of pure lines and old wheats, while five formed a distinct group of genotypes very closely related genetically, but distinct from all others. the clustering analysis also revealed a stronger genetic similarity between most sar. leucurum genotypes and three out of four ‘saragolla’ genotypes from puglia and a somewhat lesser degree of affinity with the oldest genetic resources (2024), 5 (9), 72–82 characterization of italian autochthonous ‘saragolla’ wheats 81 genotypes. expanding on the hypothesis (zeven, 1998), that factors such as geographic distance, environmental conditions and the selection made by farmers can shape the genetic composition of local wheats, it is plausible to infer that the migration of wheat commenced from sicily. in fact, the leucurum genotypes were documented in southern italy as early as the beginning of the 1900s (percival, 1921; de cillis, 1927; draghetti, 1927) and were likely among the oldest cultivated in sicily (porceddu et al, 1981). from sicily, it is plausible that these wheats initially spread to the nearby region of puglia and then reached abruzzo where the cross with indigenous wheat occurred as well as the selection of alleles improving adaptability, productivity and quality. in terms of affinities with ’kamut’, the results suggest that the sar. italicum genotypes, despite having elongated and large seeds similar to t. turanicum, formed a distinct cluster and showed a closer genetic relationship to sar. leucurum and old durum wheats. conclusions the ‘saragolla’ wheat presently grown in the abruzzo region is characterized by its rich diversity, predominantly comprising sar. italicum/apulicum and sar. leucurum-like durum wheats, some of which share close morphological and genetic traits. recently, these genotypes have been officially registered as ‘saragolla’ conservation varieties from abruzzo. the genetic distance observed among 39 ‘saragolla’ genotypes, representative of only two out of the nine previously identified botanical varieties of durum wheat, exceeded that found among the other 13 modern, or old durum wheats used in this study, which differ for age of cultivation and origin. consequently, at the maiella national park seedbank, targeted ex situ conservation measures will be implemented to preserve the currently cultivated populations. a more extensive genetic characterization will enable the assessment of existing variability within the ‘saragolla’ landrace, for adaptive and agronomically valuable traits, useful for breeding improved varieties. supplemental data supplemental table 1. geographic coordinates of the cultivation sites for the 12 saragolla wheats. supplemental table 2. passport details of old and modern wheats used in the present work. supplemental table 3. morphological traits of saragolla leucurum, italicum, and modern and old wheats. acknowledgements the authors wish to thank the farm owners who provided the studied materials, dr maurizio odoardi and dr daniela codoni (department of rural development and fisheries policies – promotion of knowledge and innovation in agriculture – dpd022) of the abruzzo regional authorities, for their valuable contribution to the ‘sarab project: characterization of ancient ‘saragolla’ populations from the abruzzo region’. the authors also wish to thank mr leonardo morrone and mr vito de gregorio for their valuable assistance in conducting the experimental trials. funding this work was in part supported by the abruzzo region. author contributions ar, study conception and manuscript draft; vds, molecular analysis; lg, analysis and interpretation of results; sp, data collection; mt, field trials; 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hasan, choudhary, naaz, sharma, & laskar, 2021; helentjaris, king, slocum, siedenstrang, & wegman, 1985; tanksley, 1983). over time, these techniques have been augmented with high-quality phenotypic data to perform genome-wide association studies (gwas) and genomic selection and prediction, further fueling breeding for quantitative or complex traits (eathington, crosbie, edwards, reiter, & bull, 2007; heffner, sorrells, & jannink, 2009; lorenzana & bernardo, 2009). while these achievements are significant, many crop species grown for human consumption and livestock feed are still unable to apply these techniques in breeding efforts. many breeders would like to adopt molecular breeding tools and techniques, but sometimes doing so is hampered by large barriers-to-entry challenges. the range of barriers and how surmountable they are, varies from species to species and is impacted by species-specific challenges in logistics, technical know-how, biology and the growing environment. alfalfa (medicago sativa l.) is the most widely grown perennial forage crop worldwide (undersander, 2021). in the united states, it was the fourth most cultivated crop in 2021 with an estimated direct value of us$11.6 billion (putnam & meccage, 2022) and ranked first among forage crops planting area with a total of 14.9 million acres in 2022 (https://www.nass.usda.gov/). alfalfa is a key nutritional component for dairy and beef production because it contains a high amount of crude protein, provides dietary fibre needed to maintain rumen health, and is an excellent source of vitamins and minerals. in addition, it is unparalleled as a component of sustainable agricultural systems because of its ability to fix nitrogen, protect water quality, interrupt pest and pathogen cycles in annual crops, and improve soil carbon storage (fernandez, sheaffer, tautges, putnam, & hunter, 2019). alfalfa is adapted to different growth environments and depending on location and management, is highly persistent. as a highly heterozygous outcrossing autotetraploid species, alfalfa features a predominant pattern of random chromosome pairing during meiosis. the four sets of chromosomes add a layer of complexity to genotyping endeavours. traditional snp marker systems, primarily designed for diploid species, often fall short when applied to alfalfa due to their inability to identify allelic dosages accurately. thus, the intricacies of alfalfa's genetic structure call for more sophisticated snp genotyping systems capable of addressing the unique challenges posed by its autotetraploid nature. the investment cost and reliance upon skilled bioinformatics support for each genotyping run, make this a high-risk technology for breeders to adopt. currently, most alfalfa cultivars are synthetic populations developed by multiple cycles of phenotypic selection for desired traits. evaluation for biomass yield, winter survival, grow back, disease resistance and forage nutritional quality among other traits is a multi-year process before cultivar registration and commercial seed production. breeding programmes have operated with half-sib populations originating from polycrosses, where only the maternal parent is known, and the paternal parent can range from a few individuals up to hundreds. unfortunately, breeding for yield gains in alfalfa using traditional phenotypic evaluation and recurrent selection methods has hit a plateau partly due to its highly heterozygous and heterogeneous population-level breeding. community genomic tools like those in the ‘tools for polyploids’ project (https://www.polyploids.org/) or developed in other polyploid outcrossing crops have shown promise in accelerating breeding and yield gains (ferrão, amadeu, benevenuto, oliveira, & munoz, 2021). the creation of genomic tools that account for the biological and logistic challenges of the crop, has the potential to significantly improve yield gains in alfalfa through breeding. the first and typically most tractable place to build capacity and tools for molecular breeding is to begin by creating a rapid genotyping pipeline that fits within both the breeding cycle and the selection cycle and can deliver on the breeder’s objectives (hawkins & yu, 2018; mejia-guerra, zhao, & sheehan, 2021). as stated here, a pipeline refers to a complete workflow starting with a genetic marker platform, vendors for service and bioinformatic tools to transform returned raw data into a usable format for breeders. there are several factors to consider when choosing a genetic marker platform: cost per data point, vendor services, turnaround times and what genetic analyses can be done with the resulting data. for alfalfa, we determined that a targeted-amplicon sequenced-based approach would be the most beneficial for breeders. unlike genotyping-by-sequencing (gbs), targeted, amplicon-based genotyping technologies such as dartag (diversity array technology dart), and capture-seq (lgc genomics) have low missing data rates and query the same exact loci in all samples across genotyping projects, allowing new data to be easily appended to existing data (darrier et al., 2019; telfer et al., 2019; wang et al., 2020). the amount of data returned is in the tens of thousands or less, rather than the millions of reads from gbs, simplifying downstream bioinformatics processing (darrier et al., 2019; milner et al., 2019). this in turn speeds up the analysis time for marker-assisted selection (mas), introgression tracking, linkage mapping and genomic prediction (darrier et al., 2019). here, we report the creation of a dartag panel of 3,000 loci distributed across the alfalfa genome for use in molecular breeding and genomic prediction. dartag is a hybridization/amplicon-based targeted genotyping platform developed by dart (blyton et al. (2023); https://www.diversityarrays.com/services/targeted-genotying/). oligos are custom designed to target known genetic variants (snps and indels less than 50bp) with its flanking genomic regions, and sequencing products of 54bp (legacy technology) or 81bp (current technology) in length are produced. the dartag assay consists of four steps based on principles described in (krishnakumar et al., 2008). briefly, the pool of 3,000 alfalfa oligos, each targeting one genetic variant, is hybridized to denatured gdna in step 1, followed by snp/indel copying into dartag molecules by dna polymerase in step 2. after ligation into circular molecule also in step 2, and nucleases treatment to remove unwanted molecules in step 3, dartag products are subsequently amplified in step 4 with simultaneous addition of sample unique barcode used downstream for demultiplexing. the products of dartag assay, after purification and quantification, are sequenced on ngs platforms (e.g. novaseq 6000, illumina) to a depth of around 350x per marker per sample, then demultiplexed and genetic variants detected using dart’s proprietary analytical pipeline. figure 1: dartag sequencing reads from locus chr1.1_000194324. each sequence is a microhaplotype detected in breeding material tested on the panel. the dartag assay was designed to detect the target locus (black rectangle) and distinguish the reference allele from the alternative allele. additional variant nucleotide positions (yellow fill) distinguish the individual microhaplotypes. indels are shown in grey fill. physpos refers to the physical nucleotide position within the sequencing read from left to right. the alfalfa dartag panel was designed on the legacy technology to produce 54bp reads but works equally well with the current technology (81bp reads) with the caveat that some residual adapter sequences may be included (read-through of the entire fragment into the adapter). after trimming of any residual adapter sequences, the reads can be used to call snps, or in the case of complex genomes like alfalfa, used to identify microhaplotypes (figure 1). sequencing reads can contain variants beyond the target snp, which allows for the detection of more than two alleles at each of the 3,000 loci. as the amplicons are very short, variants found within these reads are assumed to be in complete linkage disequilibrium and therefore can be used for phasing genotyping calls for genetic map construction. in addition, accurate allele dosage can be determined for both bi-allelic and multi-allelic haplotypes, allowing genetic effect contributions to be determined for each unique haplotype for traits of interest. as dart had not tested many polyploid species with dartag when this study was initiated, we agreed to limit the number of probes to 3,000 loci, though the optimal max may differ by species and genome complexity, and read depth required to sufficiently call genotypes (andrzej kilian, dart, personal communication). results this alfalfa 3k dartag panel was developed from a diversity panel of 40 individual alfalfa clonal genotypes, focusing on elite breeding and stress-resistant genotypes used in north america. this panel consisted of 17 elite parents with various fall dormancy levels, six samples of diploid-cultivated alfalfa, 13 genotypes with abiotic stress resistance, one genotype with aphanomyces root rot disease resistance, and three other genotypes (table 1, column 2). two biological replicates of the diversity panel were processed, where the sequencing libraries were prepared using either illumina nextera wgs library prep at cornell institute of biotechnology or nebnext ultra dna library prep kit with an average insert dna size of 300bp. the whole-genome sequencing (wgs) was done using illumina novaseq 6000 at novogene (https://en.novogene.com/). raw fastq sequences were processed by removing residual adapter sequences and low-quality bases using trimmomatic (leading:10 trailing:10 slidingwindow:4:15 minlen:30) (bolger, lohse, & usadel, 2014). cleaned reads were then aligned to the haploid set (the first set out of the four homologous chromosomes) of xinjiangdaye reference genome (chen et al., 2020) using bwa-mem (li, 2012; li, 2013) and structural variants (snps and indels) were called using the dnaseq pipeline developed by sention (https://www.sentieon.com). a total of 28m snps present in both replicates were discovered from the whole-genome re-sequencing of the diversity panel, where a high-confidence set of 10k snps (figure 2) were obtained by requiring them: (1) not located within 5bp distance to an indel, (2) qual > 30, (3) minimum and maximum read depths of 20 and 1,900, respectively, (4) for each sample, at least one read supporting reference allele and two reads supporting the alternative allele, (5) no missing genotype per snp position, (6) with a minor allele frequency greater than 0.25, (7) not located in transposable elements and (8) not within 1kb of chromosome termini. the 10k snps were assessed by dart, and from those that passed qc, a 3k snp set targeting even genomic distribution was selected to form a 3k dartag marker panel. of the 3,000 loci selected for the panel, 85% (2,542) reside in genic regions and only 15% (458) reside in non-genic regions (supplemental table 1). oligo probes were synthesized, and genotyping done at dart. the alfalfa 3k dartag marker panel was validated using a bi-parental f1 population (n = 184), a backcross (bc1) population (n = 94), and a diverse set of elite genotypes (n = 74) and individual plants from other medicago species (n = 20) (table 1, column 3). it should be noted that all 40 alfalfa lines used in the snp discovery were also included in this validation sample set. the material selected for validation was to assess (1) the panel’s ability to construct genetic (linkage) maps with the data output and (2) to define the usable limit to the panel with extant species (non-medicago sativa) germplasm. as expected, the missing data (a marker with < 10 reads in a population) is the lowest among the medicago sativa genotypes. the alfalfa lines used in snp discovery showed the least missing data (an average of ~9% of the 3k markers with no data) and the rest m. sativa lines of the validation sample set had comparable missing rates (10%) (supplemental figure 2). other medicago species had an average of 51% markers with missing data, which is approximately five times higher than the m. sativa genotypes. table 1: accessions used in the construction and testing of the alfalfa (medicago sativa l.) 3k dartag panel. germplasm used for whole genome sequencing and snp database construction are indicated by ‘y’ in the ‘snp discovery’ column. germplasm used to validate the 3k panel is indicated by ‘y’ in the ‘validation set’ column. sample id snp discovery validation set contributor note s&w fd4 y y s&w seed co. elite parent; fall dormancy 4 s&w fd5 y y s&w seed co. elite parent; fall dormancy 5 legacy fd4 y y legacy seeds elite parent; fall dormancy 4 legacy fd5 y y legacy seeds elite parent; fall dormancy 5 s&w fd6 y y s&w seed co. elite parent; fall dormancy 6 s&w fd7 y y s&w seed co. elite parent; fall dormancy 7 s&w fd8 y y s&w seed co. elite parent; fall dormancy 8 s&w fd9a y y s&w seed co. elite parent; fall dormancy 9 s&w sfd9b y y s&w seed co. elite parent; fall dormancy 9 cadl-1 y y n. young cultivated alfalfa at diploid level cadl-3 y y n. young cultivated alfalfa at diploid level cadl-4-5 y y n. young cultivated alfalfa at diploid level cadl-5-3 y y n. young cultivated alfalfa at diploid level cadl-13 y y n. young cultivated alfalfa at diploid level cadl-18 y y n. young cultivated alfalfa at diploid level umn3988-bip y y d. samac biomass type regensy27x y y d. samac regenerator; ref. genome i195 y y n. young waph5; aphanomyces root rot ut14-46 sp y y m. peel tetraploid medicago falcata ut27-62 y y m. peel elite parent; salt tolerant fl99 y y e. rios elite parent; fall dormancy 9 bulldog 505 y y a. missaoui elite parent; fall dormancy 5 gams 1403-fsh y y a. missaoui elite parent; fall dormancy 7 gams 1404-fsh y y a. missaoui elite parent; fall dormancy 8 gams 1405-fsh y y a. missaoui elite parent; fall dormancy 9 3010 y y a. missaoui elite parent; fall dormancy 3 cw1010 y y a. missaoui elite parent; fall dormancy 10 cuf 101 y y d. samac fall dormancy 10 check bip1 y y m. peel salt tolerant; 27-62 bip2 y y m. peel salt tolerant; 31-6 bip3 y y m. peel semip; 1-34 bip4 y y m. peel semip; 6-2 bip5 y y m. peel semip; 14-46 bip6 y y m. peel drought (ut7); 17-43 bip7 y y m. peel drought (ut8); 17-44 bip8 y y m. peel drought (ut9); 18-22 bip9 y y m. peel drought (ut10); 21-3 bip10 y y m. peel drought (ut11); 22-30 bip11 y y m. peel drought (ut26); 7-18 bip12 y y m. peel drought (ut30); 13-14 wilson n y l.-x. yu elite parent wa467895 n y l.-x. yu elite parent cornell ny1 n y d. viands elite parent cornell ny2 n y d. viands elite parent cornell ny3 n y d. viands elite parent cornell ny4 n y d. viands elite parent paf 13 5, 11-1 n y h. riday medicago falcata paf 13 2, 9-4 n y h. riday medicago falcata paf 13 9, 10-5 n y h. riday medicago falcata paf 13 7, 21-2 n y h. riday medicago falcata fal12 1, 11-5 n y h. riday medicago falcata fal12 4, 12-4 n y h. riday medicago falcata mav8 n y d. samac elite parent aph 2 n y d. samac elite parent mav13 n y d. samac elite parent mav14 n y d. samac elite parent mav15 n y d. samac elite parent zg9 n y d. samac elite parent zg20 n y d. samac elite parent zg21 n y d. samac elite parent zg23 n y d. samac elite parent zg25 n y d. samac elite parent aph 11 n y d. samac elite parent aph 47 n y d. samac elite parent pi 516640 n y b. irish medicago arabica pi 504540 n y b. irish medicago arborea pi 495215 n y b. irish medicago bonarotiana pi 315458 n y b. irish medicago cancellata pi 498767 n y b. irish medicago ciliaris w6 32886 n y b. irish medicago daghestanica pi 538998 n y b. irish medicago hybrida pi 498849 n y b. irish medicago laciniata pi 537186 n y b. irish medicago littoralis pi 516711 n y b. irish medicago marina pi 287999 n y b. irish medicago monspelliaca pi 537259 n y b. irish medicago murex pi 220021 n y b. irish medicago orbicularis pi 464704 n y b. irish medicago papillosa pi 253450 n y b. irish medicago pironae w6 5252 n y b. irish medicago polymorpha pi 150564 n y b. irish medicago popovii pi 577446 n y b. irish medicago prostrata pi 631912 n y b. irish medicago ruthenica pi 631715 n y b. irish medicago sativa nothosubsp. tunetana pi 631714 n y b. irish medicago sativa nothosubsp. tunetana pi 631952 n y b. irish medicago sativa nothosubsp. varia pi 631920 n y b. irish medicago sativa nothosubsp. varia pi 631923 n y b. irish medicago sativa subsp. caerulea pi 631921 n y b. irish medicago sativa subsp. caerulea pi 641405 n y b. irish medicago sativa subsp. glomerata pi 631978 n y b. irish medicago sativa subsp. glomerata pi 631869 n y b. irish medicago sativa var. viscosa pi 631870 n y b. irish medicago sativa var. viscosa pi 197356 n y b. irish medicago scutellata i195 x j432 n y d. samac f1 population (184 progeny) aphbc1 n y d. samac bc1 population (94 progeny) figure 2: filters and criteria applied to produce the 3k dartag snp panel from the whole-genome sequencing (wgs) of the alfalfa diversity panel. m, millions; k, thousands. figure 3: composite genetic maps of a bi-parental f1 and a backcross (bc1) population. a) regeneration of the eight linkage groups of alfalfa genome. scale bar is shown in cm. b) scatter plots showing the relationship of genetic distance (cm) to physical distance (mbp) for each of the eight linkage groups. using the 3k panel genotyping results, we generated linkage maps for two distinct populations, an f1 and a backcross (bc1) that share the parent i195. for the f1 population, individuals were derived from a cross between parents i195 and j432, which are resistant and susceptible to aphamomyces euteiches, respectively. meanwhile, the bc1 population was obtained through a cross between i195 and a progeny (85-209) from the above f1 population. initially, we constructed individual genetic maps for the f1 and bc1 populations. genotype dosages for both were determined using updog software (gerard, ferrão, garcia, & stephens, 2018). subsequently, updog-generated objects were fed into mappoly software (mollinari & garcia, 2019; mollinari et al., 2020) to build separate genetic maps for each population. a standard screening was performed based on missing data and mendelian segregation fit. we calculated the recombination fraction matrix between all retained markers, using this information to cluster markers into linkage groups. according to available genome information, most of these markers corresponded with specific chromosomes. notably, a few markers that were mapped outside their physical position still presented consistent linkage with the markers in their assigned group. this pattern held true across both f1 and bc1 populations. for each linkage group formed, we used mappoly’s functions, mds_mappoly and est_rf_hmm_sequential to carry out de novo ordering and phasing to obtain the final f1 and bc1 maps. from all mapped markers, only 2.55% were assigned to different chromosomes in the f1 map and 0.97% in the bc1 map. after constructing individual maps for the f1 and bc1 populations, we merged them using the genome order (figure 3a; supplemental figure 1a). the mapped markers were all consistent placed in the two maps, but a few markers were assigned to different linkage groups when comparing the linkage and physical assembly in both maps. these markers were retained in filtering because they had reasonable mendelian segregation behaviour and their association with linkage groups that do not correspond to their physical chromosome assignment could indicate potential errors in the reference assembly (figure 3b). markers mapped out of their physical positions were inserted into the genome-based map using the multidimensional scaling (mds) de novo information. we then reconstructed a joint map by employing the hidden markov model (hmm) algorithm’s extension, as mollinari and garcia (2019) detailed. the implementation for this algorithm can be found in the github repository https://github.com/mmollina/highprechmm. finally, haplotypes for all individuals across both f1 and bc1 populations were reconstructed using the same algorithm (supplemental figure 1b). conclusion this panel is now publicly available and open for any researcher or breeder to order through dart (https://www.diversityarrays.com). researchers interested in using the panel and genotyping services are encouraged to contact dart directly for pricing details. raw data in fastq can be requested as can the missing allele discovery file (madc) that indicates the read depth of each detected haplotype in each sample. the panel and its resulting data are suitable for marker-assisted selection, reconstruction of recombination patterns, allele dosage estimation, and parental confirmation in north american cultivated alfalfa, with some limited application in other medicago species. the efficacy of the panel on breeding materials outside of north america has not been tested, nor has its efficacy in gwas. single plant samples were used to create and test the panel. subsequent testing on samples that are genotyped individually and in tissue or dna bulks (dna bulks up to 30 individuals per population) have produced the same allele frequency ratios in both sample types but higher read depth in pools (esteban rios, personal communication). more testing is needed to determine the most efficient number of samples to pool to achieve population-level allele frequencies with minimal human labour and monetary costs. the dartag assay can be processed from gdna or from tissue to genotyping data extraction in a three-week turnaround time. the genotyping data report comprises allele dose calls and raw data with custom report formats available upon request. one benefit that dartag has over fixed array platforms is the ability to update and improve the panel as required over time. the panel is a pool of 3,000 oligos, one per locus, which is used to generate the sequencing libraries from the assayed material. because the pool is created from individual oligo stocks, the removal of suboptimal loci or the addition of new loci can be easily done by creating a new pool. to determine which loci should be considered for removal, extensive genotyping (> 10,000 samples) is underway to identify those loci that consistently underperform or fail and flag them for removal. independently, as new significant qtl markers and/or markers specific to other germplasm are detected, they can be targeted for inclusion in the original pool in the next version(s) of the panel. dart offers re-pooling services once per year at low or no cost, but more frequent requests could result in labour surcharges being applied (andrzej kilian, personal communication). researchers interested in initiating projects with dart are encouraged to contact dart directly for consultation. another benefit of the deep testing underway is the ability to detect and catalogue all the microhaplotypes into a fixed allele database, which will improve combining data sets across genotyping projects (manuscript in preparation). if after deep testing it is clear that there are too few markers for gwas for given traits of interest, additional panels can be made to complement this panel. the other option is to add the required loci to the existing panel up to the technical limit of 7k, which is a more cost-effective option for the routine genotyping service with scalability. we choose to create a panel of 3,000 loci due to cost and technical reasons, but smaller complementary panels can be made at lower up-front and downstream usage costs. the addition of a complementary 3k panel would nearly double the cost of genotyping per sample but would result in more granular genotyping data. data availability statement the fastq files from the whole-genome skim sequencing for the 40 medicago sativa accessions used for identifying the candidate snp variants are housed in the ncbi short read archive under the bioproject id prjna1014379. the targeted regions used to create the 3k dartag markers and the haplotypes detected as of 31 may 2023 (v17) are available on dryad (https://datadryad.org/stash/share/wjen32dfl94eoymoem00pjti6mkulipbtatsgbwjyou). the code and data for construction of the f1, bc1 and joint maps in mappoly are available in our github repository for those interested in reproducing our analysis (https://github.com/breeding-insight/alfalfa_dartag_panel_paper.git. acknowledgments breeding insight is acknowledged for project design, marker development, curation and data processing. diversity arrays technology created the oligo array, provided sequencing services and contributed to the manuscript. we thank s&w seed company, legacy seeds, debby samac, brian irish, michael peel, long-xi yu, heathcliffe riday, nevin young, esteban rios, ian ray, ali missaoui and don viands for providing germplasm and sharon mitchell for manuscript review. breeding insight was funded for this work through a cooperative agreement between usda-ars and cornell (project number: 8062-21000-043-004-a). marcelo mollinari was funded by a usda nifa-awarded afri grant (project number: 2022-67013-36269). supplemental data supplemental figure 1 . alfalfa genetic map construction for an f1, bc1, and a joint map of the consensus. supplemental figure 2. missing data rates for different grouped subsets of genetic material. supplemental table 1. final 3,000 loci selected for the dartag panel. author contributions dz, kmmg, ds and mjs contributed to experimental design and planning. dz, ds and mjs selected the diversity panel for wgs. ds, mp and bi grew and harvested all plant materials used in the study. kmmg performed all the wgs analyses, snp database creation, filtering pipelines and quality control analyses to create the 3k panel. khu managed the panel creation at diversity arrays technology. dz, mm and ds executed the data analyses and genetic mapping. dz and mjs wrote the initial draft of the manuscript. cb managed experiments and communication among all authors involved. all authors contributed to reviewing the manuscript. conflict of interest statement the authors have no conflicts of interest to report. references blyton, m. d. j., brice, k. l., heller-uszynska, k., pascoe, j., jaccoud, d., leigh, k. a. and moore, b. d. 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(2020). applications of genotyping-by-sequencing (gbs) in maize genetics and breeding. sci rep 10. https://doi.org/10.1038/s41598-020-73321-8 original article genetic resources (2024), 5 (10), 81–93 doi: 10.46265/genresj.bqtw2172 https://www.genresj.org issn: 2708-3764 checklist and prioritization of crop wild relatives in sudan and south sudan ahmed aldow *,a, joana magos brehm a, maha kordofani a,b, fatouma abdoul-latif c and nigel maxted a a school of biosciences, university of birmingham, edgbaston, b15 2tt, birmingham, uk b department of botany, the university of khartoum, 11115, khartoum, khartoum, sudan c centre of studies and research of djibouti (cerd), medicinal research institute, djibouti abstract: crop wild relatives (cwr) encompass wild plant species or subspecies closely related to domesticated crops. this study presents the first comprehensive checklist and prioritized inventory of cwr for sudan and south sudan. building on the regional cwr list for northeast africa, we identified 499 cwr taxa belonging to 44 families, with 90% of these being native species. the most prominently represented families were poaceae (148), fabaceace (72) and convolvulaceae (43), while panicum (32), eragrostis (27), ficus (24) and pennisetum (20) were the most frequent genera. a prioritized inventory of 85 cwr taxa was developed based on three criteria: economic value, utilization potential and threat status. the prioritized cwr are predominately native (78%) and encompass 12 families dominated by poaceae (38), followed by solanaceae (9), fabaceae (6) and cucurbitaceae (6). priority genera included 27, with digitaria (17), solanum (9) and cucumis (5) emerging as key genera for conservation attention. this comprehensive national cwr inventory provides a crucial foundation for developing targeted conservation strategies in sudan and south sudan. keywords: crop wild relatives, conservation, checklist of crop wild relatives, priority inventory citation: aldow, a., brehm, j. m., kordofani, m., abdoul-latif, f., maxted, n. (2024). checklist and prioritization of crop wild relatives in sudan and south sudan. genetic resources 5 (10), 81–93. doi: 10.46265/genresj.bqtw2172. © copyright 2024 the authors. this is an open access article distributed under the terms of the creative commons attribution license (cc by 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. introduction crop wild relatives (cwr) are wild ancestors of plant taxa that are closely related to domesticated crops. these wild plants possess a high reservoir of genetic diversity for improving the resilience and productivity of our cultivated crops (ford-lloyd et al, 2011). cwr possess a broader genetic diversity compared to domesticated crops because of their adaptation to various climatic conditions (dempewolf et al, 2017). this genetic richness allows cwr to share valuable genes with their domesticated counterparts, enhancing crop resistance to pests, diseases and environmental stresses (barazani et al, 2008). globally, an estimated 50,000–60,000 cwr occur, with 10,740 identified as potential contributors to future food security (maxted ∗corresponding author: ahmed aldow (ahmedalsafie@yahoo.com) and kell, 2009). recognizing their importance, a study by vincent et al (2013) established an initial global priority list of 1,392 species for conservation and utilization to ensure food security. however, cwr face numerous threats, including nitrogen deposition, landuse alterations, invasive alien species, overgrazing, urbanization and climate change (ford-lloyd et al, 2011). climate change is projected to have a detrimental impact on global crop production (lobell et al, 2011). over the past three decades, global warming has accelerated and is expected to intensify further in the years to come (ipcc, 2020). in africa, the effects of climate change on food production are already apparent, manifesting as changes in rainfall patterns, rising temperatures and an increased frequency of extreme weather events (ipcc, 2020). these changes have resulted in reduced crop yields, particularly in sub-saharan africa (ipcc, 2020), which is home to received: 10.06.2024 accepted: 17.09.2024 published online: 29.10.2024 https://www.genresj.org https://www.doi.org/10.46265/genresj.bqtw2172 https://www.genresj.org https://www.doi.org/10.46265/genresj.bqtw2172 mailto:ahmedalsafie@yahoo.com 82 aldow et al genetic resources (2024), 5 (10), 81–93 approximately 45,000 plant species (linder, 2014), and where a significant portion of the population relies on plant resources as the foundation of their diet (gollin and rogerson, 2014). for the comprehensive utilization of cwr in crop improvement programmes, it is vital to conserve, classify and make them accessible to researchers and plant breeders at national, regional and global levels (maxted et al, 2015). hence, urgent measures involving complementary protection, both in situ (on farms) and through storage in ex situ facilities (genebanks), are essential steps to preserve these genetic resources and ensure their availability to researchers and breeders (maxted and kell, 2009). the initial phase of cwr conservation planning entails the development of a checklist, defining the taxon names of existing cwr within a specific region or country (maxted et al, 1997). subsequently, the creation of a priority cwr checklist involves reducing the number on the checklist to more manageable levels. this is achieved by applying criteria such as threat status, endemicity and potential utilization (ford-lloyd et al, 2008). to assess the potential utilization of cwr in plant breeding, researchers rely on two main concepts: the gene pool concept (harlan and de wet, 1971) and the taxon group concept (maxted et al, 2006). these concepts are crucial as they help us to understand the genetic relationships between cultivated crops and their wild relatives. close relatives hold a higher likelihood of intercrossing than distant ones. the gene pool concept furnishes valuable information to plant breeders, aiding them in selecting germplasm for crossbreeding and plant improvement. it is divided into three categories: the primary gene pool (gp1), where gp1a represents the cultivated forms and gp1b the wild or weedy forms; the secondary gene pool (gp2), consisting of species that are less closely related but still capable of gene transfer, albeit with difficulty; and the tertiary gene pool (gp3), consisting of distantly related species where gene transfer is either impossible or requires advanced methods such as genetic engineering (harlan and de wet, 1971). in cases where gene pool concept information is unavailable, the taxon group concept serves as an alternative. the taxon group concept categorizes relationships as follows: taxon group 1a (tg1a) is the crop itself, taxon group 1b (tg1b) includes the same species as the crop, taxon group 2 (tg2) consists of species within the same series or section as the crop, taxon group 3 (tg3) refers to species in the same subgenus as the crop, taxon group 4 (tg4) includes species in the same genus as the crop, and taxon group 5 (tg5) includes species in the same tribe as the crop but belonging to a different genus (maxted et al, 2006). cwr checklists and prioritized inventories have been established in various countries worldwide, including mexico (contreras-toledo et al, 2019), the united states (khoury et al, 2013), spain (rubio-teso et al, 2018), portugal (magos-brehm et al, 2008), benin (idohou et al, 2013), tunisia (mokni et al, 2022), italy (ciancaleoni et al, 2021) and south africa (holness et al, 2019). a recent study by aldow et al (2023) developed the first regional cwr inventory for northeast africa. however, there has been no prior investigation into cwr diversity in sudan and south sudan. thus, the objectives of this study are 1) to prepare sudan and south sudan cwr checklist, and 2) annotate this checklist to prioritize it for active conservation using the interactive toolkit for cwr conservation planning (magos et al, 2017). geographical context sudan and south sudan, located in northeast africa between latitudes 4◦ and 22◦ n and longitudes 22◦ and 38◦ e (zaroug, 2006), are bordered by ethiopia and eritrea to the east and egypt to the north. the combined total area of both countries is approximately 2.5 million km2, with sudan covering about 1,878,000km2 and south sudan about 646,883 km2 (zaroug, 2006; world bank, 2021). pasture and forests cover 40% of the land, while cultivated area accounts for about 33% of the total area, of which only 21% is actively cultivated, resulting in very low crop yields (unep, 2007). the two countries are divided into five ecological zones: desert, semidesert, woodland savannah, flood region and montane vegetation. agriculture is the main source of income for 60–80% of the population (elgali et al, 2010). the five agricultural systems include mechanized rain-fed agricultural schemes, traditional rain-fed agriculture, mechanized irrigation schemes, traditional irrigation and livestock husbandry/pastoralism (zaroug, 2006). approximately 90% of the farming areas in the country depend on precipitation (siddig et al, 2020), while in south sudan, despite more favourable climatic conditions, agricultural output remains low (diao et al, 2012). materials and methods cwr checklists and inventories are the main starting points for effective conservation strategies (maxted et al, 2015). this study is based on the cwr diversity identified for northeast africa (djibouti, eritrea, ethiopia, sudan, and south sudan) by aldow et al (2023) to create a checklist and a priority inventory for sudan and south sudan. we excluded invasive species documented for sudan and south sudan from the global invasive species database (gisd) of iucn (http://www.iucngisd.org/gisd/) and the invasive species compendium (cabi) (http://www .cabi.org/isc/). our approach involved a three-step process: 1. starting point: we began with the comprehensive cwr checklist for northeast africa compiled by aldow et al (2023), which included 1,020 taxa. 2. regional refinement: we then used a digitalized floristic checklist of sudan and south sudan, compiled by the first author based on plants of sudan and south sudan: an annotated checklist (darbyshire et al, http://www.cabi.org/isc/ genetic resources (2024), 5 (10), 81–93 sudan and south sudan crop wild relatives 83 2015) during the establishment of the cwr checklist for northeast africa, to retain a checklist with taxa occurring only in these two countries. note that in the flora of sudan and south sudan, the compositae family is referred to as the asteraceae family. 3. a priority inventory of cwr was selected based on three criteria: (a) economic value: faostat crop valuation (fao, 2021) was used to select taxa with economic importance based on the related crop. (b) the utilization potential: priority taxa within gene pool categories 1b, 2, 3 and taxon group categories 1b, 2, 3 and 4 (with documented use in crop development only) were selected based on the information available in the germplasm resources information network taxonomy (usda, 2023) and the harlan de wet cwr inventory (vincent et al, 2013). (c) threat status: although (kell et al, 2017) recommended incorporating threat status as a prioritization criterion, its application was limited due to the scarcity of red list data for these countries (only 7% of plant species according to darbyshire et al (2015). however, based on expert consultation, the endangered south sudanese coffee species coffea neoleroyi a. p. davis (iucn, 2022), was added to the priority checklist. additional information was incorporated into the compiled cwr checklist and inventory. this supplementary data encompassed taxonomic details such as accepted taxa names, synonyms and authorities. additionally, it included the common names of related crops, native status, the utilization of the crop, the type of relatedness (gene pool or taxon group), confirmed or potential use in crop breeding, distribution status along with relevant references, and in situ and ex situ conservation statuses. results cwr checklist the cwr checklist of sudan and south sudan contains 449 taxa (including subspecies and varieties), belonging to 88 genera across 44 families. both native and introduced taxa are included. invasive taxa, like sorghum halepense (l.) pers, were removed from the checklist. sudan has 133 taxa, 59 genera, and south sudan 161 taxa and 47 genera. over 90% of the listed cwr are native to these two countries. the most common plant families include poaceae (grasses) with 148 taxa, fabaceae (legumes) with 72 taxa, and convolvulaceae (morning glories) with 43 taxa. the genera with the highest number of cwr taxa are panicum (32), eragrostis (27), ficus (24) and pennisetum (20). details on the distribution of taxa can be found in supplemental table 1 and figure 1. priority checklist the priority checklist of sudan and south sudan contains 85 taxa related to 12 families and 27 genera. figure 2 illustrates the distribution of these priority taxa across both countries. the most mentioned families were poaceae (38), solanaceae (9), fabaceae and cucurbitaceae (6 each), while digitaria, solanum, cucumis and echinochlo were the most mentioned genera (supplemental table 2 and figure 3). the closest wild relatives to the crop gp1b, tg1b, tg2 and gp2 represent about 49% and gp3 counts for about half of the priority taxa (figure 4a and supplemental table 3). the confirmed use of taxa is about 8%, potential use 17%, confirmed and potential use 14%, and unconfirmed use 61% (figure 4b and supplemental table 3). cwr in the checklist have provided a number of beneficial traits to crops such as chickpea, teff, coffee, finger millet, cassava, rice, cotton and sorghum found in sudan and south sudan (table 1). discussion the inventory conducted in sudan and south sudan reveals a rich diversity of native and introduced taxa associated with a broad range of crops. this provides a crucial foundation for the development of national policies and strategies in both countries. these strategies should prioritize the conservation of the identified cwr genetic diversity, both in situ and ex situ, with the ultimate goal of ensuring the longterm conservation of these valuable resources for the benefit of future generations (kell et al, 2017). in light of the critical importance of cwr for global food security and sustainable agriculture, researchers and scientific institutions have advocated for a coordinated global cwr conservation strategy (dempewolf et al, 2014), which should encompass both in situ and ex situ approaches for effective conservation. the checklist of cwr identified a significantly higher diversity of cwr in south sudan (37,9%) compared to sudan. interestingly, about one-third of the cwr identified are found in both countries. additionally, nearly half (45%) of the priority cwr are shared between the two nations. unfortunately, most documented plant taxa in these countries are outdated due to a lack of recent research, likely a result of longstanding civil conflicts. this highlights the urgent need for taxonomic experts to update the classification of these cwr. national cwr conservation strategies should be harmonized with regional and international initiatives. as proposed by maxted et al (2015), integrating national and regional cwr conservation strategies is highly beneficial. this recognizes that prioritization criteria for cwr conservation at the national level can often be aligned with the regional level. this overlap facilitates collaboration between various agencies (governmental, private, or voluntary) at both levels, leading to the development of more effective cwr conservation strategies. 84 aldow et al genetic resources (2024), 5 (10), 81–93 figure 1. distribution of cwr taxa in the checklist in sudan and south sudan figure 2. distribution of priority cwr in sudan and south sudan genetic resources (2024), 5 (10), 81–93 sudan and south sudan crop wild relatives 85 figure 3. diversity of taxa among cwr families within the priority cwr inventory in sudan and south sudan. figure 4. genetic relatedness and use potential for priority cwr inventory in sudan and south sudan. a) shows the genetic relatedness of the priority cwr inventory in sudan and south sudan, based on the gene pool and taxon group concepts. b) illustrates the status of the priority cwr inventory in sudan and south sudan for crop improvement, categorized as potential, confirmed, confirmed and potential, and unconfirmed. 86 aldow et al g enetic resources (2024),5 (10),81–93 table 1. relatedness of cwr taxa and their confirmed use in crop improvement in the priority inventory of sudan and south sudan. gp1, primary gene pool; gp2, secondary gene pool; gp3, tertiary gene pool. ’b’ indicates the wild or weedy form. taxon relatedness to cwr confirmed use of cwr to broaden crop improvement references coffea canephora var. gossweileri a. chev. gp2b coffee berry disease resistance; coffee rust resistance; root-knot nematode resistance anthony et al (2011); levi et al (2005); noir et al (2003); prescott-allen and prescott-allen (1988) coffea liberica hiern gp2b coffee rust resistance anthony et al (2011); prakash et al (2009); prescott-allen and prescott-allen (1988) fragaria chiloensis (l.) duchesne. gp1b fruit size; fruit quality ahmadi and bringhurst (1992) fragaria vesca l. gp3 anthracnose resistance; powdery mildew resistance; improved aroma ahmadi and bringhurst (1992); scott (1951) fragaria virginiana duchesne. gp1b fruit number; fruit size; powdery mildew resistance; scorch resistance; day neutral ahmadi and bringhurst (1992); hancock et al (2002) diplotaxis erucoides (l.) dc. gp3 alternaria blight resistance; blackleg resistance; cytoplasmic male sterility klewer et al (2003); prakash et al (2009); siemens (2002) diplotaxis harra (forssk.) boiss. gp3 gene transfer begum et al (1995) eleusine africana k. obyrne gp1b fertility trait dida and devos (2006) eleusine kigeziensis s.m. gp1b fertility trait dida and devos (2006) thinopyrum junceum (l.) á. löve gp3 soil salinity tolerance nevo and chen (2010) gossypium longicaly x hutch. & b.j.s. lee. gp2 reniform nematode resistance robinson et al (2007) ipomoea purpurea (l.) roth. gp3 gene transfer cao et al (2009) lens ervoides (brign.) grande gp2 seed size; yield improvement; anthracnose resistance; ascochyta blight resistance; stemphylium blight resistance ahmad et al (1997); kumar et al (2014); tullu et al (2011) lupinus mexicanus cerv. er lag. gp3 gene transfer busmann-loock et al (1992); clements et al (2005) malus sylvestris miller gp1b agronomic trait volk et al (2015) manihot carthagenensis subsp. glaziovii (müll. arg.) allem gp2 cassava bacterial blight; resistance; cassava mealy bug resistance; cassava mosaic virus resistance hahn et al (1980); hajjar and hodgkin (2007); nair and unnikrishnan (2007); prescott-allen and prescott-allen (1988) medicago arborea l. gp3 anthracnose resistance armour et al (2008); quiros and bauchan (1988) olea europaea subsp. cuspidata (wall. ex g. don) cif. gp2 crop ontology trait hannachi et al (2009) continued on next page g enetic resources (2024),5 (10),81–93 sudan and south sudan crop w ild relatives 87 table 1 continued taxon relatedness to cwr confirmed use of cwr to broaden crop improvement references oryza brachyantha a. chev. & roehr. gp2 bacterial blight resistance brar and singh (2011) oryza longisteminata a. chev. & roehr. gp1b drought tolerance; yield improvement; bacterial blight resistance; grassy stunt resistance brar and singh (2011); hajjar and hodgkin (2007); jena (2010) pistacia khinjuk stocks. gp2 rootstock hormaza and wünsch (2007) pennisetum purpureum schumach. gp2 cytoplasmic male sterility; fertility restoration genes; panicle length; days to maturity; yield improvement dujardin and hanna (1989) hajjar and hodgkin (2007); hanna (1997); palit et al (2014) pennisetum squamulatum fresen. gp2 fertility restoration genes dujardin and hanna (1989) phaseolus coccineus l. gp2 aluminium tolerance; yield improvement; angular leaf spot resistance; anthracnose resistance; bean stem maggot resistance; bean yellow mosaic virus resistance; common bacterial blight resistance; fusarium root rot resistance; white mould resistance de ron et al (2015); freytag et al (1982); loskutov and rines (2011); mahuku et al (2003); miklas et al (1999); porch et al (2013); schwartz and singh (2013); singh et al (2008); singh (2001); wilkinson and re (1983); zapata et al (2004) saccharum spontaneum l. gp2 cold tolerance; red rot resistance; smut resistance; sugarcane mosaic virus; early maturing cordeiro et al (2003); prescott-allen and prescott-allen (1986) setaria viridis (l.) p. beauv. gp1b triazine resistance darmency and pernes (1985) sinapis arvensis l. gp2 blackleg resistance; sclerotinia resistance; cytoplasmic male sterility hu et al (2002); snowdon et al (2000); wei et al (2010) solanum aethiopicum solan l. gp3 rootstock; yield improvement; bacterial wilt resistance; fusarium wilt resistance collonnier et al (2001); daunay (2008); frary et al (2007); rotino et al (2014); usda (2011) solanum incanum l. gp2 drought tolerance; rootstock; verticillium wilt resistance frary et al (2007); knapp et al (2013); usda (2011) solanum linnaeanum hopper & jaeger gp2 fungal wilt resistance frary et al (2007); rotino et al (2014); yin et al (2015) solanum macrocarpon l. gp3 rootstock usda (2011) solanum marginatum l. f. gp3 gene transfer borgato et al (2007) sorghum purpureosericeum (hochst. ex a. rich.) schweinf. & asch. gp3 sorghum shoot fly resistance nwanze et al (1990) vigna radiata var. sublobata (roxb.) verdc. gp1b bruchid resistance konarev et al (2002) vigna vexillata (l.) a. rich gp3 gene transfer gomathinayagam et al (1998) 88 aldow et al genetic resources (2024), 5 (10), 81–93 the development of the national checklist and inventory of cwr focused exclusively on those associated with food crops, such as rice, sorghum and finger millet. this focus is justified by the crucial role these crops play in providing nutrition and ensuring food security in these two nations. this region has historically been affected by food insecurity as a direct consequence of social conflict and warfare, making the prioritization of food security crops in the cwr inventory a strategic approach. climate change poses a significant threat to the future of food crops, including their wild relatives. jarvis et al (2008) emphasized the critical need to identify and conserve cwr that are threatened by climate change, such as cowpea (vigna), a crucial food security crop in sub-saharan africa. studies by jarvis et al (2008) estimate that 2-6% of vigna species in sub-saharan africa could face extinction by 2055, highlighting the urgency of identifying and conserving these threatened cwr. fortunately, sudan and south sudan contain three vital cwr of v. unguiculata subsp. dekindtiana (harms) verdc, v. unguiculata subsp. pubescens (r wilczek) pasquet and v. vexillata (l.) a. rich. these cwr represent a valuable genetic reservoir that could be important for developing climate-resilient cowpea varieties in the future, potentially preserving food security in the region and beyond. cwr conservation priorities are an important step in conservation planning at the national, regional and international levels. this newly developed inventory in sudan and south sudan identifies cwr associated with essential food crops such as sorghum, rice, cowpea and pearl millet. while this inventory represents a valuable resource, the number of taxa in sudan and south sudan (449 taxa) is lower compared to the checklists of other countries such as indonesia (rahman et al, 2019), china (kell et al, 2015), portugal (magos-brehm et al, 2008), usa (khoury et al, 2013) and zambia (ng’uni et al, 2019). this difference highlights the importance of continued cwr conservation and exploration efforts in sudan and south sudan. conclusion this study highlights the important role of cwr in sudan and south sudan in enriching crop diversity and promoting sustainable food production at all levels – national, regional and international. by identifying and prioritizing 85 cwr taxa from a comprehensive checklist of 449, this research provides a crucial foundation for targeted conservation efforts. the establishment of the first cwr checklist and inventory for these two countries offers the basis for further research to ensure the longterm sustainability and utilization of the prioritized cwr. key areas for future research include: • protecting genebanks during civil conflicts: the ongoing civil conflict in sudan tragically exemplifies this threat. researchers were forced to call upon the international community to intervene and protect the country’s main seedbank from the potential loss of irreplaceable crop varieties and damage to its facilities (nordling, 2024). similar situations have been observed with icarda in syria (darvish et al, 2023) and yemen (aljarmouzi et al, 2024) this incident highlights the urgent need for a comprehensive regional and global initiative to safeguard genebank during conflicts. • enhanced floras and cwr inventory validation: develop separate, comprehensive floras for sudan and south sudan, collaborating with agronomists to validate the cwr inventory accuracy. • taxonomic expeditions for new cwr discovery: conduct taxonomic research projects in remote, untapped areas, potentially leading to the discovery of new cwr, and work with national genebanks on collaborative efforts and germplasm preservation. • gap analyses: initiate in situ and ex situ conservation gap analysis for the priority cwr taxa in each country. these analyses will inform the development of comprehensive conservation plans for each cwr’s specific needs. • climate change impact assessment: assessing climate change models to evaluate potential threats to cwr populations. supplemental data supplemental table 1. checklist of cwr in sudan and south sudan supplemental table 2. priority inventory of cwr in sudan and south sudan supplemental table 3. related crop and concept level of the priority inventory of cwr taxa in sudan and south sudan author contributions ahmed aldow: conceptualization, data curation, formal analysis, investigation, methodology, resources, validation, visualization, writing (original draft, review and editing). joana magos brehm: supervision. maha kordofani: resources, validation. fatouma abdoul-latif: resources, validation. nigel maxted: supervision. conflict of interest statement the authors confirmed that no conflict of interest exists. references ahmad, m., russell, a., and mcneil, d. 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(2006). country pasture/forage resource profiles (rome: fao). https://doi.org/10.1007/s00122-009-1236-6 https://doi.org/10.1007/s00122-009-1236-6 https://data.worldbank.org/indicator/ag.lnd.totl.k2 https://data.worldbank.org/indicator/ag.lnd.totl.k2 https://doi.org/10.5194/esd-6-45-2015 introduction geographical context materials and methods results cwr checklist priority checklist discussion conclusion supplemental data author contributions conflict of interest statement original article genetic resources (2024), 5 (9), 61–71 doi: 10.46265/genresj.ryaj6068 https://www.genresj.org issn: 2708-3764 combined cytogenetic and molecular methods for taxonomic verification and description of brassica populations deriving from different origins cyril falentin*,a,†, houria hadj-arab b,†, fella aissiou b, claudia bartoli a, giuseppe bazan c, éo boudet a, lydia bousset-vaslin a, marwa chouikhid, olivier coriton a, gwenaëlle deniot a, julie ferreira de carvalho a, laurène gay e, anna geraci c, pascal glory a, virginie huteau a, riadh ilahyd, vincenzo ilardi c, josé a jarillo f, vladimir meglič g, elisabetta oddo c, mónica pernas f, manuel piñeiro f, barbara pipan g, thouraya rhimd, vincent richer a, fulvia rizzah, joëlle ronfort e, mathieu rousseau-gueutin a, rosario schicchi i, lovro sinkovič g, maryse taburel a, valeria terzih, sylvain théréné a, mathieu tiret a, imen tlilid, marie-hélène wagner j, franz werner badeckh and anne-marie chèvre a a igepp, inrae, institut agro, université de rennes, 35650, le rheu, france b faculty of biological sciences fsb, university of sciences and technology houari boumediene usthb, bp 32, 16111, bab-ezzouar, el-alia, algiers, algeria c department of biological, chemical and pharmaceutical sciences and technologies (stebicef), università degli studi di palermo, via archirafi 38, 90123, palermo, italy d laboratory of horticulture, national agricultural research institute of tunisia (inrat), university of carthage, menzah 1, 1004, tunis, tunisia e umr agap institut, université de montpellier, cirad, inrae, institut agro, montpellier, france f centro de biotecnoloǵıa y genómica de plantas, universidad politécnica de madrid (upm), instituto nacional de investigación y tecnoloǵıa agraria y alimentaria (inia)/csic), campus montegancedo upm, madrid, spain g crop science department, agricultural institute of slovenia, hacquetova ulica 17, si-1000, ljubljana, slovenia h research centre for genomics & bioinformatics, council for agricultural research and economics (crea), i -29017, fiorenzuola d’arda (pc), italy i department of agricultural, food and forest sciences (saaf), università degli studi di palermo, viale delle scienze ed. 4, 90128, palermo, italy j geves, station nationale d’essais de semences, 49071, beaucouzé, france abstract: agriculture faces great challenges to overcome global warming and improve system sustainability, requiring access to novel genetic diversity. so far, wild populations and local landraces remain poorly explored. this is notably the case for the two diploid species, brassica oleracea l. (cc, 2n=2x=18) and b. rapa l. (aa, 2n=2x=20). in order to explore the genetic diversity in both species, we have collected populations in their centre of origin, the mediterranean basin, on a large contrasting climatic and soil gradient from northern europe to southern sub-saharan regions. in these areas, we also collected 14 populations belonging to five b. oleracea closely related species. our objective was to ensure the absence of species misidentification at the seedling stage among the populations collected and to describe thereafter their origins. we combined flow cytometry, sequencing of a species-specific chloroplast genomic region, as well as cytogenetic analyses in case of unexpected results for taxonomic verification. out of the 112 b. oleracea and 154 b. rapa populations collected, 103 and 146, respectively, presented a good germination rate and eighteen populations were misidentified. the most frequent mistake was the confusion of these diploid species with b. napus. additionally for b. rapa, two autotetraploid populations were observed. habitats of the collected and confirmed wild populations and landraces are described in this study. the unique plant material described here will serve to investigate the genomic regions involved in adaptation to climate and microbiota within the framework of the h2020 prima project ‘brasexplor’. citation: falentin, c., hadj-arab, h., aissiou, f., bartoli, c., bazan, g., boudet, m., bousset-vaslin, l., chouikhi, m., coriton, o., deniot, g., ferreira de carvalho, j., gay, l., geraci, a., glory, p., huteau, v., ilahy, r., ilardi, v., jarillo, j. a., meglič, v., oddo, e., pernas, m., piñeiro, m., pipan, b., rhim, t., richer, v., rizza, f., ronfort, j., rousseau-gueutin, m., schicchi, r., sinkovič, l., taburel, m., terzi, v., théréné, s., tiret, m., tlili, i., wagner, m., badeck, f. w., chèvre, a. (2024). combined cytogenetic and molecular methods for taxonomic verification and description of brassica populations deriving from different origins. genetic resources 5 (9), 61–71. doi: 10.46265/genresj.ryaj6068. © copyright 2024 the authors. this is an open access article distributed under the terms of the creative commons attribution license (cc by 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. received: 14.09.2023 accepted: 12.04.2024 published online: 10.05.2024 mat https://www.genresj.org https://www.doi.org/10.46265/genresj.ryaj6068 https://www.genresj.org https://www.doi.org/10.46265/genresj.ryaj6068 62 falentin et al genetic resources (2024), 5 (9), 61–71 introduction agriculture has to face great challenges to overcome global climate change and improve the sustainability of agricultural systems while maintaining crop production and quality. regarding crop improvement, there are at least two main questions to consider: (i) which type of genetic diversity should we promote in breeding programmes to withstand the new climatic regime and (ii) which material to select for the development of new relevant varieties in this erratic context. intensive farming systems and particularly modern breeding techniques have led to a drastic reduction in crop genetic diversity. on the other hand, local landraces and wild plant populations are a great source of genetic diversity. however, for many crop species such plant material has either never been collected, is not available, or has been poorly analyzed and/or characterized. the two diploid species that we focused on in this study, brassica oleracea l. (cc, 2n=2x=18) and b. rapa l. (aa, 2n=2x=20), are native to the mediterranean basin (cheng et al, 2016; bird et al, 2017; qi et al, 2017; cai et al, 2021; mabry et al, 2021; mcalvay et al, 2021; cai et al, 2022), in which they grow as wild populations or as local landraces selected over several generations by farmers. they encounter a large gradient of contrasted climate, soils and biotic factors from northern europe to southern sub-saharan regions, which makes these species particularly relevant for the analysis of diversity in relation to adaptation to the climate. the new populations will be complementary to accessions available in biological resource centres (brc) as they continue to evolve under current climatic constraints. indeed, exploring these wild populations and local varieties represents a unique opportunity to identify locally adapted material for which genetic diversity and adaptive traits could be relevant to face upcoming climatic changes and disease emergences correlated to global change in the mediterranean area, thus contributing to biodiversity-based agriculture. convergent evolution has led to similar morphotypes in these two economically important vegetable species that were locally selected for a long time by farmers all over the mediterranean basin, mainly for their inflorescence at budding stage (cauliflower or broccoli for b. oleracea, broccoletto for b. rapa), leaves (cabbage, kale for b. oleracea; fodder turnip for b. rapa) or epicotyls/roots (kohlrabi for b. oleracea, turnip for b. rapa). this morphological convergence between the two species is linked to their recent common ancestor (cheng et al, 2016) as they diverged only 2–4 million years ago (cheng et al, 2014). the morphological similarity between them is one of the reasons for some confusion when identifying the species. additionally, a third species widely cultivated for seeds, resulting from the hybridization and genome doubling of the two diploid ∗corresponding author: cyril falentin (cyril.falentin@inrae.fr.) † these authors contributed equally to this work species, b. napus l. (aacc, 2n=4x=38), can also produce edible roots in swede cultivars, or leaves as forage or vegetable. as both species share many morphological characteristics with b. napus, species identification remains difficult at the seedling stage and controls are required before further analyses. in this paper, we describe the collection, along a broad climatic gradient, of more than 100 populations each of b. oleracea and b. rapa species, including both landraces and wild populations, which co-evolve under current climatic constraints. to ensure the absence of species misidentification or potential interspecific hybrids at the seedling stage before sequencing, plants of each population were assessed using different methods sequentially from the easiest to the most timeconsuming: (1) flow cytometry on all the plants based on different genome size and chromosome number (630mb for 18 chromosomes in b. oleracea, 529mb for 20 chromosomes in b. rapa) (belser et al, 2018), (2) sanger sequencing of a species-specific chloroplast genomic region on a sub-sample per population (li et al, 2017), and (3) cytogenetic approaches in the event of unexpected results from the previous analyses. after these controls, the geographical distribution and ecological environment of each population were described. this unique plant material will support further analyses from our consortium investigating the genomic regions involved in local adaptation to climate and microbiota. materials and methods plant material wild populations of both b. oleracea and b. rapa species were collected in france based on information in the national inventory of natural heritage database inpn (2024) and maggioni et al (2020). in addition, b. rapa wild populations were gathered in italy, algeria, slovenia and b. oleracea in spain (gomez-campo et al, 2005) based on local flora and long field experiences. siliques were collected from 30 plants per population (when available), depending on the size and accessibility of populations. some wild populations of b. oleracea closely related species were identified and added to the analysis: eight b. montana pourr. populations (six from france and two from italy), as well as two b. rupestris raf. (subsp. rupestris), two b. villosa biv. [subsp. drepanensis (caruel) raimondo & mazzola and subsp. tineoi (lojac.) raimondo & mazzola], one b. macrocarpa guss., and one b. incana ten., all from sicily, italy. b. oleracea and b. rapa landraces were collected in five different countries either through direct collects on farms in algeria, tunisia and italy or in brc maintaining old landraces in france (brc bracysol) and slovenia (slovene plant gene bank in slovenia, srgb kis). in agreement with each country’s policy, the nagoya protocol will be applied, pending the introduction of the relevant collected material into the multilateral system of the fao’s international treaty mailto:cyril.falentin@inrae.fr. genetic resources (2024), 5 (9), 61–71 cytogenetic and molecular methods in brassica taxonomy 63 on plant genetic resources for food and agriculture. thus, during this transition period, the material will be available after seed production upon request, either in brc bracysol and srgb for french and slovenian populations, respectively, or by contacting the partner in each country, as reported in supplemental tables 1 and 2. each collected population was named following a specific code. it starts with (1) two letters representing the species (bo for b. oleracea, br for b. rapa, bm for b. montana, bu for b. rupestris, bv for b. villosa, ba for b. macrocarpa, and bi for b. incana), followed (2) by a letter for the country of origin (f for france, i for italy, s for slovenia, e for spain, a for algeria, or t for tunisia), (3) then four letters indicating the location of the collecting site, (4) either a w for a wild population or an l for a landrace, (5) and an additional letter (a, b, c, etc.) in case of several collecting sites at the same location (i.e. br i cast w a and br i cast w b). for all these populations, a common sheet was filled for wild populations to describe the environment (supplemental table 1) and another one for landrace collects at the farm or when seeds were acquired from genetic resource centres (grc bracysol in france, kis in slovenia) (supplemental table 2). thirty plants per population were grown in the greenhouse for taxonomy assessments. for wild populations, we planted one seed of each of the 30 collected mother plants. when seeds were collected from fewer than 30 plants, we sowed several seeds per mother plant, equally represented, to reach a total of 30 seeds. for landraces, 30 seeds were sown. as controls for the different experiments, we used a known representative of b. oleracea, b. rapa and b. napus species: doubled haploid lines of b. oleracea subsp. italica (hdem) and b. rapa subsp. trilocularis (z1) (belser et al, 2018) and a pure line of b. napus subsp. oleifera, ‘darmor’. cytogenetic control and chromosome counts flow cytometry was performed on all plants to assess the chromosome number of each plant using leaves as described by leflon et al (2006). briefly, approximately 0.5cm2 of fresh leaves were harvested and transferred to a petri dish. this material was chopped using a sharp razor blade in 300µl of nuclei extraction staining buffer (from kit cystaintm uv presice p-sysmex) and incubated at room temperature for 30 to 90sec. 1.2ml of dapi staining buffer was added per sample and the solution was then filtered through a 50µm nylon mesh. estimation for each accession was obtained with flowmax software using a cyflow space cytometer (sysmex inc.). for the screening of b. oleracea and independently of b. rapa populations, the control variety, hdem for b. oleracea and z1 for b. rapa, was adjusted to a fluorescence intensity value of 300 for nuclei at g1 stage. coincidence or deviation was compared with these controls. for populations for which flow cytometer and chloroplast sequencing data were not congruent, the chromosome number was also determined from mitotic chromosomes observed on metaphasic cells isolated from root tips. root tips of 0.5–1.5cm in length were treated in the dark with 0.04% 8-hydroxiquinoline for 2h at 4◦c followed by 2h at room temperature to accumulate metaphases. they were then fixed in 3:1 ethanol:glacial acetic acid for 48h at 4◦c and stored in 70% ethanol at -20◦c until use. after being washed in distilled water for 10min, in hcl 0.25 n for 10min, then treated for 15min with a 0.01m citric acid-sodium citrate buffer (ph 4.5), root tips were incubated at 37◦c for 30min in an enzymatic mixture (5% onozuka r10 cellulase (sigma), 1% y23 pectolyase (sigma)). the enzymatic solution was removed and the digested root tips were then carefully washed with distilled water for 30min. one root tip was transferred to a slide and macerated with a drop of 3:1 fixation solution. dried slides were then stained by a drop of 4’,6-diamidino2-phenylindole (dapi). cells were viewed with an orca-flash4 (hamamatsu, japan) on axio imager z.2 (zeiss, oberkochen, germany) and analyzed using zen software (carl zeiss, germany). fluorescence in situ hybridization (fish) the bob014o06 bac clone from b. oleracea bac library (howell et al, 2008) was used as probe for ‘genomic in situ hybridization (gish)-like’ to distinguish specifically all c-genome chromosomes in b. napus (suay et al, 2014). the bob014o06 clone was labelled by random priming with alexa594 dutp (red) (thermo fisher scientific). the ribosomal probe 45s rdna used in this study was pta71 (gerlach and bedbrook, 1979) which contained a 9-kb ecori fragment of rdna repeat unit (18s-5.8s26s genes and spacers) isolated from triticum aestivum l. pta71 was labelled by random priming with biotin14-dutp (invitrogen, life technologies). biotinylated probes were immunodetected by fluorescein avidin dn (green) (vector laboratories). the chromosomes were mounted and counterstained in vectashield (vector laboratories) containing 2.5µg/ml 4’,6-diamidino-2phenylindole (dapi) (grey). fluorescence images were captured using an orca-flash4 (hamamatsu, japon) on an axio imager z.2 (zeiss, oberkochen, germany) and analyzed using zen software (carl zeiss, germany). species identification by sequencing of a chloroplast region the aim was to amplify a chloroplast genomic region containing diagnostic single nucleotide polymorphisms (snps) or indels for b. oleracea, b. rapa or b. napus. to that purpose, we first retrieved and aligned the brassica chloroplast genome sequences available for the three species from li et al (2017) using geneious prime 2022.2.2 (https://www.geneious.com). we then identified a genomic region and designed consensus primers enabling us to discriminate each species. the 64 falentin et al genetic resources (2024), 5 (9), 61–71 consensus primers allowed amplification o f 1,118pb for b. oleracea, 1,088pb for b. rapa or 1,084pb for b. napus. dna of one to three plants per population and of control lines was extracted using 50mg of fresh leaf tissue, which had previously been freezedried, and the nucleospin plant ii kit (macherey nagel). the consensus primers used were trnk-rps16 f (5’ cataaacaggtagactgctaactgg 3’) and trnkrps16 r (5’ gtattcttcctaaaggtatgaaaactaac 3’) with following pcr reagents: 1x buffer, mgcl2 2mm, dntps 0.25mm, primers 0.5µm each, taq promega 1.5u and 5ng dna of the sample analyzed. the pcr conditions were a denaturation 94◦c 2min, then 35 cycles 94◦c 30sec 59◦c 30sec 72◦c 1min 30sec, with a final elongation 72◦c 10min. the amplified region was then sequenced by sanger (genoscreen) and analyzed using geneious software (https://www.geneious.com). all amplified chloroplast sequence data have been deposited into ncbi/genbank as popset 2716368500.: pp619885 pp620127). results taxonomic verification of the collected populations among the collected populations (table 1), the first limiting factor encountered was the germination of the collected seeds, even under favourable controlled conditions applied on automated germination tools for b. rapa, in spite of seed viability confirmed by tetrazolium staining. specifically, 6.8% of the collected populations showed a poor emergence in the greenhouse with less than 30 plants per population and were not considered for further analyses. this low germination rate may be attributed to two different factors: the high level of seed dormancy (observed here in 18.2% of b. rapa wild populations) and the seed conservation of landraces collected on farms (5.2% and 14.9% of seeds showed very poor germination for b. rapa and b. oleracea landraces, respectively). to validate the correct species identification of each collected population and to verify the absence of contamination in the collected seeds, we performed flow cytometry on all the plants grown representing a population. as the investigated species have different profiles linked to their differences in dna content (630mb for 18 chromosomes in b. oleracea, 529mb for 20 chromosomes in b. rapa) (figure 1a), it was possible to determine with +/-2 chromosomes the genomic structure of each plant. due to possible contamination with species having a close chromosome number, this analysis was complemented by sequencing a chloroplast genomic region that showed species-specific differences. we chose a genomic region with a sequence specific to each species according to li et al (2017). the size of the amplified regions was 1,118bp, 1,088bp and 1,084bp for b. oleracea, b. rapa and b. napus, respectively (all these chloroplast sequences are available on ncbi/genbank as popset 2716368500.: pp619885 pp620127). after aligning the sequences, we compared the sequences obtained in the sampled populations with those of the controls for the three species. we observed four snps and six indels specific to b. oleracea, four snps and four indels specific to b. rapa and three snps and five indels specific to b. napus (examples provided in figure 2a). b. montana (2n=18) differed from b. oleracea at only three snps and one indel whereas b. villosa and b. macrocarpa differed from b. oleracea at 17 snps and six indels. b. rupestris showed exactly the same sequence as the two latter species except for one snp, indicating that these three species (b. villosa, b. macrocarpa and b. rupestris) are highly related to each other whereas b. montana seems closer to b. oleracea (figure 2b). when flow cytometer and sequencing data were not congruent, chromosome counting was performed during mitosis to identify the species. this observation was combined with gish-like allowing identification of the c chromosomes and of rdna locus number, specific to each species with four, ten and 12 rdna loci for b. oleracea, b. rapa and b. napus, respectively (figure 3). by fish, in b. rapa (a genome) (figure 3b), the 45s rdna probe (green) marks five different chromosomes. the strongest fish signal located on the a03 chromosomes reflects a large number of genes. the second gene-rich locus is located on a01 chromosome proximal to the centromere. the remaining sites are located on cytogenetically undistinguishable a05, a06 and a09 chromosomes. b. oleracea (c genome)(figure 3d) had two pairs of chromosomes (c07, c08) containing 45s rdna loci. the sites localized on chromosome c08 show extensive decondensation while loci on c07 are fully condensed. in natural b. napus, we observed twelve 45s rdna signals and the bob014o06 staining revealed that eight signals were located on a genome and four on the c genome (ksiażczyk et al, 2011) (figure 3c). all the misidentified populations were listed in supplemental table 3. the most frequent mistake was confusing b. oleracea or b. rapa with b. napus. among the 103 b. oleracea populations analyzed, only three were misidentified (one wild and two landraces) and were thereafter confirmed to belong to b. napus using flow cytometry (figure 1d). this misidentification was also validated by chloroplast sequencing (figure 2a) and chromosome counting. among the 146 analyzed b. rapa populations, 15 were misidentified, out of which 12 were identified as b. napus. nine of these 12 populations were sampled in the wild and are probably volunteers of b. napus, i.e. escaped from the fields. all these data were confirmed by the sequencing of a chloroplast genomic region (figure 2a) revealing that all carried b. napus chloroplasts except for one wild tunisian population (br t aria w a), which had a b. rapa type chloroplast. the b. napus origin of this population was confirmed by cytogenetic analyses, revealing the presence of nine c chromosomes and 12 45s rdna signals by fish, eight on a genome and four on c genome genetic resources (2024), 5 (9), 61–71 cytogenetic and molecular methods in brassica taxonomy 65 figure 1. flow cytometry profiles of brassica controls and selected populations harbouring an unexpected profile: a) brassica oleracea, b) b. rapa, c) b. napus. for the screening of b. oleracea and independently of b. rapa populations, the control variety was adjusted to 300 for fluorescence value of nuclei at g1 stage. coincidence or deviation was compared with these controls. three examples of populations misidentified (d, e, f) are presented with a fluorescence intensity of g1 nuclei close to the one of b. napus. further analyses revealed that d) was a b. napus population whereas e) and f) were b. rapa autotetraploids. figure 2. alignments of chloroplast regions showing differences between the brassica species: (a) comparison between the controls and different brassica oleracea and b. rapa populations. the lines 7 (bo f biar w a), 8 (bo f gren l a), 12 (br f fron w a), 13 (br a roua l a) and 14 (br f stgi w b) were misidentified populations with a b. napus chloroplast, (b) comparison between the controls and different b. oleracea related species, b. montana (bm), b. macrocarpa (ba), b. rupestris (bu) and b. villosa (bv), highlighting polymorphisms between the different species. 66 falentin et al genetic resources (2024), 5 (9), 61–71 table 1. origin and number of collected brassica oleracea and b. rapa wild and landrace populations, as well as five b. oleracearelated species. the number of populations, for which we obtained a germination sufficient for their multiplication, is indicated. for these latter, the number of populations for which the species was validated using flow cytometry, chloroplast sequencing, plus cytogenetic controls when required, is also given in the last column. expected species expected subspecies collected populations populations with a satisfying germination validated populations/ species-subspecies wild populations brassica oleracea oleracea 45 45 44 brassica incana 1 1 1 brassica macrocarpa 1 1 brassica montana 8 8 8 brassica rupestris rupestris 2 2 2 brassica villosa drepanensis 1 1 1 brassica villosa tineoi 1 1 1 brassica rapa sylvestris/campestris 77 73 63 landraces brassica oleracea acephala 9 9 9 brassica oleracea botrytis 6 6 6 brassica oleracea capitata 19 19 19 brassica oleracea gemmifera 1 1 1 brassica oleracea gongylodes 1 1 1 brassica oleracea italica 6 5 5 brassica oleracea medullosa 6 6 6 brassica oleracea ramosa 2 2 2 brassica oleracea sabauda 1 1 1 brassica oleracea unknown 16 8 6 brassica rapa rapa 71 68 63 brassica rapa sylvestris var. esculenta 6 5 5 (figure 3e). among the three remaining misidentified b. rapa populations, one wild population from tunisia had a cytometry value close to b. rapa but no chloroplast gene amplification was detected; further morphological observations of this population revealed that it probably belongs to the genus sinapis. the two last cases observed were b. rapa populations (one slovenian wild population br s ljub w d and one french landrace br f cond l a) having a flow cytometry value close to the one of b. napus (figure 1e and figure 1f) but a b. rapa chloroplast genomic sequence. using cytogenetics, we detected no c chromosomes after a gish-like experiment and 20 45s rdna were counted, i.e. five rdna loci per a genome (figure 3f), which led us to the conclusion that these populations were in fact b. rapa autotetraploids (aaaa, 2n=4x=40). most of the populations confirmed as belonging to a specific species had an identical chloroplast sequence. nevertheless, we observed a few snps specific to some populations. in b. oleracea, two snps were specific to only seven populations (bo f jouy l a, bo s ljub l g, bo s ljub l h, bo s ljub l l, bo s ljub l m, bo s ljub l n and bo s ljub l o) and one allele at a different snp was specific to bo f mers w a. in b. rapa, three variations differentiated a few populations, one snp in br a dell w a, one base deletion in br a seba w a and br a bome w a and one snp in br a blid w a, br a bouf w a, br a chle w a, br a bara w a. these differences were observed in all the individuals tested per population. for b. oleracea related species (b. montana, b. rupestris, b. villosa, b. macrocarpa and b. incana), all collected populations per species had the same flow cytometry value and the same chloroplast sequence. description of the populations after discarding the few populations that did not germinate or were misidentified (supplemental table 3), we further characterized the remaining populations and their respective data collected during harvest. wild b. oleracea populations were collected on cliffs on the atlantic coast in france and spain (figure 4), whereas its related species (b. montana, b. rupestris, b. villosa, b. macrocarpa and b. incana) were growing more in southern regions, on the mediterranean coast. their locations and the characteristics of each environment are described in supplemental table 1. b. oleracea landraces were selected by farmers in each country, even in very warm regions such as the south of algeria (figure 4; supplemental table 2). selection of different organs for crop production (flowers, leaves, stems or roots) has led to the divergence of highly diverse phenotypes. it is worth mentioning that some 1 genetic resources (2024), 5 (9), 61–71 cytogenetic and molecular methods in brassica taxonomy 67 figure 3. brassica chromosomes stained by fluorescence in situ hybridization (fish). chromosome number counted in mitosis with the three controls a and d) b. oleracea, b) b. rapa and c) b. napus and two populations showing an unexpected structure: e) br t aria w a with b. napus genomic structure with 18 c chromosomes and 12 rdna signals and f) br f cond l a, an autotetraploid of b. rapa with 40 a chromosomes and 20 rdna signals. the bob014o06 bac clone (red) is specific to c chromosomes allowing to distinguish a and c genomes. morphotypes were difficult to classify in one subspecies as some of them were domesticated at the same time for leaf production (such as subsp. acephala) and for head cabbage (such as subsp. capitata, e.g. bo a tazl l a). additionally, even within the same morphotype, different developmental traits can be observed such as in mugnuli populations (south of italy) with several floral heads compared to common broccoli (laghetti et al, 2005). wild b. rapa populations (figure 5; supplemental table 1) were found in locations where competition with other species is lower, such as vineyards, orchards or field margins. thus, regardless of the country, the populations were generally large. the majority of the collected b. rapa local landraces (figure 5; supplemental table 2) were turnips (subsp. rapa) with the exception of few broccoletto (subsp. sylvestris var. esculenta) selected by italian farmers. discussion in this paper, we described the sampling of wild populations and local landraces of b. oleracea and b. rapa along a large climatic and soil gradient from the north of france to the sub-saharan regions. our objective was to validate at the early stage of plant development before sequencing that the seeds collected from plants of 112 and 154 of b. oleracea and b. rapa populations (both wild and local landraces), respectively, belonged to the expected botanical species. then the origin of each population is described as a preliminary material for future botanical determination and plant adaptation genetic studies. the first limiting factor was germination. seed dormancy was only detected among b. rapa populations. in spite of seed viability confirmed by tetrazolium staining and of cold treatment, we did not succeed in getting enough seedlings per mother for four b. rapa wild populations to keep the initial genetic diversity of the populations. this trait, described in brassica as primary physiological dormancy (finchsavage and leubner-metzger, 2006), seems to be a characteristic of some wild b. rapa populations. in our case, some populations met problems of imbibition as the seed coat was impermeable. puncturing the seed coat before adding gibberellic acid improved germination for sicilian and some algerian wild populations. these results indicated a seed coat imposed dormancy in b. rapa which has not been described for brassica (baskin and baskin, 1998). the conditions of seed conservation on the other hand is a likely explanation for the low germination rate in landraces of both species. this observation highlights the importance 68 falentin et al genetic resources (2024), 5 (9), 61–71 figure 4. distribution of the brassica oleracea populations collected: 44 wild populations indicated with red dots, 56 landraces with green dots and 14 related species’ populations with pink triangles. of seed quality and storage conditions, especially in brcs (subramanian et al, 2023). because of the morphological similarity between the species at the seedling stage, our controls have revealed the importance of performing molecular and cytogenetic analyses before undertaking genetic sequencing and agronomic studies. we decided to combine a straightforward method, flow cytometry for assessment of chromosome number with a more expensive one, sequencing of a species-specific chloroplast region to validate the taxonomy. we applied more difficult and time-consuming cytogenetic methods for populations showing incongruent results with the two first methods. flow cytometry is a high throughput technique allowing dna content assessment of all plants, here 30 plants per population. yet, as several species of the brassiceae tribe have a similar dna content, this technique might not be precise enough (leflon et al, 2006) to validate the species. that is the reason why we complemented this analysis by sequencing a species-specific chloroplast genomic region taking advantage of the whole chloroplast genome sequences of many brassica species/populations published by li et al (2017). the combination with the analysis of chloroplast sequences allowed the confirmation of a misidentification for one tunisian population presenting a flow cytometry value similar to b. rapa but no chloroplast amplification as it probably belongs to the genus sinapis. however, the most frequent mistake was a confusion with b. napus, showing a higher dna content, detectable by flow cytometry. yet, among the 17 populations identified as b. napus by flow cytometry (three populations in the b. oleracea and 14 in the b. rapa collections), three had a chloroplast sequence similar to b. rapa. this conflicting result called for further cytogenetic experiments for these three populations, using gish-like on mitotic chromosomes with a bac specific to b. oleracea chromosomes (suay et al, 2014) and 45s rdna probes revealing the number of rdna loci (ksiażczyk et al, 2011). from this data, we concluded that one tunisian population was indeed a b. napus population. it could be interesting to precisely compare after chloroplast assembly with the results reported by li et al (2017). these authors reported that b. napus chloroplasts can be classified into two different clades identified from different b. rapa morphotypes. the two other populations were b. rapa autotetraploids, with 40 a chromosomes and 20 45s rdna loci as expected when doubling the a genome. such autopolyploid populations were previously reported for the production of new forage varieties (olsson and ellerström, 1980). genetic resources (2024), 5 (9), 61–71 cytogenetic and molecular methods in brassica taxonomy 69 figure 5. distribution of the brassica rapa populations collected: 63 wild populations indicated with red dots, 68 landraces with green dots. among the 100 and 131 confirmed diploid populations for b. oleracea and b. rapa respectively, chloroplast sequences revealed only a few variants snv (li et al, 2017) for some accessions in both species. the low mutation rate of the chloroplast dna in most flowering plant families can explain these variations as already reported from global chloroplast assembly. interestingly, li et al (2017) observed more snvs in the b. rapa than in the b. oleracea genotypes that they investigated, with 343 and 16 snv, respectively. by investigating an enlarged b. oleracea diversity, perumal et al (2021) described more snvs with clustering of different cultigroups. in our collected wild and landrace populations, we observed that a common variation is shared by seven populations belonging to capitata and acephala groups originating from slovenia with the exception of one french landrace. for b. rapa, snv were only observed in some wild algerian populations. further studies are in progress in order to compare the genetic diversity from chloroplast assembly and nuclear snp, taking into account the different cultigroups and their geographic origins. a large morphological diversity was observed among the b. oleracea landraces whereas wild populations were morphologically similar to forage kales. for mugnoli belonging to the same group as broccoli (subsp. italica), biancolillo et al (2023) developed a non-destructive tool based on multivariate image analysis and agro-morphological descriptors for the characterization and authentication of these local varieties. for b. rapa, landraces selected by farmers are mainly turnips, with the exception of five populations of broccoletto. in this paper, we describe the different environments in which these different populations were collected. this well-characterized material collected on a very large climatic and soil gradient opens the prospect of identifying genomic regions involved in adaptation to climatic constraints and microbiota descriptors (fungus and bacterial composition). to do so, seeds were produced at the same geographic location in order to avoid the environmental effects of the collecting site on seed quality. high-throughput sequencing for bulks of 30 plants per population is currently ongoing to capture the maximum diversity existing within the population. mapping the reference genome of each species and snp calling will allow the description of genetic diversity and the design of nested core collections. genomewide association (gwas) and genotype-environment association (gea) analyses will be possible from the project consortium to identify genomic regions involved in climate adaptation. functional analyses will be 70 falentin et al genetic resources (2024), 5 (9), 61–71 performed on the most contrasted populations to finely investigate their responses to cold and warm temperatures. field experiences of core collections in five countries will allow the validation of favourable alleles under different environmental conditions. all these data will be used (1) to promote local landraces, as several are endangered, and (2) to design crosses that could be relevant to produce pre-breeding populations, each adapted to the climatic evolution of each country. supplemental data supplemental table 1. description of b. oleracea and b. rapa wild populations supplemental table 2. description of b. oleracea and b. rapa landraces supplemental table 3. populations that did not germinate or were misidentified acknowledgements we thank the genetic resource centers bracysol (htt ps://igepp.rennes.hub.inrae.fr/l-igepp/plateformes/bra cysol) and the agricultural institute of slovenia (https ://www.kis.si/en/) for providing seeds from different landraces. we thank biogenouest (the western french network of technology core facilities in life sciences and the environment, supported by the conseil regional des pays de la loire) for access to molecular cytogenetics (h ttps://www6.rennes.inrae.fr/igepp eng/about-igepp/ platforms/molecular-cytogenetics-platform-pcmv) and genouest bioinformatic platforms (https://www.genou est.org/). we thank plant imaging platform phenotic in angers (inrae-irhs, angers university, institut agro, geves, france) for experiments on seed germination and v. blouin for tetrazolium staining. we also thank all the staff who took care of our plant material (especially l. charlon, j-p. constantin and f. letertre). all the research is funded by h2020 prima, project no. 1425, brasexplor (https://brasexplor.hub.inrae.fr/) for ‘wide exploration of genetic diversity in brassica species for sustainable crop production’ and by inrae through tsara initiative (transforming food systems and agriculture through a partnership research with africa) promoting a specific french-algerian collaboration. author contributions cf, hh, and amc designed and managed all the experiments. cf, hh, fa, cb, gb, lb, mc, gd, jfc, lg, ag, ri, vi, jaj, vm, eo, mp, mp, bp, tr, fr, jr, rs, ls, vt, st, it, fwb, amc participated to the collects and the local description of the populations. vm, bp, vr, st provided landraces and their description from brc. mt performed flow cytometer analyses. gd and mrg designed chloroplast markers and performed experiments. oc and vh performed all molecular cytogenetic experiments. mb managed the database for population description. cf, hh, mrg and mt contributed to writing the manuscript, which was finalized by amc. conflict of interest statement the authors declare that they have no financial or competing interests. references baskin, c. and baskin, j. m. 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(2024). use of plant genetic resources in yemen and suggestions for potential improvement. genetic resources 5 (10), 39–52. doi: 10.46265/genresj.vdwo8193. © copyright 2024 the authors. this is an open access article distributed under the terms of the creative commons attribution license (cc by 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. introduction over the past few decades, efforts have increased in collecting, documenting and conserving genetic resources in various countries with their different plant, animal, forestry, pastoral and aquatic components, including microorganisms and invertebrates. these resources are considered a national wealth and an indispensable economic resource for any country, not only because of their importance in food security but also because they are vital for sustainable development. the convention of biological diversity (unep, 1992) and the international treaty on plant genetic resources for food and agriculture (fao, 2004) are two binding global agreements calling the countries for concerted efforts to ensure the conservation of and access to biological resources, including plant genetic resources ∗corresponding author: maeen ali aljarmouzi (maeen669@gmail.com) (pgr), to facilitate their sustainable use, along with fair and equitable sharing of benefits arising out of their utilization. many countries, including yemen, have recognized the importance of genetic resources and have set out to establish centres, specialized units and genebanks since the 1970s, especially in the field of pgr. over the past few decades, it has been possible to carry out many surveys and collect seeds and other types of samples of many diverse plant crops and species for conservation and herbaria. these accessions show a distinctive and rich plant genetic diversity in the country reflecting topographic, geographic and climatic diversity as well as the diversity of ecosystems and farming systems. the flora surveys in yemen identified a total of 2,838 plant species, including 2,602 that grow naturally, 129 cultivated plant species, 107 introduced crops, and 608 species identified as endemic (whose presence is limited to yemen only) or semi-endemic species (whose received: 13.04.2024 accepted: 10.09.2024 published online: 27.09.2024 https://www.genresj.org https://www.doi.org/10.46265/genresj.vdwo8193 https://www.genresj.org https://www.doi.org/10.46265/genresj.vdwo8193 mailto:maeen669@gmail.com 40 aljarmouzi et al genetic resources (2024), 5 (10), 39–52 presence is limited to the arabian peninsula only) (alkhulaidi, 2013). aljarmouzi et al (2023) mentioned that the national genetic resources center (ngrc) in yemen plays a pivotal role in collecting, conserving, documenting and characterizing pgr. currently, the genebank conserves 6,849 seed accessions from 45 different crops. these crops encompass cereals such as sorghum, maize, millet, wheat and barley, totalling 4,701 accessions. moreover, the genebank safeguards an extensive collection of legumes (1,215 accessions), including lentils, beans, cowpeas, peas and fenugreek. additionally, it conserves a diverse selection of vegetables (561 accessions), such as onions, tomatoes, chilli peppers, cucumbers, zucchini, mallows, radishes, eggplant and okra. ngrc’s efforts extend to the conservation of oil-producing crops (97 accessions) such as sesame and peanuts, as well as less commonly utilized crops like black seed (nigella sativa), henna (lawsonia inermis), coriander (coriandrum sativum), caladium (caladium bicolor), arugula (eruca sativa) and mustard (brassica juncea l.). furthermore, in various regions, field genebanks have been established by agricultural research stations to complement ngrc’s endeavours, holding an extensive assortment of crops ranging from date palms, mangoes, citrus fruits, almonds, grapes, bananas, papayas, as well as forage and forest species. as aljarmouzi et al (2023) reported, a total of 917 accessions are maintained in field genebanks. these field genebanks are strategically located across different regions, including the northern, central and southern highlands, the tihama coastal plains region, the southern coast region (al-kud, abyan), and the eastern plateau region (marib and seiyun). collectively, ngrc and its associated field genebanks demonstrate a coordinated commitment to the comprehensive collection, conservation, documentation and characterization of yemen’s national pgr. the process of collecting, conserving, documenting and characterizing pgr is not a goal by itself, but it aims to achieve sustainable use of those resources. in this regard, the use of genetic resources, especially the materials conserved in seed and field genebanks as well as in vitro storage, has remained one of the most important challenges facing the management of agricultural research and genetic resource institutions (aljarmouzi et al, 2023). according to the yemen national report in the second global plan of action for plant genetic resources (gp2) of the un’s food and agriculture organization (fao, 2011), the issue of genetic resource utilization was considered one of the most prominent weaknesses in the implementation of programmes and plans for pgr in yemen (fao, 2019b). the extent to which collected and conserved seeds and other accessions of different plant species and crops in genebanks are being utilized remains unclear. this issue needs to be addressed through studying, understanding and tracking any changes that may affect it, especially considering the current conditions and transformations that the country is witnessing. identifying the challenges and obstacles is crucial to developing a clear vision for advancing the use of pgr – an objective this paper aimed to achieve. materials and methods in this study, the authors relied on historical descriptive information regarding the use of pgr. this was done by collecting and reviewing national and international documents, reports and other surveys and studies related to pgr, in addition to relying on authors’ previous works, personal knowledge and experiences, as team members directly involved in the conservation and use of genetic resources in yemen (aljarmouzi et al, 2023). to prepare this paper, the authors used the methodological approach followed by the fao commission on genetic resources for food and agriculture (cgrfa), and the guiding indicators approved for assessing the implementation of the gp2 (fao, 2011). about 50 of those indicators were used, mostly to evaluate the current state of pgr use in yemen during the period 2007–2023, according to the areas and activities included in the gp2, as well as the medium-term plan of the agricultural research and extension authority (area) in yemen, and the annual plans of ngrc, operating under area, dhamar. based on its general objective, this current study assessed the utilization state of pgr, focusing on the most relevant aspects of four main themes: 1) notable achievements, 2) major changes and trends, 3) gaps and challenges, and 4) developing proposals for future pgrfa activities. results and discussion most important achievements expanding characterization and evaluation activities and developing collecting of germplasm there is no doubt that improving the characterization and evaluation of the conserved accessions allows and increases the efficiency of their use, considering that proper documentation is required, also to register landraces and to enable their protection by property rights. area, through ngrc and its affiliated regional research stations and specialized research centres located in the different agroclimatic zones, has implemented many research programmes and projects related to the characterization and evaluation of local varieties of cereals, vegetables and fruit tree crops. the main studies carried out are summarized in table 1 and their results were published in the yemeni journal of research and studies (yjars), issued biannually by area. genetic resources (2024), 5 (10), 39–52 plant genetic resources use in yemen 41 table 1. research and studies published in the yemeni journal of research and studies (yjars) on the characterization and evaluation of germplasm accessions in the agricultural research and extension authority (area) and its affiliated research stations and centres. source: prepared by the authors based on the articles published in the yjars. title of the study crop(s) no. of accessions reference evaluation of some local germplasm of lentil, collected from some areas located in the northern and central highlands lentil 22 (lutf, 2000) comparing sorghum varieties for resistance to sorghum midge in the tehama region of yemen sorghum 5 (muharram et al, 2001) shibam -2: a new faba bean variety with small-seed for the northern highlands region of yemen faba bean 1 (lutf, 2000) genetic analysis of some traits of maize plants under water stress conditions maize 15 (muqbil and abdullah, 2002) evaluation of several local peanut germplasm accessions peanuts 9 (sodi and fouad, 2003) evaluation of grain yield of barley varieties with the participation of farmers in the northern highlands barley 8 (saif, 2004) evaluating the productivity efficiency of some introduced maize hybrids maize 11 (noman, 2004) evaluating of local landraces of eggplant in wadi hadhramaut eggplant 32 (hassan, 2005a) evaluation of local landraces of carrots in wadi hadhramaut carrot 15 (hassan, 2005b) grain yield comparison of wheat varieties in the yemeni northern highlands with farmers participation. wheat 20 (saif and al-shamiri, 2005) evaluation of yield and agricultural characters of eight wheat varieties wheat 8 (saif et al, 2005) evaluation of local landraces of sweet melon in wadi hadhramaut watermelon 44 (hassan, 2006) study of heritability percentage of some quantitative characters in f2 lentil hybrids lentil 12 (zaid and maqbool, 2006) yield efficiency of the wheat promising variety qaa elhaqal-7 in yemeni central highlands region wheat 4 (habib et al, 2007) evaluation of the local landraces of tomato in wadi hadhramaut tomato 25 (hassan and bin zeghew, 2007) purification and evaluation of the local wheat variety (arabi) wheat 1 (habib et al, 2007) evaluation of different varieties of fenugreek under rainy conditions in the northern highlands of yemen fenugreek 5 (zaid, 2007) evaluation of local genotypes of local pumpkins in wadi hadhramaut pumpkin 21 (hassan and bin zeghew, 2007) evaluation and description of local landraces of common bean beans 37 (zaid et al, 2008) evaluation of genetic landraces of okra in wadi hadhramaut okra 31 (hassan and bin zeghew, 2008) evaluation of yield and other agronomical characters of local wheat mutants under rain-fed conditions wheat 9 (saif et al, 2008) evaluation of the performance of five cowpea varieties under rain-fed conditions in the southern uplands of yemen cowpea 5 (al-duwailah, 2009) continued on next page 42 aljarmouzi et al genetic resources (2024), 5 (10), 39–52 table 1 continued title of the study crop(s) no. of accessions reference evaluation of radiated seeds of sonalika wheat variety resistant to yellow rust wheat 1 (basha et al, 2009) improving the quantitative and qualitative traits of gemmiza-9 wheat variety by mutation induction wheat 3 (saif et al, 2010) use of gamma rays to induce desirable mutants in local lentil variety (d2001) lentil 7 (zaid and saif, 2010) evaluation of the genotypes of coriander, nigella and cumin in wadi hadhramaut coriander, nigella and cumin 66 (hassan and al-saqqaf, 2011) yield and quality evaluation of seven potato varieties in wadi hadhramaut potato 7 (hassan et al, 2011) evaluation of different barley varieties for yield and other agronomical characters under rain-fed conditions barley 46 (saif and al-shamiri, 2011) evaluation and selection of some introduced forage lines of common vetch for yemeni northern highlands vetch 15 (zaid, 2011) evaluation of yield and some agronomical traits of cowpea varieties under spate irrigation at yemen southern coastal region cowpeas 15 (al-bakri and sodi, 2012) early performance of some mexican peaches and nectarines in yemen highlands peach 12 (al-dalas et al, 2012) productivity evaluation of four finger millet cultivars (eleusine coracana) under the central highlands conditions millet 4 (doss et al, 2013) effect of mutants on quantitative and qualitative characteristics of local bakkur barley under rain-fed conditions barley 10 (saif et al, 2014) effect of pure line selection on the growth and yield characterization of local sorghum variety qaira’a sorghum 1 (noman and dirham, 2014) morphological characterization of some plant genotypes of date palm in shabwa governorate, yemen date palm 9 (abdullah and saeed, 2014) genetic resources (2024), 5 (10), 39–52 plant genetic resources use in yemen 43 regarding the field collections, teams of researchers undertook the characterization of varieties of mango, coffee, date palm and grapes in different regions of yemen. as a reference standard, the international plant genetic resources institute (ipgri) descriptors were used. for example, in 2021, a team of researchers at the northern highlands research station (nhrs) characterized 45 genotypes (14 grape, 20 apricot, 3 pear, 4 fig and 4 peach genotypes). in 2022, five grape varieties and five pomegranate varieties were characterized. the results of the cluster analysis confirmed the distinct diversity among grape and pomegranate varieties both of which are among the most popular fruit crops in yemen (al-dalas et al, 2022). the same is true for coffee, as several genotypes have been described in sana’a governorate. a team of researchers at the al-wadi and al-sahra research station (wsrs) in seiyun, hadramaut, also characterized date palm varieties in the yemeni governorates where the crop is cultivated (area, 2013). as for mango, all varieties grown in yemen have been characterized, including 26 varieties introduced by the ministry of agriculture and irrigation and area (almunaifi, 2022). in this context, it is worth noting that documentation and description of mango varieties spread in yemen were also implemented by area with the support of the sultanic dewan of oman and included in the collection (area, 2013). upon reviewing the ngrc technical reports and the project reports implemented through the centre, specifically those focused on characterizing accessions stored in the genebank (table 2), significant weaknesses were identified. these included inconsistency in the data and repetition in the description of some accessions. for example, 1,967 sorghum accessions, more than 1,320 millet accessions, 883 wheat seed accessions and 918 barley seed accessions were characterized (table 2), and the most prominent results of this characterization can be summarized as follows: every report from ngrc included activities related to the phenotypic characterization of genetic resource accessions. there was a repetition in the characterization for some accessions because of the weak management operations in the genebank, especially in documentation. the number of wheat and barley accessions characterized exceeds the actual number of accessions preserved in the genebank. the inadequate quality and accuracy of characterization data were clearly demonstrated, as it was noted that these reports depend on a limited number of descriptors that were studied or documented. some technical reports were prepared in a systematic manner and were scientifically sound, but they were not published in the form of evidence or scientific articles in peer-reviewed scientific journals. the results on varietal characterization were not documented with photos highlighting the characteristics of the local landraces and genotypes that were studied or referred to verbally in the reports. in this context, it is also worth noting that some accessions of millet, wheat and sorghum were genetically characterized using molecular markers to determine their degree of genetic relatedness, through the ‘rainfed agriculture and livestock’ project (2006–2013) and the ‘agricultural biodiversity and adaptation to climate change’ project (2014–2015), both funded by the world bank in collaboration with the international center for agricultural research in the dry areas (icarda) and ngrc. encouraging crop diversity the information collected for this paper, through reviewing the main and secondary references, revealed that the optimal use of genebank holdings can take different forms to encourage crop diversity utilization in the country. some important examples of pgr use are summarized below: agricultural farming systems that rely on a limited number of crops lack resilience, and these systems can lose their production because of factors such as diseases and pests (lin, 2011). therefore, diversification in crop production must be encouraged to meet local and national needs as well as to improve prevailing dietary patterns. in cooperation between fao and area, quinoa (chenopodium quinoa) was introduced and evaluated in farmers’ fields, starting in 2013 with the introduction of several types of quinoa. many genotypes were evaluated on the research farms in sana’a, taiz, dhamar and al-kadan under different environmental conditions. in 2014 and 2015, the productivity of three varieties was evaluated at the research farm in the central highlands, to obtain a variety with high fodder and grain productivity suitable for the conditions of the region (daws and al-muallem, 2018). the wsrs in seiyun, hadhramaut governorate, has evaluated and propagated haidawan (boerhavia elegans), a naturally growing local plant with significant nutritional value. this plant is commonly used as a food ingredient in the dietary meals of the hadhramaut community, as well as in the al-mahra and shabwa governorates. one notable dish that features haidawan is the ‘aseed’ meal, a porridge made with mashed dates and sesame oil, locally known as ‘salit juljul’. haidawan is rich in bioactive compounds, including antioxidants and essential nutrients, which contribute to its health benefits. in addition to its use in aseed, haidawan seeds are often ground into flour and incorporated into various traditional recipes. the seeds are also recognized for their potential to enhance the nutritional profile of foods such as biscuits (kanzal and madhi, 2012). introducing varieties of beans, lentils, peas, wheat, and barley into governorates where the cultivation of such crops has declined because of economic and social factors, for example in dhamar governorate (in the districts of jahran, ans, utomah, and al-hada), sana’a governorate (in the bani matar district), and al-mahwit 44 aljarmouzi et al genetic resources (2024), 5 (10), 39–52 table 2. number of accessionsof the main crops in yemen, preserved in the genebank at the national genetic resources center (ngrc), dhamar, and characterized and evaluated between 2007 and 2021. source: prepared by the authors based on the technical reports of ngrc (2007–2021). crop year total 2007 2008 2009 2010 2011 2012 2013 2014 2019 2021 sorghum 638 277 110 227 57 658 1,967 millet 197 289 442 130 130 15 117 1,320 maize 73 120 50 50 4 36 333 wheat 61 202 154 97 97 35 237 883 barley 57 332 84 112 110 7 216 918 cowpea 59 170 88 18 18 353 lentils 53 108 58 17 3 78 317 peas 23 26 16 3 89 157 beans 93 93 total 523 289 2,038 238 812 110 665 142 1,524 6,341 governorate (in shibam and al-dhula’a districts), ibb governorate (in yarim district), hajjah governorate (in kuhlan af’far district) and amran governorate (in qa’a al-bawn). expanding the cultivation of legume crops is one of the priorities of agricultural policymakers at present, especially considering war and siege conditions. it is worth noting in this context that the introduction and encouragement of the cultivation of these crops and their varieties have taken place through several projects, such as the ‘food security project’, funded by the islamic development bank (isdb), the kuwait fund for arab economic development and the state of qatar. additionally, the ‘participatory conservation and sustainable use of local landraces to improve the livelihood and resilience of farmers to climate change in yemen’ project, funded by fao (fao, 2019a), has introduced and spread legume varieties in areas with very limited legume cultivation. one of the most important obstacles facing the expansion of legume cultivation is the reluctance of farmers to grow these crops that need guarding and protection because people uproot the plants and eat the green immature pods or fruits directly, especially since the number of farmers who grow these crops is limited so far. introducing varieties of almond and coffee and encouraging farmers in sana’a governorate (haraz, alhaymah and bani matar districts) to grow these crop varieties to increase crop diversity (y30, 2023). the same is also noted in the case of ibb governorate (alsaddah, al-nadira and wadi banna districts) and in taiz governorate (al-mawaset, mawiya and al-shamaytain districts) (undp, 2021). in dhamar governorate (utomah district), the agriculture office (ao) implemented many activities encouraging crop diversification. in addition to the expansion and spread of coffee and almond trees in the district stated above, new types of fruits have also been introduced, such as kiwi, blackberry, pomegranate, sa’adi grape and annona fruit crops. it was also noted that some innovative farmers in other governorates began individually to grow cardamom (sana’a governorate) and ginger (al-mahwit governorate). the ‘rainfed agriculture and livestock’ project funded by the world bank in 2006–2013 included an activity related to promoting on-farm conservation and sustainable use of landraces of cereals, food legumes and vegetables, implemented jointly by icarda and area. this project provided examples of added-value technologies and alternative sources of income along with institutional arrangements. it has established 70 seed producer associations in five governorates to multiply and distribute seeds of landraces of sorghum, millet, wheat, barley, maize, lentil and faba bean. supporting breeding and genetic improvement programmes the genetic material that has been – or is being – collected can be used to identify or select distinctive traits to develop crop varieties as needed, or to expand the genetic base of a breeding programme. such traits may include earliness of the local varieties (of cereal and leguminous crops), or drought resistance and salt tolerance in some other local varieties, and good-quality attributes needed by breeding programmes to develop new high-yielding and climate-resilient varieties. in this regard, the ngrc in dhamar provided seed samples of many local varieties in response to requests from researchers and graduate students in various yemeni agricultural regions to implement genetic improvement activities. however, these requests are still very limited compared to the available genetic material preserved in the genebank (table 3). plant breeders at area agricultural research stations have implemented many breeding programmes using mutations through external projects funded by the international atomic energy agency (iaea), or through local projects funded by the annual government budget, and the work on these programmes is still ongoing. distinctive successes have been achieved in this aspect, especially in sesame and barley. three varieties of sesame were released: hazza-1 and hazza-2, both distinguished by their red seed colour and superior genetic resources (2024), 5 (10), 39–52 plant genetic resources use in yemen 45 table 3. crops and number of genebank accessions provided by ngrc upon request for research and breeding purposes. source: prepared by authors based on the data of the genebank of the national genetic resources center in dhamar. year crop no. of accessions 2010 lentil 5 sorghum 5 wheat 2 barley 4 lentil 5 bean 2 2011 maize 10 2013 millet 20 sorghum 175 millet 40 maize 37 wheat 34 barley 33 cowpea 35 lentil 19 fenugreek 14 peas 11 bean 10 maize 5 2014 okra 35 wheat 24 cowpea 45 lentil 15 sorghum 2 beans 2 peas 1 total 590 productivity compared to local varieties, and hazza-3, noted for its white seed colour, high yield and high oil content. varieties of wheat, barley, fenugreek and lentils were also released in the northern highlands as part of the mutation programme. between 2013 and 2020, a total of 93 research activities were conducted in the field of variety evaluation and improvement across area branches, including regional research stations and national centres (table 4). in 2021, thanks to area, many varieties of several crops, including wheat, barley, corn, lentils, beans and peas were released by the high committee for registration of agricultural varieties and technologies (hcravt), which includes representatives of various relevant authorities. these varieties were delivered by area to the public corporation for improved seed multiplication (gsmc) in a special ceremony sponsored by ministry of agriculture and irrigation (table 5). the genetic resource center (grc) of the college of agriculture, sana’a university also implemented many activities such as the evaluation of landraces and breeding by selection method during the period 2014–2019, the most important of which were the following (personal communication, dr mohammed alaswadi, grc director, college of agriculture, sana’a university, 2022): production of maize hybrids improvement of wheat productivity (al-himyari variety and al-bawni variety) improvement of barley varieties productivity (sakla and al-aswad (the black)) improvement of four sorghum varieties productivity (al-lahmani, al-jameli, al-shahedhi and al-safara) improvement of local lentils products improvement of local peas productivity. these advancements are integral to genetic improvement programmes aimed at enhancing traits such as earliness, drought resistance and heat tolerance. the productivity of these varieties has been significantly improved through the purification and maintenance of local varieties well-adapted to yemen’s predominantly arid climate. the improvement process involves selecting plant heads based on distinctive characteristics such as size, weight and yield. supporting seed production and distribution to ensure the use of available pgr, effective seed systems must guarantee that the seeds adopted by farmers for cultivation are available in sufficient quantity, of high quality, and can be obtained by farmers at the right time, in the right place and at reasonable prices. during the past decade, area and gsmc received funding from fao to produce and distribute seeds to farmers as part of humanitarian aid. these seeds include locally adapted varieties such as sorghum, millet, barley and lentils, which are crucial for maintaining agricultural biodiversity and resilience. these local varieties, inherited by farmers from their ancestors, are well-adapted to climate change and represent an important genetic resource for improving agricultural production. therefore, the multiplication and distribution of these local seeds were vital for preserving genetic resources and ensuring their sustainable use. it should be noted here that after problems occurred due to distribution of low-quality seeds of cereal crops by some humanitarian aid organizations, the ministry of agriculture and irrigation stipulated that organizations and providers of seed distribution services to farmers must have the seeds screened and packaged in the stores and warehouses of the gsmc. fao’s support and funding for area and gsmc came within the framework of strengthening its capabilities to fulfil any resulting obligations or requirements from the execution of the ministry’s new instructions. however, there was an increase in cultivated areas and the production of improved seeds for certain crops during specific years (2014–2017). these seeds were distributed to farmers based on their needs and the availability of funding from supporting organizations. it is worth noting that despite fluctuations observed 46 aljarmouzi et al genetic resources (2024), 5 (10), 39–52 table 4. research activities related tothe evaluation of crop varieties and genetic enhancement implemented by the agricultural research and extension authority (area) research stations and centres (2013–2020). source: prepared by authors based on the reports of a collection of multi-year annual technical reports of the different area research stations and centres (2013–2020). chrs, central highlands research station; shrs, southern highlands research station; nhrs, northern highlands research station; wcrs, west coast research station; scrs, southern coast research station; ecrs, eastern coast research station; wsrs, wadi and al-sahra research station; errs, eastern region research station; ngrc, national genetic resources center. research activity research station/centre total chrs shrs nhrs wcrs scrs ecrs wsrs errs ngrc evaluation of cereal crop varieties (wheat, barley, sorghum, maize and millet) 21 2 6 2 2 5 6 44 evaluation of varieties of legume crops (lentils, beans, peas, chickpeas, beans and fenugreek) 8 2 5 4 19 evaluation of varieties of vegetable crops (potatoes, onions and carrots) 2 3 5 evaluation of varieties of fruit crops (almond, peach, mango, palm, lemon, banana and papaya) 2 1 2 4 1 10 evaluation of cash oil crop varieties (peanuts, sesame, quinoa, cotton, safflower and fescue) 2 3 3 2 1 4 15 total 35 4 12 7 7 2 7 9 10 93 table 5. species and varieties registered in the high committee for registration of agricultural varieties and technologies (hcravt), plant production directorate, ministry of agriculture and irrigation (mai), sana’a. source: prepared by authors based on the minutes of hcravt meetings. crop no. of varieties names of varieties wheat 10 bohouth-8, bohouth-14, bohouth-15, bohouth-37, bohouth-5, sonalika mohasan, bohouth-10, arabi, nagi, shibam-8 and naeem-1 barley 7 ashmour-2, qa’a al-haql-7, bohouth-2002, bohouth-28, bohouth-26, bohouth-7 and kawkaban-1 sorghum 1 jera’ah mohasan-98 millet 6 murakab zabid, kadan-1, kadan-2, kadan-3, kadan-4 and kadan 5 lentils 2 dhamar-1 and dhamar-2 peas 2 amran-1 and yahsub-1 beans 3 dhafar-1, dhafar-2 and shibam-1 mango 10 surdoud-5, surdoud-7, surdoud-11, surdoud-13, surdoud-18, surdoud-21, surdoud-36, kechener, totapuri, and surdoud-108 total 41 in subsequent years, there was still a notable effort to expand distribution to beneficiary farmers (table 6). in 2016, the general corporation for grain production improvement (pcgdp) played a significant role in boosting the production of improved seeds across several governorates, particularly focusing on wheat, and selling them at reasonable prices. the corporation entered into agreements with numerous farmers, providing them with seeds and essential production inputs such as fertilizers and irrigation to encourage the expansion of wheat cultivation. after the harvest, the corporation purchased the crops from the farmers, deducting the costs of the seeds and production inputs provided. the quantities distributed by the general corporation for grain production improvement increased from 52 tonnes in 2018 to 640 tonnes in 2022 (figure 1). since 2015, several international organizations in yemen have launched rapid response programmes to support farmers during the ongoing conflict. these programmes involve purchasing and distributing seeds through national partners like gsmc and area. the seeds are usually sourced directly from farmers, especially for crops like sorghum and millet, or from research and multiplication institutions. changes and trends the results of this study revealed the main changes and latest trends in the use of pgr in yemen during the study period (2007–2023) compared to the previous period which was covered by the first (fao, 1996) and second (fao, 2009) country reports of yemen on pgrfa issued by fao. the most important of these changes and genetic resources (2024), 5 (10), 39–52 plant genetic resources use in yemen 47 table 6. area (ha) and quantities of produced improved seeds (tonne) in yemen (2012–2022) for wheat, sorghum and millet. average seeding rate (kg/ha): wheat (140), sorghum (20) and millet (15). na, data not available. source: prepared by the authors based on unpublished data of the gsmc, dhamar, yemen. crop details year 2012 2013 2014 2015 2016 2017 2018 2019 2020 2021 2022 wheat cultivated area (ha) 326 444 473 292 332 392 na na na na na production (tonne) 652 888 946 584 665 785 720 718 570 385 346 area covered by improved seeds (ha) 4,664 6,350 6,757 4,171 4,750 4,906 na na na na na sorghum cultivated area (ha) 92 117 101 115 234 241 na na na na na production (tonne) 92 117 101 115 234 241 178 338 232 108.6 141.4 area covered by improved seeds (ha) 4,600 5,850 5,050 5,750 11,070 12,050 na na na na na millet cultivated area (ha) 36 5 33 27 55 42 na na na na na production (tonne) 36 5 33 27 55 42 16 153 30.3 43.5 49.8 area covered by improved seeds (ha) 2,400 333 2,200 1,800 3,666 2,800 na na na na na figure 1. quantity of wheat seeds (tonnes) distributed by the general corporation for grain production improvement (2018–2022) trends can be summarized as follows: considering the conditions of war and siege that the country has faced since the beginning of 2015, and with the growing awareness of protecting the country’s sovereignty over its pgr, there has been growing interest in protecting local landraces from potential misappropriation of intellectual property rights. this defensive protection might be developed through the registration of landraces into national catalogues (noriega, 2016). it would also be important to deposit samples in publicly accessible plant collections and document each accession with internationally recognized documentation standards, such as fao/bioversity international multi-crop passport descriptors (mcpd) (alercia et al, 2015) and ipgri/bioversity crop-specific phenotypic descriptors.1 assignment of unique identifiers 1 full collection available at https://hdl.handle.net/10568/56589 such as dois to accessions is also key to facilitating the exchange of germplasm passport information, ensuring that genetic resources are effectively managed and conserved. therefore, in their crop improvement programmes, area prioritized the documentation of genebank accessions and registration of landraces. the ministry of agriculture and irrigation also completed the steps to register many improved varieties of strategic crops. area submitted the required documents for sound registration procedures to the plant production directorate of the ministry, and the higher committee for varieties registration (hcravt) approved in its meetings the completion of their registration procedures. this included varieties of coffee, maize, wheat, barley, millet, sesame, and legumes such as lentils and peas. ngrc (dhamar), in coordination and partnership with the central highlands research station (chrs) (dhamar) and wadi and al-sahra research station 48 aljarmouzi et al genetic resources (2024), 5 (10), 39–52 (wsrs) (seiyun), characterized and evaluated varieties of sorghum, wheat, barley, lentils and cowpea (58, 35, 7, 3 and 18 accessions, respectively), which were collected from the selected districts in the governorates of dhamar, hadhramaut and al-mahra as part of the activities of the project ‘participatory conservation and sustainable use of local landraces to improve farmers’ livelihoods and their resilience in adapting to climate change in yemen’ (2019–2023). this project was funded by the itpgrfa benefit-sharing fund. in 2021, area represented by its northern highlands research station (nhrs) (sana’a), chrs (dhamar) and wcrs (al kadan), and in coordination with ngrc, implemented the largest phenotypic characterization activity – within the framework of collections – of accessions preserved in the ngrc’s genebank for cereal and legumes crops. more than 1,500 accessions were characterized, as a first step towards the characterizazion of groups with distinct characteristics. the data listed in table 7 shows the number of samples sent from ngrc’s genebank to the said research stations. table 7. number of samples sent by the national genetic resources center (ngrc) genebank to the research stations (2021). chrs, central highlands research station; nhrs, northern highlands research station; wcrs, west coast research station. source: prepared by authors based on data from ngrc’s genebank (dhamar). crop chrs nhrs wcrs total no. of samples sorghum 238 218 202 658 wheat 138 99 237 barley 98 65 163 lentil 40 36 76 peas 40 35 75 bean 43 32 75 beans 53 50 103 millet 97 97 maize 33 33 coriander 38 38 black seeds 28 28 total 650 601 332 1,583 there is no doubt that the scope of distribution and use of seeds has expanded over the past years due to the work of different organizations that worked in the country and provided farmers with seeds of different crops, which is undoubtedly a positive thing. however, this expansion was not accompanied from the beginning by any coordination with area. this has resulted in a gap between farmers, researchers and other sectoral line authorities. although diversity is desirable and required to enhance the resilience of farmers, the introduction of species and varieties must be based on field evaluation tests and on the results and recommendations of area. ngrc participated in implementing projects that encourage increasing on-farm diversity in plant species and within species. these projects, funded by various organizations, include the ‘agricultural biodiversity and climate adaptation project’ funded by the world bank, the ‘food security project’, funded by the islamic development bank (isdb), the kuwait fund for arab economic development and the state of qatar. additionally, the ‘participatory conservation and sustainable use of local landraces to improve the livelihood and resilience of farmers to climate change in yemen’ project, funded by fao. limited efforts to introduce quinoa as a food/fodder crop through testing trials and assessments that took place in various agricultural regions. the breeding programmes at area relied on the selection from nurseries provided to yemen by the international agricultural research centres, such as icarda, through regional projects and research programmes and networks for cooperation between those centres and national research institutions. many activities were conducted in the research stations and on farmers’ fields, and these projects, programmes and networks mostly targeted agricultural crops such as wheat, barley, lentils, peas, beans, cowpea, sorghum, millet and maize. over the past decade, the activities of most international centres in yemen have been disrupted due to the war, siege and later the covid-19 pandemic, making the transfer and exchange of genetic materials difficult. these challenges have persisted to the present day. in 2021, breeding research programmes began hybridization in vegetables (tomato) and cereal (wheat) crops. this is a new trend that must be noted and praised. it came in response to the conditions caused by war, blockade and global crises such as the covid-19 pandemic and the ukrainian-russian war, and the resulting tough economic conditions, especially considering the high seed import bill from abroad. this has become a major concern among decision-makers in searching for options that encourage researchers as well as the public and private sectors to move towards local seed production. the efforts in this respect are still modest because of the lack of experience and financial capabilities. in this context, it is important also to point to the efforts of the potato seed company in producing higher grades of potato tubers and tissue culture multiplied potato varieties in partnership with the private sector during the past five years, to reduce the import bill by producing seeds locally, as well as achieving self-sufficiency in this important food crop. a decrease in the number of plant breeders in agricultural research for several reasons (including retirement, death, illness and migration) in addition to the effects resulting from the war, such as the cessation and irregularity of salary payment and the absence of operational budgets. the process of exchanging genetic resources with the regional and international centres was halted due to the war and siege, and the paralysis or ineffectiveness of the various public service institutions. there have been no changes in national policies that encourage the development and trade of local varieties genetic resources (2024), 5 (10), 39–52 plant genetic resources use in yemen 49 and underutilized crops, as the agricultural seeds and fertilizers law no. 20 of 1998 is still in place and primarily focuses on the regulation, production and marketing of adopted seeds and fertilizers, but it does not specifically address the development and trade of underutilized crops (govt. of yemen, 1998). there has been a positive and active change on the part of public and commercial sectors in the trade of local varieties and underutilized crops. this is often done with the support of external organizations within the framework of the emergency response and humanitarian aid programmes/projects, despite the parallel negative points that could accompany this endeavour. an example of this is the pcgdp establishment of a specialized market (in 2022) in the heart of the capital, sana’a, to sell and trade types and varieties of different locally produced grains, including wheat, barley, legumes, and underutilized crops. this market complex still provides its services to both the private and public sectors. although limited information is available about private sector activity in seed production, it can be noted that some agricultural input companies have contracted farmers to produce seeds as required by funding from international organizations working in yemen on relief and humanitarian aid, through tenders to purchase quantities of seeds. there is also a significant activity by some companies working in propagation nurseries and producing seedlings or seeds of some crops. examples of such companies include the ‘yemen nabat’ nursery producing tomato seedlings, as well as potato seeds using tissue culture. the pcgdp was established in 2016 with the aim of achieving self-sufficiency in grains. the corporation provided financial support to area, agricultural colleges, and seed producer associations to implement many research and production activities in addition to training and awareness raising of producers at the local community level. the area cultivated with improved, high-quality varieties remains limited, not exceeding 15% of the total cultivated land. cultivation is still primarily focused on cereals and, to a lesser extent, legumes. gaps and challenges this study showed that using existing pgr in ngrc’s genebank and other genebanks in yemen still suffers from many shortcomings, gaps and challenges for important aspects. such gaps and challenges limit the process and hinder the effective use of the country’s genetic resources for food and agriculture. these obstacles can be summarized as follows: lack of trained and qualified technical personnel, especially in crop description and evaluation. weak capabilities in using computer programmes to analyze characterization and evaluation results. weak capabilities in using biotechnology, especially molecular characterization. weak documentation of data and dissemination of information on characterization and evaluation. research breeding programmes that promote crop diversification are limited. although there were initial efforts to introduce quinoa, these attempts were not sustained. yemen has a diversity of plant foods, yet agricultural research has not adequately explored these species. limited use of the genetic material preserved in genebanks for plant breeding. decrease in the number of plant breeders. poor use of modern technologies in education. weak characterization and evaluation programmes for underutilized plant species and wild food plants. limited number of crop varieties released that are well adapted to local conditions, especially sorghum and millet varieties, despite the diversity and abundance of genetic material for these two crops in the national genebank. weak capabilities of producing and distributing sufficient quantities of high-quality seeds of improved varieties of different crops by the government and private sectors, farmer community organizations, farmer groups and individual producers. absence of a comprehensive national policy to regulate seed production and trading, whether governmental, private, commercial or non-profit. weak funding for breeding, improvement and application of biotechnology programmes. great weakness in encouraging and developing the trading of seeds of unexploited crop varieties. conclusion and recommendations based on the results and indicators presented above regarding the achievements, trends and challenges affecting the use of pgr in yemen, it can be concluded that the utilization of national genetic resources remains weak and limited in contributing to food security, agriculture, the economy and sustainable development. to improve the use of these resources, several recommendations were formulated for future consideration including: characterizing and evaluating local genotype groups of basic crops, underutilized crops, and wild relatives for specific and distinct traits selected according to need, importance or priority. developing a documentation and information system for description and evaluation data and education programmes. expanding the application of genetic characterization using biotechnology tools. publishing the results of characterization and evaluation of the preserved genetic materials so that they are available to researchers, academics and those interested, and to encourage them to use and benefit from these resources. issuing guides identifying the accessions preserved in genebanks. strengthening staff technical capabilities in genetic characterization, evaluation and improvement through training and qualification programmes. 50 aljarmouzi et al genetic resources (2024), 5 (10), 39–52 giving priority to activities related to plant breeding and providing the required financial support. supporting the gsmc technically and financially to widen its coverage. reviewing and developing a special law regulating the seed production and trading sector, and developing a national seed policy and strategy that defines tasks and roles of the various public and private partners. increasing collaboration with international research institutes to leverage global expertise, share knowledge and access advanced technologies for the improvement of pgr. author contributions maeen ali al-jarmouzi prepared the study proposal, collected, organized, analyzed the data, wrote and improved the manuscript’s drafts. khalil m. alsharjabi contributed to reviewing and improving the study proposal, collecting the data, writing the manuscript, editing and improving the manuscript’s drafts at different stages. ahmed amri provided support through guidance, reviewing and improving the manuscript draft, enhancing the translation of the manuscript and providing important references. conflict of interest statement the authors declare no known conflicts of interest or any financial or personal relationships influencing the work or materials appearing in the article. acknowledgements the authors are thankful to the project ‘participatory conservation and sustainable use of local landraces to improve farmers’ livelihoods and enhance their resilience to climate change in yemen’ of the itpgrfa benefit-sharing fund, and the commission on genetic resources for food and agriculture. thanks go to all ngrc employees and the leadership of area and gsmc for their support and cooperation in providing the necessary data and information for this study. we also extend our thanks and appreciation to arshiya noorani from the commission of genetic resources for food and agriculture for her support and encouragement in completing this work. references abdullah, i. o. and saeed, a. a. 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(2010). use of gamma rays to induce desirable mutants in local lentil variety (d2001). the yemeni journal of agricultural research and studies (21), 119–126. https://www.undp.org/yemen/projects/qat-coffee-climate-resilience-and-human-security https://www.undp.org/yemen/projects/qat-coffee-climate-resilience-and-human-security https://www.undp.org/yemen/projects/qat-coffee-climate-resilience-and-human-security https://www.cbd.int/doc/legal/cbd-en.pdf https://www.cbd.int/doc/legal/cbd-en.pdf https://y30.ch/trees-of-hope-y30s-reforestation-initiative-revitalizes-yemens-landscape-and-economy/ https://y30.ch/trees-of-hope-y30s-reforestation-initiative-revitalizes-yemens-landscape-and-economy/ https://y30.ch/trees-of-hope-y30s-reforestation-initiative-revitalizes-yemens-landscape-and-economy/ introduction materials and methods results and discussion most important achievements expanding characterization and evaluation activities and developing collecting of germplasm encouraging crop diversity supporting breeding and genetic improvement programmes supporting seed production and distribution changes and trends gaps and challenges conclusion and recommendations author contributions conflict of interest statement acknowledgements short communication genetic resources (2023), 4 (7), 46–55 doi: 10.46265/genresj.alfv3636 https://www.genresj.org issn: 2708-3764 characterization of microsatellite markers for the duckweed spirodela polyrhiza and lemna minor tested on samples from europe or the united states of america jae e kerstetter a,b, andrea l reid c, joshua t armstrong a,d, taylor a zallek a, trapper t hobble a and martin m turcotte *,a a department of biological sciences, university of pittsburgh, pa, 15260, pittsburgh, usa b department of entomology, rutgers university, nj, 08901, new brunswick, usa c department of geodesy and geomatics engineering, university of new brunswick, nb, fredericton, canada d center for environmental studies, virginia commonwealth university, va, 23284, richmond, usa abstract: microsatellite primers are a valuable tool to use for both observational and experimental studies in numerous taxa. here, we develop 18 and 16 microsatellite markers for the widespread duckweeds lemna minor l. and spirodela polyrhiza (l.) schleid, respectively. all 18 l. minor primers and 12 of the 16 s. polyrhiza primers amplified polymorphic loci when tested on samples from europe or western pennsylvania, usa. keywords: lemnaceae, simple sequence repeats, genotyping, genetic identification, molecular markers citation: kerstetter, j. e., reid, a. l., armstrong, j. t., zallek, t. a., hobble, t. t., turcotte, m. m. (2023). characterization of microsatellite markers for the duckweed spirodela polyrhiza and lemna minor tested on samples from europe or the united states of america. genetic resources 4 (7), 46–55. doi: 10.46265/genresj.alfv3636. © copyright 2023 the authors. this is an open access article distributed under the terms of the creative commons attribution license (cc by 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. introduction the globally distributed duckweed family (lemnaceae) or subfamily (lemnoideae) is composed of 36 species (bog et al, 2020) of very small floating or submerged aquatic plants (landolt, 1986; sree et al, 2016). duckweeds have a long history of scientific study given their highly specialized morphology, widespread distribution, high abundance and production of the world’s smallest flowers (jacobs, 1947; hillman, 1961; landolt, 1986, 1992). more recently, there has been an explosion in research interest given their potential applied uses including for agricultural feed (cheng and stomp, 2009), bioremediation (gupta and prakash, 2013; ekperusi et al, 2019) and biofuel production (cui and cheng, 2015). furthermore, their use as a model ∗corresponding author: martin m turcotte (turcotte@pitt.edu) system to experimentally study numerous topics in ecology and evolutionary biology is quickly expanding (laird and barks, 2018). this growing basic and applied interest stems from their ability to reproduce clonally very quickly with population doubling times in as little as 1.5 days (ziegler et al, 2015). in addition, they are amenable to large-scale manipulative experiments in both the lab and field mesocosms (armitage and jones, 2019; hart et al, 2019; tan et al, 2021; o’brien et al, 2022), and have growing genomic data and tools (wang et al, 2014; ho et al, 2019; xu et al, 2019; cao et al, 2020) and characterization of their microbiome and herbivore communities (acosta et al, 2020; subramanian and turcotte, 2020). finally, duckweed express variation in numerous traits across species and among genotypes (clonal lineages) within species (van steveninck et al, 1992; hart et al, 2019; chen et al, 2020; hitsman and simons, 2020; anneberg et al, 2023). therefore, being able to identify genotypes may also be beneficial in many received: 16.10.2022 accepted: 25.02.2023 published online: 05.05.2023 https://www.genresj.org https://www.dx.doi.org/10.46265/genresj.alfv3636 https://www.genresj.org https://www.dx.doi.org/10.46265/genresj.alfv3636 mailto:turcotte@pitt.edu genetic resources (2023), 4 (7), 46–55 47 ecological studies to assess differences in traits among genotypes and to determine how these genotypes may respond to different environmental conditions. genetic markers, such as microsatellite markers, are important tools to study population genetics. microsatellites, also known as simple sequence repeats (ssr), are tandem repeats two to ten base pairs in length, that are flanked by conserved sequences and occur ubiquitously throughout eukaryotic genomes (tautz and renz, 1984). they are highly informative as locus-specific genetic markers due to their high abundance, high reproducibility, co-dominance, and polymorphic nature (morgante and olivieri, 1993; powell et al, 1996). the length of the sequence repeats can be determined through pcr amplification using primers specific to their flanking regions; variation in pcr product length is a function of the number of repeated sequences. the high levels of polymorphisms observed in ssr markers (tautz, 1989; schlötterer and tautz, 1992) and the relative ease of detection of these polymorphisms by pcr amplification have led to the wide applications of microsatellites as genetic markers (vieira et al, 2016). such within-species markers have numerous applications including quantifying biogeographic distributions, population genetic structure, evolutionary history, and mating systems. moreover, a growing number of experimental evolution studies use ssr markers to track changes in genotypic composition of asexually reproducing populations over multiple generations (turcotte et al, 2011; hart et al, 2019; agrawal et al, 2013) in large replicated experiments for which genotyping-by-sequencing remains too costly. these cost savings are magnified when several loci can be multiplexed and genotyped in the same reaction (markoulatos et al, 2002). with the growing interest in duckweed, microsatellite markers have been developed for a few duckweed species. wani et al (2014) developed nine polymorphic and 24 monomorphic haplotype chloroplast dnabased microsatellite primers for l. minor. xu et al (2018) developed 60 microsatellite primers for spirodela polyrhiza, 19 of which were polymorphic within three populations of s. polyrhiza from china. feng et al (2017) developed three microsatellite primers for the identification of s. polyrhiza and landoltia punctata (g. mey.) les & d. j. crawford haplotypes. more recently, fu et al (2020) developed 70 microsatellite primers within coding regions for l. gibba l. it is important to continue developing and reporting new microsatellite markers as populations can differ in which markers function (e.g. due to null alleles) and are polymorphic chapuis and estoup (2007). here, we report on the successful development of 18 and 16 new microsatellite markers, respectively, for two commonly studied and widespread duckweed species: the common duckweed lemna minor l. and the greater duckweed spirodela polyrhiza (l.) schleid. a small subset of these microsatellite primers was used to differentiate genotypes in our experimental studies on evolutionary coexistence (hart et al, 2019). in addition, we report genotyping results using these markers on individuals sampled in europe and the united states of america (usa). we thus provide new tools and evidence that they function, which can be utilized by the growing community of duckweed researchers (laird and barks, 2018). materials and methods sample collection our objective when sampling was not to genetically characterize duckweed populations, but instead find genotypes that differ in ecologically relevant traits to use in various experiments. thus, we genotyped few individuals from numerous bodies of water in various locations. primers were developed at eth zurich (europe) and the university of pittsburgh (usa), and thus were tested on different collections of duckweeds. we collected duckweeds from numerous still bodies of water (e.g. ponds, lakes, wetlands) primarily in switzerland and western pennsylvania (usa); however, a few samples were also collected from the netherlands and germany. in addition, some european duckweed samples included in the set were obtained from the landolt duckweed collection (formerly in zurich, switzerland, see supplemental tables s1 and s2 for collection locations). given the two-part development of the primers, some duckweed samples were only tested on the primers developed in that country (as noted in supplemental tables s1 and s2). duckweeds mostly reproduce clonally via meristematic pockets from which clonal daughters emerge, creating clonal clusters of one to eight individuals that eventually split into smaller clusters (landolt, 1986). we sampled single duckweed clusters and established isofemale laboratory colonies from these clusters. we then sterilized each colony using sodium hypochlorite following a method adapted from barks et al (2018). from each colony, we put single individuals into individual sterile petri dishes (one individual per dish) containing sterile 0.5 strength schenk and hildebrandt growth medium (schenk and hildebrandt, 1972) supplemented with sucrose (6.7g/l), yeast extract (0.067g/l), and tryptone (0.34g/l) for 24 hours to encourage algal and bacterial spore germination. then each individual was exposed to one of an array of concentrations of sodium hypochlorite (0.3% or 0.5%) for varying amounts of time (3 or 6 minutes for l. minor, 4 or 7 minutes for s. polyrhiza respectively), then rinsed with autoclaved distilled water and allowed to grow (barks et al, 2018). sterile colonies were maintained in sterile 0.25 strength schenk and hildebrandt media (schenk and hildebrandt, 1972) without the additional supplements in room temperature laboratories or growth chambers under plant grow lights. these collections do not reproduce sexually under lab conditions. spirodela polyrhiza and lemna minor microsatellite markers 48 kerstetter et al g enetic resources (2023),4 (7),46–55 table 1. lemna minor microsatellite markers and motifs including the location of initial primer development (usa or europe), optimized mgcl2 concentrations, and annealing temperatures (ta). in addition, we report marker success rate, which is the number of samples successfully genotyped divided by those attempted, the number of unique alleles, and number of unique genotypes for each primer. average heterozygosity (h) is the fraction of individuals that are heterozygotic for each primer. see supplemental table s1 for specific allele values. allele lengths with an * denote that these lengths include the m13 tail sequence. primers forward primer (5’-3’) reverse primer (5’-3’) motif location of development mgcl2 (mm) ta (◦c) observed product length (bp) marker success rate unique alleles unique genotypes average h lmr.1.a f: gttcctaaggattcatcacc r: tacgaggagggacacgag aag europe 2.0 60 178–185* 75/81 2 2 0 lmr.4.a f: agtggctacgaacggaagag r: agaggaacgttgtgtctggg aag europe 0.9 63 219–234 28/28 5 5 0.036 lmr.4.b f: cttattggatcttcgcgccg r: aagatatctgacggcgttgg ag europe 1.2 63 366–392 28/28 6 6 0.071 lmr.5.c f: gatgccagtagatccggc r: acgcctgaacacgattgatg agat europe 2.0 60 320–444 104/109 25 41 0.846 lmr.8.b f: tgtactcatctgtgggcgag r: aacaatttggccaccgtcag agat usa 1.2 63 306–376 28/28 10 9 0.036 lmr.8.c f: gacaacttagggtgcacgc r: ggagtgagagctgaggactg agg usa 1.2 60 435–450 28/28 3 3 0 lmr.10.a f: tcctttctcgtgtctcccag r: atgcccgacctagtcc ag europe 2.0 60 222–254* 31/81 4 5 0.032 lmr.10.c f: ctctcctttctcctccacgg r: atcgcaaccctctagccg agat europe 2.0 60 179–254* 79/81 4 4 0.278 lmr.12.b f: tctctgctgaccgactcaag r: gccgttggatctttctcacg at usa 1.2 60 274–320 27/28 8 9 0.111 continued on next page g enetic resources (2023), 4 (7), 46–55 49 table 1 continued primers forward primer (5’-3’) reverse primer (5’-3’) motif location of development mgcl2 (mm) ta (◦c) observed product length (bp) marker success rate unique alleles unique genotypes average h lmr.14.a f: tcgcactagagagatgggtg r: tcccattaccaggatgcgag aat usa 1.2 60 261–270 24/28 3 3 0.042 lmr.14.b f: catgccaggtaaatgccctc r: tcgagctccttctccaaacc atc usa 0.9 63 430–440 28/28 3 3 0 lmr.14.c f: ttcgtcgagggtatgagctg r: tctcttatttgacacgcgcg ag usa 0.9 63 162–178 28/28 7 7 0.036 lmr.15.a f: gtgacagcgtatccttgtgc r: cagcggcaagatcatcaag atc europe 1.2 60 222–285 109/109 13 15 0.578 lmr.15.b f: tcgagctaatcagtggagcc r: gagtgctcggcttgactttc ag europe 1.2 60 170–210 104/109 13 25 0.692 lmr.15.c f: catgttcccacccacttgac r: aaggaagagggagcaaggg at europe 1.2 60 368–400 109/109 14 26 0.743 lmr.26.b f: gtgtctccgagagcctacag r: tttaaagctcggtgggtccc ag usa 1.2 63 283–329 28/28 10 7 0.964 lmr.31.a f: ggtgatctcaggtagccgag r: tgagatcaccactgtctgcc aag usa 0.9 63 402–432 26/28 5 6 0.077 lmr.31.b f: agtcggcatagtacttcccg r: cttcttcaagaccgttccgc aag usa 1.2 63 155–239 28/28 7 9 0.071 spirodela polyrhiza and lem na m inor m icrosatellite m arkers 50 kerstetter et al g enetic resources (2023),4 (7),46–55 table 2. spirodela polyrhiza microsatellite markers and sampling results as described in table 1 with allele calls in supplemental table s2. primers forward primer (5’-3’) reverse primer (5’-3’) repeat motif location of development mgcl2 (mm) ta (◦c) observed product length (bp) marker success rate unique alleles unique genotypes average h sp.1035 f: tgcttggtcactcttgtctg r: cgattcctagctcctctgc at europe 1.2 60 361–369 42/42 4 5 0.381 sp.1467 f: agttgaggaagcttcatgg r: attacctccagcacctctcc ag europe 2.0 58 386–411* 9/20 5 4 0.444 sp.2597 f: tcccattcaccacagtctcc r: tcattccaccacgtcccac at europe 2.0 58 397–399* 14/20 2 2 0.071 sp.5050 f: attaaccttgggcgcagag r: tagcagcagagtgtgaggg aat europe 2.0 58 287* 14/20 1 1 0 sp.5250 f: aaacgagacctcctacgcc r: gcctgcgagtaatatgtgc atgccc europe 2.0 58 385* 19/20 1 1 0 sp.7286 f: cgaatatgccgaggaatgc r: tcctcgatctgccgctttag cg europe 1.2 60 386–394 42/42 5 7 0.310 sp.7688 f: aatggttgactcgacgctg r: tcacaccgccataatttcgc agc europe 2.0 58 199–211* 19/20 2 2 0.158 sp.7814 f: agtgtagggtgcagctgtg r: ttcgtgaaaggcctagcac ag europe 1.2 60 220–228 42/42 5 6 0.095 sp.7908 f: gagacacatcattgccagc r: taatgcaggccacacaacc ag europe 2.0 58 234–236 20/20 2 2 0.850 sp.8563 f: gtattgggtgggcaaatcg r: aagggatagggtcgtgtcc ag europe 2.0 58 350–354* 14/20 3 4 0.071 continued on next page g enetic resources (2023), 4 (7), 46–55 51 table 2 continued primers forward primer (5’-3’) reverse primer (5’-3’) repeat motif location of development mgcl2 (mm) ta (◦c) observed product length (bp) marker success rate unique alleles unique genotypes average h sp.8910 f: ccttccctacgttgactccc r: gcgtttctctgatcagcacc acg –> cgt europe 2.0 58 358 20/20 1 1 0 sp.9307 f: gggagcgagctgtatgaag r: tttcaacaccctcaccatgc ag europe 2.0 58 450–452* 9/20 2 3 0.444 sp.9311 f: gtgagaaaggaaaggtggc r: tgctcaggattctatgggcc ag europe 2.0 58 253–255* 10/20 2 3 0.400 sp.pso27 f: aagggtttcagtgcggacg r: ctcgccttctcgtacatcatc aag europe 2.0 58 133* 9/20 1 1 0 sp.pso31 f: tccaccgtctccctgtaatg r: ccactccctcgtcgtgaag aag europe 1.2 60 240–270 32/42 7 7 0.406 sp.pso32 f: tgctggcgatgtcaatgttg r: cttcagcaccaagagagctc atc europe 2.0 58 377–380* 19/20 2 3 0.895 spirodela polyrhiza and lem na m inor m icrosatellite m arkers 52 kerstetter et al genetic resources (2023), 4 (7), 46–55 microsatellite marker development a total of 18 l. minor and 16 s. polyrhiza microsatellite markers were developed across europe or the usa. we downloaded the whole genome shotgun sequence data for s. polyrhiza strain 7498 from the national center for biotechnology information’s genbank database (accession atdw01000001.1) deposited by wang et al (2014). for l. minor, a draft genome (strain 8627) was downloaded from www.lemna.org on 16 october 2015 (genome draft lm8627.asmv0.1). a recent study using tubulin-based polymorphism suggests that this lineage is in fact an interspecific hybrid of l. japonica landolt and l. turionifera landolt both closely related to l. minor (braglia et al, 2021). the species identity for most samples on which we report below has been confirmed using morphology and/or barcoding (fazekas et al, 2012; barks et al, 2018). while some microsatellite markers are known to amplify across more than one duckweed species (xu et al, 2018), we have not yet explicitly tested these markers against other species. using msatcommander (version 1.0.8, faircloth 2008), we identified microsatellite loci using the default settings, avoiding mononucleotide repeat motifs. we then selected loci that would produce products of different lengths, had different motif lengths and were found on different contigs. the 5’ end of forward primers were labelled with one of several fluorescent dyes from various suppliers. primers developed at eth zurich were m13-tailed to reduce cost during development (boutin-ganache et al, 2001). this entailed adding the full or a partial m13 sequence of tgtaaaacgacggccagt for the s. polyrhiza primers and ggaaacagctatgaccat for l. minor primers to the 5’ end of the forward primer. the m13-labelled forward primers were used in combination with an m13 primer that had the same sequence but was fluorescently dye-labelled at its 5’ end. some primers amplified loci that were fully or mostly monomorphic or did not amplify as consistently as others. for these primers, we only have fragment lengths that include the m13 tail (see table 1 and table 2), and we estimate that this lengthens the pcr product by 12–19 base pairs. for most primers, however, following initial testing with m13, we ordered new labelled primers that did not include the m13 tail. at least 20 duckweed samples were tested using each primer. european duckweed samples were tested across 7 l. minor and 16 s. polyrhiza primers, and usa duckweed samples were tested across 15 l. minor and 4 s. polyrhiza primers (see supplemental tables s1 and s2 for details). each duckweed sample was tested with each primer using at least two independently extracted dna samples. we only report allele lengths that were consistent in both samples. microsatellite amplification and optimization all duckweed collections were extracted and genotyped at least twice by first sampling four to ten individuals from each monoclonal collection and lyophilizing them for 24 hours. we then extracted dna using a modified ctab-based method by healey et al (2014). for primers developed in europe, the conditions were the following: pcr amplification was conducted in 15µl volume reactions containing 3µl of template dna, 3µl of 5x colorless gotaq flexi buffer (promega, usa), 2.0mm mgcl2, 0.2mm dntp mix, 0.05µm of forward primer, 0.2µm of reverse primer, 0.2µm of m13 primer tagged with a fluorescent probe (e.g. 5’ 6-fam or 5’ hex), and 1 unit of gotaq g2 flexi dna polymerase (promega, usa). dna concentrations were rarely quantified as amplification was successful across a range of values (e.g. 2–40ng/µl). thermocycling conditions for both s. polyrhiza and l. minor from europe that were m13 tagged were: initial denaturing at 94◦c for 5 min, followed by 30 cycles of 1 min at 94◦c, 1 min at 60◦c, 1 min at 72◦c, followed by eight m13 cycles consisting of 1 min at 94◦c, 1 min at 53◦c, 1 min at 72◦c, followed by a final extension at 72◦c for 10 min. for all primers developed in the usa, the conditions were the following: pcr amplification was conducted in 15µl volume reactions containing 3µl of template dna, 3µl of 5x colorless gotaq flexi buffer (promega, usa), 1.2mm mgcl2, 0.2mm dntp mix, 0.08µg/µl of bovine serum albumin (bsa), 0.2µm of each forward and reverse primer, and 1 unit of gotaq g2 flexi dna polymerase (promega, usa). thermocycling conditions for s. polyrhiza were: initial denaturing at 94◦c for 5 min, followed by 34 cycles of: 1 min of denaturing at 94◦c, 1 min of annealing at 60◦c, and 1 min of extension at 72◦c, followed by a final extension at 72◦c for 10 min. for l. minor, touchdown pcr was employed with an initial denaturation of 94◦c for 5 min, followed by five cycles of denaturation (94◦c, 1 min), annealing (67◦c, 1 min; decreasing by 1◦c per cycle), and extension (72◦c, 1 min). then 25 cycles of 1 min at 94◦c, 1 min at 63◦c, and 2 min at 72◦c, followed by a final extension at 72◦c for 15 minutes. primers were then optimized for annealing temperatures and mgcl2 concentration (table 1 and table 2 ). fragment length analyses for all primers were conducted on abi 3730 genetic analyzers (applied biosystems) at either the eth zurich genetic diversity center (switzerland), keck dna sequencing lab at yale university (usa), or the university of pittsburgh genomics research core (usa), using either genescantm 500 or 600 liztm dye size standards (applied biosystems). allele calls were made using either geneious (version 9.1.6, kearse et al (2012)) or genemarker software (version 3.0.0, softgenetics, state college, pennsylvania). results and discussion we successfully developed 18 l. minor and 16 s. polyrhiza microsatellite primers (table 1 and table 2) which were tested on samples of duckweeds from europe or western pennsylvania (usa). some markers were more successful than others (table 1 and table 2). all markers amplified in some samples; of these, all genetic resources (2023), 4 (7), 46–55 53 18 l. minor primers and 12 of the 16 s. polyrhiza primers amplified polymorphic loci, having more than one allele. moreover, these polymorphic loci differed in product length and can be used in multiplex reactions to increase efficiency and lower genotyping costs. we also found that some loci were much more polymorphic than others. for l. minor, these included loci amplified by primers lmr.5.c, lmr.8.b, lmr.15.a, lmr.15.b, lmr.15.c and lmr.26.b, some of which showed high allele richness even when tested on only 28 samples (table 1). for s. polyrhiza these included loci amplified by primers sp.1467, sp.7286, sp.7814, and sp.pso31 (table 2). monomorphic loci may still be useful in different duckweed populations (chapuis and estoup, 2007). many microsatellite loci also showed heterogeneity (supplemental tables s1 and s2), which helps make the primers more informative to distinguish genotypes. we note that some primers developed in one continent were not tested on samples from the other continent (see caption in supplemental tables s1 and s2); we suspect these primers will work across continents given patterns observed in the others, but this remains to be tested. comparing between species, we saw that s. polyrhiza has lower allelic and genotypic richness across most primers, although we also tested fewer samples of this species. this is consistent with our own recent largescale sampling (hobble et al. in preparation) as well as other studies using different genotyping methods, that similarly found low genetic diversity in s. polyrhiza (bog et al, 2015; xu et al, 2015; feng et al, 2017). it has been hypothesized that this low genetic variation in s. polyrhiza is due to its low mutation rate (xu et al, 2019). in addition, primers differed greatly in average observed heterozygosity, but species had similar mean heterozygosities (0.256 for l. minor and 0.283 for s. polyrhiza). given that our sampling was designed to find unique genotypes (shallow and widespread) and not characterize populations, we limit our discussion of population genetic indices. the primers we developed can help researchers address various ecological and evolutionary questions as well as better identify and catalogue genotypes for the expanding applied uses of duckweed in bioremediation, biofuel production and as a forage crop. supplemental data supplemental table s1: lemna minor sample collection sites and allele lengths. supplemental table s2: spirodela polyrhiza sample collection sites and allele lengths. acknowledgements we thank walter lämmler for sharing duckweed lineages from the landolt duckweed collection. we are grateful to the former levine plant ecology group at eth zurich and the turcotte lab for their assistance in maintaining collections. we thank eth zurich genetic diversity center and mary janecka for help troubleshooting primer development. m.m.t. was supported by the eth zurich center for adaptation to changing environments and now by an nsf grant deb1935410. author contributions all authors contributed to testing, optimizing, and evaluating marker data, and contributed to reviewing the manuscript. jek and mmt wrote the initial 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(2015). relative in vitro growth rates of duckweeds (lemnaceae) the most rapidly growing higher plants. plant biol 17, 33–41. doi: https://doi.org/10.1111/plb.12184 spirodela polyrhiza and lemna minor microsatellite markers https://doi.org/10.1139/b72-026 https://doi.org/10.1139/b72-026 https://doi.org/10.9755/ejfa.2016-01-038 https://doi.org/10.9755/ejfa.2016-01-038 https://doi.org/10.1111/een.12932 https://doi.org/10.1111/een.12932 https://doi.org/10.1038/s41559-021-01406-2 https://doi.org/10.1038/s41559-021-01406-2 https://doi.org/10.1111/j.1461-0248.2011.01676.x https://doi.org/10.1111/j.1461-0248.2011.01676.x https://doi.org/10.1590/1678-4685-gmb-2016-0027 https://doi.org/10.1590/1678-4685-gmb-2016-0027 https://doi.org/10.1038/ncomms4311 https://doi.org/10.1038/ncomms4311 https://doi.org/10.3732/apps.1300099 https://doi.org/10.3732/apps.1300099 https://doi.org/http://dx.doi.org/10.1002/aps3.1153 https://doi.org/http://dx.doi.org/10.1002/aps3.1153 https://doi.org/10.1038/s41467-019-09235-5 https://doi.org/10.1038/s41467-019-09235-5 https://doi.org/10.1007/s10750-014-2163-3 https://doi.org/10.1007/s10750-014-2163-3 https://doi.org/10.1111/plb.12184 introduction materials and methods sample collection microsatellite marker development microsatellite amplification and optimization results and discussion supplemental data acknowledgements author contributions conflict of interest statement original article genetic resources (2024), 5 (10), 25–38 doi: 10.46265/genresj.hynz9140 https://www.genresj.org issn: 2708-3764 genetic diversity and intraspecific mitochondrial dna variations in the georgian mountain breed of bos taurus reveal admixture, introgression and potential parallel vs. convergent evolution patterns givi basiladze a,b, leila tabatadze a, ekaterine gabashvili c, mariam osepashvilid, marine murskhvaladzed and mamuka kotetishvili *,e,f a scientific research center of agriculture, 36b marshal gelovani ave, 0159, tbilisi, georgia b agricultural university of georgia, 240 david aghmashenebeli alley, 0159, tbilisi, georgia c institute of biodiversity, friedrich schiller university jena, 159 dornburger strasse, d07743, jena, germany d school of natural sciences and medicine, ilia state university, 1 giorgi tsereteli exit, 0162, tbilisi, georgia e school of science and technology, one health institute, university of georgia, 77a m. kostava st. tbilisi, 0171, georgia f g. natadze scientific research institute of sanitary, hygiene and medical ecology, 78 d. uznadze st. 0102, tbilisi, georgia abstract: this study elucidates the haplotype diversity and mechanisms of evolutionary divergence for a broad population of the georgian mountain breed (gmb) of bos taurus, using the sequencing and analysis of its mitochondrial dna (mtdna). in the evolutionary analyses, sequences of the targeted mtdna region, involving the d-loop, cytb, trna-thr, and trna-pro encoding genetic loci were analyzed using mega11, dnasp, and splitstree software packages. a total of 25 haplotypes were determined among 82 individuals of gmb, belonging predominantly to the haplogroups t (t3, t1, t2, t4) or q (q1). ten singleton haplotypes could also be determined in the gmb population. in the maximum likelihood evolutionary analysis, the singleton haplotype sngt-9 appeared to be most closely related to the bos indicus sub-haplogroup i1a. the haplotype diversity (0.997), nucleotide diversity (0.00636) and the overall mean distance within a population (0.01) calculated for gmb were greater as compared to the respective estimates (0.930, 0.00482 and 0.00) determined for its closest cattle relatives globally, suggesting stronger selection. it is suggested that the gmb diversity has been shaped by both parallel and convergent evolution, as well as by possible introgression, while pinpointing this breed’s ancient origin collectively. keywords: georgian mountain breed, cattle, haplotype, haplogroup, genetic diversity, population structure, mtdna citation: basiladze, g., tabatadze, l., gabashvili, e., osepashvili, m., murskhvaladze, m., kotetishvili, m. (2024). genetic diversity and intraspecific mitochondrial dna variations in the georgian mountain breed of bos taurus reveal admixture, introgression and potential parallel vs. convergent evolution patterns. genetic resources 5 (10), 25–38. doi: 10.46265/genresj.hynz9140. © copyright 2024 the authors. this is an open access article distributed under the terms of the creative commons attribution license (cc by 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. introduction climate change, driven by global warming, threatens agricultural animals’ survival in the anthropocene (elayadeth-meethal et al, 2018). smaller body ∗corresponding author: mamuka kotetishvili (m.kotetishvili@ug.edu.ge) size in mammals is thought to aid adaptation to warmer climates (pacifici et al, 2017), as there appears to be an interplay between body size reduction and increased livestock tolerance to warming (elayadeth-meethal et al, 2018). in this light, preserving biodiversity, conserving endemic cattle breeds of small body size, and deciphering their population structures and evolutionary mechreceived: 17.05.2024 accepted: 08.08.2024 published online: 10.09.2024 https://www.genresj.org https://www.doi.org/10.46265/genresj.hynz9140 https://www.genresj.org https://www.doi.org/10.46265/genresj.hynz9140 mailto:m.kotetishvili@ug.edu.ge 26 basiladze et al genetic resources (2024), 5 (10), 25–38 anisms are important, given global food security pressures and climate change (mitchell et al, 2018). according to the archeozoological and genetic evidence, mainly relying on the results of mitochondrial dna (mtdna) analyses, modern cattle are thought to have emerged via just two independent and geographically distinct domestication events of aurochs (bos primigenius), both of which occurred in southwest asia: one event is thought to have occurred in the fertile crescent, resulting further into modern taurine breeds of b. taurus, while the other event took place in the indus valley, leading to the emergence of modern zebuine breeds of its subspecies b. indicus (achilli et al, 2008). the diversity of modern cattle has been structured largely into three major groups represented by eurasian taurine, african taurine and asian indicine cattle; these include different types of crosses, all combinations and intricacies (kim et al., 2020), with various breeds exhibiting predominantly the macro-haplogroup t of b. taurus (achilli et al, 2008). while the genetic diversity of many cattle breeds from the above groups has been well characterized, there remains a significant lack of information specifically concerning the georgian mountain breed (gmb) of b. taurus endemic to the caucasus region. gmb individuals are very small in body size, with the live weight varying from 220 to 280kg for mature cows, and from 270 to 370kg for bulls (kunelauri et al, 2019). the coat colours of gmb individuals are black, blackand-white or red-and-white. compared with other breeds from this region, gmb not only demonstrates stronger endurance and enhanced sustainability towards the above conditions but is also less susceptible to impoverished food (kunelauri et al, 2019). this breed has been well adapted to the harsh climate and other conditions of the caucasus mountains, and to its grazing lands with slopes that sometimes reach an angle of 45 degrees (kunelauri et al, 2019). two initial studies by kunelauri et al (2019, 2022) analyzing the sequences of mtdna loci versus a mitogenome in a very limited number of cattle individuals, revealed b. taurus haplogroups t and q (branch q1) and suggested the presence of some unknown haplotypes as well in this breed. our study aimed to characterize the haplotypes across broad gmb populations from the khevsureti and adjara regions of georgia, and to gain initial insights into their evolutionary mechanisms by analyzing mtdna genetic loci. materials and methods animal sampling a total of 82 gmb individuals were sampled across the khevsureti and adjara regions of georgia, as these regions have a high concentration of this breed, in 2019–2022, selecting unrelated animals based on pedigree information to minimize kinship. only one individual was selected and sampled per village across the above regions. specifically, hair follicle samples from the selected khevsurian (n = 36) and adjarian (n = 46) cattle were obtained for mtdna extraction, pcr amplification, and sequencing. primer design, pcr amplification and sequencing of targeted mtdna the tissue and hair extraction kit, coupled with the dna iqtm kit (promega, inc., madison, wi, usa), and the quick-dna microprep plus kit (zymo research, inc., irvine, ca, usa), were used for the extraction of mtdnas from the hair follicle samples. the primerquesttm tool (integrated dna technologies, usa) was used to design primers for pcr amplification and sequencing of the targeted mitochondrial genome region. the designed forward and reverse primers (5’-ccaacaaactaggaggagta3’ and 5’-cgcggcatggtaattaag-3’) allowed us to amplify the 810-bp mtdna region encompassing the genes encoding for trna-thr, trna-pro, as well as the cytb gene and the d-loop loci across the above mitochondrial region from the selected gmb individuals. pcr conditions were 94◦c for 5min, followed by 35 amplification cycles, each consisting of sequential incubation at 94◦c (30s), 51◦c (30s), and 72◦c (1min and 20s), with the final 72◦c (5min) extension. the dna sequencing of the pcr-amplified products was performed in both directions, using the bigdye terminator v3.1. cycle sequencing kit (applied biosystems, inc., foster city, ca or thermo fisher scientific) or the brilliantdyetm terminator (v3.1) cycle sequencing kit (nimagen, nijmegen, the netherlands). the postcycle sequencing reaction contaminants were removed by applying the zr dna sequencing clean-up kit (zymo research, inc., irvine, ca, usa). the 3100xl genetic analyzer (applied biosystems, inc., foster city, ca, usa) was used to separate the labelled dna fragments by size; the geneious prime v. 7.0.9 (biomatters, inc., boston, ma, usa) and sequencher v. 5.4.6 (gene codes, corp. ann arbor, mi, usa) were utilized to edit and assemble the consensus sequences. all low-quality sequences were trimmed from both forward and reverse dna sequence reads. the representative mtdna sequences, obtained from the above dna sequencing experiments, were submitted to, and are available under the accession numbers or412787-or412811, the genbank database of the national center for biotechnology information (ncbi, https://www.ncbi.nlm.nih.gov/). evolutionary analyses the mega11 (v. 11.0.13) and dnasp (v. 6.12.03) software packages were used to determine the population structures and evolutionary features of gmb individuals and their genetically closest cattle from different breeds of b. taurus and b. indicus. for these analyses, the respective mtdna regions of 77 genetically closest cattle (supplemental table 1) were selected in the ncbi nucleotide database, using megablast with the default parameters (expected threshold 10, word size 28, genetic resources (2024), 5 (10), 25–38 genetic diversity of georgian mountain cattle 27 match/mismatch scores 1,-2, gap costs linear, extension 2). the evolutionary analysis included representatives of each haplotype from gmb and its genetically closest cattle individuals preselected based on dna identity, query coverage and e values from the blast analysis. specifically, for evolutionary analyses, we selected the 491-bp mtdna region, encompassing the cytb and d-loop loci, as well as the genes encoding for trnathr and trna-pro, which were shared exclusively by all these organisms. in this subset, we performed a random selection of the breed representatives from their country of origin, when they exhibited the blast-generated multiple identical hits to the query dna sequences in the ncbi genbank database. mega11 was used to determine specifically the shape parameter for the discrete gamma distribution to model evolutionary rates, as well as nucleotide substitution patterns and rates across the targeted mtdna genetic loci as recommended previously (tamura and nei, 1993). in the mega analyses, maximum likelihood (ml) estimates of the transition/transversion bias were also determined, by applying the kimura 2-parameter model (kimura, 1980). maximum composite likelihood (mcl) estimates of the pattern of nucleotide substitution were also determined as described by tamura et al (2004). the ml method and the tamura-nei model (tamura and nei, 1993) were used to infer the evolutionary relationships between the gmb individuals and their genetically closest cattle individuals. the haplotypes and their diversity, as well as polymorphic (segregating) sites, nucleotide diversity (pi), invariable (monomorphic) versus variable (polymorphic) sites, and singleton variable versus parsimony informative sites across the 491-bp mtdna region were determined using dnasp. the haplogroup was determined for each gmb individual according to the haplogroup classification described previously (xia et al., 2021). the degree of linkage disequilibrium (ld) (including the zz-value) was assessed as recommended by rozas et al (2001). as the standardized measure of ld, the zz-value was derived from the squared correlation coefficient (r2) between pairs of polymorphic sites, and the variance of the r2 values across multiple loci. the two-tailed fisher’s (f) exact test and the chi-square (chi-sq) test were applied to determine whether the associations between polymorphic sites were significant, with bonferroni correction additionally employed for these tests as implemented in dnasp. in the genetic recombination analysis, a minimum number of putative recombination events (rm), and the coordinates of recombination hotspots were determined when detected by dnasp across the targeted mtdna regions of the cattle individuals. for re-examining recombination inferences, we also applied the splitstree (version 4.14.4) and rdp4 (beta 4.96) software packages. particularly, we used the method of split decomposition method, implemented in splitstree, to detect parallel nucleotide substitutions with conflicting evolutionary signals in the targeted mtdna region in the targeted cattle populations. every subset of the mtdna sequences that encompassed such parallel changes conjointly displayed across a splitstree-generated parallelogram(s) was subjected to the phi (pairwise homoplasy index) test (bruen et al, 2006) for measuring homoplasy. in the rdp4 analyses, searching for genetic recombination events, the rdp, geneconv, bootscan, maxchi, chimaera, siscan, and 3seq recombination detection algorithms were employed. we used the stringent approach: the predetermined bonferroni-corrected p-values only in a range of ≤ 0.05 were considered statistically significant for the significant breakpoint clusters (99%) if detected in these analyses. results dna sequence polymorphism and phylogenetic analyses based on sequencing of a 810bp sequence of mtdna a total of 25 haplotypes were determined among 82 gmb individuals, using dnasp. the haplotypes, haplogroups, genetic sub-lineages/clusters, and geographic distribution of these cattle individuals are presented in table 1. as shown, a great majority of the gmb individuals belonged to the haplogroup t3 (42.6%) followed by q1 (12.1%), t1 (3.6%), t2 (15.8%) and t4 (1.2%), as well as ten unique sngts not associated with any previously described haplotype of b. taurus. in figure 1, we display the ml tree constructed using the nucleotide analysis of the 491-bp mtdna region, elucidating the population structures of the gmb haplotypes, their genetically closest cattle individuals of b. taurus worldwide, and their genetic relationships. the ml analysis resulted primarily in the distribution of the gmb haplotypes, including several sngts, and their genetically closest cattle individuals across 13 major haplogroup-specific genetic clusters i-xiii (figure 1 and table 1). as shown, the mtdna sequence profiles of specific gmb haplotypes represented by individuals 6k, 7k, 15k and 31a were identical to those of various breeds from the european, african and southeast asian regions, belonging to t3 haplogroup with t3119 included. the phylogenetic inferences, for the gmb singletons, exhibited their broad scattering patterns across the ml tree, demonstrating their close genetic relationships with different breeds from different cattle groups globally. these groups included collectively the turano-mongolian, central european, podolic, european dairy, balkan, british mountain, south asian and shorthorned zebu (bos indicus) cattle respectively from haplogroups t1-t4, q, and the specific sub-lineage i1a of the lineage i1. it is important to indicate that while many gmb haplotypes could not be distinguished globally from various b. taurus breeds within the t and q sub-lineages, certain sngts of the above georgian breed (e.g. sngt-1, sngt-4, sngt-7, sngt-9, sngt-10) either clustered separately within or fell outside these sub-lineages (figure 1). 28 basiladze et al genetic resources (2024), 5 (10), 25–38 table 1. sample designations and geographic regions for the georgian mountain breed (gmb) individuals, genbank accession numbers for the sequenced mtdna region of their representative haplotypes, as well as haplogroups and major genetic clusters. sngt, singleton; n/d, not determined; genbank accession # for a representative from each haplotype; qnsg, q new subhaplogroup. no. gmb id region genbank id haplotype haplogroup major genetic cluster 1 33ksh khevsureti or412798 t1-1 t1 vi 2 18k khevsureti or412803 t1-2 t1 ix 3 36ksh khevsureti t1-2 t1 ix 4 13a adjara or412802 t2-1 t2 ix 5 12a adjara t2-1 t2 ix 6 37a adjara or412800 t2-2 t2 viii 7 23a adjara or412811 t2-3 t2 xiii 8 35ksh khevsureti t2-3 t2 xiii 9 29ksh khevsureti t2-3 t2 xiii 10 28ksh khevsureti t2-3 t2 xiii 11 26ksh khevsureti t2-3 t2 xiii 12 36a adjara t2-3 t2 xiii 13 32a adjara t2-3 t2 xiii 14 14a adjara t2-3 t2 xiii 15 22k khevsureti t2-3 t2 xiii 16 11k khevsureti t2-3 t2 xiii 17 15k khevsureti or412808 t3-1 t3 xii 18 13k khevsureti t3-1 t3 xii 19 39ab adjara t3-1 t3 xii 20 1a adjara t3-1 t3 xii 21 7k khevsureti or412806 t3-2 t3 xi 22 31ksh khevsureti t3-2 t3 xi 23 6k khevsureti or412794 t3119-1 t3119 iii 24 31a adjara or412795 t3119-2 t3119 iv 25 20a adjara t3119-2 t3119 iv 26 10a adjara t3119-2 t3119 iv 27 5a adjara t3119-2 t3119 iv 28 14k khevsureti t3119-2 t3119 iv 29 48ab adjara t3119-2 t3119 iv 30 44ab adjara t3119-2 t3119 iv 31 43ab adjara t3119-2 t3119 iv 32 47ab adjara t3119-2 t3119 iv 33 12k khevsureti t3119-2 t3119 iv 34 2a adjara t3119-2 t3119 iv 35 9a adjara t3119-2 t3119 iv 36 18a adjara t3119-2 t3119 iv 37 28a adjara t3119-2 t3119 iv 38 40ab adjara t3119-2 t3119 iv 39 46ab adjara t3119-2 t3119 iv 40 10k khevsureti t3119-2 t3119 iv 41 21k khevsureti t3119-2 t3119 iv continued on next page genetic resources (2024), 5 (10), 25–38 genetic diversity of georgian mountain cattle 29 table 1 continued no. gmb id region genbank id haplotype haplogroup major genetic cluster 42 8a adjara t3119-2 t3119 iv 43 17a adjara t3119-2 t3119 iv 44 27a adjara t3119-2 t3119 iv 45 45ab adjara t3119-2 t3119 iv 46 1k khevsureti t3119-2 t3119 iv 47 17k khevsureti t3119-2 t3119 iv 48 6a adjara t3119-2 t3119 iv 49 16a adjara t3119-2 t3119 iv 50 26a adjara t3119-2 t3119 iv 51 34ksh khevsureti t3119-2 t3119 iv 52 24a adjara or412796 t4-1 t4 v 53 2k khevsureti or412788 q1-1 q1 i 54 8k khevsureti q1-1 q1 i 55 4k khevsureti q1-1 q1 i 56 19k khevsureti q1-1 q1 i 57 20k khevsureti q1-1 q1 i 58 15a adjara q1-1 q1 i 59 22a adjara q1-1 q1 i 60 29a adjara q1-1 q1 i 61 24ksh khevsureti q1-1 q1 i 62 27ksh khevsureti q1-1 q1 i 63 3a adjara or412789 qi-3 qnsg* i 64 41ab adjara qi-3 qnsg* i 65 23ksh khevsureti qi-3 qnsg* i 66 21a adjara or412791 ii-1 n/d ii 67 19a adjara ii-1 n/d ii 68 38a adjara ii-2 n/d ii 69 30a adjara or412792 ii-2 n/d ii 70 42ab adjara or412805 t3-3 t3 xi 71 4a adjara t3-3 t3 xi 72 34a adjara t3-3 t3 xi 73 3k khevsureti or412787 sngt-1 q i 74 25a adjara or412801 sngt-2 t viii 75 30ksh khevsureti or412810 sngt-3 t2 xiii 76 35a adjara or412790 sngt-4 n/d n/d 77 33a adjara or412793 sngt-5 n/d n/d 78 25ksh khevsureti or412797 sngt-6 t1 vi 79 9k khevsureti or412799 sngt-7 t3 vii 80 5k khevsureti or412807 sngt-8 t3 xii 81 16k khevsureti or412804 sngt-9 n/d x 82 32ksh khevsureti or412809 sngt-10 t2 xiii 30 basiladze et al genetic resources (2024), 5 (10), 25–38 moreover, according to the ml analysis, certain genetic groups from the same sub-haplogroups of t (except t4), determined previously (xia et al., 2021), fell into two or more distantly related phylogenetic clades. in table 2, we describe polymorphisms of the targeted mtdna region of the gmb haplotypes exhibiting previously unknown mutations within the b. taurus global populations. specifically, a total of 11 unique dna polymorphisms could be identified in this subset, being mainly g↔a and t↔c nucleotide substitutions. in addition, table 3 displays the ml estimates of the substitution matrix, as well as the mcl estimates of the pattern of nucleotide substitution. in table 4, we provided the ml estimates of transition/transversion bias (r) and respective evolutionary distance values for the gmb haplotypes, their genetically closest cattle individuals, and these two cattle groups combined. as shown, we only found some differences in the transition/transversion bias between the gmb haplotypes and their genetically closest cattle individuals worldwide: for gmb, the ml estimates (for a↔g and t [u] ↔ c) were collectively slightly higher in contrast to the mcl estimates (for t [u] ↔ c) being slightly lower as compared to these estimates determined for the group of its genetically closest cattle individuals worldwide. similarly, some slight differences could be also found between the above cattle groups in the mega-generated values (table 3) determined for id, the overall average composition and pairwise distances, the discrete gamma distribution, and the other parameters including average nucleotide composition, as well as nucleotide frequencies. however, as demonstrated, these values were still almost always greater for the gmb haplotypes as compared to their genetically closest cattle individuals. furthermore, the average genetic distances within the population of the gmb and its genetically related cattle group were 0.01 and < 0.00, respectively. the calculated average distance between these two groups was 0.00557. in the analysis of the targeted mtdna region, using dnasp, we could identify a 160-bp conserved region (the coordinates according to the b. taurus reference genome [nc 006853.1]: 15629-15788) exhibiting the genetic loci involved in coding for cytochrome b and trna-thr. the other dnasp-generated statistics are presented in table 5. as shown, in the dnasp analysis, we could detect 12 polymorphic mutations across this mtdna region collectively in the mgb haplotypes, which, in contrast, appeared to be monomorphic in the population of their genetically closest cattle individuals; and vice versa, the dnasp identified four mutations that were polymorphic in the latter, while exhibiting the monomorphic patterns in the group of gmb haplotypes; a total of 12 mutations were shared by the above two groups; the average number of nucleotide differences (k) versus that of the figure 1. maximum likelihood (ml) tree showing the genetic relationships between randomly selected representatives of each haplotype of the georgian mountain breed and their genetically closest cattle individuals from the bos taurus global populations identified by their corresponding genbank accession ids. i1a*, the sub-lineage of the i1 lineage of bos taurus indicus. t3*, haplogroup t3 exhibiting holstein dairy cow described previously (kinoshita et al, 2018). t1†, t3, t3119‡, t2, cow individuals sharing the same sub-haplogroups (t1,/t3/t3119/t2) according to a previous study (xia et al., 2021), although being clustered indifferent genetic groups across the ml tree. genetic resources (2024), 5 (10), 25–38 genetic diversity of georgian mountain cattle 31 table 2. previously unknown polymorphisms identified across the targeted mtdna region of the georgian mountain breed haplotypes. the coordinates were determined according to the b. taurus reference mt genome (nc 006853.1) available in the ncbi eukaryotic genome database. haplotype allele frequency polymorphisms and their coordinates across the targeted mtdna region 15789 15813 15846 15878 15917 15919 15954 15961 15966 16055 16057 t3-3 3 c t t c a g g g g t g ii-1 2 c c t c g a g g g t g ii-2 2 c t t c g a g g g t g qi-1 13 c t t c a a g a g t g sngt-1 1 c t t c a a g g g c g sngt-2 1 t t t c a a g g g t g sngt-3 1 c t t c a a g g a t c sngt-4 2 c t c c a a g g g t g sngt-5 1 c t t c a a a g g t g sngt-8 1 c t t t a a g g g t a sngt-10 1 c t t c a a g g g t t table 3. maximum likelihood (ml) and maximum composite likelihood (mcl) estimates calculated for the nucleotide substitutions of the targeted mtdna region across the georgian mountain breed (gmb) haplotypes, their genetically closest cattle individuals globally, and both cattle groups combined. rates of different transitional substitutions are shown in bold and those of transversal substitutions are shown in italics. targeted population ml estimate of substitution matrix mcl estimate of the pattern of nucleotide substitution a t/u c g a t/u c g gmb a 1.13 0.98 10.5 2.07 1.79 10.14 t/u 1.83 22.25 0.56 3.36 18.6 1.02 c 1.83 25.66 0.56 3.36 21.45 1.02 g 33.05 1.13 0.98 33.34 2.07 1.79 gmb closest cattle relatives globally a 2.20 1.90 9.37 1.97 1.71 9.57 t/u 3.57 19.59 1.08 3.21 20.03 0.97 c 3.57 22.62 1.08 3.21 23.13 0.97 g 30.90 2.20 1.90 31.56 1.97 1.71 both cattle groups combined a 1.25 1.08 10.54 2 1.73 9.74 t/u 2.03 20.80 0.61 3.25 19.61 0.99 c 2.03 24.01 0.61 3.25 22.64 0.99 g 34.72 1.25 1.08 32.09 2 1.73 nucleotide substitutions per site (dxy) between gmb and the group of its genetically closest cattle individuals were 2.717 and 0.00553 respectively, while the net nucleotide substitutions (da) per site between these two groups was -0.00006. analysis of evolutionary divergence mechanisms to determine the mechanisms of evolutionary divergence of the gmb haplotypes and their genetically closest cattle individuals, the ld degree across the targeted mtdna region was assessed. the scatter graphs (a-c), shown in figure 2, provide the ld values plotted across the nucleotide distance estimates for both the above two groups separately and these groups combined. among the multiple ld-linked polymorphic sites in the mtdna sequence alignment, different positions were identified across the cattle groups examined: for the gmb haplotypes (n = 26), the 12-338 site positions were highly supported by a strong f-generated p-value (< 0.001) and a chi-square estimate of 26.000 (p < 0.001) after bonferroni correction. for the cattle group genetically most closely related to gmb (n = 75), the sites 12-306, 12-338, 407-435 and 427-478 could be determined, strongly supported by the f-produced p32 basiladze et al genetic resources (2024), 5 (10), 25–38 table 4. results of the mega analyses of the targeted mtdna region, elucidating the evolutionary patterns for the georgian mountain breed (gmb) haplotypes, their closest cattle individuals, and both cattle groups combined. evolutionary characteristics targeted population gmb gmb genetically closest cattle globally both cattle groups combined overall average disparity index (id) 0.0023750587 0.0012190602 0.0014805106 overall average composition distance 0.0072881090 0.0048344067 0.0054175153 average nucleotide composition for t(u)/c/a/g 25.1/21.8/40.8/12.4 25.1/21.7/40.8/12.4 25.1/21.7/40.8/12.4 nucleotide frequencies (%) respectively for a/t(u)/c/g 40.76/25.09/21.76/12.39 40.78/25.11/21.74/12.37 25.1/21.7/40.8/12.4 ml estimate of transition/transversion bias (r) 6.23 4.01 7.69 overall average pairwise distance 0.0064039174 0.0048494716 0.0052283806 overall mean distance 0.01 0.00 0.01 discrete gamma distribution 0.0500 0.1000 0.1000 table 5. dnasp-generated evolutionary statistics obtained from the nucleotide sequence analyses of the targeted mtdna region for the georgian mountain breed (gmb) haplotypes, the group of their genetically closest cattle individuals, and both cattle groups combined. singleton variable sites (2 variants)* site positions, in the dna sequence alignment, for: gmb (174, 198, 231, 263, 304, 339, 346, 351, 389, 427, 440, 470, 472); genetically most closely related cattle (309 345 389 441); both cattle groups combined (174, 198, 231, 263, 304, 309, 339, 345, 346, 351, 440, 441, 472). parsimony informative sites (2 variants)* site positions, in the dna sequence alignment, for: gmb (12, 302, 338, 407, 434, 435, 443, 478,); genetically most closely related cattle (12 306 338 407 427 434 435 443 470 478); entire population (12, 302, 306, 338, 389, 407, 427, 434, 435, 443, 470, 478). parsimony informative site (3 variants)* site position (442), in the dna sequence alignment, for a group of gmb genetically closest cattle group. the dna sequence alignment is provided in supplemental figure 1. evolutionary characteristics targeted population gmb gmb closest cattle globally both cattle groups combined no. of polymorphic sites 22 15 26 total no. of mutations 24 16 28 average no. of nucleotide differences (k) 3.123 2.367 2.551 nucleotide diversity (pi) 0.00636 0.00482 0.00520 theta (per site) from eta 0.012 0.00667 0.008 invariable (monomorphic) sites 469 476 465 variable (polymorphic) sites 22 15 26 singleton variable sites 13 4 13 parsimony informative sites 9 11 13 singleton variable sites (2 variants)* 13 4 13 parsimony informative sites (2 variants)* 8 10 12 singleton variable sites (3 variants) 0 0 0 parsimony informative sites (3 variants) 0 1 0 singleton variable sites (4 variants) 0 0 0 parsimony informative sites (4 variants) 1 0 1 sequence conservation (c) 0.947 0.947 0.947 no. of haplotypes (h) 25 20 34 haplotype diversity (hd) 0.997 0.930 0.950 variance of haplotype diversity 0.00014 0.00013 0.00007 standard deviation of haplotype diversity 0.012 0.011 0.008 dna conserved region 13-173 13-173 13-173 genetic resources (2024), 5 (10), 25–38 genetic diversity of georgian mountain cattle 33 figure 2. the ld patterns of the polymorphisms of the targeted mtdna region for the georgian mountain breed (gmb) haplotypes (a), their genetically closest cattle individuals (b), and these two cattle groups combined (c). values (< 0.001) and chi-square estimates (26.172, 75.00, 42.391, 48.340; p < 0.001) after bonferroni correction. for these two cattle groups combined (n = 101), the interlinked site positions 12-338, 407435, and 427-478 could be determined and verified by the f-produced p-values (< 0.001) and the robust chi-sq estimates (101.000, 43.148, 61.137; p < 0.001 respectively) after bonferroni correction. the dnaspgenerated zz values, calculated for inferring intragenic recombination separately between the gmb haplotypes, their genetically most closely related conspecifics, and the total population, were -0.0236, -0.0044, and -0.0157 respectively. in the dnasp analysis, rm = 1, with the detected recombined regions located between the following sites for the above cattle groups respectively: 407-435, 427-478 and 389-434. in contrast, in the rdp4 analysis of the targeted mtdna region, using rdp, geneconv, bootscan, maxchi, chimaera, siscan and 3seq, we could not detect genetic recombination events that would be supported by statistically reliable values across the above entire population examined in this study. when applying the method of split decomposition, in the splitstree analysis of the same mtdna region, we could determine parallel nucleotide substitutions consolidated into five parallelograms shared by multiple individuals from the populations of the gmb haplotypes and their genetically closest cattle individuals. importantly, as shown in figure 3, while the highest fit value of 100 was obtained for the above split decomposition inferences, the bootstrap values, calculated for the nodes of these five parallelograms, were significantly lower, being ≤ 63.6. moreover, the phi test, when measuring homoplasy across the targeted mtdna region for the above subset of cattle individuals, resulted in a very insignificant p-value of 0.4093. discussion haplotype diversity and population structures of gmb and its genetically closest cattle individuals most studies investigating the haplotype diversity and evolution of b. taurus have concentrated on the highly variable d-loop region of mtdna (colominas et al, 2015; kunelauri et al, 2019). although the d-loop is the most diverse functional region of the mitochondrial genome, several other genetic loci of the mitochondrial genome also show significant polymorphism. nevertheless, when analyzing the dna sequences of the d-loop region, some studies have struggled to consistently identify certain haplotypes (e.g. p and t5) in different b. taurus populations globally (cubric-curik et al., 2021). furthermore, the analysis of the d-loop hypervariable loci has sometimes failed not only in distinguishing between specific breeds but also between some ancient branches (achilli et al, 2009; xia et al, 2019). dna sequencing and analyses of the complete mitogenomes provided new and important insights into the genetic 34 basiladze et al genetic resources (2024), 5 (10), 25–38 figure 3. the splitstree-generated parallelograms showing the parallel nucleotide substitutions across the targeted mtdna region, shared by some individuals of the georgian mountain breed (gmb) and their several genetically closest cattle individuals. the numerical values along the nodes of the parallelograms represent their bootstrap estimates obtained from 10,000 bootstrap replications. fit = 100 for the above split decomposition inferences. in the splitsgraph, the gmb individuals are represented by the sample names assigned to these individuals (see table 1), while their genetically closest relatives from the global cattle populations are displayed by their respective genbank accession ids. diversity and evolution of b. taurus (achilli et al, 2008; xia et al, 2019; xia et al., 2021; cubriccurik et al., 2021). however, the cost of dna sequencing for complete mitogenomes is still not easily affordable for most lowand middle-income countries. in earlier studies, the dna sequencing of cytb loci also appeared very instrumental in revealing high haplotype variability (tarekegn et al, 2018), genetic differences between specific breeds from different countries (kim et al., 2013), and even male-mediated introgression (kikkawa et al, 2003) in b. taurus. in our study, the dna sequencing and analysis of the d-loop, cytb, trna-thr, and trna-pro encoding genetic loci could not differentiate, in many instances, between gmb and multiple other breeds from the turano-mongolian, european taurine, and some other cattle within the t and q sub-lineages. although highlighting the necessity for examining complete mitogenomes, as discussed further, our findings also offer new insights into the haplotype diversity of gmb and the molecular-genetic mechanisms governing the evolution of this breed and its genetically closest cattle individuals. interestingly enough, according to the results obtained from our analysis of the targeted mtdna region, the gmb haplotype diversity was notably greater than previously determined in two pilot studies. however, these pilot studies used a very limited sample size, with the initial investigation including 17 individuals (kunelauri et al, 2019), and the subsequent one selecting 5 individuals from the original 17 (kunelauri et al, 2022). nevertheless, these authors (kunelauri et al, 2022) reported on several gmb haplotypes falling outside of the known taurine diversity in their analysis of the complete mitogenomes of these cattle individuals. in our study, while the haplotype diversity estimate was comparatively smaller (0.997 versus 0.9995), the pi value (0.00636) appeared to be notably greater than the mt genome-wide nucleotide diversity estimates (0.0015 and 0.0010-0.0020) calculated earlier respectively for taurine cattle from southeast europe and some other european regions (cubric-curik et al., 2021). thus, at least three different possible scenarios can be considered when attempting to explain the observed differences between gmb and the european cattle breeds: as compared with the latter, gmb may have an older evolutionary history, accumulating over time more genetic changes, and/or has evolved at higher evolutionary rates; it is also possible that, compared with gmb, the southeast european breeds might have undergone more extensive selection. these observations could pave a new avenue for future research to better understand the selection-driven evolutionary differences that can potentially exist between these two groups of cattle. the domestication of b. primigenius, which took place around 10,000 years ago, marked a significant neolithic advancement (bonfiglio et al, 2010). this process involved cattle breeding over various periods and had profound socioeconomic impacts on old world populations (clutton-brock, 1989), possibly including early tribes living in georgia. it’s worth noting that historically, dating back 1.8 million years, the territory of georgia was inhabited by a variant(s) of homo erectus (lordkipanidze et al, 2013; schwartz et al, 2014). in this light, it is possible that the above geographic region, being the habitat for certain early tribes, could be one of the oldest cattle domestication sites in the old world. this scenario gains plausibility given the ancient traditions of winemaking (ieri et al, 2021), honey production (kvavadze et al, 2007) and wheat cultivation (gogniashvili et al, 2021) in georgia. unfortunately, there is no clear scientific information available about ancient animal husbandry practices and traditions in georgia. here, we show that the gmb khevsurian and adjarian populations belong predominantly to the t1, t2, t3 and t4 sub-haplogroups. it should be noted that the investigations, examining complete mitogenomes, revealed significant diversity across modern cattle within t (cubric-curik et al., 2021). the t subhaplogroups primarily reflect specific geographical structuring, with t1 being most common in african breeds, t2 prevalent in near eastern and mediterranean breeds, t3 predominantly found in european breeds, genetic resources (2024), 5 (10), 25–38 genetic diversity of georgian mountain cattle 35 and t4 most frequently characteristic of east asian breeds (carvajal-carmona et al, 2003; chen et al., 2010). the earlier studies reported that, in europe, t3 demonstrated a reduced diversity relative to near eastern cattle – consistent with the patterns found in european cattle in the near east (troy et al, 2001; carvajal-carmona et al, 2003). in our study, two unique haplotypes, representing the sngts, could be identified among the gmb individuals that belonged to t3. interestingly, t4 could not be found previously in near eastern breeds within t (troy et al, 2001). here, we show that, similar to the east asian and some other cow breeds, the gmb adjarian population also can carry the sub-haplogroup t4 characteristics. according to our phylogenetic analysis, certain gmb sngts demonstrated their closest genetic affinities with some sub-linages within t and q, while some others appeared to represent previously unidentified haplogroups of b. taurus. importantly, the haplogroup q, which is likely of near eastern origin, has been considered to be rare in the global populations of modern taurine cattle (bonfiglio et al, 2010; xia et al., 2021; cubriccurik et al., 2021). here, we provide strong amplifying evidence for the growing existence of cattle individuals belonging specifically to q1 – the sub-lineage of q – across the gmb populations, strengthening the earlier findings of two pilot studies (kunelauri et al, 2019, 2022). moreover, our analysis of the targeted mtdna region of the gmb populations revealed the possible presence of an unknown sub-haplogroup within q. thus, the present findings point out that georgia should be added to the list of countries (such as egypt, china, turkey and several european countries), where the b. taurus populations exhibit q along with other haplogroups (achilli et al, 2009). among the typical representatives of the gmb adjarian population, we could identify as well the mtdna pattern that was most closely related to the subhaplogroup i1a. i1a is a relatively novel sub-haplogroup of b. indicus (chen et al., 2018), which originated in indus valley about 8,000 years ago, and further spread eastwards to southeast asia and southern china < 400 years ago (loftus et al, 1994; chen et al., 2010). indicine cattle were found to be dominant in southern china (li et al., 2013), which is considered to be one of the domestication centres. interestingly, it is suggested that within i1 cattle found in guangxi, i1a represents a unique and dominant sub-haplogroup while being absent in india, demonstrating the dominant status for local cattle in yunnan located in the southwestern part of china (xia et al, 2019). thus, similar to earlier observations on cattle (edwards et al., 2007; achilli et al, 2008), the closest genetic affinity of the specific sngt of gmb with i1a suggests the existence of local admixture populations in this breed, likely influenced by introgression from wild aurochs. besides, the above highlights can be also collectively suggestive of the complex patterns of the domestication process contributing to shaping the gmb population structure; these findings are in strong agreement with the early studies on the complexity of the domestication process as being a phenomenon influencing the genetic diversity and divergence of cattle populations globally (achilli et al, 2009; bonfiglio et al, 2010; olivieri et al., 2015; xia et al, 2019). hence, considering all the above findings and observations, we suggest that gmb may have an ancient origin, playing an important role in the evolution of b. taurus globally. more extensive research, using at least the dna sequencing of complete mitogenomes with significantly larger sample sizes, is needed to gain more in-depth insights into the evolution of gmb. mtdna polymorphisms and mechanisms of evolutionary divergence of gmb examining the mtdna polymorphisms of gmb, we could unravel the presence of multiple hitherto undescribed mutations, clearly exhibiting a transition bias, the phenomenon observed in mesolithic wild aurochs (b. primigenius) (edwards et al., 2007), as well as in some of the ancient and modern bovine populations (stock et al, 2009). these mutations, identified across 11 sites of the targeted mtdna region, appeared to be characteristic predominantly to certain gmb sngts, distinguishing them from other haplotypes determined previously in the b. taurus global populations. from the evolutionary patterns, it becomes clear that the observed heterogeneity is pronounced primarily across both the sngt variable sites with two variants, and the parsimony informative sites with two and four variants, being coupled with the above evolutionary estimates including, but not limited to, h, hd, id, r and various overall average and mean distances values calculated. this is the first study offering some important initial insights into the mechanisms of evolutionary divergence of the gmb populations. it should be noted that using the large-scale mitogenome sequence analysis of b. taurus, the first attempts aimed at detecting genetic recombination events in domestic taurine cattle revealed no evidence for this phenomenon in these animals (cubriccurik et al., 2021). while investigating the mechanisms of evolutionary divergence with split decomposition, we identified parallel nucleotide substitutions with some illuminating the conflicting evolutionary signals across the targeted mtdna region in the populations of both gmb and its genetically closest cattle individuals identified worldwide. while usually such splitstree-derived signals can frequently exhibit lateral genetic transfer events, these putative homologous recombination inferences could not be strongly supported by the fit and bootstrap values in the subsequent analysis using the above software package. the scenario of homologous recombination was neither supported by the phi test estimates when measuring homoplasy for the same mtdna subset, suggesting, instead, most likely the presence of parallel or convergent evolution in these b. taurus populations. the lowered ld rates, calculated for the targeted mtdna region of the above populations, could 36 basiladze et al genetic resources (2024), 5 (10), 25–38 be additionally linked to possible recombination events across the populations of gmb and its genetically closest cattle individuals globally. however, it is important to consider that by lowering ld, homoplasy can sometimes mimic such recombination events (tibayrenc and ayala, 2017). importantly, this and alternatively the other genetic recombination inferences (e.g. chi-sq and rm estimates) – the scenario of intragenic recombination – were also rejected when the negative zz values were considered in the dnasp analyses. such a similar conflicting scenario, depicting the chi-sq and rm positive inferences versus the negative zz estimates, was described previously, suggesting that at least rm can be sometimes inflated by parallel mutations not necessarily associated with genetic recombination (rozas et al, 2001). interestingly enough, in the earlier study (groves and shields, 1997), the cytb genes of the takin (budorcas taxicolor) and muskox (ovibos moschatus) from the family bovidae were assumed to have been impacted by convergent evolution. also importantly, the yak/bison mitochondrial transfer, in light of the parallel accumulation of unique mutations of mtdna, was also suggested (zeyland et al, 2012). therefore, our findings conjointly with the above observations strongly lead to the scenario(s) of parallel and/or convergent evolution in the populations of gmb and its genetically closest cattle individuals. these scenarios, versus the scenario of genetic recombination, become even more plausible if we also consider the negative outcomes received from the rdp4 analyses revealing the absence of recombination breakpoints in the mtdna region of the targeted b. taurus populations. conclusions the majority of gmb individuals from the khevsureti and adjara regions of georgia belong to the subhaplogroups t1, t2, t3 (including t3119), t4 and q1. they also exhibit multiple novel haplotypes, largely represented by sngts. some of these sngts may belong to currently unidentified sub-haplogroups or even to previously unknown haplogroups of b. taurus. notably, the haplogroup q is common in the gmb populations, unlike many other breeds and populations of b. taurus worldwide. additionally, the adjarian population of gmb includes the sngt-9 that is genetically closest to the sub-haplogroup i1a of b. indicus. it is suggested that parallel and/or convergent evolution, along with introgression, have shaped the gmb population structure in these regions of georgia. further in-depth research, particularly through the dna sequencing of complete mitochondrial genomes from a significantly larger number of gmb individuals, is needed to better understand the origin of this breed and its potential role in the evolution of b. taurus global populations. supplemental data supplemental table 1. the genetically closest cattle individuals of the georgian mountain breed included in the evolutionary analyses. supplemental figure 1. clustalx-generated multiple dna sequence alignment utilized in the evolutionary analyses of the georgian mountain breed and its genetically closest cattle individuals. acknowledgments this study was supported by the shota rustaveli national science foundation of georgia (srnsfg) (grant no.: fr-19-21496). the lugar center for public health research at the national center for disease control contributed to the dna sequencing experiments described in this study. author contributions givi basiladze and leila tabatadze: conceptualizing the research, and performing the sampling and selection of gmb-specific types of cattle individuals, also contributing to drafting the manuscript; ekaterine gabashvili and mariam osepashvili: performing the dna sequencing and sequence assembly procedures; marine murskhvaladze: performing the nucleotide quality analysis, 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(2022). the state of conservation of animal genetic resources in slovakia. genetic resources 3 (6), 49–63. doi: 10.46265/genresj.xrhu9134. © copyright 2022 the authors. this is an open access article distributed under the terms of the creative commons attribution license (cc by 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. introduction biodiversity for food and agriculture is indispensable to food security, sustainable development and the supply of many vital ecosystem services (fao, 2019). animal genetic resources (angr) are also sources of social and cultural benefits. their contribution to a country’s economy is not just in the form of animal products but also of employment opportunities for people in rural regions. angr are also an important part of landscape management and the agrotourism sector. despite all their roles and characteristics, according to the second report on the state of the world’s animal genetic resources for food and agriculture of the food and agriculture organization of the un (fao), 17% of all breeds are classified as being “at risk of ∗corresponding author: ján tomka (jan.tomka@nppc.sk) extinction” (fao, 2015). in the case of local breeds, the main reason for rapid erosion is the lack of economic profitability (gandini and oldenbroek, 1999). this is closely related to the import of specialized and highly productive breeds, and their cross-breeding with local breeds. recently, local breeds are getting more attention thanks to their adaptability to local environmental conditions, their suitability for extensive agriculture and their expected roles in climate change adaptation. the increasing interest of consumers in animal production brings challenges but also opportunities to breeders. advances in biotechnologies lead to more intensive research on the genetic level, increasing demands for establishing genebanks and providing angr material from already existing genebanks (groeneveld et al, 2016). received: 25.11.2022 accepted: 01.07.2022 published online: 09.09.2022 https://www.genresj.org https://www.dx.doi.org/10.46265/genresj.xrhu9134 https://www.genresj.org https://www.dx.doi.org/10.46265/genresj.xrhu9134 mailto:jan.tomka@nppc.sk 50 tomka et al genetic resources (2022), 3 (6), 49–63 countries adopt different policies and implement different approaches to address the conservation and use of angr, including local and endangered breeds, and to raise awareness of the need to conserve them. the main guidance is provided by the global plan of action (gpa) for animal genetic resources (fao, 2007) and implementation progress in the four priority areas is monitored. the latest results show that globally, the greatest progress in the last years was achieved in the field of policies, institutions and capacity-building, while the lowest progress was achieved in conservation (cao et al, 2021). this review aims to provide a summary of the activities related to angr conservation in the slovak republic, with examples of the development and conservation of several breeds in the country. the information provided here should serve as a starting point in the preparation of the national strategy for the conservation of angr in the slovak republic. history of angr in the slovak republic slovakia is located in the heart of europe and its relatively small area has made it difficult to maintain its local livestock breeds. in the past, intensive imports and crossbreeding contributed to forming the actual livestock diversity in the country. the main drivers for change in livestock diversity were improving performance and adaptedness of local breeds, often directed by landowners or the government. in the second half of the 20th century, the intensification of animal production and planned agriculture led to the creation of a list of recognized and allowed breeds (act no. 110/1972 coll. on livestock breeding, edict 108/1974 on act no. 110/1972), which could be kept by cooperatives and farmers, and contributed to a narrowing of livestock diversity. the massive planned use of holstein bulls for crossing with slovak spotted and slovak pinzgau cows led to a decrease in both populations (kadleč́ık et al, 2013). unifying existing breeds contributed to the loss of within-breed diversity. for example, in the case of the valachian sheep, which was considered undeveloped in the starting period of intensification, the planned unification and improvement of the population led to the loss of diversity of exterior animal characteristics (colour varieties, horn shapes). similar to other countries, mechanization in agriculture and decline of their use in the army during the second half of the 20th century had a significant effect on horse populations. their number decreased dramatically and has not recovered to this day. in those times, there was no national interest in the conservation of local breeds and some of them became completely extinct in the country (e.g., red carpathian and gray carpathian cattle). to improve production and adaptedness, new specialized breeds were created. in poultry, the oravka breed was developed for adaptation to the colder regions of northern slovakia following a request from the government. the breed became a favourite among small breeders, spreading around the whole country, and its current population is estimated to be up to 9,000 animals. on the other hand, some of the new breeds created were outperformed by other specialized and well-established breeds or their breeding process failed in later phases and these breeds became extinct (e.g. slovak white and slovak meaty pig). in horses, the creation of three breeds started in this period, including noric of murany, slovak sport pony and slovak warmblood. after the political changes in the 1990s in central europe, agriculture and animal production started to transform and the overall number of livestock decreased rapidly in these countries (oravcová et al, 2004). for example, at the breed level, in 30 years the number of slovak pinzgau cattle decreased from more than 90,000 at the end of the 1980s to just 11,000 at present. the improved valachain sheep population decreased from almost 200,000 in the mid-1990s to 100,000 animals, currently. some breeds became endangered according to effective population size (table 1). breeders started to import exotic specialized breeds to improve production and be competitive at the international level. these changes put more pressure on the breeds with a long tradition in the country. while most of the breeders were improving production, some turned their attention to endangered breeds and also breeds that were already extinct in the country. thanks to common history (czechoslovakia, austro-hungarian monarchy), animals from these breeds could still be found in neighbouring countries and the exchange of these animals allowed the recovery of their populations in slovakia. in the case of the almost extinct valachian sheep, in the 1990s, breeders started to select phenotypically acceptable animals from the improved valachian population and have continued to import animals from breeders in the czech republic, who have maintained the rest of the original valachian population. occasional imports of valachian sheep from germany, where a part of the valachian sheep population was exported in the 1990s, have also occurred. since the exchange of breeding animals between slovakia and neighbouring countries continues, most of the breeds can be considered as being transboundary in terms of fao definitions (fao, 2005). due to common history and changes in animal production, including intensive crossbreeding, it is quite difficult to strictly distinguish between native and non-native breeds. there is no legal definition of an autochthonous/native/traditional breed in the country and no legal criteria are set for the recognition of an endangered breed. however, references to generally accepted autochthonous or traditional breeds can be found across the literature (oravcová et al, 2006; weis and hrnčár, 2009; šidlová et al, 2015; kadleč́ık et al, 2017). while a common understanding of autochthonous breeds was also reported in poland, austria has a more precise definition, which includes breeds having a herdbook in austria before 1938, or ample evidence that they have always been present and bred in a part of austria, or stem from the austrogenetic resources (2022), 3 (6), 49–63 angr conservation in slovakia 51 hungarian monarchy and have had relations to regions of austria (kompan, 2014). in poultry, big producers use hybrids, while continuous breeding activities of small farmers have led to the creation of new colour and dwarf types. for example, in the case of oravka chicken, three new colour types and dwarf types have been created since the original type’s official recognition in 1990. the number of registered breeders, which can be considered a reliable source of breeding animals, is small and thus the number of breeding animals is also small compared to the population size (table 2). similarly in rabbits, big producers use hybrids. breeding activities of small farmers are considered a hobby and are focused on the stabilization of existing and newly created breeds’ characteristics as well as the creation of new breeds. the estimated size of rabbit populations and the number of breeding animals reflect the length of a breed’s existence (table 2). while populations of breeds recognized before 1990 are estimated to be more than 200 animals strong (blue of holic, nitra, slovak greyblue rex, zemplin), populations of breeds recognized after this year are smaller. legislation and policies the slovak republic signed the convention on biological diversity (cbd, 1992) in 1993 and became a party to the cbd after approval in the following year. issues related to biodiversity and its protection fall under the ministry of environment of the slovak republic. after affirmation of its commitments in 2007 (interlaken declaration on animal genetic resources (fao, 2007)), the slovak republic started to implement the gpa for animal genetic resources. according to strategic priority 20 of the gpa for animal genetic resources (fao, 2007), countries should periodically review their national policies in order to investigate their direct and indirect effects on the use, development and conservation of angr. at present, the main responsibility for angr conservation in the country lies on the ministry of agriculture and rural development of the slovak republic and nppc – research institute for animal production nitra (national focal point for animal genetic resources in slovakia). in the slovak republic, the most important legislative measure is act no. 194 of 1998 coll. (last amended in 2020) on livestock breeding, which, in general, specifies the rights and duties of authorized breeders’ associations to carry out their professional activities connected with animal breeding. it also addresses endangered breeds to a limited extent. similar to the eu regulation 2016/1012 (eu, 2016), the national act no. 194/1998 under paragraph 3 section 1e) lists the protection of endangered populations and provision of livestock genetic reserves as an important part of breeding development. although recently not strictly followed, under paragraph 2 section 7 of act no. 194/1998, protected farms are defined as farms keeping animals of endangered breeds that are used for the conservation of the breed’s genepool and maintenance of its historical value. according to paragraph 13 section 2 of act no. 194/1998, the transfer of breeding stock and genetic reserves originating from such farms should be controlled and permitted only after approval of the state. contrary to this general approach, there are legislative acts of neighbouring countries, where the legislative basis for angr conservation, especially the setting of a national conservation programme, is part of the breeding acts (e.g. czech republic – paragraph 14 of act no. 154/2000 coll. (last amended in 2021) (breeding act), or poland – art. 34 of coll. of laws 2021, item 36, act on the organization of breeding and reproduction of farm animals). based on the slovak national act, there are authorized breeders’ associations responsible for breeding the main livestock species including cattle, sheep, goats, horses, pigs, rabbits and poultry. these associations maintain breeding books, studbooks and breeding registers and are providing information to the national focal point for animal genetic resources in slovakia as part of the regular angr monitoring. authorized breeders’ associations differ in organization and number of breeds managed. for instance, the slovak pinzgau cattle are represented by an association solely responsible for this breed, thus making it easier to promote and take actions aimed at this particular breed. on the other hand, valachian sheep are represented by an association responsible for several breeds of sheep and goats. all the activities are undertaken by the association. a group of breeders has been created within this association to improve collaboration and the breeding process of valachian sheep. the same applies to the breeders of poultry (different groups according to species and breeds) and rabbits. in horses, national stud is responsible for managing the studbook of lipitsa, hutsul, arab and shagya arab. studbooks of other breeds, except for english thoroughbred, are managed by one horse breeders’ association. regarding angr cryoconservation, the national legislation addresses only insemination centres with no considerations or exceptions for endangered breeds conservation and genebank activities. a description of the technical requirements for establishing genebanks is lacking, and so far, only scientific experience and rules for insemination centres are followed. the same applies to requirements for the acquisition and use of angr samples stored in genebanks. the actual status of the national veterinary legislation (act no. 39/2007 coll. (last amended in 2021) on veterinary care) therefore does not address and reflect the actual needs of angr conservation in the country and further steps are needed to fill this gap. the slovak republic became a party to the nagoya protocol (cbd, 2011) in 2016. the responsibility for the nagoya protocol implementation in the slovak republic lies with the ministry of environment. in order to facilitate access to its angr and taking into account that almost all livestock breeds in slovakia are considered transboundary, slovakia decided not to control access to its angr. following the eu regulation 511/2014 eu (2014b), the slovak national law (act. no. 263/2015 52 tomka et al genetic resources (2022), 3 (6), 49–63 table 1. numbers of purebred registered breeding males and females and endangerment class of supported breeds of livestock in the slovak republic according to 2020 monitoring data. endangerment class was set according to effective population size calculated according to the formula ne = (4 × m × f)/(m + f) (falconer and mackay, 1996) and assuming mass selection ne(sel) = 0.7 × ne (santiago and caballero, 1995). the degree of endangerment was assessed according to the following limits: critically endangered (ne ≤ 50), endangered (50 < ne ≤ 200), monitored (200 < ne ≤ 1,000) and not endangered (ne > 1,000) species breed males (m) females (f) endangerment class cattle slovak pinzgau 31 2,025 endangered sheep valachian 45 907 endangered tsigai 360 5,613 monitored improved valachian 616 8,175 not endangered askanian merino 6 68 critically endangered goat white shorthaired 50 708 endangered brown shorthaired 9 81 critically endangered horse lipitsa 6 165 critically endangered shagya arab 16 154 critically endangered hutsul 7 133 critically endangered furioso 17 161 critically endangered nonius 7 28 critically endangered noric of murany 18 137 critically endangered slovak sport pony 7 84 critically endangered slovak warmblood 16 857 critically endangered table 2. estimated population size, purebred breeding males and females of registered breeders and endangerment class of poultry and rabbit breeds in the slovak republic according to 2020 monitoring data. endangerment class was set according to effective population size calculated according to the formula ne = (4 × m × f)/(m + f) (falconer and mackay, 1996) and assuming mass selection ne(sel) = 0.7 × ne (santiago and caballero, 1995). the degree of endangerment was assessed according to the following limits: critically endangered (ne ≤ 50), endangered (50 < ne ≤ 200), monitored (200 < ne ≤ 1,000) and not endangered (ne> 1,000) species breed population males (m) females (f) endangerment class chicken oravka 9,000 59 465 endangered goose suchovy 250 19 33 critically endangered slovak white 270 22 40 critically endangered rabbit blue of holic 420 30 80 endangered liptovsky lysko 160 20 42 critically endangered nitra 1,250 38 200 endangered slovak pastel rex 105 15 40 critically endangered slovak greyblue rex 600 28 92 endangered zemplin 280 15 55 critically endangered zobor 60 8 18 critically endangered strbsky gepardi rex 100 15 35 critically endangered chrabrany 120 19 38 critically endangered coll. on competences in the area of access to genetic resources and sharing of benefits arising from their utilization) sets only rules for the users of genetic resources falling under the nagoya protocol. the national legislation pays limited attention to endangered breeds and their conservation and lacks direct strategies for angr conservation. these are partly compensated for by a few cross-sectorial strategies that refer to angr. as part of biodiversity, some actions related to angr conservation were included in the updated national strategy for biodiversity protection 2020 (mzp sr, 2013). these were broadly defined, and included monitoring of angr, support for in situ conservation of traditional breeds, genetic analysis of diversity, support for ex situ conservation, including the development of a genebank, and identification of ecosystem services provided by angr. to a certain extent, the inclusion of these activities into the strategy may be considered a formality since they have been carried out even before the strategy was adopted, and the funds for most of them were allocated regardless of the strategy. additional issues related to angr have been also addressed in the updated version of the adaptation strategy of the slovak republic to climate change (mzp sr, 2018). the disadvantage of including angr conservation in genetic resources (2022), 3 (6), 49–63 angr conservation in slovakia 53 multiple strategies is that some activities may be duplicated, for example, the monitoring of livestock species. moreover, the number of angr conservation activities has increased over time and new conservation approaches are being applied. therefore, cross-sectoral policies are no longer sufficient to address specific activities related to angr conservation, as demonstrated in the case of poultry breeders. while cross-sectoral policies have been grouping the main responsible angr stakeholders from different biodiversity sectors, the involvement of small stakeholders that are important from an angr perspective has been very limited. while the overall angr monitoring was included in the strategy, no attention was paid to gaps and the limited registration system of local poultry breeds and farmers in the country. this led to further problems in providing financial support to these farmers. the limited implementation of the actions listed in the cross-sectoral policies resulted also from the lack of funding since not all actions have financial cover granted. in contrast to this approach, functional conservation programmes have been implemented across european countries, which have been closely tied with funds provided by the eu (ligda and zjalic, 2011). monitoring the livestock monitoring system in the slovak republic is based on the use of already existing data. population data for monitoring purposes come from two sources. the first is the breeding services of the slovak republic, a state enterprise which provides actual sizes of cattle, horse, goat and sheep populations at the breed level. these data represent the number of obligatory registered livestock animals according to the breed. the reliability of these data is very high since this obligation applies to all farmers of the four mentioned species and the portion of non-registered animals is assumed to be negligible from a population size perspective. horse breeding has some specificities and discrepancies exist in their registration, therefore the final number of animals is estimated based on data from the register and numbers from studbooks. the second source of data is the authorized breeders’ associations which maintain the herdbooks/studbooks and breeding registers, and provide relevant information on registered breeding animals. although it does not cover the whole population, this information is of high interest, because these animals represent the part of the populations that can actively participate in the breeding process and serve as a basis to create future generations. it provides the numbers of purebred and crossbred animals and a picture of crossbreeding in the populations. to compare between years and exclude fluctuations during the year, actual numbers relevant for the last day of the previous year are used in the monitoring. the reliability of information on cattle, horses, goats, sheep and pigs is high since it comes from reliable existing registration sources. on the other hand, information on poultry and rabbits is less reliable since the registration of animals of these species is limited and some information is based on estimates. this arises from the fact that while registration is obligatory for every animal from big livestock species, only farmers with more than 350 poultry animals are obliged to register them. furthermore, due to the already mentioned fact that purebred animals of local breeds are mostly kept by small farmers and in small numbers as a hobby activity, limited information on the population size is available. a similar situation can be observed in the registration of purebred animals of these species, as only a few small farmers keep track of their animals’ pedigree and performance. similar obstacles were reported in the czech republic (mze cr, 2017). in this situation, since the identification of eligible farmers/animals is very complicated, the breeding process becomes difficult and financial support from the state is limited. although population data are available for most breeds, active monitoring is performed only for those breeds that are considered established in the country. this is due to the high fluctuation of exotic breeds in the slovak republic and to the limited information about small populations of those breeds in the country. the examples of tarantaise cattle or lincoln sheep show the farmers’ enthusiasm for exotic breeds. however, they were unable to keep breeding them for different reasons, and currently, there are less than ten animals of these breeds in the country. in 2020, more detailed monitoring included 15 cattle, 11 horse, 27 sheep, 5 goat, 6 pig, 49 rabbit, 21 chicken, 3 duck and 3 goose breeds. among those breeds, 34 were considered autochthonous or traditionally kept in the country, 6 new and 100 exotic. results of monitoring were used to evaluate the status of endangered breeds tomka et al (2016); tomka and huba (2019) and to update population data in fao’s domestic animal diversity information system. in addition to population monitoring, communicating with breeders of endangered breeds to understand their needs and expectations is important. this was already shown by wanner et al (2021) who interviewed farmers of german-speaking parts of the alps and found that the opinions and expectations of farmers differ from studies and opinions of other stakeholders. a short survey among the registered breeders of valachian sheep (11 out of 14) and askanian merino sheep (1 out of 1), slovak white goose(5 out of 8) and suchovy goose (9 out of 11), was conducted by nppc in 2017 and 2018. the survey aimed to investigate the breeders’ motivation for the choice of breed, their breeding purposes and future plans for endangered breeds in slovakia. the results of the survey can help predict trends in these populations since it provides a glimpse of the breeders’ attitudes towards monitored endangered breeds. some outputs of this survey are presented and discussed in this review to show the breeders’ motivation and attitudes. conservation the angr conservation activities in the slovak republic are primarily oriented toward measures supporting 54 tomka et al genetic resources (2022), 3 (6), 49–63 in situ breeding of live animals by private bodies (cooperatives, farmers). this way, endangered breeds can be further developed and continually adapted, e.g., to changing climate conditions (fao, 2013). there are no restrictions on breeding goals compared to other european countries, where breeders have to commit to maintaining the traditional characteristics of the animals. for instance, for the slovak pinzgau cattle, part of the population has already been transformed from dual-purpose to beef production (cow–calf system) (pavĺık et al, 2013). the reason for changing to a cow–calf system may be explained by the fact that the slovak pinzgau cattle is still predominantly kept in big herds owned by cooperatives and expected to make a profit primarily from milk production. therefore, cooperatives have to either improve the milk performance of animals (through e.g. crossbreeding, choice of a different breed) or change the orientation of production. a positive change trend toward beef production is shown by the numbers of living cows registered in different sections of the herdbook (dual-purpose vs. beef). while 1,030 purebred dual-purpose cows and 667 beef cows were reported in 2012 in the population of slovak pinzgau cattle, the opposite ratio was reported in 2020, i.e. 841 purebred dual-purpose cows and 1,183 beef cows. at the moment, animals of both groups are eligible for financial support. although this free approach allows breeds to change their breeding purposes and become more competitive, it can also bring tensions within breeding organizations (lauvie et al, 2011). at the moment, there are no disagreements on the two different types of slovak pinzgau in the association. however, this may change in the future if beef animals divert significantly from the dual-purpose type. a different approach to in situ conservation of angr with defined requirements is applied in the czech republic, where conditions for animal breeding must be as similar as possible to those in which the breed was developed, and modern breeding technologies should be used to a limited extent. most importantly, the selection of animals is not aimed at improving their performance, but at stabilizing their characteristics and/or maintaining their original characteristics (mze cr, 2017). an approach based on maintaining the animals’ original characteristics can be found in the valachian sheep population in slovakia. at the moment, the selection of animals is based primarily on exterior characteristics, since the breed is known for its different colours of wool and different types of horns. less pressure on selection for productivity traits in this breed may be explained by their ownership. these animals are mostly kept by small farmers as a hobby and for agrotourism, or by cooperatives along with other more productive sheep breeds. the results of the short survey conducted by nppc showed that resilience, low requirements for feeding and adaptation to the local environment were the most mentioned advantages of these animals. a similar situation can be observed in poultry and rabbit populations. in oravka chicken, selection is based primarily on exterior characteristics. this comes as no surprise since the breeding of these animals is considered a hobby activity or is done to ensure small-farmers self-sufficiency. one of the disadvantages of in situ conservation, especially in small populations, is the risk of increasing inbreeding. the responsibility for following breeding programmes and maintaining low inbreeding levels lies with breeders’ associations. therefore, cooperation between breeders’ associations and research and academic institutions is crucial for the effective development and implementation of breeding programmes. recently, several national research projects have been carried out to study the actual status of local breeds populations, and published papers showed different levels of inbreeding in several cattle and horse populations. while pedigree-based inbreeding under the acceptable level of 1% was estimated in slovak spotted and slovak pinzgau populations of cattle (kukučková et al, 2017; kasarda et al, 2019a), genomic analyses showed higher levels of inbreeding and thus increasing trends of inbreeding are expected in both populations. a low level of pedigree-based inbreeding (0.23%) and genomic inbreeding (0.11%) was observed in the relatively small population of noric of murany (kasarda et al, 2019b). sufficient levels of variability were observed in the populations of lipitsa, furioso and nonius (kasarda et al, 2018). in older studies (pjontek et al, 2012), which investigated populations of horses, higher levels of relatedness and expected higher levels of inbreeding based on pedigree information were reported in the population of hutsul (6.26%) and slovak sport pony (2.67%). a preliminary assessment of inbreeding trends in valachian sheep (oravcová and marget́ın, 2011) showed a lack of pedigree information in animals resulting in unclear coefficients of inbreeding, while a more recent analysis (pavĺık et al, 2017) showed a low average coefficient of inbreeding (0.85%). a low level of pedigree-based inbreeding (0.69%) was also calculated in the population of white shorthaired goats (oravcová, 2013). other conservation activities include in situ and ex situ conservation of live animals by state organizations. while horse breeds (noric of murany, lipitsa, hutsul, shagya arab) are maintained in situ, live sheep (valachian) and chicken (oravka) are conserved ex situ. conservation activities of the state organizations are aimed mostly at stabilizing and maintaining the original characteristics of these breeds. additionally, two of these organizations also receive national funds for the long-term storage of angr samples. while national stud provides long-term storage of horse breeds samples (samples are stored as a reserve and are not distributed to breeders), the genebank of the national agricultural and food centre (gb nppc-vuzv) stores samples of other livestock species and breeds. this is similar to other european countries, where the long-term storage of angr is mostly carried out by public research institutes (passemard et al, 2018). genetic resources (2022), 3 (6), 49–63 angr conservation in slovakia 55 samples stored in gb nppc-vuzv are owned by the state and are expected to be available for cooperation with breeders and research purposes. in the past, private and public insemination centres used to cooperate on the storage of samples for conservation purposes. this is the case of the slovak pinzgau cattle, whose old bull lines were kept by a private insemination centre for commercial and long-term storage purposes. due to costs associated with cryoconservation, the slovak pinzgau samples were later transferred to gb nppcvuzv. there was no involvement of slovak pinzgau breeders in the process of animal and sample selection. currently, there is no information available on the number of long-term samples stored by these private insemination centres. the participation of the private sector in long-term conservation activities, however, should be restored because it can reduce collection development costs (pizzi et al, 2016). moreover, it is assumed that the private sector storing samples of transboundary commercial breeds can allow the public sector to focus more on the country’s local and endangered breeds. in december 2021, gb nppcvuzv reported 3,058 samples, from which the majority is represented by semen samples from 12 breeds and 4 species (cattle, sheep, chicken, rabbit). these represent mainly endangered local breeds including slovak pinzgau cattle, valachian sheep, oravka chicken, blue of holic, nitra, slovak greyblue rex and zobor rabbit. in the case of valachian sheep and oravka chicken, samples are primarily collected from animals kept ex situ in vivo by nppc-vuzv. this means, that there is no involvement of breeders in the process of sample selection and only limited participation of breeders in providing samples to the genebank. the predominant storing of semen is understandable here and across other countries (leroy et al, 2019) and may be explained by the long and routine use of artificial insemination in some breeds. the collection of samples for long-term storage in gb nppc-vuzv is rather random, not following any conservation plan or breed-specific attributes (e.g. endangerment, economic return). such an opportunistic approach was already presented by blackburn (2009), who described the initial phase of germplasm collection development in the usa. in 2021, gb nppc-vuzv has become a member of the european genebank network for animal genetic resources (eugena). this step should help to increase the genebank visibility at the national level and improve cooperation at the international level. from the transboundary breeds perspective, joining the network in combination with strategic collection and storage planning can lead to saving cryoconservation costs in the future (silva et al, 2019). on the other hand, joining the network does not imply that overlapping of collected angr samples should be strictly avoided (danchinburge et al, 2011). funding and valorization in order to identify endangered breeds requiring support, breed definitions by fao (2005) have been followed and endangerment of breeds has been evaluated by the national focal point for angr in agreement with the rules laid down by the current european legislation. as a result of this approach, different breeds have been supported during the last decades based on their needs. the open approach of the slovak republic is demonstrated, for instance, by the askanian merino sheep breed. although not originating in slovakia, this breed has become eligible for subsidies because of its long breeding tradition in the country, its unique wool performance and its status of critically endangered breed not just at country level but also in the eu. with such an approach, immediate actions can be taken to support critically endangered breeds. in the mid-1990s, state incentives were provided for live purebred females, but the support varied in the amount per head and number of breeds, because public funds were limited (oravcová et al, 2004). in 2003 these incentives supported two cattle breeds (slovak spotted, slovak pinzgau), seven horse breeds (hutsul, lipitsa, furioso, nonius, shagya-arab, noric of murany, slovak sport pony), three sheep breeds (valachian, improved valachian, tsigai) and three poultry breeds (oravka hen, slovak white goose), including japanese quail. after joining the european union in 2004, a new funding scheme for in situ conservation of endangered breeds was applied. support was provided through the rural development plan (mp sr, 2003) as in many other european countries (ligda and zjalic, 2011). in the period 2004–2006 this support was provided only to one sheep (valachian), one goat (white shorthaired), eight horse (slovak warmblood, hutsul, furioso, nonius, slovak sport pony, lipitsa, shagya arab, noric of murany) and nine poultry breeds (oravka hen, plymouth rock hen, rhode island red hen, new hampshire hen, vlaska hen, sussex hen, slovak white goose, suchovy goose and bronze turkey). due to significant changes in the list of supported breeds, one can argue there have been inconsistencies in the approach and big pressure from breeders to include other breeds on the list of supported ones. the main changes in the list of supported breeds were the consequence of the transition from very limited national funds to european funds. also, new conditions for support were set after joining the eu. during this period, the number of registered purebred females in herdbooks decreased in slovak pinzgau cattle (table 3). unfortunately, no data were available to present the trend of registered females of valachian sheep and white shorthaired goat, which were supported in this period. however, it can be assumed that this number increased in valachian sheep and decreased in white shorthaired goats (table 3). in horses, the number of registered females increased in lipitsa, shagya arab, hutsul, furioso and slovak sport pony, decreased in noric of murany and stayed at low levels in nonius. 56 tomka et al genetic resources (2022), 3 (6), 49–63 table 3. development of purebred breeding females registered in herdbooks. *, year 2003, source: (oravcová et al, 2004); **, data for 2007; n.a. – data not available. species breed registered purebred females (monitoring data) 2003∗ 2006 2010 2012 2014 2016 2018 2020 cattle slovak pinzgau 2,500 1,600 1,969 1,697 1,491 n.a. 1,479 2,024 sheep valachian 50 n.a. 65 225 349 553 820 907 tsigai 13,000 13,000∗∗ 11,406 12,734 7,277 5,571 5,433 5,613 improved valachian 24,000 21,000∗∗ 15,724 14,682 9,412 7,641 6,971 8,175 askanian merino n.a. n.a. n.a. 34 27 39 52 68 goat white shorthaired 1,000 n.a. 864 832 1,031 634 752 708 brown shorthaired 3 n.a. 29 102 67 116 152 81 horse lipitsa 60 105 145 186 352 165 181 165 shagya arab 85 102 165 197 359 128 151 154 hutsul 50 115 120 110 280 106 141 133 furioso 40 80 211 162 150 175 158 161 nonius 40 35 39 26 32 28 26 28 noric of murany 115 70 219 104 106 114 119 137 slovak sport pony 42 60 145 92 70 109 98 84 slovak warmblood 320 n.a. 1,794 868 925 863 836 857 eu regulation 1698/2005 (eu, 2005) allowed to provide support for in situ conservation (under article 39 (2,4)) and ex situ conservation (under article 39(5)) of genetic resources in agriculture. financial support for in situ conservation was defined as compensation of additional costs and income foregone resulting from the commitment made. eu regulation 1974/2006 (eu, 2006) and, in its annex iv, defined the thresholds for endangered local breeds eligible for in situ conservation support. these criteria included a number of purebred breeding females of local breeds in all eu member countries registered in the herdbook kept by an appropriate breeding organization. within the period 2007–2013, support for endangered breeds from the rural development programme (mp sr, 2007) was provided to one cattle breed (slovak pinzgau), one sheep breed (valachian), one goat breed (white shorthaired), eight horse breeds (slovak warmblood, hutsul, furioso, nonius, slovak sport pony, lipitsa, shagya arab, noric of murany) and eight poultry breeds (oravka hen, plymouth rock hen, rhode island red hen, new hampshire hen, vlaska hen, sussex hen, slovak white goose, suchovy goose). contrary to other european countries, subsidies were paid solely for livestock unit, without any differences made between sexes or level of endangerment or any further required goals (ligda and zjalic, 2011). during the next period (2014–2020), similar support for endangered breeds from rural development programme (mp sr, 2015) continued based on eu regulation 1305/2013 (eu, 2013). some changes were introduced in slovakia regarding the list of eligible breeds based on the experience from the previous period, while the same conditions for support were applied. since almost no support was provided to poultry breeds during the previous period, these breeds were excluded from the support scheme. on the other hand, three more sheep breeds (askanian merino, improved valachian, tsigai) and one goat breed (brown shorthaired) were included in the support scheme. eu regulation 807/2014 (eu, 2014a), introduced transitional provisions and the role of a relevant scientific body to identify endangered breeds. in slovakia, the assessment of endangerment was based partly on the criteria used in the previous period (number of registered purebred breeding females), as well as on effective population size calculated following falconer and mackay (1996). one of the main goals of angr conservation is to stop the loss of biological diversity of livestock. thanks to funding from rural development programmes, the negative trends of population sizes of endangered breeds from the 1990s were more or less stabilized in the 2000s (oravcová et al, 2010), and these trends were repeatedly reported by tomka et al (2016) and tomka and huba (2019). the number of registered purebred slovak pinzgau cows decreased in the period 2003–2006 (table 3), but it recovered to 2,000 females by 2010. the stabilized trend with small fluctuations may be observed both in population size (table 4) and the number of registered purebred females in the recent period 2010–2020 (table 3). it should be noted again that, while the number of registered females has been more or less stabilized at around 2,000 heads, the transfer of cows from milk production to cow–calf system is ongoing. this means breeders are trying to increase the competitiveness and profit generated from these cows. at the moment, it is unclear whether the number of registered purebred females would decrease if the subsidies were lowered for more competitive cows in cow–calf systems. the continuous decrease of improved valachian and tsigai populations and increase of the valachian population are significant (table 4). the number of regisgenetic resources (2022), 3 (6), 49–63 angr conservation in slovakia 57 tered purebred improved valachian and tsigai females decreased in the period without subsidies (2003–2016). this number levelled off in 2016 (table 3). this can be attributed to the start of subsidies provided to the breeders of these breeds. the decreasing trend of population size and stabilized number of registered purebred females after the implementation of subsidies suggest that further decrease could occur if the financial support is terminated. it may be assumed that in such a case, these populations will not become extinct, but their possible reduced sizes will lead to problems due to the availability of a smaller number of purebred breeding animals. on the other hand, the case of the original type of valachian sheep shows positive trends in the population size and number of registered purebred females. while only 50 females were registered in 2003 (table 3), recently, 900 females were registered in the herdbook. although these trends may be attributed to subsidies provided for this breed, breeders did not state that subsidies were the motivation for choosing and breeding this breed in the short survey conducted by nppc. the stable trend of askanian merino may be explained by the fact that there has been only one registered breeder of this breed in slovakia. two breeds of goats are eligible for financial support in slovakia. however, the trends in their population sizes are different (table 4). while the larger population of white shorthaired goats can be considered stable, the smaller population of brown shorthaired slightly increased during the period 2010–2020. different patterns may be observed in the number of registered females. the number of registered white shorthaired goats has been slightly decreasing since 2003, despite subsidies. on the other hand, a small increase of registered brown shorthaired goats can be observed even before support was provided (2003–2012) and the number stabilized in the following period 2012–2020. these findings show that providing financial support in slovakia does not automatically lead to an increase in population size or in the number of registered females. these findings also support the fact that the motivation of farmers plays an important role. in horses, the estimated population sizes are stable (table 4). the number of registered purebred females increased compared to the year 2003 (table 3) and can be considered stable in the last years (2016–2020). however, some of them (nonius, slovak sport pony) have been staying at very low levels since then and there is a big risk of losing these populations. while the nonius horse is a transboundary breed and cooperation with neighbouring countries can help to prevent the loss of the breed in the country, the population of slovak sport pony is extremely vulnerable due to its local distribution and the presence of only a few breeders. a positive effect of subsidies on the demographic development of endangered breeds in europe was presented by gicquel et al (2020) who argue that correctly setting the conditions and level of subsidies are crucial elements of a subsidiary system. based on previously published data (kompan, 2014), it can be speculated that while some population sizes increased over time thanks to a sufficient level of subsidies (e.g. in hungary and poland) and/or also thanks to the support only to active breeding animals in some countries (e.g. austria), subsidies had a stabilizing or even no effect in other countries including slovakia. on the other hand, an insufficient level of subsidies was identified as a possible reason for the continual decrease of supported animals, especially in horses (e.g. in austria). this may be explained by limited economic income from horse breeding, which requires higher compensation for income loss. stabilized trends of registered animals and the number of subsidy applicants in slovakia suggest that the support for in situ breeding of endangered breeds is set correctly to prevent population sizes, and most importantly the number of registered animals, from decreasing in most breeds. this means that the value of a subsidy is not so high to attract speculative breeders, who would primarily keep animals of endangered breeds only to profit from the subsidies and not for breeding and developing endangered breeds. this can be illustrated in the case of askanian merino. one could expect that farmers of traditional merino sheep would switch to breeding askanian merino since they are phylogenetically closely related breeds. however, after the introduction of financial support for the askanian breed, no dramatic change in the number of farmers and animals has been observed. the number of existing breeders of supported endangered breeds is not decreasing, so it can be assumed that, at the moment, subsidies provide sufficient support for these breeders to keep endangered breeds. such an approach is in line with strategic priority 8 of gpa, i.e. in the long-term perspective, the emphasis should be put on the sustainable use of local breeds without the need for support from public funds or extra funding. however, it is questionable whether the support would be efficient with lower subsidies and reflect the level of endangerment of breeds. it is also questionable whether the number of animals would increase if some requirements were implemented, e.g. only active breeding animals eligible for subsidies. strong tools to improve the competitiveness of local breeds are the products related to the breed (verrier et al, 2005). unfortunately, no breed-related animal products exist in slovakia. therefore, new ways of promoting products from endangered breeds have to be exploited. as an example of efforts, activities of the sheep and goat breeders’ association resulted in the official certifications of animal products that originate exclusively from slovak sheep and goat milk and meat. the aim of the ’golden sheep’ and ’golden goat’ certificates is to ensure the quality of animal products originating from sheep and goats, and fair price for producers. such efforts, however, are not aimed at the breed level, but rather at species. at the moment, slovakia has no animal products registered as protected designation of origin. however, there 58 tomka et al genetic resources (2022), 3 (6), 49–63 table 4. development of slovak breed populations in the period 2010–2020. species breed population size (monitoring data) 2010 2012 2014 2016 2018 2020 cattle slovak pinzgau 9,883 11,384 10,166 9,895 12,004 10,982 sheep valachian 2,391 2,372 2,231 2,437 2,834 2,906 tsigai 122,253 142,944 127,071 123,660 113,135 91,239 improved valachian 130,207 143,757 134,403 131,573 121,807 100,235 askanian merino 350 541 341 345 418 440 goat white shorthaired 8,389 6,798 7,685 8,036 8,166 7,576 brown shorthaired 1,485 812 1,137 1,627 1,912 1,901 horse lipitsa 750 500 575 775 900 900 shagya arab 600 500 500 650 700 700 hutsul 500 450 400 550 500 500 furioso 450 450 375 475 475 475 nonius 110 110 93 105 105 125 noric of murany 400 400 350 475 475 450 slovak sport pony 200 200 225 250 250 250 slovak warmblood 2,500 2,500 2,150 3,250 2,500 2,500 are several cheese products registered as a protected geographical indication, four of which are related to certain regions and the rest is related to the whole country. the connection of local breeds to these regions may bring some interest in consumers. the promotion of animal products coming from local breeds raised in protected areas may have a similar impact. this approach, however, needs the involvement of the environmental sector and the facilitation of protected areas used for sustainable grazing. this is in line with recent european strategies (the european green deal, biodiversity strategy for 2030, farm to fork strategy), which are calling for environment-friendly agriculture and high-quality animal products while referring also to local angr in this regard. more straightforward use of non-productive services of livestock may be found in some cases. while national legislation sets strict rules on farming in protected areas, the environment sector is already searching for livestock species and breeds that are adapted to specific natural conditions in order to use them for maintaining valuable ecosystems in the country. these measures may be presented by including extensive pasture of different livestock species as a management tool in the projects ’restoration and management of danube flood plain habitats (life14 nat/sk/001306)’ and ’restoration of natura 2000 sites in cross-border bratislava capital region (life10 nat/sk/000080)’. livestock animals in these projects are primarily used to maintain the traditional character of meadows and pastures while preventing the spread of invasive plant species and securing the wide biodiversity related to these ecosystems. in some cases, the production roles of livestock are neglected in favour of these nonproductive ecosystem maintenance services. in the longterm perspective, such an approach may lead to the loss of the breeds’ productive characteristics. some authors have pointed out that this non-productive approach may lead to the use of livestock solely for environmental reasons without any production benefits, and may be vulnerable to lack of external payments, making the sustainability of such measures questionable (wilson, 1996; evans and yarwood, 2000). recently, however, studies suggested that conservation should aim beyond genetic and production goals since the distribution of breeds is changing in favour of more productive environments, where the diversity of breeds is lower and areas with higher breed diversity are being abandoned (velado-alonso et al, 2020). awareness raising angr conservation has its specific features compared to wild biodiversity conservation. the most important one is the ownership of angr. while the government is committed to conserving angr under its jurisdiction, the animals are owned by private bodies. since the decision to keep animals lies on the private person, awareness raising of breeders about the characteristics, roles and benefits arising from breeding endangered livestock breeds is crucial. this is because in many cases, breeders of endangered breeds are hardly aware of the value of their animals as genetic resources (herold et al, 2012). economic aspects and modern technologies, which increase availability and enable the exchange of angr across europe and the world, make it very difficult to motivate breeders to prefer raising endangered and less productive breeds. in some cases, breeders follow ’fashion trends’ and decide to prefer an exotic breed because of its unique appearance or assumption of high profits from selling its progeny and products. they are often not aware of whether the breed is fit for their environment, and this may lead to low production. even in cases when only non-productive livestock services are expected, exotic breeds are preferred. of genetic resources (2022), 3 (6), 49–63 angr conservation in slovakia 59 table 5. swot analysis of angr conservation activities in slovakia strengths weaknesses • breeding activities of each breed in slovakia are covered by breeders’ associations. • data from herdbooks/studbooks and data from animal registration system are available. • angr sector has been a part of cross-sectoral policies. • open system of conservation allows to include new breeds in the list of supported breeds. • lack of national legislation and policies that specifically address conservation of angr. • lack of registration in poultry and rabbits. • limited inclusion of stakeholders and farmers in planning conservation activities and cryoconservation of angr. • limited transfer and implementation of results from research. opportunities threats • cooperation between breeders’ associations of neighbouring countries. • new technologies, establishment of genebanks and public funding to improve cryoconservation of angr. • new european strategies • introduction of new ways of valorization of angr products and services. • small number of breeders of local breeds. • conservation measures may be at risk if the funding is limited. • lack of awareness and personal motivation of breeders and their successors. • trends of preferring extensive systems and decreasing livestock numbers. course, local breeds are not the only ones able to provide services related to landscape and ecosystem maintenance (leroy et al, 2018), but some studies showed their specific abilities in harsh environments. in this case, awareness raising can help to promote local and endangered breeds, which can perform better in more diverse conditions. the already described short survey conducted by nppc showed that one of the main reasons to raise local breeds in the country is patriotism. this finding suggested that choosing a local breed depended on breeders’ enthusiasm and thus small populations may be vulnerable to changes in their motivation. results of the survey also suggested that breeding these animals is strongly connected to the breeder and low interest from the breeder’s successors can cause a further decrease in the local breed population. similar findings were already presented by yarwood and evans (1998) in the uk, who suggest that the motivation of new breeders is important. this can be primarily achieved by financial support, but in many cases improving and acknowledging the status of breeders of local breeds can increase the attractiveness of local breeds. in slovakia, breeders’ associations also have limited awareness of cryoconservation activities; the recent initiatives for cryoconservation are coming from state organizations, differently from neighbouring countries (czech republic, poland), where the management of preserved breeds’ sample acquisition and conservation involves farmers and their associations. limited involvement of breeders’ associations in decisions on angr sample selection is not considered a big problem in the initial phase of sample collection, but it may result in limited harmonization of cryoconservation and breeding programmes of endangered breeds in the future. therefore, angr cryoconservation should be promoted widely among breeders as a complement to in situ conservation. new schemes of cryoconservation funding should be investigated to improve the active involvement of breeders and the overall state of long-term conservation of angr. the predominant presence of big cooperatives and the separation of farmers from their land until the 1990s resulted in a weak connection between farmers and consumers at the national and local levels. after the 1990s, people started to return to their land. however, the establishment of direct channels between farmers and consumers is still underway. in this situation, raising public awareness about angr, their roles and products is very important for generating demand. general promotion at the national level brings attention to angr. at the local level, it is very important to also acknowledge the breeders who keep endangered breeds to facilitate the creation of local niche markets. swot analysis a swot analysis of angr conservation activities in slovakia was conducted in order to facilitate the preparation of a national conservation strategy (table 5). the absence of a legal basis for angr conservation makes it difficult to adopt any long-term conservation programme, clearly define breed categories and criteria to assess endangerment and provide related financial support, and complicates the operation of genebanks. there are also problems with records in poultry and rabbits, which hamper the identification and support of eligible farmers and animals. the adoption of a specific national strategy could facilitate this support. cross-sectoral policies allow closer cooperation with the environment and wild biodiversity sector and this increases awareness of agricultural biodiversity as part of overall biodiversity. they can also help in achieving 60 tomka et al genetic resources (2022), 3 (6), 49–63 common goals, including ecosystem maintenance and adaptation to climate change in the future. breeding activities are well organized in slovakia. this gives a good baseline for appropriate management of populations and population structure monitoring. breeders should not only act as keepers of angr and providers of their samples but should be involved in the decision-making of national cryoconservation goals and related activities. moreover, participation of private insemination and cryoconservation centres could bring new opportunities, e.g. long-term storage of samples from commercial breeds. the majority of livestock breeds in slovakia are considered transboundary as defined by fao. this creates an opportunity for breeds at risk to be recovered from resources coming from neighbouring countries. in this light, data from dad-is as a tool to provide information on transboundary breeds in different countries are very important. in some cases, even a sign of the presence of a breed in a country presented in dad-is may serve as a starting point to search for animals and farmers. on the other hand, a very small number of breeders of local breeds, like slovak sport pony, are considered a risk. big efforts should be made to attract new breeders to such breeds to spread the population among more breeders and lower the risk of losing the whole population when old breeders quit breeding or decide to change breed. early identification of and financial support to endangered breeds can serve well as a preventive tool. on the other hand, the number of animals of some endangered breeds may decrease despite financial support. this applies in particular to horse breeds, where economic income from breeding is limited. awareness raising and personal motivation of breeders and their successors are in many cases the key to keeping endangered breeds. modern trends of acquiring popular exotic breeds and the decreasing interest of breeders’ successors in animal breeding are a big challenge for the conservation of angr. connection of products to the environment, acknowledgement of breeders and monetary expression of non-productive services related to landscape maintenance may improve the valorization of angr products. changing attitudes of the public toward animal breeding and production, and corresponding european strategies create opportunities for more research activities in the field of non-productive roles of angr in extensive livestock systems with a positive impact on the environment and studies of unique traits that are linked to adaptability (and resilience) of local breeds. on the other hand, these european strategies may lead to favour extensive systems and decreasing livestock numbers to reduce emissions. this may also have a harmful effect on angr in the country. for example, local breeds, which are at present kept in bigger herds by traditional cooperatives, may be abandoned without or with limited replacement if smaller farmers decide not to keep them or to keep them in smaller herds in more extensive systems. author contributions ján tomka contributed to the concept and design of the manuscript. all authors contributed to drafting and reviewing the manuscript. conflict of interest statement the authors declare that no conflict of interest exists. acknowledgements this study was supported by the ministry of agriculture and rural development of the slovak republic, slovak republic (mprv sr), task of expert assistance (no. 2170-2100058): breeding and monitoring of animal genetic resources in the slovak republic. the authors are thankful to mrs zuzana salagova (mprv sr) for her involvement in this study. references blackburn, h. d. 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(1998). new places for ‘old spots’: the changing geographies of domestic livestock animals. society and animals 6(2), 137–165. doi: https://doi.org/10.1163/156853098x00122 https://doi.org/10.1038/s41598-020-77878-2 https://doi.org/10.1017/s1014233900005538 https://doi.org/10.1017/s1014233900005538 http://dx.doi.org/10.15414/afz.2015.18.02.25-29 https://doi.org/10.1016/j.jort.2021.100405 https://doi.org/10.1016/j.jort.2021.100405 https://doi.org/10.1016/0016-7185(96)00010-3 https://doi.org/10.1163/156853098x00122 introduction history of angr in the slovak republic legislation and policies monitoring conservation funding and valorization awareness raising swot analysis author contributions conflict of interest statement original article genetic resources (2024), 5 (10), 1–16 doi: 10.46265/genresj.pcwy8016 https://www.genresj.org issn: 2708-3764 collecting mediterranean wild species of the brassica oleracea group (brassica sect. brassica) lorenzo maggioni *,a, smiljana goreta ban b,c, sokrat janid, nenad jasprica e, simone treccarichi f, nina išić c and ferdinando branca f a european cooperative programme for plant genetic resources (ecpgr), c/o bioversity international, via di san domenico, 1, rome, 00153, italy b centre of excellence for biodiversity and molecular plant breeding, zagreb, croatia c institute of agriculture and tourism, k. huguesa 8, 52440, poreč, croatia d institute of plant genetic resources, agricultural university of tirana rruga ‘siri kodra‘, tirana, albania e institute for marine and coastal research, university of dubrovnik, kneza damjana jude 12, 20000, dubrovnik, croatia f dipartimento di agricoltura, alimentazione e ambiente (dia3), università di catania, via valdisavoia 5, 95123, catania, italy abstract: within the framework of the project eubraswild (capturing brassica wild relatives diversity in southeastern europe), several collecting missions were organized, targeting wild brassica complex species (2n = 18), belonging to the gene pool of brassica oleracea l. these crop wild relatives have repeatedly shown their potential to contain useful alleles for biotic and abiotic stress resistance, and nutritional or health-beneficial traits that can be easily intercrossed with the related crop. the missions described in this paper aimed to collect taxa that are poorly represented in public genebanks or databases for long-term conservation. this report describes missions carried out by national teams in albania, croatia and italy (ponza and sicily), including highlights of newly discovered locations. keywords: brassica cretica, brassica drepanensis, brassica incana, brassica macrocarpa, brassica montana, brassica oleracea, brassica rupestris, brassica villosa, crop wild relatives, collecting missions citation: maggioni, l., goreta ban, s., jani, s., jasprica, n., treccarichi, s., ǐsić, n., branca, f. (2024). collecting mediterranean wild species of the brassica oleracea group (brassica sect. brassica). genetic resources 5 (10), 1–16. doi: 10.46265/genresj.pcwy8016. © copyright 2024 the authors. this is an open access article distributed under the terms of the creative commons attribution license (cc by 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. introduction the cole crops (brassica oleracea l.) comprise several types of commercially important vegetables, including cabbage, cauliflower and broccoli (branca, 2008). these crops share the same c genome with 2n = 18 chromosomes and easily intercross with several wild species native to the mediterranean area, such as b. cretica lam., b. drepanensis (caruel) damanti, b. incana ten., b. insularis moris, b. macrocarpa guss., b. montana pourr., b. rupestris raf., b. villosa ∗corresponding author: lorenzo maggioni (l.maggioni@cgiar.org) biv., included in brassica section brassica (snogerup et al, 1990; bothmer et al, 1995). these crop wild relatives are potential providers of agronomically useful traits and can serve as a source of suitable alleles that may have been lost during the domestication process. for example, in b. incana, resistance against various fungal diseases has been identified, such as verticillium wilt (verticillium longisporum) and sclerotinia sclerotiorum (happstadius et al, 2003; mei et al, 2011; taylor et al, 2018); in b. insularis against leptosphaeria maculans (mithen and magrath, 1992) and pyrenopeziza brassicae (light leaf spot disease) (bradburne et al, 1999); in b. villosa against downy mildew (hyaloperonospora brassicae) (coelho received: 11.03.2024 accepted: 25.06.2024 published online: 29.07.2024 https://www.genresj.org https://www.doi.org/10.46265/genresj.pcwy8016 https://www.genresj.org https://www.doi.org/10.46265/genresj.pcwy8016 mailto:l.maggioni@cgiar.org 2 maggioni et al genetic resources (2024), 5 (10), 1–16 et al, 2018). recently, resistance to xanthomonas campestris was identified in one accession of b. montana collected in 2005 by l. maggioni on the island of ponza, italy, differently from what was reported in sheng et al (2020) (f. branca, pers. comm.). various types of insect resistance were studied in b. villosa, b. incana and b. montana (palaniswamy and bodnaryk, 1994; ellis et al, 1999; pelgrom et al, 2015; vosman et al, 2015). furthermore, zhang et al (2016) carried out a comparative transcriptome analysis to evaluate the resistance against plasmodiophora brassicae in broccoli and b. macrocarpa. their analysis revealed the activation of different metabolic pathways in b. macrocarpa, which enabled it to overcome the pathogen infection. the opportunity to breed for higher content of potentially beneficial (antioxidant and anti-cancer) glucosinolates has been studied (picchi et al, 2020; arena et al, 2022; bianchi et al, 2024) and used for the development of the ‘beneforte’ broccoli, resulting from a cross with b. villosa from sicily (mithen, 2014). the wide genetic variability expressed by brassica wild relatives has also facilitated the detection of distinct allelic patterns associated with morphometric traits in wild relatives compared to their cultivated counterparts (treccarichi et al, 2023). the importance of wild relatives of b. oleracea and the high priority for their seed conservation were highlighted as far back as 1981 in a plan of action report prepared by international experts for the international board for plant genetic resources (ibpgr), with the title genetic resources of cruciferous crops (ibpgr, 1980). a plan of action was subsequently accepted and supported by ibpgr as the ‘germplasm conservation of wild (n = 9) mediterranean brassicas project’ (gustafsson, 1982; gustafsson et al, 1985). six collecting missions were funded between 1982 and 1988 targeting cyprus, france, greece, italy, spain, tunisia, turkey and the united kingdom (gómez-campo et al, 1994). an agreement was signed in 1981 between ibpgr and the genebank of the universidad politécnica, madrid (upm), to hold a global collection of wild relatives of cruciferous crops, as part of the register of base collections, with agreed responsibilities for long-term conservation and distribution to bona fide users (maggioni, 2010). therefore, all samples were deposited at upm, as well as safety-duplicated in the country of origin and other genebanks. despite these arrangements, a survey of the status of brassica wild relative accessions (maggioni et al, 2013) revealed that most species and localities were underrepresented in collections. the main geographical gaps were the adriatic coasts (albania, croatia and italy), the aegean coasts (greece and turkey), northern cyprus and the coastal cliffs of tunisia. moreover, the availability of accessions with a standard material transfer agreement (smta) was very limited, owing to the lack of sufficient seed, since brassicas are laborious and expensive to multiply. in other cases, there was reluctance to share wild brassicas, even though the entire brassica complex is fully included in annex i of the international treaty on plant genetic resources for food and agriculture (fao, 2009). in particular, upm became unable to guarantee sufficient multiplication and distribution of samples (gonzalez-benito, 2010). similar difficulties were faced by other genebanks. additionally, some geographic areas had not been covered by the international collecting missions, such as the croatian coast, observations of new sites have constantly expanded the known distribution range of these wild relatives, and new species were proposed, such as b. tyrrhena giotta, piccitto & arrigoni (giotta et al, 2002) and b. trichocarpa c. brullo, brullo, giusso & ilardi (brullo et al, 2013). the most comprehensive distribution map and list of observations of all the c genome wild brassica species still remains the one prepared by snogerup et al (1990), although it is today outdated and would require a thorough revision. a small project titled ‘capturing brassica wild relatives diversity in southeastern europe (eubraswild)’, funded by the european cooperative programme for plant genetic resources (ecpgr), was recently set up under the coordination of smiljana goreta ban, institute of agriculture and tourism, poreč, croatia, involving partners from seven countries of the south balkan area and italy. among the objectives of this project, which was dedicated to monitor, collect and characterize various populations of crop wild relatives in the brassicaceae family, there was also the exploration, collecting and multiplication of (2n = 18) wild brassica populations. this paper describes the collecting missions carried out in albania, croatia and italy, including background information, methodology and results, focusing on b. cretica, b. drepanensis, b. incana, b. macrocarpa, b. montana, b. rupestris and b. villosa. knowledge about the distribution range and existing collections in europe and their gaps for the above species is briefly summarized below. taxonomy names and synonyms are according to the grin taxonomy (wiersema and schori, 2022) and euro+med (2006) for taxa not treated by grin. brassica cretica lam. [synonym: brassica oleracea l. subsp. cretica (lam.) gladis & k.hammer] the distribution range of this species covers mainly coastal areas of albania, greece, west turkey, central and south lebanon (possibly introduced) and israel at mount carmel (also possibly introduced) (snogerup et al, 1990; barina et al, 2011; flora ionica working group, 2016). collections documented in the european search catalogue for plant genetic resources (eurisco, http://eurisco.ecpgr.org), consist of 120 accessions, conserved in greece (43), spain (42), albania (17), the united kingdom (10), germany (5), israel (2) and italy (1). these originate from greece (85), albania (17), turkey (10), israel (3) lebanon (3) and unknown (2). accessions from albania have recently been added to the albanian genebank as a result of the eubraswild activity, whereby a thorough exploration genetic resources (2024), 5 (10), 1–16 collecting wild brassica in the mediterranean 3 has filled gaps in knowledge and material, as described in this paper. other areas of greece and turkey are not well covered by the documented collections of this species. brassica drepanensis (caruel) damanti [synonym: brassica villosa biv. subsp. drepanensis (caruel) raimondo & mazzola] this taxon is endemic to northwest sicily, italy, and limited to four locations in the province of trapani, with an ‘endangered’ status, according to the iucn red list of threatened species (maggioni and domina, 2020). urbanization and fires are likely to threaten all known subpopulations of this species, especially below the cliffs of mount erice (snogerup et al, 1990). only thirteen accessions clearly referable to this taxon are documented in eurisco. four of these are conserved at ipk in germany, four at upm madrid, spain and one in warwick, united kingdom, all collected around the town of erice. one sample is conserved at the university of catania, italy, received as a duplicate from ipk, germany. other three accessions, with undisclosed location of collecting, are conserved in hungary (2) and the united kingdom (1). two accessions documented under the name b. villosa subsp. drepanensis, conserved at upm, spain, were collected outside the recognized distribution area of this taxon, which raises doubts about their correct identification. the population in erice has been the most collected, which is justified by the fact that it is relatively small and probably the most threatened due to human activities. other small populations are included in protected areas (zingaro nature reserve and monte cofano, capo san vito e monte sparagio special protection area) (raimondo et al, 1991). however, they are not subject to active conservation and are not regularly monitored. therefore, they would deserve to also be secured in a genebank. although local collections of these populations may exist in sicilian universities or botanic gardens, the italian national inventory displayed in eurisco is deficient as far as b. drepanensis is concerned. brassica incana ten. [synonyms: brassica botteri vis.; brassica cazzae ginzb. & teyber; brassica mollis vis., brassica taurica (tzvelev) tzvelev] this species is distributed in tyrrhenian coastal areas of central and south italy, northeast and east sicily, adriatic coastal localities in puglia and croatian islands, south albanian coast and greek ionian islands (snogerup et al, 1990; baldini, 1995; castellano and bazan, 2009; anzalone et al, 2010; barina et al, 2011; maggioni, 2015; flora ionica working group, 2016; bartolucci et al, 2018). one population growing in crimea, farther apart from the rest of the distribution range, has been considered as an introduced sample (snogerup et al, 1990) and was interpreted as a feral lineage by mabry et al (2021). based on eurisco, 53 accessions are conserved in european genebanks in spain (28), the united kingdom (11), germany (7), albania (5) and italy (2). overall, these samples originate from italy (37, of which 17 from campania, 16 from sicily, 2 from tuscany, 1 from lazio and 1 from puglia), albania (5), ukraine (3), croatia (1) and unknown (7). this collection appears largely unrepresentative of the diversity and geographic distribution of this species, with several gaps from the italian distribution area, as well as from croatia and greece, the latter two totally missing, except for one sample from korčula, croatia. particularly poor is the collection maintained in italy, which declares to conserve only two samples, even though the country hosts the widest number of existing populations within its territory. brassica macrocarpa guss. [synonyms: brassica oleracea l. subsp. macrocarpa (guss.) gladis & k. hammer; eruca macrocarpa (guss.) caruel] this is an endemism of egadi islands, sicily, which is present only on the two islands of favignana and marettimo, while it has no longer been found on the island of levanzo (maggioni et al, 1996). this species is classified as critically endangered in the iucn red list of threatened species, due to its very narrow extent of occurrence, area of occupancy and risk of decline due to the possible human disturbance of its habitat (branca and tribulato, 2011). according to eurisco, 23 accessions are conserved in germany (2), spain (4) and the united kingdom (17). most of them were collected in favignana and two of them are from marettimo. brassica montana pourr. [synonyms: brassica robertiana j. gay; b. oleracea l. subsp. robertiana (gay) rouy et foucaud] b. montana is distributed along the northern mediterranean coasts of spain (gerona province), south france and italy (liguria and northern tuscany) (snogerup et al, 1990). it was formerly present in the apennine mountains of emilia romagna, but its occurrence there could not be confirmed recently (maggioni and alessandrini, 2019). it reappears further east in the republic of san marino near the italian adriatic coast and then in ancona and at monte conero (maggioni and alessandrini, 2019). on the tyrrhenian side of italy, it grows on the pontine islands (anzalone et al, 2010). punctual observations have been reported also further south in campania, basilicata and calabria (bartolucci et al, 2018), however, current presence and actual distribution in southern italy would require a thorough investigation. collections documented in eurisco with at least approximately known and reliable collecting locations comprise 57 accessions. these originate from gerona province in spain (16), south france (26) and italy (15, of which 9 from liguria, 4 from tuscany and 2 4 maggioni et al genetic resources (2024), 5 (10), 1–16 figure 1. collecting sites in italy (1), croatia (2) and albania (3). map created with qgis v3.28.11-firenze, (cc by-sa 3.0) from marche regions). gaps in ex situ collections are especially evident for central and south italy. the recent account that one sample from ponza was highly resistant to xanthomonas campestris (sheng et al, 2020) raised increased interest and the need to secure the conservation of populations from the pontine islands. brassica rupestris raf. [synonym: brassica oleracea l. subsp. rupestris (raf.) gladis & k. hammer] it is distributed mainly in north and west sicily (snogerup et al, 1990) and also found in southeastern calabria (hammer et al, 1987). more recently, few sites have been identified also in eastern sicily (branca pers. comm.) and southwestern calabria (maggioni, pers. comm.). according to eurisco, european genebanks only hold 22 accessions from western sicily, conserved in spain (13), germany (4), the united kingdom (3) and italy (2). the genetic diversity of a few localized populations from the provinces of palermo and reggio calabria has been studied extensively (maggioni et al, 2014). otherwise, a thorough analysis of the geographic distribution and diversity of this species is lacking and serious gaps exist in the ex situ european collections. brassica villosa biv. [synonyms: brassica villosa biv. subsp. tinei (lojac.) raimondo & mazzola; brassica villosa biv. subsp. bivoniana (mazzola & raimondo) raimondo & mazzola; brassica villosa biv. subsp. villosa] b. villosa is endemic to the northwestern and central part of sicily, in a few cases coastal, otherwise mostly found at inland cliffs localities (snogerup et al, 1990). various taxonomic treatments exist for this taxon, which is structured into various subspecies according to malfa et al (2020), also including b. drepanensis as a subspecies of b. villosa. according to eurisco, only 26 accessions of b. villosa are conserved in european genebanks, excluding those samples referring to b. drepanensis. these are conserved in the united kingdom (11), spain (7), germany (5), italy (1), the netherlands (1) and sweden (1). in some cases, the location of the collecting is not indicated, or it is placed in eastern sicily, which makes the taxonomic attribution doubtful. based on these data, the overall european ex situ collection of b. villosa is probably underrepresented. a thorough taxonomic revision including molecular markers and a better definition of the actual distribution range of b. villosa would also be beneficial and enable defining gaps in the existing collections. collecting missions – methods and results collecting missions were carried out by project partner teams in albania, croatia and italy (lazio and sicily). the composition of the teams, preparatory steps, itineraries and results are summarized below for each mission. all the collection sites are shown on a map of europe in figure 1. information on collected accessions is summarized in table 1. g enetic resources (2024),5 (10),1–16 collecting w ild b rassica in the m editerranean 5 table 1. summary of surveyed populations and collected accessions. accessibility: a, the entire population or up to 80% of the plants are accessible; b, between 20% and 80%; c, less than 20% accessible and d, the entire population is inaccessible unless special equipment is used. taxon fao country code locality collecting date collecting numbers gps coordinates receiving genebank (fao code) genebank accession numbers collected material status of the population brassica cretica lam. alb vlore, tuneli, depo ujit jul 2020 sj;nh;lf019, sj;nh;lf020, sj;nh;lf021 40.421485; 19.488230 alb026 agb4451; agb4452; agb4453; seed small population; accessibility: b alb vlore, uji ftohtë, moli ujit jul 2020 sj;nh;lf022, sj;nh;lf023 40.421293; 19.487295 alb026 agb4454; agb4455 seed small population; accessibility: c alb vlore/barrestaurant kala jul 2020 sj;nh;lf024, sj;nh;lf025, sj;nh;lf026, sj;nh;lf027 40.412923; 19.480489 alb026 agb4456; agb4457; agb4458; agb4459 seed few individuals; accessibility: c alb vlore/resort marina bay jul 2020 sj;nh;lf029, sj;nh;lf030 40.415465; 19.482669 alb026 agb4461; agb4462 seed few individuals; accessibility: b; endangered by constructions alb radhimë jul 2020 sj;nh;lf028 40.412221; 19.481160 alb026 agb4460 seed few individuals; accessibility: b alb sazan island – south side jul 2021 sj;nh;lf039, sj;nh;lf040, sj;nh;lf041 40.480726; 19.286550 alb026 agb4471; agb4472; agb4473; seed large population; accessibility: b alb sazan island – north side jul 2021 sj;nh;lf042, sj;nh;lf043 40.506662; 19.276134 alb026 agb4474; agb4475; seed large population; accessibility: b alb alican – karabuni jul 2021 40.315463; 19.379214 not collected relatively large population; accessibility: d alb palermo rock jul 2021 40.051141; 19.792803 not collected small population; accessibility: b brassica drepanensis (caruel) damanti ita erice 25 jul 2021 38.03505556; 12.59147222 ita331 unict 5288 seed abundant; accessibility: b continued on next page 6 m aggioniet al g enetic resources (2024),5 (10),1–16 table 1 continued taxon fao country code locality collecting date collecting numbers gps coordinates receiving genebank (fao code) genebank accession numbers collected material status of the population brassica incana ten. alb himarë /potam beach jul 2020 sj;nh;lf032, sj;nh;lf033, sj;nh;lf034, sj;nh;lf035, sj;nh;lf036 40.089685; 19.752504 40.088730; 19.750805 alb026 agb4464; agb4465; agb4466; agb4467; agb4468 seed relatively large population; accessibility: a/b alb gijpe 40.128361; 19.672164 not collected small population; accessibility: b hrv sušac jul 2022 ipt521 42.752716, 16.490663 hrv050 ipt521 seed <20 plants; accessibility: b hrv kosor jul 2022 ipt518 42.901346, 16.761636 hrv050 ipt518 seed 20 plants; accessibility: a hrv stupa jul 2022 ipt522 42.894560; 16.786568 hrv050 ipt522 seed 10 plants; accessibility: a hrv obljak jul 2022 ipt520 42.904644, 16.749480 hrv050 ipt520 seed 20 plants; accessibility: a hrv koločep oct 2021 ipt517 42.668715, 18.014589 hrv050 ipt517 seed 30 (–40) plants; accessibility: b hrv svetac may 2023 ipt 618 43.019725, 15.728069 hrv050 ipt 618 seeds and small plants 20 plants; accessibility: b hrv vis (oključina) may 2023 ipt 619 43.074649, 16.102745 hrv050 ipt 619 seeds and small plants 15 plants; accessibility: b hrv vis (gradac) may 2023 ipt 620 43.075623, 16.134977 hrv050 ipt 620 seeds and small plants 10 plants; accessibility: b hrv vis (st. duh) may 2023 43.036415, 16.114780 not collected 3 plants; accessibility: b hrv vis (hiking trail from st. duh church to st. nikola church) may 2023 ipt 621 43.034960, 16.110665 hrv050 ipt 621 seeds 5 plants; accessibility: b hrv vis (crvene stijene) may 2023 ipt 622 43.047342, 16.107393 hrv050 ipt 622 seeds and small plants 20–30 plants; accessibility: a continued on next page g enetic resources (2024),5 (10),1–16 collecting w ild b rassica in the m editerranean 7 table 1 continued taxon fao country code locality collecting date collecting numbers gps coordinates receiving genebank (fao code) genebank accession numbers collected material status of the population brassica incana ten. hrv vis (st. mihovil church) may 2023 ipt 623 43.048160, 16.112421 hrv050 ipt 623 seeds 5 plants; accessibility: b ita sortino 9 june 2022 37.133350, 15.031676 ita331 unict 5310 shoots for cutting very few individuals; accessibility: c ita francavilla 20 jul 2021 37.90494444; 15.12491667 ita331 unict 5311 seed, shoots for cutting and small plants abundant; accessibility: c brassica juncea (l.) czern. alb himarë june 2020 sj;nh;lf037, sj;nh;lf038 40.101337; 19.741662 alb026 agb4469; agb4470 seed brassica macrocarpa guss. ita favignana 25 jul 2021 37.92108333; 12.31330556 ita331 unict 5289 seed abundant; accessibility: c ita favignana 25 jul 2021 37.93400000; 12.31694444 ita331 unict 5308 seed abundant; accessibility: c ita marettimo 25 jul 2021 37.94880556; 12.08336111 ita331 unict 5309 not collected very few individuals; accessibility: d brassica montana pourr. ita ponza june 2021 lm21-01; lm21-02 40.888889; 12.953056 ita331 and orto botanico roma unict 2306 unict 5316 seed very few individuals; accessibility: a; endangered by landslides ita ponza june 2021 40.879751; 12.953690 not collected very few individuals; accessibility: d continued on next page 8 m aggioniet al g enetic resources (2024),5 (10),1–16 table 1 continued taxon fao country code locality collecting date collecting numbers gps coordinates receiving genebank (fao code) genebank accession numbers collected material status of the population brassica rupestris raf. ita corleone 25 may 2021 37.798463, 13.307139 ita331 unict 5383 unict 5326 unict 5327 unict 5328 seed abundant; accessibility: b ita palermo 25 may 2021 38.16830556; 13.35211111 ita331 unict 5290 seed abundant; accessibility: b brassica rupestris subsp. tardarae (ilardi & al.) raimondo ita sambuca di sicilia 25 may 2021 37.615472; 13.053333 ita331 unict 5286 seed less than 100 individuals; accessibility: c brassica villosa biv. ita caltabellotta 25 may 2021 37.57669444; 13.16616667 ita331 unict from 5329 to 5335 seed few individuals; accessibility: c brassica villosa subsp. tineoi (lojac.) raimondo & mazzola ita butera 23 mar 2022 37.21597222; 14.00886111 ita331 unict 5291 shoots for cuttings abundant; accessibility: c ita marianopoli 23 mar 2022 37.597965; 13.923801 ita331 unict 5292 shoots for cuttings very few individuals; accessibility: b ita butera 20 oct 2022 37.21577778; 14.01016667 ita331 unict 5312 seed endangered; accessibility: c genetic resources (2024), 5 (10), 1–16 collecting wild brassica in the mediterranean 9 albania under the leadership of sokrat jani, the institute of plant genetic resources, agricultural university of tirana, organized a mission targeting the exploration, identification and collection of seed samples of wild relatives of brassica (b. cretica lam. and b. incana ten.) in ionian coast area and sazan island. the collecting team was composed of sokrat jani, institute of plant genetic resources, tirana, nexhip hysolakoj, regional agency of protected areas of vlora and lavdosh ferruni, organic agriculture association of albania. the travel target areas were the ionian coast of vlora and himarë, and sazan island, a small uninhabited island with an area of 5.7km2 and a coastline of about 21km, at the gulf of vlora. during the years 2019–2021, nine field missions were undertaken, three each year respectively around 2–20 april, which coincides with the blooming phase; 10–20 may, which coincides with the fruiting period and 20 june–10 july, which coincides with the seed ripening period. to reach sazan island, the team used the boat of the regional agency of protected areas of vlora. there are no regular roads for driving on the island, therefore the group explored the area on foot. information available about the sites to visit was based on literature, herbarium specimens and personal knowledge. the first documentation about the presence of two maritime brassica species in the ionian coastal region of albania was published in flora of albania for two localities, spile (himarë) for b. incana, and vlora for b. oleracea subsp. oleracea (paparisto and qosja, 1976). a brief description of b. incana at himarë is given in flore de l’albanie (qosja et al, 1996). based on the material collected during their field trips to albania from 2008 to 2010, barina et al (2011) presented some confirmations for chasmophytic brassica species in three localities, of which two are the same as those of paparisto and qosja (1976). in spile bay (potam beach) at himarë, they confirmed the occurrence of b. incana, while b. cretica was observed near vlora, about 4km north of the village of radhimë, and in the porto palermo peninsula. subsequently, during a field trip on 4 april 2019 a group consisting of lorenzo maggioni (ecpgr secretariat), eva thörn (ecpgr executive committee), imke thormann (federal office of agriculture and food, germany), fetah elezi and sokrat jani from agricultural university of tirana, surveyed the presence of a small population of 12 reproductive plants and some vegetative individuals of b. cretica in the front part of the cliff, on both sides of the coast road tunnel at uje i ftohtë, as well as a small population on the southwest side of the coast, near bar kalaja. further south, by the potam beach (himarë), at the base of the cliff, on its northwestern side, a mediumsized population of about 30 reproductive plants and some vegetative individuals of b. incana was surveyed by the same group. both species were then in the flowering phase. these expeditions aimed at gathering information, exploring more localities for possible other population sites, finding and identifying species, assessing the situation, evaluating vegetation status and habitats, and collecting seed material for conservation and use. samples were identified in the field, based on morphological characters. during the ripening period, seed samples were collected from most populations. this was done according to a collection protocol trying to capture the maximum variability of the population (from as many plants as possible), while avoiding harm to the populations themselves (harvesting only a small part of the seeds produced by any plant). mature siliquas were collected from individual plants and kept separately in cotton bags. the size of the populations was estimated, distinguishing vegetative and reproductive individuals, as well as human and livestock accessibility. gps coordinates were taken with a gpsmap 60csx. after return from the field, the collected seed samples, after being processed, were included in the albanian genebank, tirana, for longterm storage. as the terrain was difficult, the working group was supported with the necessary tools and equipment, such as cars, boats, drones, professional cameras, etc., by the regional agency of protected areas, vlora. results out of 23 sites visited with a suitable habitat, wild brassicas were observed in 13 of them. the results obtained from the exploration are described below. ionian coast area of vlora the presence of b. cretica was observed in five sites in the vlora bay area (two sites near the tunnel area in uje i ftohtë, and respectively one each in marina bay resort, bar-restaurant-kala, and radhimë, part of kala road) and one on the karaburun peninsula (in the western part, from alicani to bristani bay): tunnel area of uje i ftohtë, in front of the cliff, northwest side: two populations of b. cretica were identified there, respectively to the left and right of the tunnel entrance, called depo ujit and moli ujit, with 12 and eight reproductive plants, respectively, and very few vegetative individuals (new plants). these sites were respectively rated average and slightly accessible by humans and livestock. seed was collected from three and two plants, respectively, separately in cotton bags. bar-restaurant kala area, south side: the site was first surveyed in 2019, and it was surprising in 2020 to note the disappearance of a small population of b. cretica from every accessible site along the base of the cliff, due to mowing of the area by resort gardeners. not a single plant remained on the cliff, except at the bottom of it, in the inaccessible part, where only ten reproductive plants and very few young plants could be counted. seeds were collected in 2020 from four plants, separately in cotton bags, with the help of two young climbers. radhimë, part of kala road, west side: it seemed to be a newly established population, with 18 small reproductive plants and very few mature siliquas and 10 maggioni et al genetic resources (2024), 5 (10), 1–16 many new plants, possibly formed by migrations from a now extinct b. cretica population of jonufra (4km north of radhimë), which was an old population documented by paparisto and qosja (1976) and later confirmed by barina et al (2011). seed was collected from only one plant. resort marina bay area, north side: at this site, two populations of b. cretica were located very close to each other in the same area and only 200–300m away from each other (marina bay and resort gjyla), with overall 12 reproductive plants and very few young plants; these were moderately accessible to humans and endangered of disappearance due to constructions in the tourist resort. seed was collected from the two matured plants. karaburuni peninsula, west side/alican – brisani bay: a site was explored with an area of 4,678m2 and a relatively large population of b. cretica, with a density of 1–2 reproductive plants/100m2. the first individual plants were identified in the alican site, on vertical rocks, an area isolated and inaccessible to humans or livestock. its extent, with few individual plants, was linear to the coast of the entire gulf of brisan. no seeds were collected, as the entire population was located on steep cliff tops and therefore inaccessible unless special equipment is used. skele neighbourhood of vlora: in addition to the target species (b. cretica and b. incana), a small population of eight to ten plants initially identified as b. rupestris raf. was found in a home garden in the coastal neighbourhood skele of the city of vlora. from the conversation with the owner of the home garden, it was learned that that population may have originated from a maritime limestone rock area about 7–8km away, called kuzumbaba, where he had taken soil five years before to use for the home garden; but with the soil, it seems, the seeds also came. however, following a survey of the described area by our team, no plants of b. rupestris could be found. seed was collected from home garden plants and sent to the albanian genebank, tirana. after closer observation, these plants more probably belong to a domesticated type of leafy kale. ionian coast area of himarë three sites were found with a good presence of b. incana, respectively in potam, gjipe bay and possibly b. cretica in the porto palermo peninsula. potam beach: in the southern part of the bay of spile (himarë), in a rocky strip with a length of about 2–3km, on maritime limestone rocks, the presence of a relatively large population of b. incana was reconfirmed, linear to the coast, with 28 reproductive plants and many new vegetative individuals, which was previously observed and documented by paparisto and qosja (1976) and later confirmed by barina et al (2011); every part of it was accessible along the base of the cliff. seed was collected from five plants. himarë seaport, south side: besides the two target species (b. cretica and b. incana), in a home garden, near the himarë seaport, a small population of four plants of b. juncea (l.) czern, apparently spontaneously grown was explored. seed was collected from two plants. unfortunately, our monitoring in the spring of 2022 revealed that this population had disappeared. other localities: other parts of the coast of himarë were surveyed, north and south of the bay of spile (himarë), but new brassica sites were found only in the gjipe bay (near gjipe canyon), about 15km north of himarë and on the peninsula of porto palermo, about 9km south of himarë, at a site known as the rock of porto palermo. it was surprising to notice the disappearance of the b. cretica population in the cave site of porto palermo, which was described in 2010 by barina et al (2011). however, a new and smaller population was found, about 1km northwest, in a segment with straight and steep rocks, in the place known as the rock of porto palermo. it seems that livestock may have influenced the migration of brassica vegetation from one site to the other. we estimated that in gjipe bay and the rock of porto palermo, near the falling rocks beach, 18 and 30 reproductive plants of b. incana and b. cretica (to be verified) were growing, and some vegetative individuals, but it was not possible to collect seeds from either site. sazan island area, east and northeast side the large presence of b. cretica on the island was surprising, mainly concentrated in two sites, the south cape and the north cape. the largest area with more reproductive plants and young plants was located in the south, in the gully of jehnem site, where the density was 30–40 reproductive plants/100m2. seeds were collected from the two sites, respectively from three and two plants. croatia under the leadership of nenad jasprica, institute for marine and coastal research, university of dubrovnik, and smiljana goreta ban, institute of agriculture and tourism (iptpo), poreč, two expedition missions were organized, targeting several wild brassica species in the southern croatian islands and islets. the goals of the expeditions were to check the status of the brassica species populations, to collect seeds or young plants from previously known sites and to estimate the possible losses that happened during previous periods. apart from n. jasprica and s. g. ban, the collecting team also consisted of nina ǐsić and dean ban from the institute of agriculture and tourism, poreč, branka salopek sondi from the ruer bošković institute, zagreb, and mirta tkalec and nataša bauer from the faculty of science, university of zagreb. preliminary information on the distribution, habitat, ecology and phenology of insular brassicas in croatia was obtained from the flora croatica database (nikolić (2005) and onwards). this database contained data from the literature, field observations and references to herbarium specimens, and allowed further detailed study with the aim of completing knowledge on the subject. genetic resources (2024), 5 (10), 1–16 collecting wild brassica in the mediterranean 11 the first expedition started on 5 july 2022, travelling to split and taking the ferry to vela luka located on the korčula island. the exploration of the island’s localities took place over two days, 6-7 july 2022. on 6 july, the team visited the islands sušac (lastovo islands nature park), and islets obljak, kosor and stupa (korčula archipelago) by speedboat, and on 7 july the collecting team visited two previously targeted localities (the pebble beach of vaja and the bay of samograd) near the račǐsće village on the northern coastline of korčula island. in addition, the island of koločep near the city of dubrovnik was also previously visited on 7 july 2020, 1 october 2021 as well as on 15 july 2022. during the first expedition, mature siliques were collected from four localities: the island of sušac, and the islets of obljak, kosor and stupa. gps coordinates were determined with an android device. seeds from one plant were collected on sušac island, while from each of the other three islands, seeds were collected from approximately ten plants. siliquas were kept in paper bags. the seeds were counted with contador optical seed counter and weighted. the second expedition took place between 22 and 26 may 2023. the expedition included localities previously mentioned in the literature on the island of vis and the island of svetac (locally named st. andrija) (nikolić (2005) and onwards). the exploration of the two islands took place over two days, 23-24 may 2023. on 23 may, the collecting team visited the svetac island by speedboat, followed by several localities on the vis island, mainly north-facing cliffs and small coves. on 24 may, the third day of the expedition, the team visited previously mentioned inland localities of vis, the first one being beneath st. duh church. next, the team followed a hiking trail from st. duh church to st. nikola church towards komiža city where the presence of a b. incana population was previously recorded. the team then travelled to a location known as crvene stijene (red rocks), a sport climbing area, where an abundant b. incana population was found. the next location was near st. mihovil church, while the presence of a military zone prohibited the members from visiting the location above podhumlje. the expedition members spent the fourth day travelling home. gps coordinates were determined with an android device. during the second expedition, unripe green siliquas were collected from six localities: the island of svetac, two coastal localities on the island of vis (oključina and gradac) and three inland localities on the island of vis (hiking trail near st. duh church, crvene stijene and st. mihovil). young plants for vegetative propagation were collected from the island of svetac, two coastal localities on the island of vis (oključina and gradac) and one inland locality on the island of vis (crvene stijene). siliquas were kept in paper bags. results koločep island the population of b. incana (locally named b. mollis) was quite numerous on the island (more than 50 individuals). it mainly inhabited the vertical sea cliffs oriented towards sw-se, reaching up to 60m a.s.l. on the vertical profile of the cliffs, it colonized the halotolerant vegetation of the saline areas up to the bushy vegetation and aleppo pine near the tops of the cliffs. sušac island, southwest side the population of b. incana (locally named b. cazzae) was reduced to a single reproductive plant seen by the expedition team near the sušac lighthouse. since the plant was on an inaccessible cliff, no seed was collected from this plant. vegetative individuals were found on the island in the halophilous vegetation of the salt-sprayed rocky cliffs at 90–110m a.s.l., but also in the maquis on the cliff top. sušac island, northwest side one reproductive plant and several young ones were found near the trail leading to the sušac lighthouse. mature siliquas were collected in a paper bag and one entire plant was taken for vegetative propagation. obljak, kosor and stupa islets on each of these three islets, the team found more than 20 individuals of b. incana. the mature siliquas of about ten plants from each islet were collected in paper bags. on obljak and kosor, the plants were found on the entire surface of the islets. most of the brassica population on stupa was on the northern slope of the islet. the species occured mainly in the herbaceous vegetation of the salt-sprayed low rocky coasts, but also in the maquis in the upper belt, which is only weakly influenced by the sea aerosol. two small bays near the village of račišće, korčula island the team found no individuals of b. incana at these two previously known sites, a result of habitat loss due to anthropogenic impacts. these sites consist of gravel shorelines exposed to wave action, resulting in a thicker or shallower layer of pebbles at the surface. nitrogenrich humus often forms beneath the surface. svetac island the b. incana population on the west coast of svetac island consisted of about 20 plants, a good portion of them in reproductive stage. the team collected unripe, green siliquas from three mature plants and two young plants. the siliquas were collected in paper bags. vis island in the locality oključina the team found about 15 plants, several of them in reproductive stage. the team collected unripe, green siliquas from three plants and took two young plants. in gradac cove the team found around ten b. incana plants and one plant morphologically similar but with smooth, hairless leaves and stems. several b. incana plants were in the reproductive stage and siliquas were taken from two plants along with two entire young plants. seeds were taken also from the morphologically similar species, and one young plant was collected just beneath the reproductive one. 12 maggioni et al genetic resources (2024), 5 (10), 1–16 in the inland location of st. duh the team only found three young b. incana plants in vegetative stage. due to the lack of siliquas and a very small number of individuals, no siliquas or plants were taken. on the hiking trail from st. duh church to st. nikola church in komiža, the team found five b. incana plants. several plants were in reproductive stage, so the team collected a small portion of unripe green siliquas from two plants. because it was only a small population, no young plants were taken. the locality crvene stijene hosted the most abundant population in the vis island, counting more than 20 b. incana plants, several of them in reproductive stage. the team collected a small portion of green siliquas from five plants and two young plants. around five plants were found near st. mihovil church with only two plants in reproductive stage. the team collected a small portion of green siliquas from the two plants and no young plants were collected. italy island of ponza, lazio, italy lorenzo maggioni, ecpgr secretariat, rome, carried a mission to the island of ponza on behalf of the university of catania (unict), targeting b. montana. travelling from rome to formia and then taking the ferry to ponza on 13 june 2021, the exploration of the island took place over two days, 14 and 15 june 2021 by foot and using the public bus. selected dates were suitable to find mature siliquas, based on previous experience. information available about the sites to visit was based on literature, herbarium specimens and personal knowledge. in their map of vegetation series of ponza, stanisci et al (2005) indicated the presence of b. montana as a companion species at localities monte guardia and faro. anzalone et al (2010) indicate the presence of b. montana in the pontine islands. herbarium specimens in the anzalone herbarium in rome [ro-ha] were collected from localities monte guardia in 1966 and forna in 1974. during a previous survey in march 2004, from the trail midway up to the top of monte guardia, northwest side, maggioni could count more than 200 flowering plants in a gully facing the famous cliff and beach of chiaia di luna. this gully was subsequently subject to a rock collapse which probably destroyed a large part of this population and the trail became almost inaccessible. other individual plants could also be observed on formerly cultivated abandoned terraces, closer to the top of the mountain. in july 2005, at the southwest side of monte guardia, maggioni surveyed the presence of a relatively large population at the base of the cliff, all along the trail reaching the lighthouse at punta della guardia. the objective of the present survey was to collect seeds from sites already known and explore the rest of the island for possible other population sites. mature siliquas were collected from individual plants and kept in separate paper bags. the size of the population was estimated, distinguishing vegetative and reproductive individuals. gps coordinates were taken with a cellular phone. after return from the field, siliquas were manually threshed in paper trays and the number of seeds was approximately counted (data not shown). results monte guardia, northwest side: it was confirmed that the relatively large population observed in 2004 had almost disappeared and only two reproductive plants and very few vegetative individuals could be found on or along the trail. seed was collected from the two plants [collecting numbers lm21-01 and lm21-02]. paper envelopes containing respectively ca. 200 and 100 seeds were sent to unict. one envelope with ca. 50 seeds of lm21-01 was also sent to the botanic garden of the university la sapienza in rome, italy (care of giuseppe fabrini). monte guardia southeast side: it was surprising to note the disappearance of b. montana from any accessible site along the base of the cliff. no plants could be identified on the cliff either, except at the very end of the trail, on the small promontory punta della guardia, hosting the lighthouse. here, less than ten vegetative plants could be observed in inaccessible positions. in recent years, this area has been subject to landslides that have disturbed the base of the cliff. also, the abundant presence of dittrichia viscosa (l.) greuter, widely colonizing the area, might have become a competitor to b. montana. other localities: other parts of the island were surveyed (descent to punta del fieno; piana d’incenso up to punta incenso; fortino del papa and surroundings; and descent to scogli della cantina) but no new sites were found. the locality forna was not thoroughly surveyed and might deserve a more careful check, considering that one herbarium sample was collected at this locality in 1974 by anzalone, but with no detailed site indications. sicily under the leadership of ferdinando branca, unict, italy, organized three missions targeting several wild brassica species growing in sicily. the unict team, represented also by the phd students maria concetta di bella, simone treccarichi, donata arena and giulio flavio rizzo, collected several samples in natural areas during three expeditions, also evaluating the status of the populations. in total, six expeditions were carried out, travelling by car from catania, between may 2021 and october 2022, targeting the provinces of palermo and agrigento (i), erice and egadi islands (ii), butera and marianopoli, province of caltanissetta (iii), sortino, province of siracusa (iv), francavilla di sicilia, province of messina (v) and butera and mount muculufa, province of caltanissetta (vi). the aim of the expeditions was to increase the availability of seeds for the biomorphological, biochemical and genetic characterization of these populations which were not available in genebanks. based on previous personal knowledge and local information, the team genetic resources (2024), 5 (10), 1–16 collecting wild brassica in the mediterranean 13 explored the above-mentioned sites to observe the status of the populations (distribution and estimated number of plants) and seed samples were collected based on the biological status of the plants. information regarding the collecting sites was obtained through google earth and by interviews with local residents. the collecting methodology encompassed either harvesting of siliquas from individual plants, which were kept separate in the case of b. rupestris of corleone and b. villosa of caltabellotta, or bulked from several plants for all other populations for which seeds were collected. additionally, vegetative cuttings were also collected. siliqua samples were stored in paper bags to avoid mould formation. subsequently, siliquas were cleaned at the university of catania, specifically in the lab of biotechnology of vegetable crops belonging to dipartimento di agricoltura, alimentazione e ambiente (di3a). as concerns the cutting method, it consisted of oblique cutting carried out with professional pruning shears. fresh material was stored in plastic bags to maintain high moisture conditions. results the first expedition (i) was carried out at the end of may 2021 and five different accessions were collected: two accessions of b. rupestris from corleone and monte pellegrino (palermo province), one b. rupestris subsp. tardarae (ilardi & al.) raimondo at gole della tardara, near sambuca di sicilia (agrigento province), one accession of b. villosa from caltabellotta (agrigento province). the first site is located south of corleone on the rocca cliffs, where an abundant population of b. rupestris is widespread. with regard to mt. pellegrino, an important population of b. rupestris was found around the cliffs of the monastery of santa rosalia in palermo, which is the type site for b. rupestris. the population of the subsp. tardarae was not abundant or much diversified in terms of plant age, whereas the population of b. villosa identified around pizzo telegrafo and the surrounding areas near caltabellotta was abundant. during the second expedition (ii) at the end of july 2021 in the province of trapani, three different accessions were collected. two accessions of b. macrocarpa were collected on the islands of favignana, in two different stations. of these two populations, the more abundant was located on monte santa caterina, spreading from the top of the mountain down to the sea coast. on the other hand, on the island of marettimo, a few individuals were noted in cliffs that were not well accessible around punta bassana. another population in marettimo was located on the top of monte falcone, but this was not visited. finally, one accession of b. drepanensis was collected on mount erice. the third expedition (iii) was carried out in march 2022 at mount muculufa, near butera and marianopoli, caltanissetta province, monitoring two populations of b. villosa subsp. tineoi (lojac.) raimondo & mazzola and collecting shoots for establishing rooted cuttings. of the two identified populations, the former was abundant whereas the latter was represented by few individuals. the fourth expedition (iv) was organized at sortino, siracusa province, in june 2022, monitoring the populations of b. incana and collecting shoots for establishing rooted cuttings. the fifth expedition (v) was organized at francavilla di sicilia, messina province, in july 2022, collecting b. incana seeds and cuttings for rooting. finally, the sixth and last expedition (vi) was organized near ravanusa exploring monte muculufa, caltanissetta province, in october 2022, where a population of b. villosa was found in a chalk quarry, endangered by human activities (caves for salt extraction) and fires. conclusion although the distribution of the wild populations belonging to the gene pool 2n = 18 of b. oleracea has been intensely investigated, starting in the 1970s, a full overview of all the existing populations is far from complete. even for well-known populations, major gaps remain in the available documentation of the european genebanks and access to documented populations is often uncertain or not guaranteed, owing to limited seed availability, difficult and expensive multiplication or other limitations. the ecpgr-funded eubraswild project enabled collecting activities focused on some of the less documented locations within the distribution range of these wild brassicas. each mission was carried out by local teams within their country, with the intention to eventually make both information and genetic material publicly available. as indicated in table 1, all accessions have been deposited in the respective genebanks with an assigned accession number. requests from potential users can be immediately honoured in case of sufficient seed availability. the missions carried out in albania were for the first time specifically dedicated to these taxa and allowed to describe with a good degree of accuracy several populations of b. incana and b. cretica, including some locations that had never been described before (b. cretica in the sazan island and karaburun peninsula and b. incana in the gjipe bay). in other cases, habitat loss due to direct or indirect human intervention is affecting the size or the existence of some populations. for the first time, seed samples were collected for long-term conservation, deposited in the albanian genebank and publicly documented in the eurisco catalogue. the croatian missions were successful in monitoring already known populations of b. incana and collecting seed for long-term conservation and further characterization. the possible disappearance of two populations on the island of korčula due to habitat loss was an unpleasant observation. the collected populations will be documented in the eurisco catalogue. the expeditions carried out in italy also enabled monitoring of existing populations and discovering previously unknown sites, such as the case of b. villosa subsp. tineoi near butera and to monitor the populations of the already-known sites. a few sites were observed, where the populations are endangered by human 14 maggioni et al genetic resources (2024), 5 (10), 1–16 activities. on the island of ponza, it was significant to notice the severe reduction, close to disappearance, of b. montana, possibly due to a change of habitat determined by natural causes. the few seeds collected may deserve careful attention if it is confirmed that resistance to xanthomonas has been found in a plant from ponza. the very poor documentation in eurisco of the populations conserved in italy is improving as a result of this activity. overall, the activities carried out in this project have added important pieces of information on the status and trends of a few mediterranean wild brassica populations, have increased the level of collaboration across countries and obtained material which will be useful for further collaboration regarding its characterization and possible use. given the importance and immense diversity potential of the gene pool of b. oleracea, which is a unique resource of the european and mediterranean region, it is hoped that more systematic investigations with larger breadth and funding can be planned in the future (including to sort out a complicated taxonomy which is currently only based on very unstable and unreliable morphological characters). gathered information should also be useful to raise the awareness of national and local authorities about the need to take action for in situ conservation of the most endangered populations. acknowledgements collecting expeditions to sicily and croatia were partly funded through the ecpgr grant scheme activity eubraswild. collection in ponza was funded by the ecpgr secretariat travel funds. the albanian missions for exploration and collecting seed samples in the ionian coast of vlora and sazani island were financed by oaa (organic agriculture association). expeditions in croatia were partly financed by the project ‘agrobiodiversity – the basis for adapting and mitigating the consequences of climate change in agriculture (kk.05.1.1.02.0005)’. the authors wish to thank filip varga for the creation of the maps. author contributions fb, lm, sgb and sj contributed to the overall conception and planning of the collecting missions. these were carried out by sj and colleagues in albania, sgb, nj, ni and colleagues in croatia, lm in italy (ponza), fb, st and colleagues in italy (sicily). the first draft of the manuscript was written by lm and all authors commented on previous versions of the manuscript. all authors read and approved the final manuscript. 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(2023). phenotypic diversity between and within harar and ogaden cattle breeds in eastern ethiopia: the first step for conservation. genetic resources 4 (7), 56–67. doi: 10.46265/genresj.ixpj9541. © copyright 2023 the authors. this is an open access article distributed under the terms of the creative commons attribution license (cc by 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. introduction the cattle population size of ethiopia, 70.3 million head (central statistical agency, 2021), is the largest in africa and more than double the cattle population registered by chad (32.2 million) which is the second country with high cattle population (statista, 2020). cattle genetic resources, the major contributor to the livestock sector, serve as sources of milk, meat, draught power, hide, manure, nutrient recycling and foreign exchanges for ethiopia (central statistical agency, ∗corresponding author: amine mustefa (aminemustefa32@gmail.com) 2021). due to the very important role cattle genetic resources play in the economy of the country, various diversity and genetic improvement studies have been made so far to ensure their sustainable utilization. diversity studies in animal genetic resources are important to better understand the breed and design appropriate breeding programmes for current and future research and development works (fao, 2012). variation within and among breeds is among the key inputs in genetic improvement and conservation programmes; the more diverse the population the more likely it leads to bring genetic improvement (falconer, 1989). identification, phenotypic and genetic characterization, and advanced performance evaluation can help received: 27.01.2023 accepted: 09.06.2023 published online: 04.07.2023 https://www.genresj.org https://www.dx.doi.org/10.46265/genresj.ixpj9541 https://www.genresj.org https://www.dx.doi.org/10.46265/genresj.ixpj9541 mailto:aminemustefa32@gmail.com genetic resources (2023), 4 (7), 56–67 characterization of cattle in eastern ethiopia 57 us understand the diversity of a given population. the first diversity assessment of ethiopian cattle genetic resources was made by the international livestock centre for africa (ilca), now the international livestock research institute (ilri), in 1992 (rege, 1999; rege and tawa, 1999). it sought to determine the status and compile information on the characteristics of cattle. it does not constitute the level of assessment required to make decisions on use and conservation, as the information was incomplete due to its very broad brush survey. however, it provides a basis for additional, targeted surveys (rege, 1999; rege and tawa, 1999; mustefa et al, 2021). according to ethiopia’s focal point for genetic resources, the ethiopian biodiversity institute, ethiopia possesses 28 indigenous cattle breeds; abergelle, anuak, adwa, afar, ambo, arado, arsi, bale, begait, begaria, borena, fogera, gofa, gojam highland zebu, guraghe, hammer, harar, horro, irob, jem-jem, jijiga, kereyu, medenes, mursi, ogaden, raya, sheko, and smada (rege and tawa, 1999; ebi, 2016). among the listed cattle breeds, some (adwa, ambo, bale, guraghe, hammer, harar, jem-jem, jijiga, and smada) do not have a detailed description of their physical appearance. therefore, it is important to consider filling the missing information to capture a country-wide picture. according to getachew et al (2014), ogaden cattle are thought to be a variety of the borena cattle found in the ogaden area of the somali region of ethiopia and bordering east hararghe. they have a well-developed hump, large dewlap and short horns. they are mainly kept for milk production but are good beef animals. the ogaden cattle were also reported to be distributed up to jigjiga and parts of east hararghe (rege and tawa, 1999; mengesha, 2019). on the other hand, according to the reports of rege and tawa (1999), the harar cattle are found in the east and west hararghe plateau. they have short, thick horns and well-developed dewlaps. the common coat colours are black, roan and red and are used primarily for draught. these two cattle breeds are found adjacent to one another. to quantify the level of relationship between them as well as to know the available cattle diversity in that area, phenotypic characterization is required. moreover, phenotypic characterization is a crucial first step for in situ conservation. the current study aims to phenotypically characterize these two cattle breeds and to quantify the level of relationship between them. materials and methods study areas this study was conducted in five districts of three zones in two regions: tulo district of west hararghe zone, jarso and fedis districts of east hararghe zone of oromia region and jigjiga and kebri beyah districts of fafen zone of somali region (figure 1). the weather conditions, altitude, ethnicity, and human and cattle population sizes of the sampled districts are presented in table 1. site selection according to rege and tawa (1999), harar cattle were found in east and west hararghe zones of the oromia region. similarly, getachew et al (2014) reported the distribution of ogaden cattle to be the somali region and adjacent areas of eastern oromia. therefore, the current study took representative samples from the two breeds of cattle. accordingly, for the harar cattle, tulo district was randomly selected from west hararghe zone while jarso and fedis districts were randomly selected from east hararghe zone. on the other hand, jigjiga and kebri beyah districts were selected randomly from fafen zone to represent ogaden cattle. the kebeles (sampling sites) within each district were categorized into highland and lowland areas. one kebele from the highland and one kebele from the lowland areas were selected randomly. households were also randomly selected from each kebeles (sampling sites) and 2–3 adult animals (four years old and above) were sampled randomly from each household. data collection data collection procedures were adapted from the fao guidelines for the phenotypic characterization of animal genetic resources (fao, 2012). sampled cattle were carefully handled by trained labourers and their owners. to avoid measurement error, the cattle were made to stand properly on flat grounds with parallel legs. four researchers were involved in the data collection: two for the quantitative and two for the qualitative data recording. measurements were carried out by the same researcher throughout the study to minimize subjectivity errors. data recording was carried out early in the morning before the animals were fed and watered. textile measurement tape in a centimetre unit was used to record quantitative data. eight morphometric/quantitative (table 2) and fourteen morphological/qualitative traits (table 3, figure 2) were recorded on a total of 456 adult cattle (354 females and 102 males). data analysis univariate analysis data entry and management were performed using microsoft excel© worksheet (microsoft office 2016). univariate procedure of statistical analysis software (sas) 9.0 (sas institute, 2002) was used to test the normality of the morphometric data. data on morphological/qualitative traits were subjected to chisquare (χ2) tests of the frequency (freq) procedure of sas 9.0 software (sas institute, 2002) . qualitative data analysis was performed using the following model by fitting sex, breed and location as class variables. yijk = µ + si + bj + lk + eijk where yijk is an observation, µ is the overall mean, si is the fixed effect of ith sex, bj is 58 mustefa et al genetic resources (2023), 4 (7), 56–67 figure 1. map of the study areas the fixed effect of jth breed, lk is the fixed effect of kth location, and eijk is the random error attributed to the nth observation. on the other hand, morphometric data analysis was performed separately for males and females by fitting location and breed as fixed variables. morphometric data were analyzed using the general linear model (glm) procedure of sas 9.0 software, with adjusted tukey-kramer test to separate the least square means (lsm). morphometric data analysis was performed using the following model: yi = µ + li + ei where yi is an observation, µ is the overall mean, li is the fixed effect of ith location, and ei is the random error attributed to the nth observation. yij = µ+ bi + lj + eij where yij is an observation, µ is the overall mean, bi is the fixed effect of ith breed, lj is the fixed effect of jth location, and eij is the random error attributed to the nth observation. multivariate analysis quantitative measurements that better discriminate the cattle populations from different locations were identified using the forward selection method of the stepwise discriminant function analysis (stepdisc) procedure of sas 9.0. the discriminant function analysis (discrim) procedure of sas 9.0. was also used to assign observations to locations and evaluate probabilities of misclassifications. a linear combination table 1. weather and population-related information of the selected districts (city population, 2007; keskes et al, 2013; ehzlfdo, 2018; whzlfdo, 2018; ayana, 2019; unhcr, 2020; kebede and utta, 2021; abdi and shiferaw, 2022) agroecology tulo jarso fedis jigjiga kebri beyah human population projection 2022 215,337 165,712 161,214 417,688 242,880 area (km2) 430.6 515.1 720.8 2,859 4,839 temperature(oc) 17–32 12–25 14–28 16–29 16–27 rainfall (mm) 600–900 400–900 516.3 300–500 582.4 altitude (m.a.s.l.) 1,600–2,400 1,500–3,060 1,702 500–1,600 1,530 cattle population 131,643 76,873 ethnicity oromo oromo oromo somali somali genetic resources (2023), 4 (7), 56–67 characterization of cattle in eastern ethiopia 59 table 2. description of the collected morphometric traits. adapted from fao (2012). measurement was performed using a centimetre (cm) unit. no. morphometric traits definitions 1 body length horizontal length from the point of the shoulder to the pin bone 2 heart girth measurement around the animal right behind its front legs 3 height at withers height from the bottom of the front foot to the highest point of the withers 4 pelvic width horizontal distance between the extreme lateral points of the hook bone (tuber coxae) of the pelvis 5 muzzle circumference circumference of the mouth a little above the nostrils and around the point where the dewlap meets the chin 6 ear length length of the back side of the ear from the root to the tip 7 horn length the longest distance from the root of the horn to its tip along the outer curvature 8 cannon bone length distance from the lateral tuberculum of the os metacarpale iv to the fetlock joint of morphometric measurements that provide maximal separations between locations was performed using the canonical discriminant function analysis (candisc) procedure of sas 9.0. the scored canonical variables were used to plot pairs of canonical variables to get a visual interpretation of location differences. pairwise squared mahalanobis distances between locations were computed as: d2 (i|j) = (xi − xj) ′ cov−1 (xi − xj). where d2 (i|j) is the distances between locations i and j, cov−1 is the inverse of the covariance matrix of measured variables, xi and xj are the means of variables in the ith and jth populations. results morphological traits the qualitative characteristics of both sexes (male and female) and both breeds (harar and ogaden) along with their chi-square values and levels of significance are presented in table 3. accordingly, sex and breed significantly (p < 0.05) affected the qualitative characteristics of the cattle populations. the majority of the males had straight-shaped lateral and upright horn orientation, shaded body colour pattern, large hump size located at thoracic position, and large dewlap width. on the other hand, the majority of the females had curved and forward-oriented horn, uniform body colour pattern, small hump size located at cervicothoracic position, and medium dewlap width. moreover, the studied cattle breeds showed significantly different qualitative characteristics. the majority of harar cattle had wide, curved and forward-oriented horn. they also possessed a sloppy rump with a small to medium hump located at the cervivo-thoracic position (figure 3a). on the other hand, the ogaden cattle possessed both narrow and wide horn spacing, straight and curved horn shapes, as well as flat and sloppy back profiles at an equivalent ratio. they also possessed an upright-oriented horn and a small hump placed at thoracic position. the majority of the ogaden cattle also had a long tail with no naval flap (figure 3b). the qualitative characteristics of the cattle populations from the different locations along with their chisquare values and levels of significance are presented in table 4. all the studied qualitative characteristics of the cattle populations were significantly (p < 0.05) affected by location differences. accordingly, almost all the cattle populations from tulo and jarso districts possessed wide horn spacing, while this was comparably narrow in cattle from jigjiga and kebri beyah districts. the horn shape of the majority of the cattle populations was curved while it was straight in cattle from kebri beyah district. upright horn orientation was dominantly observed in cattle from jigjiga and kebri beyah districts while it was oriented forward in cattle from tulo and jarso districts. the hump of the cattle populations from jigjiga and kebri beyah districts was located at the thoracic position while the hump of most of the cattle populations from the other locations was located at the cervico-thoracic position. flat-rump profile was observed in the majority of the cattle populations from kebri beyah district while all the cattle from tulo, jarso and fedis districts had a sloppy rump. the majority of the cattle populations possessed a uniform body colour pattern, erected small hump, and straight back profile. the body colour of the cattle populations disaggregated into male and female is presented in figure 2a. higher proportion of grey, red, white, and red + white body colours were observed in females than the males. on the other hand, black body colour and its mix with other colours (i.e. white + black and red + black) were observed frequently in the males. body colour disaggregated by cattle breeds (harar and ogaden) is presented in figure 2b and representative examples shown in figure 3. grey body colour was observed predominantly in ogaden cattle while several body colours were observed frequently in harar cattle. the body colour of the cattle populations from different locations is presented in figure 2c. location affected the body colour of the cattle populations significantly (p < 0.0001). grey body colour was dominantly observed in jigjiga and kebri beyah districts while cattle in tulo and jarso districts were predominantly red and the cattle population from fedis district had white body colour followed by grey. 60 mustefa et al genetic resources (2023), 4 (7), 56–67 figure 2. a: effect of sex on body colour (chi-square value 93.2, p < 0.0001); b: effect of breed on body colour (chi-square value 161.5, p < 0.0001); c: effect of location on body colour (chi-square value 275.8, p < 0.0001). genetic resources (2023), 4 (7), 56–67 characterization of cattle in eastern ethiopia 61 table 3. percentages of qualitative characteristics of cattle populations by sex and breed. n, number of animals sampled; *, p < 0.05; **, p < 0.01; ***, p < 0.0001; ns: not significant. qualitative traits sex breed male female x2 value p harar ogaden x2 value p n 102 354 293 163 horn spacing narrow 38.2 22.0 10.9 ** 11.6 50.9 84.9 *** wide 61.8 78.0 88.4 49.1 horn shape straight 65.3 24.0 61.9 *** 26.6 45.4 16.6 *** curved 34.3 76.0 73.4 54.6 horn orientation lateral 42.1 12.2 56.5 *** 25.2 7.4 126.3 *** upright 36.3 33.6 15.7 67.5 forward 19.6 44.9 50.2 19.6 dropping 2.0 9.3 8.9 5.5 colour pattern uniform 26.5 71.7 78.3 ** 55.0 73.6 20.3 ** spotty 2.0 3.7 5.1 0 pied 9.8 4.8 6.1 5.5 shaded 61.7 19.8 33.8 20.9 hump shape erect 86.3 100 50.1 *** 95.2 100 8.0 ** dropping 13.7 0.0 4.8 0.0 hump size small 12.7 78.0 232.7 *** 55.6 77.3 50.7 *** medium 35.3 21.5 35.2 5.5 large 52.0 0.5 9.2 17.2 hump position thoracic 85.3 44.1 54.1 *** 30.0 95.1 178.1 *** cervico-thoracic 14.7 55.9 70.0 4.9 back profile straight 96.1 88.1 5.5 * 89.1 91.4 0.6 ns curved 3.9 11.9 10.9 8.6 rump profile flat 29.4 12.7 16.1 *** 0.0 46.0 161.4 *** sloppy 70.6 87.3 100 54.0 tail length short 6.9 7.9 5.3 ns 10.2 3.1 31.4 *** medium 24.5 35.9 40.3 20.9 long 68.6 56.2 49.5 76.0 naval flap width absent 41.0 na na 30.0 61.3 44.4 *** small 42.4 45.7 36.3 medium 12.4 17.8 2.4 large 4.2 6.5 0.0 perpetual sheath absent 7.8 na na 0.0 20.5 50.1 *** small 27.5 11.1 53.9 medium 62.7 88.9 20.5 large 2.0 0.0 5.1 dewlap width small 2.0 22.9 65.7 *** 2.4 46.6 143.6 *** medium 43.1 59.6 63.1 43.0 large 54.9 17.5 34.5 104 morphometric measurements least square means with their respective standard errors (lsm ± se) and pairwise comparison of the morphometric measurements of both cattle breeds disaggregated by sex are presented in table 5. overall, males of each breed and location had higher sizerelated linear body measurements than their female counterparts. both the ogaden male and female cattle had higher measurements for most of the morphometric parameters (i.e. heart girth, height at withers, pelvic width, muzzle circumference, and canon bone length). some traits (horn, ear and body length) of the two breeds were sex dependent. within the females, harar cows had longer horns while in ogaden cattle, males possessed longer horns. similarly, harar cows’ body length was significantly higher than their counterparts from ogaden while males’ body length did not differ significantly. moreover, ogaden oxen’s ear length was significantly higher than their counterparts from harar while the value was not significantly different between the cows. 62 mustefa et al genetic resources (2023), 4 (7), 56–67 table 4. percentages of qualitative characteristics of cattle populations from different locations. n, number of animals sampled; *, p < 0.05; **, p < 0.01; ***, p < 0.0001; ns, not significant. qualitative traits tulo jarso fedis jigjiga kebri beyah x2 value p n 95 103 95 97 66 horn spacing narrow 2.1 5.8 27.4 46.4 57.6 106.1 *** wide 97.9 94.2 72.6 53.6 42.4 horn shape straight 21.0 20.0 39.0 38.1 56.1 31.9 *** curved 79.0 80.0 61.0 61.9 43.9 horn orientation lateral 25.3 20.4 30.5 10.3 3.0 152.5 *** upright 10.5 13.6 23.2 62.9 74.2 forward 61.0 56.3 32.6 20.6 18.2 dropping 3.2 9.7 13.7 6.2 4.6 colour pattern uniform 50.5 46.6 68.4 74.2 72.7 33.8 ** spotty 5.3 5.8 4.2 0 0 pied 8.4 6.8 3.2 7.2 3.0 shaded 35.8 40.8 24.2 15.6 24.3 hump shape erect 89.5 97.1 99.0 100 100 24.2 *** dropping 10.5 2.9 1.0 0 0 hump size small 56.9 66.0 43.1 82.5 69.7 74.5 *** medium 34.7 22.3 49.5 4.1 7.6 large 8.4 11.7 7.4 13.4 22.7 hump position thoracic 27.4 26.2 36.8 91.8 100 181.8 *** cervico-thoracic 72.6 73.8 63.2 8.2 0 back profile straight 93.7 97.1 75.8 86.6 98.5 34.7 *** curved 6.3 2.9 24.2 13.4 1.5 rump profile flat 0 0 0 38.1 57.6 172.1 *** sloppy 100 100 100 61.9 42.4 tail length short 12.6 4.9 13.7 3.1 3.0 40.4 *** medium 45.3 39.8 35.8 19.6 22.7 long 42.1 55.3 50.5 77.3 74.3 naval flap width absent 23.4 32.4 34.2 70.5 45.6 62.3 *** small 57.1 43.3 36.7 28.2 50.0 medium 16.9 16.2 20.2 1.3 4.4 large 2.6 8.1 8.9 0 0 perpetual sheath absent 0 0 0 31.6 10.0 70.0 *** small 33.3 3.5 0 42.1 65.0 medium 66.7 96.5 100 15.8 25.0 large 0 0 0 10.5 0 dewlap width small 2.1 2.9 2.1 57.7 30.3 207.8 *** medium 76.8 70.9 41.1 29.9 62.1 large 21.1 26.2 56.8 12.4 7.6 least square means with their respective standard errors (lsm ± se) and pairwise comparison of the morphometric measurements disaggregated by the five locations for both sexes are presented in table 6. most of the morphometric measurements were significantly affected by the location of the cattle populations. significantly higher heart girth, pelvic width, muzzle circumference and cannon bone length values were observed for the populations from jigjiga and kebri beyah districts while the horn length of tulo and jarso cows was significantly higher than the others. the shortest horn was registered in oxen from fedis district. multivariate analysis for discrimination of cattle populations cannon bone length, horn length and pelvic width were the three most important morphometric variables used in discriminating the cattle populations from different locations (table 7). these results were confirmed by wilk’s lambda test where the selected variables made a highly significant (p < 0.0001) contribution in discriminating the cattle populations (table 7). results of a location-wise discriminant function analysis (table 8) show the overall classification of individual animals into their location (population). the genetic resources (2023), 4 (7), 56–67 characterization of cattle in eastern ethiopia 63 table 5. least square means (lsm ± se) and pairwise comparisons of the morphometric measurements of both cattle breeds under both sexes. n, number of animals sampled; bl, body length; hg, heart girth; hw, height at withers; pw, pelvic width; mc, muzzle circumference; el, ear length; hl, horn length; cbl, cannon bone length. *, p < 0.05; **, p < 0.01; ***, p < 0.0001; ns, not significant. traits females males harar ogaden p harar ogaden p n 230 124 63 39 bl 106.1 ± 0.38 104.2 ± 0.52 ** 108.6 ± 0.91 110.6 ± 1.16 ns hg 140.0 ± 0.53 149.1 ± 0.72 *** 143.8 ± 1.17 165.6 ± 1.49 *** hw 112.3 ± 0.30 113.6 ± 0.40 * 115.2 ± 0.73 120.9 ± 0.93 *** pw 35.3 ± 0.16 38.5 ± 0.22 *** 33.5 ± 0.38 40.6 ± 0.48 *** mc 38.6 ± 0.13 40.0 ± 0.17 *** 40.1 ± 0.30 44.6 ± 0.38 *** el 17.9 ± 0.11 17.8 ± 0.16 ns 18.0 ± 0.19 17.4 ± 0.24 * hl 20.8 ± 0.54 17.2 ± 0.73 *** 13.1 ± 0.72 15.9 ± 0.92 * cbl 20.6 ± 0.12 27.7 ± 0.16 *** 21.0 ± 0.22 27.6 ± 0.28 *** table 6. least square means (lsm ± se) in centimetre units and pairwise comparisons of the morphometric measurements of the cattle populations from different locations by sex. n, number of animals sampled; bl, body length; hg, heart girth; hw, height at withers; pw, pelvic width; mc, muzzle circumference; el, ear length; hl, horn length; cbl, cannon bone length. *, p < 0.05; **, p < 0.01; ***, p < 0.0001; ns, not significant. traits location p tulo jarso fedis jigjiga kebri beyah females n 77 74 79 78 46 bl 106.7 ± 0.66a 106.1 ± 0.67ab 105.5 ± 0.65ab 104.0 ± 0.65b 104.5 ± 0.85ab * hg 142.8 ± 0.90b 139.0 ± 0.92c 138.3 ± 0.89c 147.9 ± 0.89a 151.2 ± 1.16a *** hw 112.9 ± 0.51ab 112.4 ± 0.52ab 111.6 ± 0.50b 113.4 ± 0.51ab 113.9 ± 0.66a * pw 35.5 ± 0.27c 35.6 ± 0.28c 34.7 ± 0.27c 37.7 ± 0.69b 39.7 ± 0.35a *** mc 38.9 ± 0.22b 38.49 ± 0.22b 38.3 ± 0.22b 39.9 ± 0.22a 40.1 ± 0.28a *** el 18.3 ± 0.19a 17.5 ± 0.20bc 18.0 ± 0.19ab 18.1 ± 0.19ab 17.1 ± 0.25c ** hl 25.0 ± 0.86a 21.9 ± 0.88a 15.8 ± 0.85b 17.8 ± 0.86b 16.0 ± 1.12b *** cbl 21.1 ± 0.20c 20.7 ± 0.20cd 20.1 ± 0.20d 28.1 ± 0.20a 27.0 ± 0.26b *** males n 18 29 16 19 20 bl 107.9 ± 1.69 110.4 ± 1.33 106.1 ± 1.79 109.16 ± 1.64 111.95 ns hg 146.9 ± 2.10b 145.2 ± 1.66bc 138.0 ± 2.23c 163.6 ± 2.05a 167.5 ± 1.99a *** hw 115.2 ± 1.34b 116.9 ± 1.05ab 112.3 ± 1.42b 120.5 ± 1.3a 121.3 ± 1.27a *** pw 33.6 ± 0.70b 34.1 ± 0.55b 32.3 ± 0.74b 40.0 ± 0.68a 41.2 ± 0.66a *** mc 40.8 ± 0.55b 40.5 ± 0.43b 38.8 ± 0.58b 44.6 ± 0.53a 44.6 ± 0.52a *** el 18.6 ± 0.35a 17.8 ± 0.28ab 17.8 ± 0.37ab 17.8 ± 0.34ab 16.9 ± 0.33b * hl 16.1 ± 1.19a 14.6 ± 0.93a 6.9 ± 1.26b 16.2 ± 1.15a 15.7 ± 1.12a *** cbl 21.7 ± 0.39c 20.8 ± 0.30c 20.4 ± 0.41c 28.6 ± 0.38a 26.7 ± 0.37b *** table 7. order of traits used in discriminating the cattle populations from different locations. step variables entered partial r-square f value pr > f wilks’ lambda pr < lambda 1 cannon bone length 0.7927 431.02 < 0.0001 0.2073 < 0.0001 2 horn length 0.1445 19.00 < 0.0001 0.1773 < 0.0001 3 pelvic width 0.1495 19.73 < 0.0001 0.1508 < 0.0001 4 body length 0.1080 13.56 < 0.0001 0.1345 < 0.0001 5 ear length 0.0884 10.83 < 0.0001 0.1226 < 0.0001 6 heart girth 0.0436 5.08 0.0005 0.1173 < 0.0001 7 height at withers 0.0362 4.18 0.0025 0.1130 < 0.0001 8 muzzle circumference 0.0159 1.79 0.1290 0.1112 < 0.0001 64 mustefa et al genetic resources (2023), 4 (7), 56–67 figure 3. a: representative harar cattle bull; b: representative ogaden cattle cows. photos: amine mustefa overall analysis shows medium (61%) classification of individuals into their corresponding location with an error rate of 39%. furthermore, the highest (69.7%) classification of individuals into their location was observed in kebri beyah district while the lowest (45.6%) classifications were recorded in jarso district. results of a breed-wise discriminant function analysis (table 9) show the overall classification of individual animals into their breed. the overall analysis shows high (99%) classification of individuals into their corresponding breed with a small error rate of 1%. furthermore, a higher (99.39%) classification of individuals into their breed was observed in ogaden cattle than harar cattle (98.63%). location-wise pairwise squared mahalanobis distances are presented in table 10. the distances were highly significant (p < 0.0001). the shortest distance (0.77) was obtained between tulo and jarso populations, while fedis and jigjiga populations were most distantly related (27.34). the breed-wise analysis also showed long mahalanobis distance between the two breeds (22.15, p < 0.0001). location wise, multivariate statistics outputs showed the significance of can 1 due to its high eigenvalue (5.3) and proportion (93%) to discriminate the cattle populations from the different locations. can 2–can 4 combined had only 7% proportion in discriminating the cattle populations with significantly low eigenvalue (0.37 combined). similarly, can 1 significantly discriminated the breeds during the breed-wise analysis with high eigenvalue (5.1) and 100% proportion. therefore, the outputs of can 1 are indicative and significant. locationwise and breed-wise plot of the first two canonical varifigure 4. location-wise (a) and breed-wise (b) plot of canonical discriminant analysis. districts in a) are indicated by numbers: 1, tulo; 2, jarso; 3, fedis; 4, jigjiga; 5, kebri beyah. breeds in b) are indicated by ha, harar and og, ogaden. ables to discriminate the cattle populations is presented in figure 4. the cattle populations from tulo, jarso and fedis districts were inseparable and in the same group; similarly, the populations from jigjiga and kebri beyah districts were also inseparable and placed in the same group. however, those two groups were clearly separated from each other. discussion qualitative morphological traits can help to easily differentiate breeds. the observed qualitative characteristic similarities among the cattle populations from tulo, jarso and fedis districts support the presence of a unique cattle breed (the harar cattle) in east and west hararghe zones of oromia region. this is also backed by the report of rege and tawa (1999), which stated the east and west hararghe zones of oromia region as the breeding tract of harar cattle. similarly, morphological similarities shared between the cattle populations from jigjiga and kebri beyah districts and their variation from the previous group were also reported by getachew et al (2014) who characterized them as ogaden cattle. these qualitative results confirm the presence of two cattle breeds (harar and ogaden) in the eastern part of the country. in line with different publications including mustefa et al (2021) on raya cattle and terefe et al (2015) on mursi cattle, the results of the current study also confirm the existence of within-breed variations besides the between-breed differences. the ogaden cattle possess a relatively unique body colour compared to the harar cattle, which has multiple body colours. the presence of multicolour (red, red + genetic resources (2023), 4 (7), 56–67 characterization of cattle in eastern ethiopia 65 table 8. number (and percent) of observations classified into locations. from district tulo jarso fedis jigjiga kebri beyah total tulo 54 (56.84) 27 (28.42) 12 (12.63) 1 (1.05) 1 (1.05) 95 (100) jarso 28 (27.18) 47 (45.63) 27 (26.21) 0 1 (0.97) 103 (100) fedis 15 (15.79) 17 (17.89) 63 (66.32) 0 0 95 (100) jigjiga 0 0 1 (1.03) 64 (65.98) 32 (32.99) 97 (100) kebri beyah 0 0 0 20 (30.30) 46 (69.70) 66 (100) error rate 0.4316 0.5437 0.3368 0.3402 0.3030 0.3911 priors 0.2 0.2 0.2 0.2 0.2 table 9. number (and percent) of observations classified bybreed. from breed harar ogaden total harar 289 (98.63) 4 (1.37) 293 (100) ogaden 1 (0.61) 162 (99.39) 163 (100) error rate 0.0137 0.0061 0.0099 priors 0.5 0.5 table 10. pairwise squared mahalanobis distances between locations. ***, p < 0.0001 from district tulo jarso fedis jigjiga kebri beyah tulo 0 jarso 0.77 *** 0 fedis 2.09 *** 0.87 *** 0 jigjiga 22.17 *** 24.13 *** 27.34 *** 0 kebri beyah 20.23 *** 21.20 *** 24.47 *** 1.80 *** 0 white, red + black, white + black, white, and grey) cattle in harar might be due to the relatively highlanddominated areas of its distribution, especially the tulo and jarso districts. the frequently observed grey body in ogaden cattle was due to the agropastoralists’ preferences and selection of criteria for that particular coat colour (getachew et al, 2014). getachew et al (2014) also related the uniformly patterned grey body colour with their adaptation mechanism to the arid and semi-arid agroecologies of the ogaden rangelands. this was in line with the report of titto et al (2016), who reported animals with light coat colouring absorb less heat than those with darker coats. alongside the most observable qualitative characteristics, morphometric measurements can also produce more reliable information in characterizing and differentiating cattle breeds. the above grouping made by the qualitative characteristics of the cattle populations was also supported by the results of the quantitative measurements. significantly higher values of heart girth, pelvic width, muzzle circumference and cannon bone length with shorter horns were observed in the ogaden cattle (jigjiga and kebri beyah districts) than in the harar cattle. like for the qualitative results, within-breed variations were also observed in the morphometric measurements. within ogaden cattle, values of heart girth and pelvic width measurements were significantly higher for the population from kebri beyah district than jigjiga district. these results made the population from jigjiga district relatively closer to the harar cattle. this might be due to the presence of midand high-altitude areas of jigjiga district compared to the kebri beyah district, as well as the closeness in ground distance of the jigjiga district to the distribution areas of harar cattle. in comparison to other ethiopian indigenous cattle breeds, the morphometric measurements of both harar and ogaden breeds were found to be significantly lower than some lowland cattle breed like begait cattle (mulugeta, 2015). similarly, body length, height at withers, ear length and horn length measurements of both breeds from the current study were lower than those of raya cattle, while the reverse was true for heart girth and pelvic width measurements (mustefa et al, 2021). compared to the adjacent afar cattle, the ogaden cattle had higher values for height at withers and hearth girth while the afar cattle had a longer body than both harar and ogaden cattle breeds (tadesse et al, 2008). the observed higher size-related linear body measurements of the males in each breed and location follow rensch’s rule (rensch, 1950), which states that males are usually larger than females. such differences between males and females might be due to the testosterone secreted in males which causes the growth of muscle mass and skeletal development (baneh and hafezian, 2009). estrogen secreted in females has a limited effect on growth (chriha and ghadri, 2001; baneh and hafezian, 2009). the current results were comparable with the reports of mustefa et al (2021) on raya cattle, terefe et al (2015) on mursi cattle, and genzebu 66 mustefa et al genetic resources (2023), 4 (7), 56–67 et al (2012) on arado cattle. some size-linked morphological parameters (i.e. hump size and dewlap width) were also larger for males than females, as these traits are associated with the overall size of the cattle. the morphometric variables, which discriminated the cattle populations, were ranked according to their importance. the inclusion of horn length within the top-three discriminatory variables is comparable with the reports of mustefa et al (2021), who also classified it among the top-three variables to discriminate raya cattle from other highland cattle breeds. the high error rate of the discriminant function analysis among the different districts shows a lack of uniqueness within each location. on the contrary, some similarities were shared among locations. the cattle populations from tulo, jarso and fedis districts shared similarities justifying their belonging to the same group (the harar cattle group). similarly, the cattle populations from jigjiga and kebri beyah districts shared some similarities, which support their categorization into the same group (the ogaden cattle group). this confirms the idea of previous studies which state the presence of harar and ogaden cattle breeds in the eastern part of ethiopia (rege and tawa, 1999; getachew et al, 2014; mengesha, 2019). this grouping was also supported by the morphometric and morphological results of the current study. the pairwise squared distance results between locations confirmed the already known differences between harar and ogaden cattle breeds, supporting the morphological, morphometric and multivariate results. however, these distances showed only the relative size differences between each population. such differences might not necessarily be due to genetic differences (zechner et al, 2001; mustefa et al, 2021, 2022). therefore, further diversity studies using molecular techniques are recommended to understand the level of genetic diversity within and between each breed. in conclusion, two cattle breeds in eastern ethiopia listed under the fao domestic animal diversity information system (dad-is), were phenotypically characterized to obtain and quantify the withinand among-breed diversity. strong within-breed similarities and large between-breed differences (distance) were observed. thus, the current study confirmed the presence of two cattle breeds (the harar cattle and the ogaden cattle) in eastern ethiopia. besides breed differentiation, this study will be used to design conservation and genetic improvement programmes for each breed. data availability the data that has been used is confidential. acknowledgments the authors are highly grateful to the ethiopian biodiversity institute (ebi) for funding the work. our special appreciation also goes to the farmers for providing their animals to this work for free. we also take this opportunity to appreciate the animal science experts at zonal, district and kebele level for their endless help during the data collection. a special word also goes to our friend and work partner mr. tadesse hunduma for mapping the study area. conflict of interest statement the authors declare that they have no conflict of interest. author contributions all authors contributed to the study conception and design. material preparation and data collection were performed by amine mustefa, tesfalem aseged, seble sinkie, fasil getachew, tesfu fekensa, and manaye misganaw. amine mustefa performed the data 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however, its phenotypic diversity has not yet been fully explored. this study determined the phenotypic variability of ñuña conserved in the germplasm bank of the national institute of agrarian innovation of peru. the analysis considered qualitative and quantitative traits, using multivariate statistics and comparison of means. results revealed high phenotypic variability in both qualitative and quantitative traits. in qualitative traits, multiple correspondence analysis found that the dark and light colours of the seed heads contributed most significantly to the variability of the accessions. phylogenetic hierarchical analysis formed four clusters, representing 37% (i), 4% (ii), 7% (iii), and 52% (iv) of the accessions, respectively. for quantitative traits, principal component analysis showed no discrimination between regions of origin but indicated a highly positive correlation between leaf length and width, and between pod length and width, as well as seed length, width, thickness and weight. hierarchical analysis of quantitative characters also formed four clusters, representing 22% (a), 16% (b), 30% (c), and 31% (d) of the accessions, respectively. these clusters, analyzed for means comparison, showed significant differences (p < 0.05) with higher values in cluster b for pod length and width, and seed length, width, thickness and weight. understanding the variability of the qualitative and quantitative traits of ñuña is crucial for future genetic improvement studies aimed at achieving cultivars with desirable characteristics. keywords: qualitative traits, quantitative traits, hierarchical analysis, legume, germplasm citation: santa cruz-padilla, a. e., vásquez-orrillo, j. l., bardales-lozano, r. m., murga-orrillo, h. (2025). assessment of phenotypic diversity of ñuña, a local common bean (phaseolus vulgaris l.) from the northern andes in peru. genetic resources 6 (11), 1–13. doi: 10.46265/genresj.pmky4140. © copyright 2025 the authors. this is an open access article distributed under the terms of the creative commons attribution license (cc by 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. introduction bean (phaseolus vulgaris l.) is the most consumed grain legume worldwide. its production has spread mainly to developing countries (de ron et al, 2016; nassary ∗corresponding authors: angel esteban santa cruz-padilla (asantacruz@inia.gob.pe), jorge luis vásquez-orrillo (jorge.vasquez.orrillo@gmail.com) et al, 2020; uebersax et al, 2023), for its contribution to dietary protein for more than 300 million people in rural and urban communities in east africa and latin america (petry et al, 2015). the areas dedicated to bean production are 36 million hectares worldwide, of which 6 million are located in latin america (faostat, 2022). in latin america, ecuador and peru are considered to be the places of origin of the bean (kami et al, 1995). this gene pool was disseminated through independent received: 02.07.2024 accepted: 16.12.2024 published online: 07.02.2025 https://www.genresj.org https://www.doi.org/10.46265/genresj.pmky4140 https://www.genresj.org https://www.doi.org/10.46265/genresj.pmky4140 mailto:asantacruz@inia.gob.pe mailto:jorge.vasquez.orrillo@gmail.com 2 santa cruz-padilla et al genetic resources (2025), 6 (11), 1–13 domestication events, creating diverse landraces (iwataotsubo et al, 2016). within this genetic variability, one particular type is the ñuña bean (beem et al, 1992; fernández et al, 2014), produced on a small scale by local producers in northern peru. the ñuña plant is characterized by its indeterminate and climbing growth habit (gamarra, 2021), reaching a height of more than 2 metres at the end of flowering (gamarra et al, 2007). the pods are usually distributed along the entire length of the plant (melo and ligarreto, 2010) and contain between five and seven grains. these beans stand out for their protein content, which varies between 18% and 23% (rodŕıguez et al, 2014). as with common beans, ñuña presents high levels of other nutrients such as vitamins and minerals (beem et al, 1992; melo and ligarreto, 2010). the main characteristic of the ñuña kernel when roasted for consumption is to explode and expand like popcorn, acquiring a soft consistency and a pleasant flavour similar to peanuts. this characteristic is associated with the presence of occluded intracellular and intercellular spaces that are forced to expand by the generation of water vapour during roasting (beem et al, 1992). the ñuña has diversified under geographical restrictions, with production concentrated continuously in the andean zone (national research council, 1989; tohme et al, 1995; otálora et al, 2006). it develops between the latitudes of 7◦30’ s and 19◦30’ s, and altitudes ranging from 2,000 to 3,000masl (pearson et al, 2012). temperatures in this region fluctuate between 10 and 30ºc (llique, 1993), with a relative humidity of 60 and 75% (hernández-lópez et al, 2013), annual rainfall of 500 to 3,000mm and a photoperiod of 8 to 14 hours (llique, 1993). in peru, the ñuña has a wide phenotypic variability that is distributed in the regions of cusco, ancash, huánuco, apuŕımac, ayacucho, la libertad and cajamarca. the most significant genotypic variability is found in the latter region (tohme et al, 1995), particularly in the province of cajabamba, where there is a superior gene pool with varied productivity levels (debouck, 1986; franco and hidalgo, 2003; santacruz-padilla et al, 2021). despite the existence of morphological and molecular characterization studies of ñuña (cruz-balarezo et al, 2009), there are few reports on the morphology of genotypes in northern peru. considering that ecogeographic factors influence the phenotypic and genetic characteristics of a species (knight et al, 2005; herben et al, 2012; d́ıez et al, 2013), it is hypothesized that the morphological characteristics of flowers, pods and grains of ñuña contribute to discriminating the morphological variability of the accessions conserved in the germplasm bank of the national institute for agrarian innovation (inia) in peru. furthermore, it is considered that there is phenotypic variability related to the geographical area of origin. therefore, this research aimed to determine the phenotypic variability of ñuña conserved in the germplasm bank of inia in peru. material and methods origin of the ñuña accessions the ñuña samples were collected from plots belonging to producers located in eight districts within the regions of la libertad, cajamarca, and ancash in peru, as detailed in table 1 and illustrated in figure 1. most of the ñuña germplasm collected in northern peru was from the cajamarca region (88 accessions), followed by the la libertad region with 31 accessions and the ancash region with 3 accessions (table 1). location of the study the study covered the period from december 2019 to july 2020 and was carried out at the cochamarca experimental annex of the baños del inca agricultural experimental station, cajamarca (7.2756 s, 78.2186 w, 2,820masl, figure 1). the study area exhibits a climatic classification corresponding to the tropical low montane dry forest (bs-mbt) category, determined according to the methodology of holdridge (1947). throughout the research period, mean, minimum and maximum temperatures were 14.3◦c, 7.6◦c and 21◦c respectively, with a rainfall of 117.4mm (senhami, 2020). soil chemical properties a composite sample was extracted from the experimental area using an auger at a 0 to 30cm depth. this sample was treated at the baños del inca inia soil, water and foliar laboratory, where it underwent a process of air drying, followed by grinding and sieving through a 2mm mesh. subsequently, organic matter was determined using the walkley and black (1934) method, while phosphorus (p) was evaluated according to the olsen et al (1954) protocol, and potassium (k) was determined using the silver thiourea method. soil analysis revealed a ph of 6.5, with a 1% organic matter content, 3.82ppm p and 295ppm k. soil preparation the experimental area covered 0.16 hectares, where soil preparation was carried out to a depth of 30cm using a disc plough coupled to an agricultural tractor (new holland, 110 hp, model: ts6.110). to correct soil fertility, amendments such as island guano (1,450kg/ha), diammonium phosphate (150kg/ha) and potassium chloride (100kg/ha) were applied. subsequently, plots of 6m2 (6m × 1m) were demarcated, resulting in a total of 122 plots of one row each, corresponding to the 122 ñuña accessions. planting was carried out in december 2019, with an arrangement of plants at distances of 0.5m between plants and 1.0m between rows, totalling 12 plants per row, equivalent to 20,000 plants per hectare. g enetic resources (2025),6 (11),1–13 phenotypic diversity ofñ uña bean in peru 3 table 1. geographical origin and coding of 122 accessions of ñuña beans from the inia germplasm bank, cajamarca, peru. district, province, region location latitude (s) longitude (w) altitude (masl) no. of accessions accession code sanagoran, sánchez carrión, la libertad angasmarquilla 7.72510 78.15520 3,130 9 per002014, per002015, per002016, per002017, per002018, per002019, per002020, per002021, per002022 yanac 7.78020 77.95590 2,989 12 per002023, per002024, per002025, per002026, per002027, per002028, per002029, per002030, per002031, per002032, per002033, per002071 huamachuco, sánchez carrión, la libertad olichoco 7.81480 78.05000 3,183 10 per002034, per002035, per002064, per002065, per002066, per002067, per002068, per002036, per002037, per002038 cajabamba, cajabamba, cajamarca chanshapamba 7.66750 78.05180 2,889 13 per002039, per002040, per002041, per002042, per002043, per002044, per002045, per002046, per002047, per002048, per002049, per002050, per002051 chanshapampa 7.63040 78.02699 3,069 9 per017536, per017538, per017548, per017557, per017558, per017559, per017571, per017577, per017593 shitabamba 7.67480 78.03730 2,821 10 per002059, per002060, per002061, per002062, per002063, per002072, per017545, per017578, per017580, per017583 churgapampa 7.65645 78.06936 2,828 1 per017547 huanza 7.66693 8.07693 2,596 11 per017537, per017539, per017549, per017550, per017553, per017564, per017565, per017587, per017588, per017589, per017590 callash 7.63970 78.06299 2,756 5 per017540, per017551, per017552, per017594, per017595 cajabamba 7.62190 78.04450 2,685 2 per002069, per002070 colcabamba 7.64897 78.03058 2,899 1 per017561 chanshe 7.67867 78.06359 2,660 2 per017562, per017563 sitacocha, cajabamba, cajamarca lluchubamba 7.52039 77.96881 3,023 13 per017541, per017543, per017568, per017574, per017581, per017582, per017591, per017592, per017546, per017570, per017573, per017584, per017576 continued on next page 4 santa cruz-padilla et al g enetic resources (2025),6 (11),1–13 table 1 continued district, province, region location latitude (s) longitude (w) altitude (masl) no. of accessions accession code condebamba, cajabamba, cajamarca cauday 7.57435 78.07074 2,815 7 per017535, per017556, per017560, sc 7280, per017572, per017575, per017586 ogosgon 7.56360 78.09330 2,697 8 per002052, per002053, per002054, per002055, per002056, per002057, per002058, per017555 huarasullo 7.57815 78.10208 2,588 2 per017566, per017567 cachachi, cajabamba, cajamarca el aliso 7.44810 78.26905 3,233 3 per017542, per017544, per017554 llacanora, cajamarca, cajamarca la paccha 7.19240 78.42650 2,629 1 per002073 bambas, corongo, ancash bambas 8.60247 77.99639 2,931 3 per018011, per018012, per018027 genetic resources (2025), 6 (11), 1–13 phenotypic diversity of ñuña bean in peru 5 cultivation treatments manual weeding was carried out 45 days after planting (dap). subsequently, trellising was carried out at 50 dap by installing 2.5m high posts at the ends of the plots. on these posts, a galvanized wire (n◦ 16) was stretched along 6m, which supported the plants. a phytosanitary control against boring larvae (lepidoptera: noctuidae) was also implemented, using alphacypermethrin (25ml/20l of water). determination of variables evaluations were carried out from the beginning of flowering until harvest, recording data from 10 plants per accession. descriptors adapted from the international plant genetic resources institute for phaseolus vulgaris (ipgri, 2001) were studied (table 2). the royal horticultural society colour chart (rhs, 2001) was used to assign the colours of flowers, pods and seeds. the study included consideration of 12 qualitative descriptors (table 2) and 9 quantitative descriptors (table 3). statistical analysis characterization information was subjected to descriptive and multivariate statistical analysis. multivariate statistics included multiple correspondence analysis (mca) for qualitative traits and principal component analysis (pca) for quantitative traits. in both cases, decision trees were constructed using hierarchical and phylogenetic dendrograms. the euclidean distance and the ward.d2 (ward, 1963) method were used as a similarity measure to carry for grouping between accessions. for the clusters of the quantitative traits, a comparison of means was carried out using tukey’s test (p < 0.05). the analyses were carried out with the packages factoextra (kassambara and mundt, 2020) and factorminer (lê et al, 2008) for mca and pca. the dendrograms were elaborated with the cluster (maechler et al, 2021) and circlize (gu et al, 2014) packages, while the visualization of the results was performed with ggplot2 (wickham, 2016). comparison of means was run with the agror package (shimizu et al, 2023). all analyses were performed using rstudio statistical software (r core team, 2023). results the phenotypic data for 12 qualitative and 9 quantitative descriptors collected on 122 accessions of ñuña beans from the inia collection are summarized in supplemental table 1 and were used in statistical analyses to assess and describe their genetic diversity. multiple correspondence analysis of qualitative characters the qualitative characters evaluated were subjected to mca, the results of which are presented in figure 2. a marked association was observed between several characters, such as darker colour of seeds (dcs) and lighter colour of seeds (lcs) which presented the highest contributions to the variability of the 122 accessions of ñuña. seed coat pattern (scp), flower wing colour (wic) also associated and had similar contributions to the clustering of the accessions. there was a joint association between standard colour (stc), dry pod colour (dpc) and colour of immature pods (cip). this pattern suggests that certain characteristics share discernible similarities, while others show a less prominent relationship. these findings provide further insight into the interrelationships between the qualitative variables assessed. hierarchical analysis of qualitative characteristics the analysis of the phylogenetic hierarchical tree of the 122 accessions of ñuña is shown in figure 3. it is possible to observe the formation of four morphological clusters, grouped according to their most similar characters. cluster i represents 37% of the accessions (45 accessions), which showed similarity in seven characters associated with different morphological stages: darker colour of seeds, between white-tinged black, grey-brown and greyish purple-tinged white to brown; lighter colour of seeds between white tinged purple, yellow, greyish orange, brown, purple to absent; seed coat pattern between mottled, spotted, around the hilum; standard colour between green yellow and yellow green pigmented; wing colour between white, white pigmented violet to violet blue; colour of immature pods between green, yellowish green and green pigmented; and dry pod colour between yellow and orange. cluster ii represents 4% of the accessions (five accessions), which showed similarity in nine characters associated with different phenological stages: darker colour of seeds and lighter colour of seeds dyed purple; striped seed coat pattern; purple colour of immature pods; pod curvature curved; seed shine matt; oval seed shape; intermediate leaf persistence and absent vein in seeds. cluster iii represents 7% of the accessions (nine accessions), which showed similarity in eight characters associated with different phenological stages: in the presence of a dark colour stripe moving from the hilum towards the top of the grain, the standard colour between yellow-green pigmented with purple to purple violet; the darker colour of seeds between purple with white lateral stripe and violet blue with orange-white lateral stripe; lighter colour of seeds between greyish orange with white lateral stripe to greyish purple with white lateral stripe; longstripes seed coat pattern; wing colour between purple to purple violet; colour of immature pods between green and yellowish green; pod curvature between slightly curved to curved; dry pod colour between yellow and orange. cluster iv represents 52% of the accessions (63 accessions), which showed similarity in seven characters associated with different phenological stages: darker colour of seeds between greyish orange, greyish red, 6 santa cruz-padilla et al genetic resources (2025), 6 (11), 1–13 figure 1. map of collecting locations of 122 accessions of ñuña beans collected in three regions in peru and conserved in the inia germplasm bank. table 2. qualitative morphological characteristics assessed, codes and period of assessment. ipgri descriptor code adapted from descritores de phaseolus vulgaris (ipgri, 2001). ipgri descriptor code characteristic codes period of assessment 4.2.4 standard colour stc flowering 4.2.5 wing colour wic flowering 4.2.6 colour of immature pods cip immature pods expanded 4.2.9 pod curvature poc immature pods expanded 6.2.17 dry pod colour dpc harvest 6.1.8 leaf persistence lep when 90% of pods are dry 4.3.2 darker colour of seeds dcs grain dry 4.3.3 lighter colour of seeds lcs grain dry 4.3.1 seed coat pattern scp grain dry 4.3.4 seed shine ssh grain dry 6.3.2 veins in seeds vis grain dry 4.3.5 seed shape ses grain dry grey brown, purple, violet-blue and black; lighter colour of seeds between greyish orange, brown grey, greyish purple, purple, purple or greyish red and green yellow; seed coat pattern between absent, striped, to pattern around the hilum; standard colour between purple, purple violet and yellow-green pigmented with purple violet; wing colour between white to purple violet; colour of immature pods between yellowish green and green pigmented and dry pod colour between yellow, orange and purple. principal component analysis (pca) of quantitative traits the pca of quantitative traits explained 71.6% of the total variability and is presented in figure 4. in this analysis, the accessions did not show differentiation of their quantitative traits according to their origin. leaflet length and leaflet width showed a high positive correlation, as indicated by the direction of the arrows in figure 4. the same, but to a lesser degree, occured for the characters: immature pod length, pod width, pod genetic resources (2025), 6 (11), 1–13 phenotypic diversity of ñuña bean in peru 7 table 3. quantitative characteristics assessed, codes and period of assessment. ipgri descriptor code adapted from descritores de phaseolus vulgaris (ipgri, 2001). n/a, not available in descriptor list. ipgri descriptor code characteristics codes period of assessment units 4.2.7 immature pod length ipl fully expanded immature pods cm 6.2.13 pod width pwi fully expanded immature pods mm 6.2.14 pod beak length pbl fully expanded immature pods mm 6.3.3 seed weight (100 units) sew in dry grain, 12 to 14% moisture content g 6.3.5.1 seed length sel in dry grain, 12 to 14% moisture content mm 6.3.5.2 seed width sw in dry grain, 12 to 14% moisture content mm 6.3.5.3 seed height seh in dry grain, 12 to 14% moisture content mm 4.1.1 leaflet length ll at 50% flowering, on the third trifoliate leaf cm n/a leaflet width lw at 50% flowering, on the third trifoliate leaf cm figure 2. multiple correspondence analysis of qualitative variables of the germplasm of ñuña (phaseolus vulgaris). dcs, darker colour of seeds; lcs, lighter colour of seeds; scp, seed coat pattern; wic, wing colour; stc, standard colour; dpc, dry pod colour; cip, colour of immature pods; ssh, seed shine; vis, seed veins; poc, pod curvature; ses, seed shape and lep, leaf persistence. beak length, hundred seed weight, seed length, seed width and seed height, respectively. hierarchical analysis of quantitative characteristics the hierarchical cluster analysis based on the quantitative traits (figure 5) revealed the formation of four clusters, established according to their most similar characteristics. table 4 shows the mean values of the characters of clusters a, b, c and d, highlighting significant differences (p < 0.05) between clusters. cluster a, representing 22% of the accessions (27 in total), was characterized by lower values for immature pod length, pod width, pod beak length, hundred seed weight, seed length, seed width and seed height compared to the rest of the clusters (table 4). cluster b, comprising 16% of the accessions (20 in total), was significantly superior to the other clusters showing higher values for immature pod length, pod width, pod beak length, hundred seed weight, seed length, seed width and seed height. however, cluster b had lower values for leaflet length and leaflet width compared to the other clusters. cluster c, comprising 30% of the accessions (37 in total), showed higher mean values than cluster a, but lower than clusters b and d for the character’s immature pod length, pod beak length, hundred seed weight, seed length, seed width and seed height. for width of immature pod trait, cluster c had a higher mean than clusters a and d, but lower than cluster b. cluster c had a lower mean for the leaflet length trait, than the rest of the clusters. for the leaflet width character, the mean of cluster c was lower than clusters a and d, but higher than the mean of cluster b. finally, cluster d, comprising 31% of the accessions (38 in total), was characterized by higher values than clusters a and c for immature pod length, pod beak length, hundred seed weight, seed length, seed width and seed height. as for pod width, its mean value was higher than that of cluster a, but lower than the means of clusters b and c. cluster d stood out with higher values for leaflet length and leaflet width compared to clusters a, b and c. there was a marked diversity in the distances between clusters, particularly between cluster a and clusters c and d. this suggests that the accessions in these clusters show significant morphological differences. discussion qualitative characterization qualitative analysis revealed that characters associated with seed colour showed the greatest morphological variability, followed by seed coat pattern and, to a lesser degree, flower-related characters (figure 2). this indicates that seed-related characters are fundamental for the identification and differentiation of ñuña bean accessions, establishing associations between seed morphology and the phenotypic diversity observed among them. this result is consistent with previous studies by martirena-ramı́rez et al (2017) and espinosapérez et al (2015), who highlighted that flower colour and seed colour are essential to assessing phenotypic variability of common bean. similarly, morales-morales et al (2019) have determined the same for cowpea 8 santa cruz-padilla et al genetic resources (2025), 6 (11), 1–13 figure 3. hierarchical clustering based on multiple correspondence analysis of qualitative variables of the germplasm of ñuña (phaseolus vulgaris). dcs, darker colour of seeds; lcs, lighter colour of seeds; scp, seed coat pattern; wic, wing colour; stc, standard colour; dpc, dry pod colour; cip, colour of immature pods; ssh, seed shine; vis, veins in seeds; poc, pod curvature; ses, seed shape and lep, leaf persistence. the photographs inserted next to each group show the representative qualitative traits of each group. figure 4. projection of nine quantitative characters in the first two dimensions of the principal component analysis of 122 ñuña (phaseolus vulgaris) accessions from the germplasm collection of inia, estación experimental agraria baños del inca, cajamarca, peru. accessions are colour coded by origin regions. the arrows indicate the inertia of the contribution of the characters ipl, immature pod length; pwi, pod width; pbl, pod beak length; sew, seed weight; sel, seed length; sw, seed width; seh, seed height; ll, leaflet length; and lw, leaflet width. and the distance between them implies their correlation. genetic resources (2025), 6 (11), 1–13 phenotypic diversity of ñuña bean in peru 9 figure 5. hierarchical dendrogram of ñuña (phaseolus vulgaris), ward.d2 method. euclidean distance based on nine quantitative characters from the germplasm collection of inia, estación experimental agraria baños del inca, cajamarca, peru. table 4. descriptive analysis and comparison of means between clusters for quantitative traits. cv, coefficient of variation; msd, minimum significant difference; ipl, immature pod length; pwi, pod width; pbl, pod beak length; sew, seed weight; sel, seed length; sw, seed width; seh, seed height; ll, leaflet length; and lw, leaflet width. *means followed by the same letter in the rows do not differ statistically from each other, according to tukey’s test (p < 0.05). character average character values * msd cv(%) cluster a cluster b cluster c cluster d ipl (cm) 10.38 c 13.88 a 11.59 b 12.34 b 0.77949 9.46 pwi (mm) 12.55 c 15.35 a 14.59 b 14.29 b 0.58016 5.93 pbl (mm) 9.34 d 12.55 a 10.59 c 11.55 b 0.91175 12.08 sew (g) 41.10 c 73.43 a 55.85 b 60.08 b 6.18127 15.77 sel (mm) 8.99 d 13.40 a 10.24 c 11.53 b 0.74487 9.91 sw (mm) 7.50 c 9.10 a 8.35 b 8.56 b 0.27129 4.71 seh (mm) 6.76 c 7.97 a 7.45 b 7.55 b 0.27629 5.40 ll (cm) 11.82 b 11.75 b 11.67 b 13.66 a 0.93886 11.02 lw (cm) 8.48 b 7.90 b 8.15 b 9.73 a 0.68477 11.43 10 santa cruz-padilla et al genetic resources (2025), 6 (11), 1–13 (vigna unguiculata l. walp). murga-orrillo et al (2024) affirmed that in cowpea seed colours mark the preferences of producers and consumers since the dark colour of the testa presents greater antioxidant activity. from this perspective, a generalized tendency in bean research to use morphological characters of the seed as primary indicators of genetic variability is confirmed, underlining its importance in the classification and differentiation of accessions. the phylogenetic tree presented in figure 3 showed that the qualitative characters related to seed, flower and pod are valid phenotypic traits to discriminate morphologically the different accessions of ñuña; these characteristics, which vary among accessions, could be related to their areas of origin. in addition, it highlighted that there were no duplicate accessions in the studied collection at the phenotypic level. these results are valid for genetic diversity conservation since they indicate that the 122 accessions could represent unique genetic resources, which should be studied in detail to determine the variations of these accessions due to environmental and anthropogenic factors. also, cruzbalarezo et al (2009) conducted clustering analyses on 24 accessions of ñuña, which determined that there was no duplication of germplasm, even in cases where the accessions shared similar characters. on the other hand, in common bean, vásquez et al (2024) identified four distinct morphological groups in 58 accessions with promising characteristics for breeding programmes. these studies highlighted the importance of characterizing ñuña bean accessions for conservation and breeding initiatives. quantitative characterization regarding the spatial distribution of the 122 ñuña accessions, based on quantitative traits, no defined clustering pattern for geographic origin was identified (figure 4). this could be due, in part, to similar climatic conditions in the andean region or common agricultural practices (figure 1). mishra et al (2010) have shown that genetic diversity among bean genotypes did not show a direct relationship between clustering patterns and geographic origin. consequently, the maintenance of ñuña bean genetic characteristics from different geographic origins is crucial to preserving its biodiversity and genetic improvement. the pca presented in figure 4 shows strong positive correlations between leaf, immature pod and seed traits, demonstrating that these traits are mutually dependent. this could be related to shared developmental processes or the expression of common genes that regulate the size and structure of the mentioned plant organs. in common bean, lescaybatista et al (2017) and garćıa-fernández et al (2023) found a close association between pod characters and seed weight. also, in lima bean (phaseolus lunatus l.), a strong correlation was found between yield and stem length, number of seeds per pod, hundred-seed weight, primary leaf length and width, pod weight and pod length (akande and balogun, 2007; lópez-alcocer et al, 2016). the four clusters identified in figure 5 were evaluated through a mean comparison test (p < 0.05), as detailed in table 4. cluster b presents significant differences, with higher means in the traits related to productivity, compared to the means of clusters a, c and d. the accessions of cluster b will be valuable in genetic improvement programmes because they could provide higher yields. also, pesantes-vera and soto (2013) in ñuña, and kinhoégbè et al (2020) in pigeonpea (cajanus cajan (l.) huth), used characters related to productive yield in the selection of promising individuals. however, it is essential to conduct further studies on the 122 accessions of ñuña to identify relevant traits, such as resistance to abiotic and biotic factors, as well as nutritional aspects, in order to enrich future breeding programmes. conclusions in the qualitative traits of ñuña, mca determined that the most significant contributions to the variability of the accessions were the seed heads’ dark and light colours. in the phylogenetic hierarchical analysis, four clusters were formed, represented by 37% (i), 4% (ii), 7% (iii) and 52% (iv) of the accessions respectively. in the quantitative characters of ñuña, pca showed no discrimination between regions of origin of the accessions, but a high positive correlation between length and width of leaves, as well as between length and width of pods, and length, width, height and weight of seeds. in the hierarchical analysis of the quantitative characters, four clusters were also formed, represented by 22% (a), 16% (b), 30% (c) and 31% (d) of the accessions. subjected to comparative analysis of means, these clusters showed significant differences (p < 0.05) with higher values in cluster b for pod length and width, length, width, height and seed weight. understanding the variability of qualitative and quantitative traits of ñuña, and classifying them, is the starting point for subsequent genetic improvement studies aimed at obtaining ñuña cultivars with better yields and high nutritional value, resistant to biotic and abiotic factors. authors contribution angel esteban santa cruz padilla: conceptualization, formal analysis, writing – original, research, data curation, resources, methodology, proofreading and editing. jorge luis vásquez-orrillo: conceptualization, formal analysis, writing – original, research, data curation, resources, methodology, proofreading and editing. ricardo manuel bardales-lozano: formal analysis, writing – original, research, methodology, proofreading and editing. genetic resources (2025), 6 (11), 1–13 phenotypic diversity of ñuña bean in peru 11 hipolito murga-orrillo: formal analysis, writing – original, research, methodology, proofreading and editing. conflict of interest statement the authors have declared that no competing interests exist. acknowledgements this work was supported by the subdirección de recursos genéticos de la estación experimental agraria baños del inca of the instituto nacional de innovación agraria inia. the authors would like to thank armando linares estrada and sebastián llico sánchez for their support in the field. supplemental data supplemental table 1. agromorphological characterization data of 122 ñuña accessions from the inia germplasm bank peru. references akande, s. r. and balogun, m. o. 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(2016). ggplot2: elegant graphics for data analysis. url: https://ggplot2.tidyverse.org. https://doi.org/10.3390/nu7021144 https://doi.org/10.3390/nu7021144 https://www.r-project.org/ https://journal.upao.edu.pe/index.php/pueblocontinente/article/view/265/233 https://journal.upao.edu.pe/index.php/pueblocontinente/article/view/265/233 https://repositorio.inia.gob.pe/handle/20.500.12955/1404 https://repositorio.inia.gob.pe/handle/20.500.12955/1404 https://www.senamhi.gob.pe/servicios/?p=estaciones https://www.senamhi.gob.pe/servicios/?p=estaciones https://agronomiar.github.io/agror_package/index.html https://agronomiar.github.io/agror_package/index.html https://doi.org/10.1007/bf02862280 https://doi.org/10.1002/leg3.155 https://doi.org/10.51372/bioagro362.1 https://doi.org/10.1080/01621459.1963.10500845 https://doi.org/10.1080/01621459.1963.10500845 https://ggplot2.tidyverse.org introduction material and methods origin of the ñuña accessions location of the study soil chemical properties soil preparation cultivation treatments determination of variables statistical analysis results multiple correspondence analysis of qualitative characters hierarchical analysis of qualitative characteristics principal component analysis (pca) of quantitative traits hierarchical analysis of quantitative characteristics discussion qualitative characterization quantitative characterization conclusions authors contribution conflict of interest statement acknowledgements supplemental data genebank report genetic resources (2025), s2, 13–28 doi: 1046265/genresj.fcuw9498 https://www.genresj.org issn: 2708-3764 history and current status of plant genetic resources conserved and maintained by the hungarian central genebank zoltán áy *, attila simon, adrienn gyurkó, evelin fekete, balázs horváth and borbála baktay national centre for biodiversity and gene conservation, h-2766 tápiószele, külsőmező 15, hungary abstract: the predecessor of the national centre for biodiversity and gene conservation (nbgk) was established in hungary in 1959. the 1950s were hectic times for hungarian plant breeders, and many new genetic materials were registered in the national list of varieties. in order to save obsolete genetic resources, in 1959 the government founded the agrobotanical institute at tápiószele to prevent plant genetic erosion in the pannonian region. the centre started its operation with 16,596 accessions. their quantity continuously increased thanks to collecting missions and international connections. the nbgk collection is composed of cereals (37.3%), vegetables (18.7%), legumes (17.4%), industrial crops (5.29%), fruits and grapes (3.85%) and others. nbgk has operated under its current name since 2019 with the same mission as when it was first established. today, it is the seventh largest genebank in europe with 57,381 accessions of 1,745 plant species across 605 genera. almost 95% of samples are maintained in the form of seeds in 15 cooled storage rooms (at temperatures of 5–8◦c or -18◦c), while the others are conserved in vitro, in the form of tubers or field c ollections. s haring g enetic m aterials has been a crucial part of the institute’s activities since the beginning. between 2019 and 2023, a total of 92,100 samples were distributed to a variety of partners, mainly gardeners and farmers (83.85%) and ngos (14.63%). researchers, breeders and universities account for only 1.52% of seed requests, which is the opposite of what is observed in other genebanks. keywords: plant genetic resources, gene conservation, ex situ, seed bank, pannonian region citation: áy, z., simon, a., gyurkó, a., fekete, e., horváth, b., baktay, b. (2025). history and current status of plant genetic resources conserved and maintained by the hungarian central genebank. genetic resources s2, 13–28. doi: 10.46265/genresj.fcuw9498. © copyright 2025 the authors. this is an open access article distributed under the terms of the creative commons attribution license (cc by 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. foundation of the hungarian genebank the history of the national centre for biodiversity and gene conservation (hungarian abbreviation nbgk) and its legal predecessor institutes started in the 1880s. dr lajos szelényi, a hungarian doctor born in kismarton (today eisenstadt, austria), moved to budapest after finishing his university studies in vienna. besides his healing activities, he was famous for his charity work. he donated large sums of money to austrian and hungarian medical and agricultural science. dr szelényi purchased 400 acres of land in tápiószele, in the central hungarian region, which he donated to the national hungarian ∗corresponding author: zoltán áy (*ay.zoltan@nbgk.hu) economic association in his 1885 will, specifying that the land was to be used by future generations for agricultural experiments and vocational education in agriculture. the work of the first few decades was destroyed several times by the world wars, revolutions and political transformations. the 1950s were hectic times for hungarian plant breeders, and many new genetic materials were registered in the national list of varieties. in order to save the old and obsolete varieties, collection departments were established at the breeding institutions. the agrobotanical institute was established on the land in tápiószele left by dr szelényi under the leadership of dr andor jánossy (1908–1975) based on a government initiative in 1959, with the aim of integrating the genetic resource collections of the country and preventing genetic erosion (jánossy, received: 17.10.2024 accepted: 28.11.2024 published online: 08.01.2025 https://www.genresj.org https://www.doi.org/10.46265/genresj.fcuw9498 https://www.genresj.org https://www.doi.org/10.46265/genresj.fcuw9498 mailto:*ay.zoltan@nbgk.hu 14 áy et al genetic resources (2025), s2, 13–28 1971). despite several institutional reorganizations and changes of responsible organizations, the hungarian genebank operated under its current name and form since 2019, and its function and scope of duties have changed little over the last 65 years. currently, the nbgk operates as a central budgetary institution fulfilling public functions. it is maintained by the hungarian ministry of agriculture, with 50% of its annual budget financed by the government and the other 50% covered through grants. the majority of these grants come from national sources, but some are financed by the european union. composition and expansion of the collection the institute started its operation with 16,596 accessions of 871 plant species. according to our database, the starting number of accessions included both hungarian and foreign materials. the number of accessions has continuously increased thanks to wide international scientific connections and collecting expeditions in hungary and across the carpathian basin. since its establishment, the institute has exchanged genetic materials with around 400 other institutes, but development through collecting missions also has a long tradition in the genebank (guerrant et al, 2014). our founder and first director, dr andor jánossy, recognized already at the beginning of the 1950s that landraces were going to disappear from public production due to the spread of large-scale industrialized agriculture. he was one of the first in the world to collect cereals, fodder crops, maize and vegetable landraces and local varieties in cooperation with his colleagues before they became lost from production, replaced by high-yielding, intensively farmed improved varieties. this activity has always been regarded as an important duty of the institute, and it is still carried out today, for example in the case of fig genotypes. in 2023, we collected 22 fig (ficus carica l.) genotypes from the northern shore of lake balaton, one from budapest and another one from west hungary. during the collecting trips, we measured and characterized fruits, leaves and branches. the collected twigs are propagated in our nursery. as part of the pannon seed bank project (a life+ programme, life08 nat/h/000288), 2,064 accessions were collected between 2010 and 2014. these are vascular wild plants of the pannonian biogeographical region and wild relatives of cultivated plant species (hay and probert, 2013; walters et al, 2018). in the last 70 years, plant genetic resources have been collected from a total of 1,504 locations (figure 1), adding 13,785 accessions of hungarian origin to the collection of nbgk (table 1). today, collection work is also carried out abroad, thanks to collecting trips jointly organized with the genebanks of neighbouring countries (slovakia and romania). this has resulted in 2,477 landraces, local varieties, populations and ecotypes being collected from 340 locations. in addition, a further 237 accessions collected by foreign genebanks have been shipped to our institute through seed exchange. with regard to the amount of conserved genetic material, nbgk is the seventh largest genebank in europe today, preserving 57,381 accessions of 1,745 species from 605 genera. our oldest accessions date back to the early 1950s. both the number of accessions and taxa was continuously increased until the 2010s, with a levelling off in the 2020s (figure 2). regarding the composition of the different plant groups (table 2), our collection is dominated by cereals with 21,376 accessions, making up 37.3% of the collection. besides the major spiked cereals like wheat (triticum spp.) and barley (hordeum spp.), maize (zea spp.) and sorghum (sorghum spp.), pseudocereals like amaranths (amaranthus spp.), certain buckwheat species (fagopyrum spp.), finger milet (eleusine coracana (l.) gaertn.) and teff (eragrostis tef (zucc.) trotter) also belong to this group. vegetables and grain legumes constitute 18.7% and 17.4% of the collection, respectively. the former group consists of the collections of tomato (lycopersicon spp.), paprika (capsicum spp.), cucurbitaceae, onions (allium spp.), root and leafy vegetables and other vegetables (e.g. physalis spp.). almost half of the grain legumes collection (4,322) is composed of phaseolus species accessions originating from the american centre of origin and diversity, but pea (pisum spp.), chickpea (cicer spp.), lentil (lens spp.) and soybean (glycine spp.) accessions are also present. the three above-mentioned groups constitute 73.4% of the whole collection. the group of industrial crops containing 3,040 accessions includes genera such as sunflower (helianthus spp.), flax (linum spp.), poppy (papaver spp.) and the neglected camelina (camelina spp.) used for oil production. the group of forage legumes includes the accessions of 113 species from 20 genera, the most important of which are clovers (trifolium spp.), alfalfas (medicago spp.) and vetches (vicia spp.) constituting 80.7% of this group. in terms of the number of taxa, poaceae species are the second most diverse group (the first being herbs like medical plants from lamiaceae and asteraceae families) including 171 species of 51 genera. almost half of the 2,298 accessions of grasses have been collected. accessions of the pannon seed bank are listed as a separate group. the accessions of the above-mentioned utilization groups are stored generatively as seeds in the genebank, equating to 94.44% of the collection. besides this method, the institute has other ways of preserving plant genetic resources. although such samples are only a small part of the collection, they are very valuable. in recent years, the ratio of collections preserved in the form of field collections has increased. woody fruit crops, grapes and ornamentals are in this group, composing 3.85% of the collection. the collection of potato (solanum tuberosum l.) and its wild relatives are preserved in vitro (engelmann, 2011), constituting 1.24% of the genebank collections. from the group of tuber crops, jerusalem artichoke (helianthus tuberosus l.), sweet potato (ipomoea batatas l.) and onions genetic resources (2025), s2, 13–28 hungarian genebank – history, status and outlook 15 (allium spp.) are preserved vegetatively, making up 0.47% of the collections. multiplication and maintenance of genebank accessions genotypes stored in the form of seeds, tubers, bulbs or in vitro are reproduced in the field if the minimal number of seeds required for genebank storage has to be reached, viability has declined, taxonomic analyses are necessary, the accession needs to be multiplied for distribution purposes, or experiments are required to be performed. accessions conserved in the form of field collection are kept always outside in the fields. three professional teams carry out the work related to such duties: the department of arable crops, the department of horticultural crops and the department of fruit crops. the annual sowing plan is defined by the number of accessions requiring regeneration for the above-mentioned reasons and the number of spatially isolated field plots available for sowing. the number of accessions regenerated by growing new individuals in the field in the last 10 years is shown in table 3. the number of accessions sown from the different plant groups fluctuates yearly. this is not only because the different collections contain a different number of accessions, but also due to the diversity of factors that need to be considered during multiplication. between 2014 and 2023 plant groups with large collections – such as grain legumes, cereals, herbs and onions – dominated the multiplication process every year. consequently, members of the phaseolus, triticum, allium, lycopersicon, origanum and capsicum genera reached the highest levels of multiplication in the last ten years. in this period, common bean (phaseolus vulgaris l.) and winter wheat (triticum aestivum l.) were the most frequently multiplied species. the genebank established its fruit crop collection in 2013. due to its continuous expansion, today it contains 1,212 accessions. besides the members of the maloideae subfamily (apple (malus domestica borkh.), pear (pyrus communis l.), medlar (mespilus germanica j.b. phipps), quince (cydonia oblonga mill.)), stone fruits (plum (prunus domestica l.), sour cherry (prunus cerasus l.), cherry (prunus avium l.), peach (prunus persica l.), apricot (prunus armeniaca l.)) and other fruit species – such as cornelian cherry (cornus mas l.) and service tree (sorbus spp.) – are also found in the almost 9ha plantation. furthermore, the collection is complemented by a 1ha walnut (juglans regia l.) plantation and a 0.3ha grape (vitis vinifera l.) plantation. usually, three individual plants are conserved for each genotype. the first plants started to produce fruit in 2020. our institute has conserved a vegetative collection of ornamental plant varieties (iris, hemerocallis, hibiscus, hosta spp.) of 976 accessions in the nurseries of the genebank since 2017. there is also a rare and old woody plant stand in the central site of the genebank, which functions as a locally protected arboretum. some of the oak trees of the garden have been planted around the mansion (currently the main building) already in the time of dr lajos szelényi. unfortunately, the majority of the original english park died. the current garden was planted by the employees of the institute in the 1960s and 1970s. about 350 tree and bush species live here, including several rare ones like the californian white oak (quercus lobata née), the oregon cypress (hesperocyparis bakeri bartel), the algerian fir (abies numidica de lannoy), the trojan fir (abies nordmanniana subsp. equi-trojani spach), the cilician fir (abies cilicica carrière), the spanish fir (abies pinsapo boiss.), the turkish fir (abies bornmuelleriana coode & cullen), the lebanon cedar (cedrus libani subsp. libani a. rich.) and the mountain pine (pinus uncinata turra). the agrobotanical analysis and the assessment of the morphological biodiversity of plants sown in the fields are carried out by taxonomists according to hungarian and international guidelines (table 4). the descriptors used for characterization are based on the descriptors lists of the international union for the protection of new varieties of plants (upov, 2005), the international board of plant genetic resources (ibpgr) (thormann et al, 2018), the european cooperative programme for plant genetic resources (ecpgr) and in the case of grapes the international organisation of vine and wine (oiv) (hannin et al, 2006). for some plant species, we have refined or complemented the criteria system for agrobotanical analysis. for example, in the case of carrot, the upov guidelines have been integrated with the ibpgr descriptors and as a result, the following features are also recorded: colour of core, colour of cortex, root diameter of core relative to total diameter, and homogeneity of flesh colouring throughout root length. today these characterization data are almost always confirmed by photos as well. data are documented electronically and then assessed and stored in the database of the institute. the digitalization of the former paper-based agrobotanical analyses is a great challenge for us. human resources, infrastructural developments the hungarian genebank started its operation with 81 permanent workers in 1959. the number of employees exceeded 200 within ten years. our first director, dr andor jánossy, put great emphasis on scientific research, so he hired many researchers. being a member of the genebank’s staff was prestigious in the 1970s. political transformations that occurred in hungary in 1990 led to the decline of the institute. due to financial reasons, the number of employees was reduced to only 40 people within a few years, and the survival of the institute was at risk. fortunately, since 2010 the hungarian government has considered conservation of plant genetic diversity as an important issue once again, with a public function of strategic importance. today the institute has 130 employees (figure 3), six of which are scientists (dealing with research and development). more than 35% of workers have a university degree. 16 áy et al genetic resources (2025), s2, 13–28 figure 1. collection activity of the national centre for biodiversity and gene conservation (nbgk) in hungary (1950–2023). black, cultivated plants; red, wild plants (pannon seed bank). end of data collection: 31 december 2023 (google maps). table 1. division of collected accessions by place of origin places of collection nbgk pannon seed bank total accessions locations accessions locations accessions locations hungary 11,802 1,322 1,983 416 13,785 1,504 neighbouring country 2,396 319 81 21 2,477 340 other country 237 166 0 0 237 166 total 14,435 1,807 2,064 437 16,499 2,010 figure 2. the increasing number of genera, species and accessions maintained by the national centre for biodiversity and gene conservation (nbgk) from the date of its founding until now. end of data collection: 31 december 2023. genetic resources (2025), s2, 13–28 hungarian genebank – history, status and outlook 17 table 2. division of the national centre for biodiversity and gene conservation (nbgk) collection by utilization groups. *, roots and tubers, ornamentals and fruit crops. method of conservation plant utilization group accessions ratio of the whole collection (%) generative (94.44%) cereals 21,376 37.3% vegetables 10,739 18.7% grain legumes 10,002 17.4% industrial crops 3,040 5.3% fodder legumes 2,823 4.9% grasses 2,298 4.0% herbs 1,163 2.0% others * 687 1.2% wild species (pannon seed bank) 2,064 3.6% vegetative (0.47%) tuber crops 63 0.1% onions 209 0.4% in vitro (1.24%) potato and its wild relatives 709 1.2% plantation (3.85%) woody fruit crops 1,051 1.8% ornamentals 996 1.7% grapes 161 0.3% total 57,381 100,0% table 3. yearly number of accessions regenerated in the field between 2014 and 2023 divided by plant groups. dac, department of arable crops; dhc, department of horticultural crops. plant group 2014 2015 2016 2017 2018 2019 2020 2021 2022 2023 dac grain legumes 2,984 2,286 2,647 2,166 1,717 1,952 2,040 2,550 2,320 1,879 cereals 1,657 1,071 1,712 1,445 2,542 2,163 2,049 2,089 2,725 2,582 fodder legumes 720 566 484 382 646 583 491 360 367 330 grasses 395 364 367 478 827 621 499 239 235 296 industrial crops 296 207 214 231 314 384 418 274 229 289 cucurbitaceae 370 155 218 126 120 261 238 140 98 130 root and tuber crops 96 98 110 128 158 120 102 79 98 100 pseudocereals 34 69 78 51 16 18 52 27 20 48 total 6,552 4,816 5,830 5,007 6,340 6,102 5,889 5,758 6,092 5,654 dhc onions 235 294 286 321 288 441 403 346 327 345 tomato 67 56 82 51 49 102 138 70 50 71 paprika 84 54 53 47 44 80 112 53 26 53 other solanaceae 66 26 56 7 11 17 30 4 8 6 root vegetables 123 81 85 99 90 181 184 132 114 128 leafy vegetables 161 100 51 60 61 160 160 98 87 124 brassicaceae 50 27 56 45 29 52 67 30 41 30 cucurbitaceae 149 42 19 5 14 51 47 72 39 39 herbs 53 69 234 221 207 270 708 561 602 467 ornamentals 33 12 23 25 43 65 52 192 140 151 other vegetables 9 3 0 0 0 0 8 0 0 0 total 1,030 764 945 881 836 1,419 1,909 1,558 1,434 1,414 18 áy et al genetic resources (2025), s2, 13–28 table 4. descriptors for morphological diversity – an example using the agrobotanical description of a pear landrace according to the upov guidelines (2023). name of variety ‘hidegkúti nyári’ summer pear shoot shape straight, the internodes are long shoot colour brown-red on the sunny side with few lenticels vegetative bud rounded, markedly held out from shoot bud support size medium average shoot length 62cm average internode length/thickness 27.4mm/4.24mm average leaf length/width 61.2mm/32.9mm average petiole length/thickness 36mm/0.56mm flower bud short, mainly on spurs petals long, overlap, small in size, ovate in shape position of stigma mostly above the level of the anthers time of maturity end of july – beginning of august fruit size short in height, small in diameter, the height-to-diameter ratio is approximately 1:1 average fruit height/diameter 51.9mm/55mm average fruit weight 68.1g fruit shape the maximum diameter is in the centre of the fruit, the fruit is longitudinally symmetrical and the lateral shape is convex fruit ground colour yellow fruit cover colour absent area of russet small around the eye basin, none on the cheeks and around the stalk attachment stalk shape moderately long and thick, slightly curved, straight in relation to the axis of the fruit average stalk length/thickness 28.5mm/9.5mm average stalk cavity depth/width 1.1mm/12.8mm average eye basin dept /width 3.2mm/16.4mm flesh soft, medium juicy, fine structure sugar content 17.4bx◦ seed elliptic figure 3. staff of the national centre for biodiversity and gene conservation (nbgk) in summer 2022. photo by gergely gócsa, w ww.gocsafoto.hu genetic resources (2025), s2, 13–28 hungarian genebank – history, status and outlook 19 the infrastructure of the genebank – including the buildings and equipment – has continuously developed over time. during the first decade, seed conservation was carried out by storing the seeds in paper bags at room temperature. the first cold storage room – operating at 4◦c – was built in 1971. at the time, this was one of the first seed storage rooms of its kind in europe. in order to make seed storage more effective, paper bags were replaced by aluminium bags and glass jars. as a result of large investment in recent years, one of our old buildings has been completely renovated (figure 4). from 2024, all the cold storage rooms, seed drying rooms, and germination testing laboratory are located in the same place. currently, 15 cold storage rooms are operating for direct seed conservation purposes. there are nine active storage rooms running at a temperature of 5–8◦c ensuring medium-term conservation, and we have six base storage rooms cooling seeds to -18◦c for long-term storage of genebank accessions of orthodox species (dickie et al, 1990; nagel and börner, 2010). from the latter base storage rooms, three rooms have a special status: the national base storage room, the pannon seed bank and the safety duplicate storage room. the national base storage room has been used since 1996, with the aim of maintaining safety duplicates from the collections of the other hungarian gene conservation institutes at no cost. so far, 26 hungarian gene conservation institutes have sent genetic materials totalling 16,966 accessions of 219 plant species from 124 genera. the pannon seed bank project was a life+ programme running between 2011 and 2015, aiming at the long-term conservation of native plant species of the pannonian biogeographical region whose seeds can be stored with this technology. after a transition period, we rethought the project, and continued the monitoring and collection of wild plant species from the region in 2017. this cold storage room contains 2,064 accessions of 921 species from 430 genera today. in 2014 our genebank built the safety duplicate storage room with the support of the hungarian ministry of agriculture and the aggtelek national park in the strictly guarded passage of a dripstone cave. the role of this storage room is to duplicate the seeds of the most important plant genetic resources for food and agriculture (25% of the whole collection), including landraces, local populations and ecotypes collected from the carpathian basin and also old varieties that have disappeared from public production. so far, 6,733 accessions of 289 species from 159 genera have been put in the safety duplicate storage room. our equipment has been continuously modernized over the last 10 years, with the decreasing availability of physical labour force, driving us to purchase modern machines. our former plot seeder and harvester have been replaced by new machinery in the last 1–2 years. for decades, we used russian tractors and implements, but today we work with those produced in western europe and hungary. our germination testing laboratory has been upgraded by purchasing new incubators. the analytical, genetic and tissue culture laboratory has been equipped with state-of-the-art devices (figure 5). in the future, we plan to renovate the greenhouses built in 1961 and also to obtain phenotyping systems. breeding and maintenance of varieties since its establishment, nbgk has regarded plant breeding as an important secondary activity. our institute used to be one of the sites of the national variety testing network, where the performance testing of candidate varieties (plant materials under a 3year registration process) took place. by applying the methods of positive individual selection and crossbreeding, the researchers of the genebank have developed 28 new varieties (table 5). the qualifying certificates of our listed varieties are kept in our library (figure 6). many of them have been on the national list of varieties for decades. the maintenance and propagule production of these varieties also take place in tápiószele. today, three jerusalem artichoke (helianthus tuberosus) varieties (‘tápiói korai’, ‘tápiói sima’, ‘tápiói piros’) and one sweet potato (ipomoea batatas) variety (‘tápiói 96’) are included in this process. in 2023, our genebank applied for the registration of 17 candidate varieties on the national list of varieties in the following categories: variety, landrace and variety developed for growing under particular conditions. our candidate varieties are: one peanut (arachis hypogaea l.), two chickpea (cicer arietinum l.), two cowpea (vigna unguiculata (l.) walp.), one teff (eragrostis tef), one coracan (eleusine coracana). our landrace candidates are: one sunflower (helianthus annuus l.), one sorghum (sorghum bicolor (l.) moench.), one millet (panicum miliaceum l.), two flax (linum usitatissimum l.), one barley (hordeum vulgare l.), one maize (zea mays l.), one safflower (carthamus tinctorius l.), one fodder watermelon (citrullus amarus schrad.), two tomato (lycopersicon esculentum mill.), one paprika (capsicum annuum l.). our candidate variety developed for growing under particular conditions is: one lentil (lens culinaris medik.). our tomato and paprika landrace candidates were selected from genebank collections of 2,097 accessions and 3,615 accessions, respectively. we also perform preparatory and monitoring activities for variety development for other species, such as winter wheat, rye, common bean, kidney vetch (anthyllis vulneraria l.), crested wheatgrass (agropyron cristatum (l.) gaertn.), timothy grass (phleum pratense l.), smooth brome (bromopsis inermis (leyss.) holub), poppy, onion, beetroot (beta vulgaris l. subsp. vulgaris), parsley (petroselinum crispum (mill.) fuss), carrot (daucus carota subsp. sativus (hoffm.) arcang.), calendula (calendula officinalis l.), summer savory (satureja hortensis l.), dill (anethum graveolens l.) and oregano (origanum vulgare l.). in the case of these species, we currently do not have enough seeds for variety certifying analyses and for 20 áy et al genetic resources (2025), s2, 13–28 figure 4. ceremonial handover of the new genebank building of the national centre for biodiversity and gene conservation (nbgk) on 10 july 2024. we plan to use this new building for at least 50 years for professional conservation of plant genetic resources in hungary. photo by anikó gál soltész, nbgk figure 5. (a) oat (avena sativa l.) accessions in the active storage room at a temperature of 5–8◦c in 2015; (b) sowing of spiked cereals with a plot seeder in autumn 2019; (c) measuring crude protein content in the biochemical laboratory in 2022; (d) dna isolation from tetraploid wheat accessions in 2022. photos by attila simon, lajos horváth and dóra bárdos. genetic resources (2025), s2, 13–28 hungarian genebank – history, status and outlook 21 figure 6. qualification certificate of the soybean variety called ‘pannonia 10’ from 1967. the breeder was viktor ferenczi. digitalized by nbgk library. super-elite multiplication, and the different varieties still need to be fully described. we plan to apply for the registration of these candidate varieties on the national list of varieties within the next five years. the registration of landraces on the national list of varieties is difficult in hungary due to bureaucratic reasons, since these varieties cannot fulfil the criteria of uniformity during the dus tests (distinctness, uniformity and stability), or they often do not exceed the yield level of modern improved varieties used as control varieties during performance tests. hungarian and international scientific and social relations the institute has always worked to meet international professional requirements (cromarty et al, 1982; fao, 2014). director dr andor jánossy organized the eucarpia congress in budapest in 1974 (jánossy and lupton, 1974), during which the participants also visited the genebank in tápiószele, which by that time already had an international reputation (figure 7). in addition, our institute published the scientific journal agrobotanika between 1959 and 1975, presenting the results of research colleagues and describing collecting trips. hungary signed the convention on biological diversity (cbd), ratified the international treaty on plant genetic resources for food and agriculture (itpgrfa), and joined the quality assurance programme of a european genebank integrated system (aegis). our institute is member of the promoting a plant genetic resources for europe (pro-grace) consortium, created within the horizon europe programme in 2023. we participate in ecpgr working group activities, and our collection is available in the ex situ database of the european search catalogue for plant genetic resources (eurisco). nbgk provides the presidency of the hungarian plant genebank council, a consultative platform established in 2011. it works as an independent professional advisory board besides the minister of agriculture. it is responsible for the professional representation of plant gene conservation, as well as related research and development issues in hungary. additionally, it provides expert opinions on legislation, applications and national programmes related to plant gene conservation, including to the minister of agriculture. we have contacts with several universities and around 30 university students spend their professional practices in the genebank every year, who may become future employees of the institute. we participate in joint research and development projects together with universities and innovative enterprises from the commercial sector. we perform field experiments with drought-resistant alternative species, edible grain legumes, fodder cereals, oil crops and jerusalem artichoke, winter and spring lentil, chickpea and poppy. we study the chemical composition of apple, pear, apricot, pumpkin, beetroot, celeriac, carrot, tomato and herb accessions stored in the genebank by analytical methods (table 6). in our genetic laboratory, we analyze tetraploid wheats (röder et al, 1998) and the genetic relationship between in vitro conserved potatoes by using ssr (simple sequence repeats or microsatellite) markers. 22 áy et al g enetic resources (2025), s2, 13–28 table 5. registered plant varieties in hungary bred/maintained by the national centre for biodiversity and gene conservation (nbgk) (1954–2024). year, year of registration in hungary. denomination of species scientific name common name hungarian name of variety breeders year avena sativa l. oat tápláni csupasz mr miklós deutsch, mrs zsuzsa wesel 1973 capsicum annuum l. sweet pepper kocsolai zöldhúsú nbgk 1970 glycine max l. soybean pannonia 10 mr viktor ferenczi 1967 glycine max l. soybean tápláni takarmány mr árpád szücs, mrs zsuzsanna kanyó 1968 helianthus tuberosus l. jerusalem artichoke tápiói korai mr lajos horváth, mrs ágnes bárdy, mr lászló holly, mr józsef barta 2003 helianthus tuberosus l. jerusalem artichoke tápiói sima nbgk 2003 helianthus tuberosus l. jerusalem artichoke tápiói piros nbgk 2022 hordeum vulgare l. spring barley tápláni tavaszi mr miklós deutsch, mr andor jánossy dr, mrs józsefné németh 1968 ipomoea batatas l. sweet potato tápiói 96 nbgk 2003 lycopersicon esculentum mill. tomato tápláni konzerv mr jános ávár, mrs jánosné ávár 1970 medicago sativa l. alfalfa bánkúti mr andor jánossy dr, mr zoltán csák, mr zoltán böjtös 1961 medicago sativa l. alfalfa békésszentandrási mr andor jánossy dr, mr zoltán csák, mr zoltán böjtös 1961 medicago sativa l. alfalfa nagyszénási mr andor jánossy dr, mr zoltán csák, mr zoltán böjtös 1961 medicago sativa l. alfalfa szarvasi mr andor jánossy dr, mr zoltán csák, mr zoltán böjtös 1961 medicago sativa l. alfalfa tápiószelei 1 mr andor jánossy dr, mr árpád szücs 1970 melilotus albus medik. sweet clover kecskeméti kétéves nbgk 1969 oryza sativa l. rice nucleoryza mr zoltán sajó, mr józsef simon 1979 panicum miliaceum l. millet topáz nbgk 1986 phaseolus vulgaris l. bean nagykállói étkezési mr ambrus szabó, mrs ambrusné szabó 1979 phaseolus vulgaris l. bean tápiói cirmos étkezési mr árpád szücs, mrs árpádné szücs, mrs józsefné németh 1980 phaseolus vulgaris l. var. nanus bean tápiószelei barnabab mr árpád szücs, mrs zsuzsanna kanyó 1967 continued on next page g enetic resources (2025), s2, 13–28 h ungarian genebank – h istory, status and outlook 23 table 5 continued denomination of species scientific name common name hungarian name of variety breeders year phaseolus vulgaris l. var. nanus bean tápiószelei fürjbab mr árpád szücs, mrs zsuzsanna kanyó 1967 phaseolus vulgaris l. var. nanus bean tápiói gyöngybab mr árpád szücs, mrs zsuzsanna kanyó 1970 trifolium incarnatum l. crimson clover kemenesaljai mr andor jánossy dr, mr miklós deutsch 1968 trifolium pratense l. red clover táplánszentkereszti diploid nbgk 1954 trifolium pratense l. red clover táplánszentkereszti mr miklós deutsch 1955 trifolium pratense l. red clover hungaropoly tetraploid mr andor jánossy dr, mr miklós deutsch, mrs lajosné horváth dr, mr árpád szücs, mr lászló bányai 1966 trifolium pratense l. red clover tápiói tetraploid mr andor jánossy dr, mr miklós deutsch, mr árpád szücs, mrs lajosné horváth dr, mr istván sulyok 1970 24 áy et al genetic resources (2025), s2, 13–28 additionally, we coordinate our own on-farm network. within a given landscape and agricultural district, plant populations adapted to local biotic and abiotic factors are often the most stable varieties (holly et al, 2009). the on-farm programme was launched with four farmers in 2018. the network is expanding; in 2024, we are working with 20 farmers. collecting seeds in their fields or gardens and recording information concerning the motivation of farmers in growing these varieties has contributed to our knowledge of agricultural biodiversity. landraces of crops such as maize, vetches, cucurbits, beans, paprika, rye (secale cereale m. bieb.) and some underutilized species (i.e. safflower) are used in on-farm conservation in various regions within hungary. farmers and gardeners taking part in the programme can try those landraces which had been collected from their area decades ago. our partners worked with 430 landraces between 2018 and 2023. since they report on their experience, the genebank gets useful first-hand information on the actual producibility and marketability of these plant genotypes. our results are published in an open-access format (gyurkó et al, 2023; kis et al, 2023). besides international scientific articles, our hungarian popular publications are also well-known among the local people. we also organize and take part in seed swaps run by ngos in different parts of the country. our institute is open for groups of visitors by prior arrangement. every year we present the genebank to hundreds of professionals and lay visitors in tápiószele. distribution the national gene conservation strategy sets measures for the accessibility, mobilization and distribution of genebank samples. increasing and keeping this activity at a high level has become the most important tool for the utilization of the collections. distribution has been one of the goals of our institute since the beginning (figure 8). in the first ten years of operation, the genebank disseminated about 5,000 seed samples for plant breeding purposes. between 2019 and 2023, we registered 10,136 seed requests, from which 9,915 have been fulfilled by shipping 92,100 samples. we operate a separate website for this activity (w ww.mintakeres.hu), which works as a webshop. there are two seed dissemination campaigns each year (one in spring and the other in autumn). the majority of seed distributions (83.85%) are directed to hobby gardeners and farmers, with the remaining part to ngos, breeding and research institutes (table 7). according to the research coordinated by the centre for genetic resources, the netherlands (cgn) within the pro-grace consortium in december 2023, the situation is the opposite for the majority of european genebanks for which the majority of distributions are directed to breeding and research institutes (van hintum, unpublished). for scientific, research-related, educational or cultural seed requests, our whole collection is available in addition to those varieties listed in the webshop. however, in this case, a standard material transfer agreement (smta) (correa, 2006) needs to be signed, and the number of stored propagules should not fall below our critical threshold limit as a result of the request (2,000 seeds on average). the most distributed species in the last five years were paprika (10,289 samples), tomato (9,234 samples), maize (4,225 samples), basil (ocimum basilicum l.; 1,916 samples) and common bean (1,865 samples). we shipped 425 samples abroad. about 60% of them were requested by research institutes, while the remaining others by hobby gardeners. the number of seed requests reached its peak in 2023, while the number of distributed samples peaked in 2021. we aim to maintain these same high levels of distributions in the future. this is not a profitable activity for our institute since those requesting seeds only have to pay a 5 eur handling fee. seed dissemination for research and educational purposes is free of charge. the fruit gardener agreement – ‘agreement on cooperation in the conservation of fruit varieties long cultivated in the carpathian basin and adapted to local circumstances’ – coordinated by nbgk is connected to our fruit varieties collection. this initiative aims at the reintroduction of old fruit varieties, landraces and local varieties to municipal sites, church gardens and schoolyards. fruit saplings are provided free to the applicants by nbgk. so far a total of 329 gardens have been established throughout the country using 13,728 fruit tree scions. children are also involved in the process of planting trees, and in this way, the next generations will be more engaged in the conservation of genetic resources (figure 9). future plans our new seed cold storage facilities, opened in 2024, provides the basis for further expansion of our collection. we aim to make full use of this infrastructure and store at least 70,000 accessions within the next few years. we will increase the current number of employees from 130 to 150. we plan to invest in a new building dedicated to in vitro conservation activities and in a new greenhouse suitable for performing research activities and plant physiology analyses. we intend to decrease the average age of our machines, especially tractors, and to obtain new equipment for our laboratory, including a capillary electrophoresis instrument and a huller for glumaceous cereals. these would actively contribute to increasing the number and quality of our scientific publications. we will apply for the registration of many new candidate varieties on the national list of varieties in the next few years. we would like to further develop our database and elaborate a quality assurance system that is consistent with other european genebanks. acknowledgements our activities are supported by the ‘tkp2021-nka03’ project and the ‘promoting a plant genetic genetic resources (2025), s2, 13–28 hungarian genebank – history, status and outlook 25 figure 7. participants of the international eucarpia conference study hemp (cannabis sativa l.) plots in the nursery of the genebank in tápiószele in june 1974. photo: nbgk archives. table 6. an example of biochemical diversity – sugar, crude protein and crude fibre content – of some celeriac varieties maintained in tápiószele from the harvest of 2022 (all data is the average of three replications, and they refer to dry matter). name of variety fructose (g/l ) glucose (g/l) sucrose (g/l) fructose + glucose + sucrose (g/l) crude protein (%) crude fibre (%) albin 0.172 28.175 17.369 45.716 11.78 7.76 apia 0.138 33.404 27.683 61.225 11.42 8.18 balder 0.131 34.826 22.008 56.964 14.23 7.40 brilliant 0.332 30.419 20.702 51.453 10.10 8.80 bükkzsérci 0.289 30.334 19.081 49.704 13.29 8.98 erdőhorváti 0.634 30.494 18.268 49.396 11.58 8.56 frigga 0.052 33.547 28.188 61.788 9.56 8.87 hegykői 0.130 32.694 25.698 58.523 12.63 7.39 imperator 0.252 34.446 23.667 58.365 13.26 8.46 kecskeméti 0.133 31.141 22.745 54.018 11.02 7.99 kéki 0.219 32.215 17.727 50.161 10.76 7.82 kisteleki 0.129 36.822 20.417 57.367 11.23 8.04 maxim 0.027 30.111 27.605 57.744 11.93 8.23 neon 0.042 29.174 22.858 52.074 10.79 8.14 nýıregyházi 0.925 37.688 20.200 58.813 11.72 8.15 prágai óriás 0.157 29.142 16.604 45.903 11.04 8.49 sótonyi 0.236 33.636 25.449 59.320 9.97 8.90 taktaharkányi 0.313 43.278 23.438 67.029 13.19 9.27 tarpai 0.644 32.368 18.054 51.067 12.09 8.94 trizsi 0.123 31.512 20.378 52.013 12.60 7.72 26 áy et al genetic resources (2025), s2, 13–28 figure 8. propagules disseminated from the national centre for biodiversity and gene conservation (nbgk) between 1973 and 2023. end of data collection: 31 december 2023. table 7. number of propagule requests and distributed samples by type of requesters between 2019 and 2023. hobby gardeners farmers research institutes educational institutes ngos museums municipalities total public private 2019 requests 1,157 17 15 4 5 5 2 0 1,205 samples 9,474 58 148 41 31 2,638 1 0 12,391 2020 requests 1,376 21 3 2 6 3 0 0 1,411 samples 10,919 99 39 16 191 2,945 0 0 14,209 2021 requests 2,394 24 2 4 4 1 4 1 2,434 samples 19,150 259 3 53 58 2,500 42 3 22,068 2022 requests 2,287 30 3 5 9 4 2 0 2,340 samples 16,615 134 34 276 321 3,964 25 0 21,369 2023 requests 2,481 29 8 1 2 1 1 2 2,525 samples 20,027 487 133 1 58 1,250 2 105 22,063 total requests 9,695 121 31 16 26 14 9 3 9,915 samples 76,185 1,037 357 387 659 13,297 70 108 92,100 resource community for europe – prograce’ project (identification number: 101094738) carried out within the horizon europe programme. declaration the authors declare that the photos do not infringe on any personal or property rights, and that all people shown in the pictures have given their consent for publication. author contributions zoltán áy edited and wrote the paper. attila simon conceived, designed and performed the analysis. adrienn gyurkó collected the data on fruit crops. evelin fekete collected the data on horticultural crops. balázs horváth collected the data on arable crops. borbála baktay collected data on the history of the institute. conflict of interest statement the authors certify that they have no affiliations with or involvement in any organization or entity with any financial interest (such as honoraria; educational grants; participation in speaker’s bureaus; membership, employment, consultancies, stock ownership, or other equity interest; and expert testimony or patent-licensing arrangements), or non-financial interest (such as personal or professional relationships, affiliations, knowledge or beliefs) in the subject matter or materials discussed in this manuscript. this statement indicates that the above information is true and correct. genetic resources (2025), s2, 13–28 hungarian genebank – history, status and outlook 27 figure 9. even the youngest ones can be engaged in gene conservation. planting of landrace fruit saplings provided by the national centre for biodiversity and gene conservation (nbgk) in sokorópátka (northwest hungary, december 2021). photo by balázs csapó, 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(2018). genebank conservation of germplasm collected from wild species. in north american crop wild relatives, ed. greene, s. et al. volume 1, 245-280. https://doi.org/10.1007/s10722-018-0715-5 https://doi.org/10.1007/s10722-018-0715-5 https://www.upov.int/en/publications/tg-rom/tg014/tg_14_9.pdf https://www.upov.int/en/publications/tg-rom/tg014/tg_14_9.pdf foundation of the hungarian genebank composition and expansion of the collection multiplication and maintenance of genebank accessions human resources, infrastructural developments breeding and maintenance of varieties hungarian and international scientific and social relations distribution future plans acknowledgements declaration author contributions conflict of interest statement original article genetic resources (2022), 3 (6), 74–88 doi: 10.46265/genresj.ppuf5169 https://www.genresj.org issn: 2708-3764 where access and benefit-sharing comes from: a historical overview aysegül sirakaya *,a,b a faculty of law, lund university, sweden b abyss consulting, sweden abstract: the international legal system of access and benefit-sharing of genetic resources (or abs) under the convention on biological diversity (cbd) is an ever-evolving field as its material, temporal and activity scope is still under discussion to meet the needs of the advancement of research and development activities as well as the questions of fairness and equity that evolve with them. activities, such as research and development with digital sequence information (dsi), currently take considerable space in the negotiations and the lack of consensus between the global north and the global south continues. this paper gets its raison d’être from this lack of consensus and aims to provide a better understanding of the debate around the fair and equitable sharing of benefits arising from genetic resources as well as the sovereignty of states over their natural resources. as such, the paper provides an analysis of all relevant documents at the international level, starting from the un charter to the final text of the cbd with the hope of reminding the ongoing negotiations over the cbd why we have abs in the first place and what the international community historically aimed for when regulating genetic resources at the international level. looking back at why we had the first legally binding abs instrument in the first place, and why we thought this instrument would achieve fairness and equity in dealing with genetic resources, will serve the interests of all parties to the cbd and will hopefully enable them to interpret the provisions based on their overarching aim and reasoning. keywords: cbd, abs, access and benefitsharing, convention on biological diversity, benefitsharing, global multilateral benefitsharing mechanism, nagoya protocol, plant treaty negotiations, itpgrfa, genetic resources, plant genetic resources citation: sirakaya, a. (2022). where access and benefit-sharing comes from: a historical overview. genetic resources 3 (6), 74–88. doi: 10.46265/genresj.ppuf5169. © copyright 2022 the authors. this is an open access article distributed under the terms of the creative commons attribution license (cc by 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. introduction international law provides for mechanisms for biodiversity conservation and restoration to compensate for the utilization of nature by humans. one of the major mechanisms is the fair and equitable sharing of benefits arising from the use of genetic resources (unep, 1992), which is a bilateral legal instrument based on state sovereignty over natural resources. the system of access and benefit-sharing (abs) aims to fairly distribute benefits between the providers of genetic resources (such as biodiversity-rich countries) and users of genetic resources (such as biotechnology or pharmaceutical companies, universities, collections such as botanical gardens or genebanks) deriving from scientific research ∗corresponding author: aysegül sirakaya (aysegul.sirakaya@jur.lu.se) and development on genetic resources (gr). the abs system prescribes the parties to the convention on biological diversity (cbd) and to the nagoya protocol (secretariat of the convention on biodiversity, 2011) to implement national legislation on providing fair access to gr users while receiving fair and equitable benefits. states are then encouraged to channel benefits into biodiversity conservation and sustainable use. next to the abs system established under the cbd, specialized abs instruments exist on specific types of gr and their specific types of use. one of them is the international treaty on plant genetic resources for food and agriculture (itpgrfa) dealing with the conservation and sustainable use of all plant genetic resources for food and agriculture (fao, 2004). the itpgrfa facilitates access to the genetic materials of 64 crops in the multilateral system for research, breeding and training for food and agriculture. received: 03.08.2022 accepted: 31.10.2022 published online: 29.11.2022 https://www.genresj.org https://www.dx.doi.org/10.46265/genresj.ppuf5169 https://www.genresj.org https://www.dx.doi.org/10.46265/genresj.ppuf5169 mailto:aysegul.sirakaya@jur.lu.se genetic resources (2022), 3 (6), 74–88 where abs comes from: a historical overview 75 another specialized abs instrument is the world health organization pandemic influenza preparedness framework (or who pip framework). its purpose is the sharing of h5n1 and other influenza viruses with human pandemic potential, access to vaccines and sharing of other benefits (who, 2021). both the itpgrfa and the pip framework function on a multilateral basis, meaning that these two specialized abs instruments serve as pools of gr and operate under standardized agreements each party uses. lastly, there currently is an ongoing discussion at the international level regarding the provisions related to the bioprospecting activities taking place in areas beyond national jurisdiction under the united nations convention on the law of the sea. the negotiations are yet to be finalized, however, the issue of benefit-sharing deriving from the utilization of marine genetic resources in areas beyond national jurisdiction will be a part of this new international legal instrument (united nations general assembly a/res/72/249, 2017). abs under the cbd, as a bilateral instrument for creating incentives for conserving biodiversity within national jurisdiction, has been an active legal concept subject to national implementation since 1992. its success is being measured by the global biodiversity outlook (secretariat of the convention on biological diversity, 2020). additionally, abs has found its place in several targets within the united nations sustainable development goals, such as goal 10 target 10a, goal 15 target 6, and goal 17 target 6. it is also highly likely that targets related to abs will be an indispensable part of the post-2020 global biodiversity framework under the cbd (cbd/post2020/ws/2019/8/3, 2019). therefore, abs provides a tangible contribution to the achievement of international targets related to biodiversity conservation. the report published by the intergovernmental science-policy platform on biodiversity and ecosystem services (ipbes) in may 2019 states that our nature and ecosystems are in rapid and constant decline (ipbes, 2019). with the current trajectories, we cannot meet global targets such as the sustainable development goals. likewise, the fifth global biodiversity outlook, conducted under the auspices of the cbd to assess whether international conservation goals (such as the aichi global biodiversity targets) have been met, warns us that none of our targets has been met due to the lack of effective restoration and conservation initiatives (secretariat of the convention on biological diversity, 2020). with the success of all global environmental targets jeopardized, it emerges the need to review international legal obligations that aim to halt biodiversity loss and thereby contribute to achieving these targets, including how abs – being one of the major instruments in financing and incentivizing biodiversity conservation – was negotiated. this requires looking back at the original reasons and principles behind the implementation of an international access and benefit-sharing system. additionally, the current ongoing discussions on the scope and mechanisms of the international abs system, such as whether gr include digital sequence information (dsi), make such a retrospect even more necessary. this paper provides a historical review of the developments at international fora that led to the negotiation and adoption of abs provisions under the cbd, to serve as a guide for why the international community needed a bilateral abs system in the first place. additionally, the paper can serve for further evaluations on why abs may or may not have achieved its anticipated objectives regarding establishing the norms of fairness and equity in dealing with gr while ensuring sustainable use and biodiversity conservation. the scope of the paper is limited to the historical events and negotiations up until the adoption of the cbd. this choice is motivated by two reasons. firstly, the paper aims to elaborate on the discussions deriving from the concept of state sovereignty over natural resources and how sovereignty affected the use and provision of gr. because of this, the paper solely focuses on the bilateral abs framework under the cbd, and only refers to the abs regime under the food and agriculture organization of the united nations (fao) as a reference point in the historical events that led to the adoption of the abs mechanisms under the cbd. secondly, the paper analyzes the negotiations that led to the adoption of the cbd and not the nagoya protocol. this is because the paper does not aim to provide a fully comprehensive analysis of all concepts within the abs regime generated under the cbd. it rather aims to provide a glimpse into the history of the dynamics and needs that led to the generation of the international abs framework under the cbd starting from its establishment by the united nations and the development of the concept of the sovereignty of states over their natural resources. these were fuelled by the aftereffects of colonization, which palpably affected the dynamics between the global north and the global south. therefore, this paper does not include the analysis of the negotiations that led to the adoption of the nagoya protocol, nor does it include the negotiation processes and adoption of other specialized abs instruments. nonetheless, i wholeheartedly agree with the importance of also reviewing the post-cbd negotiations which aimed at clarifying the concept of abs, genetic resources as well as the activities of access and benefit-sharing, their legal provenance, and their purpose. for this reason, i have previously conducted research specifically on the postcbd negotiations that led to the adoption of the nagoya protocol (sirakaya, 2022). however, the scope of the present paper is not related to the clarification of the concepts generated by the cbd, but it is related to the historical reasons why we needed these concepts in the first place. 76 sirakaya genetic resources (2022), 3 (6), 74–88 sovereign rights over natural resources vs common heritage of mankind united nations biosphere conference until the 1950s there existed no discussion on the provenance of gr and their utilization under international law. the first time the international community took on the subject was in 1950 during the united nations scientific conference on the conservation and utilization of resources. at that time, delegates acknowledged that states varied considerably in their political, economic and social institutions. additionally, it was pointed out that world resources were not distributed proportionally to states’ populations or national boundaries. therefore, the free and full exchange of resources was seen as key for each nation to specialize in those products derived from such resources for which it enjoys the greatest comparative advantage or least comparative disadvantage. consequently, tariffs, duties, cartels, quotas, monetary manipulations and various other political and economic devices were generally recognized as constituting the major obstacles to improved utilization of global resources. the conference had a demonstrable preference towards unrestricted access to the world’s resources (as the term gr was not yet pronounced at the international level), thus neither the limits to utilization nor the subject of conservation of these resources made it on the agenda of this conference. the introduction of the concept of states’ rights over their natural resources dates to the charter of the united nations. the permanent sovereignty of states over their natural resources has been under discussion within the united nations general assembly starting from 1952. ten years later, the united national general assembly resolution 1803 (xvii) (1962) was adopted, which articulated that states and international organizations shall strictly and conscientiously respect the sovereignty of peoples and nations over their natural wealth and resources in accordance with the charter of the united nations and the principles contained in the resolution (united nations general assembly a/res/3171, 1973). these principles were set out in eight articles concerning, inter alia, the exploration, development and disposition of natural resources. the resolution further detailed that in cases where authorization is granted for the exploration, development or deposition of these resources, the profits derived must be shared in the proportions freely agreed upon, between the investors and the recipient state. the resolution, therefore, was the first international legal document that initiated the conversation on the states’ sharing in the benefits derived from the exploration or exploitation of natural resources within their national jurisdiction. the concerns over the conservation of the earth’s resources started building up during the 1960s when technological advances enabled humankind to develop a more enhanced understanding of the finiteness of the biosphere. the apollo 8 mission of 1968 demonstrated to humankind the vulnerability of our planet by displaying the first photograph of the earth ever taken from space suggesting the earth had no other place like it anywhere close and thus was the only place life existed. this realization provided a wake-up call and moved environmentalism to mainstream international discussions (attenborough and hughes, 2020; meadows and randers, 2013) as a response to this mainstream awakening, the united nations educational, scientific and cultural organization (unesco) convened the international biosphere conference (ibc) in 1968. next to being one of the first international high-level conferences that emphasized the importance of nature conservation, it was also the first international conference that introduced the concept of ’genetic resources’ to policymakers. more specifically, in its recommendations related to gr utilization, the ibc suggested that special efforts had to be taken urgently to preserve the rich gr that evolved over millions of years and were being irretrievably lost as a result of human actions. recommendations included the preservation of samples of all significant ecosystems, the establishment of special protected areas and living collections for both remnant and endangered species as well as long-domesticated species such as cereals and cattle. the ibc recommended the member states of unesco, as well as fao, to take vigorous efforts in implementing these recommended measures to avoid the loss of gr which could never be recovered. stockholm conference and declaration 1972 during the same year as the biosphere conference in 1968, the club of rome, an informal organization consisting of scientists, members of academia, economists and civil servants started conducting a study on the factors that limited global growth. the outcome document, limits to growth, produced future projections arguing that the economic system had to be significantly altered to address the ecological capacity of the earth (meadows et al, 1972). following these efforts, combined with the growing public awareness of global environmental problems, the united nations general assembly, in its meeting in 1968, agreed to organize the first international high-level conference to bring together all the united nations bodies and member states to generate the first global agenda for the environment. the general assembly, in its resolution 2398, stipulated that it was “desirable to provide a framework for comprehensive consideration within the united nations of the problems of the human environment in order to focus the attention of governments and public opinion on the importance and urgency of this question and also to identify those aspects of it that can only or best be solved through international cooperation and agreement.” (united nations general assembly resolution 2398 (xxiii), 1968) with this consensus, policymakers proceeded with the preparations for the first global conference on the environment, also known as the conference that led to the estabgenetic resources (2022), 3 (6), 74–88 where abs comes from: a historical overview 77 lishment of the united nations environment programme (unep). during the preparation of the stockholm conference on the human environment, the divide between developed and developing nations regarding their perception towards environmental problems and their willingness to take part in limiting growth became apparent to the global forum. this demonstrated that the developing world suffered from environmental problems due to poverty, and the developed world’s environmental problems were related to the increased use of natural resources. the developing world initially approached the first global conference on the environment with suspicion, as they were afraid that environmental measures would result in reduced development aid and increased tariffs for products from developed countries. nonetheless, it was apparent that the developing world also suffered from environmental problems, especially related to urbanization, and unlike the developed world, they did not have the means to deal with them. therefore, the stockholm conference was the first time both parties acknowledged the interdependence of their economic development and environmental resilience (johnson, 2012). on top of demonstrating the interdependence of the two worlds, the stockholm conference produced the first document introducing sovereign rights related to natural resources to the international legal arena. indira ghandi’s plenary speech during the conference served as a mirror to this interdependence between developing and developed nations. the speech stressed the delicate balance between environmental protection and restriction of industrial activities proposed by the developed world versus the need for economic and industrial development emanating from the developing world. ghandi argued that, let alone conservation, the developing nations had no means of providing incentives to limit harm to nature: ”on the one hand the rich look askance at our continuing poverty – on the other they warn us against their own methods. we do not wish to impoverish the environment any further and yet we cannot for a moment forget the grim poverty of large numbers of people. are not poverty and need the greatest polluters? for example, unless we are in a position to provide employment and purchasing power for the tribal people and those who live in or around our jungles, we cannot prevent them from combing the forest for food and livelihood; from poaching and from despoiling the vegetation. when they themselves feel deprived, how can we urge the preservation of animals? how can we speak to those who live in villages and in slums about keeping the oceans, the rivers and the air clean when their own lives are contaminated at the source? the environment cannot be improved in conditions of poverty. nor can poverty be eradicated without the use of science and technology.” waldheim et al (1972) this speech perfectly described the need to ascertain a balance between both the needs of developing and developed countries as well as the cruciality of sharing the benefits of science and technology to eradicate poverty while conserving nature and its resources at a global scale. within this atmosphere, the sovereign rights of states over their natural resources became an integral part of the stockholm declaration. principle 21 of the stockholm declaration notes that states have the sovereign right to exploit their own resources pursuant to their own environmental policies, and the responsibility to ensure that activities within their jurisdiction or control do not cause damage to the environment of other states or of areas beyond the limits of national jurisdiction (united nations doc. a/conf. 48/14, 1972). in addition, recommendation 39 of the stockholm declaration (united nations doc. a/conf. 48/14, 1972) requested governments, in cooperation with fao, to agree to an international programme on preserving the world’s gr by establishing an international network to survey international conservation efforts through in situ and ex situ methods. fao took this mandate to further expand its seed collections and later to initiate the first access mechanism to these collections. the next section provides an overview of the historical development of access to gr within fao, predating the cbd. cgiar centres and fao conference of 1981 the rapid global population increase after the second world war revealed a novel need for a smarter way of ensuring food security. many countries started suffering from food shortages and some even famine. the indian subcontinent had undergone severe famines during the 1940s which emphasized the need for countries to be self-sufficient in food production. this resulted in increased efforts in research on major cereal crops such as maize, wheat and rice that enabled the establishment of the consultative group on international agricultural research (cgiar). additionally, the cgiar centres’ objective was to take on the task fao was mandated regarding establishing an international network to survey international conservation efforts through in situ and ex situ methods, as prescribed by recommendation 39 of the stockholm declaration. in other words, cgiar centres were established as centres that conserve gr and ensure the genetic diversity of crops. in addition, the cgiar centres started research on developing new varieties, improving the yield of cereal crops as well 78 sirakaya genetic resources (2022), 3 (6), 74–88 as irrigation techniques, pesticides and fertilizers. this clustered research effort led to the ‘green revolution’, a movement started by the plant breeder norman borlaug, a nobel prize laureate who developed dwarf maize varieties that could be adapted to various climates (mooney, 1983). this success in plant breeding did however generate its drawbacks. the shift from traditional to industrial agriculture, based on the use of a limited number of high-yielding varieties, generated what experts called ’genetic erosion’. as a response, cgiar centres started establishing their own genebanks and collections to ensure the conservation of varieties for research (moore and tymowski, 2005). as the cgiar centres genebanks and collections kept expanding in the early 1980s, questions and concerns regarding access to and ownership of the conserved varieties were increasingly raised (rose, 2004; mooney, 1983). even though cgiar centres claimed that they were freely accessible, there existed no legal basis at the international level that ensured this. in 1981, the fao conference stated that there was a need to regulate access to plant genetic resources for food and agriculture (pgrfa) at the international level. this movement resulted in the adoption of the international undertaking of pgrfa (iupgrfa) by the fao conference in 1983. with this, the commission on genetic resources for food and agriculture (cgrfa) was created to manage the operations related to plant genetic resources (pgr) (fao, 1983). the international undertaking of pgrfa was the first international – yet voluntary – instrument aiming to conserve and sustainably use agricultural crops, which would then be made available for scientific research and plant breeding purposes. the international undertaking stated that gr are a heritage of mankind but did not use the full legal terminology ‘common heritage of mankind’. it emphasized, however, that pgrfa should be made available without restriction. more specifically, the undertaking stated that pgrfa, which include those kept within the premises of cgiar centres, were subject to the “universally accepted principle that plant genetic resources are a heritage of mankind and consequently should be available without restriction.” (sullivan, 2004) the reference made within iupgrfa to ‘heritage of mankind’ as opposed to ‘common heritage of mankind’ received criticism, mainly from the global south, as to whether the international undertaking followed the international law principle fully and whether pgrfa were meant to be managed as public goods (helfer, 2003). this confusion was addressed by resolution 3/91 of the fao conference, stating that the heritage of mankind principle established under the international undertaking does not contradict the states’ sovereign rights over their gr, clarifying that the states are not giving up on their sovereign rights by granting unrestricted access to their pgrfa under the international undertaking. in its original wording, it recognized that “the concept of mankind’s heritage, as applied in the international undertaking on plant genetic resources, is subject to the sovereignty of the states over their plant genetic resources.” (fao resolution 3/91, 1991) the iupgrfa was also the first instrument which stipulated that governments or institutions holding pgr were expected to adopt measures that would allow access to them and permit their export for the purposes of scientific research, plant breeding or conservation, adding that the samples should be made available based on mutually agreed terms. the international undertaking presented measures both related to in situ and ex situ conservation of pgrfa, while also emphasizing the need to establish an international cooperation structure that enabled all countries to make use of these pgrfa for the benefit of their agricultural development. the aim of making pgrfa available for further research and breeding led to the creation of an international network of genebanks and a need to clarify their legal status as well as those of cgiar centres. this goal would also lead to the negotiations for the adoption of the multilateral system within the itpgrfa, which came into force in 2004. it is worthwhile noting that pgrfa as well as the negotiations that led to the creation of the iupgrfa and later itpgrfa, require a further in-depth study of the dynamics that connect pgr with farmers’ rights, food security, securing genetic diversity, as well as the intellectual property regime surrounding the iupgrfa. the establishment of cgiar centres and adoption of iupgrfa have been included in this article, to the extent that clarifies the mandate provided to fao by the stockholm declaration. furthermore, even though the dynamics of the fao multilateral system and the cbd bilateral system differ to a great extent, a historical overview of the development of the abs system under the cbd would have contained a gap without the mention of the iupgrfa, the first international instrument dealing with facilitated access to and benefitsharing of pgrfa. convention on biological diversity negotiations following the introduction of the north-south debate at the international environmental forum during the stockholm conference, developing countries expanded their request for social and economic development. they perceived the necessity of emphasizing sovereignty over natural resources within their national jurisdiction, for they aimed to free themselves from the aftereffects of the economic order during colonization. not long after the stockholm conference, the group of 77, a block of developing countries established during the negotiations of the united nations conference on trade and development in 1964, put forth their declaration on the establishment of the new international economic order during the un general assembly of 1974 (united nations general assembly a/res/sgenetic resources (2022), 3 (6), 74–88 where abs comes from: a historical overview 79 6/3201, 1974). while acknowledging that the “interests of the developed countries and those of the developing countries can no longer be isolated from each other, that there is a close interrelationship between the prosperity of the developed countries and the growth and development of the developing countries, and that the prosperity of the international community as a whole depends upon the prosperity of its constituent parts,” the declaration requested the following to be crystallized at the international level: “full permanent sovereignty of every state over its natural resources and all economic activities. in order to safeguard these resources, each state is entitled to exercise effective control over them and their exploitation with means suitable to its own situation, including the right to nationalization or transfer of ownership to its nationals, this right being an expression of the full permanent sovereignty of the state. no state may be subjected to economic, political or any other type of coercion to prevent the free and full exercise of this inalienable right”. united nations general assembly a/res/s-6/3201 (1974) the declaration proved impactful as the general assembly adopted resolution 3281 (xxix) containing the charter of economic rights and duties of states, reinstating the sovereignty of states over their natural resources (united nations general assembly resolution 3281 (xxix), 1974). the repeated affirmations over the sovereign rights of states, as well as bringing the provenance of gr within cgiar centres to a legal basis via the iupgrfa, seemed to have provided temporary confidence to the developing world regarding how fair the global system on gr was (shackelford, 2008). the industrialization of agriculture in developed countries resulted in the privatization of the sector, which began relying on the sales of seeds and other agricultural products for profit. as a result, the importance of intellectual property (ip) rights in the agricultural sector gained increasing importance. the international union for the protection of new varieties of plants (upov) convention, adopted in 1961 (upov, 1961), followed this trend, aiming at encouraging plant breeding by means of breeder’s rights, a sui generis form of an ip right specifically designed for plant breeders. in the case of a variety protected by a breeder’s right, the breeder’s authorization is required to propagate the variety for commercial purposes except for when the variety is utilized for further breeding (also known as breeder’s exemption), for experimental purposes and private and non-commercial purposes as specified in article 15(1) as compulsory exceptions. states are also invited to consider allowing for an optional exception for farmers saving seeds as specified in article 15(2), also known as farmer’s privilege (lawson, 2015). the revisions to upov in 1972 and 1978 were argued to strengthen breeders’ rights and diminish farmers’ rights to sell, exchange or harvest seeds from protected varieties, which further raised concerns within the global south (tripp et al, 2007). the international undertaking was implemented specifically to curb these concerns. while serving to do so, the iupgrfa instead raised concerns in the developed world as the seed industry expressed its worry about the definition of pgrfa. according to the seed industry, the broad definition of pgrfa would result in the necessity of making privately owned plant varieties and special genetic stocks available without restrictions. following these concerns, a group of developed countries including canada, france, germany, japan, new zealand, switzerland, the united kingdom and the united states of america made reservations about the iupgrfa arguing for the recognition of intellectual property therein (ten-kate and diaz, 1997). at the same time, developing countries, under the auspices of fao, exclaimed that the iupgrfa did not recognize nor reward the contributions of developing countries and their farmers to the conservation and availability of pgr (moore and tymowski, 2005). in an attempt to calm the waters and satisfy both sides, fao adopted a resolution with an amendment to the iupgrfa. the resolution stated that “plant genetic resources are a common heritage of mankind to be preserved and to be freely available for use, for the benefit of present and future generations” (fao resolution 4/89, 1989). it also clarified that this would not extend to the protection of plant breeders’ rights within upov, allowing the industry to exclude their varieties from the common heritage system of the iupgrfa. this caused the need for an additional resolution from fao (fao resolution 5/89, 1989) to accentuate that farmers in all countries should be able to “participate fully in the benefits derived, at present and in the future, from the improved use of plant genetic resources, through plant breeding and other scientific methods”. the cbd negotiations, therefore, began in a tense atmosphere escalating both in the global north and the global south. on the one hand, the north aimed at conserving biodiversity via cgiar centres, as well as conserving the rights of their rapidly evolving biotechnology sector. on the other hand, the global south expressed its concerns regarding the ip rights over gr gaining power while establishing the initial global understanding of the need to create a mechanism to share in the benefits of development achieved through the use of gr. the global south believed that the common heritage of mankind over gr allowed the global north to rely on the resources of the global south to maintain their economic prosperity. in 1988, the ad hoc working group of experts on biological diversity (ahwg), mandated by unep, convened for the first time to discuss the desirability and feasibility of an international framework agreement 80 sirakaya genetic resources (2022), 3 (6), 74–88 on the conservation and sustainable use of biological diversity. the working group agreed that the question of access, including the question of free access, to gr should be studied, yet they did not reach a consensus on the notion of biological diversity as a common resource of mankind (unep/bio.div.1/3, 1989). additionally, the working group agreed that the question of placing an economic value on biological resources should be examined in detail. the second meeting of the ahwg, convened in 1990, was opened by dr m. k. tolba, the executive director of unep. in his speech, he paid due attention to the preferential treatment for those having jurisdiction and control over gr with respect to genebanks containing them and to essential newly developed varieties obtained through breeding. he also emphasized the international transfer of and favourable access to biotechnology that could be usefully applied or adapted to developing countries needs (unep/bio.div.2/3, 1990). it is visible from his speech that access to gr and access to technology were regarded as two separate subjects, yet interdependent, to be dealt with rather than access to technology as a result of or deriving from access to gr (unep/bio.div.2/3, 1990). in fact, at this point, access did not only relate to access to gr but also to technology. the ahwg emphasized that “accessibility to biological diversity, including new varieties, and to related technologies, including conservation technologies, are two sides of one and the same coin and must be an integral part of the planned legal instrument.” subsequently, it became clear to the working group that the issue of ip rights relating to the ownership of biotechnology and both the provision of access to gr from biodiversity-rich countries and the provision of access to technology from technology-rich countries needed to be reviewed. dr tolba stipulated that “any new international agreement should not infringe upon the sovereignty of nation states over their natural resources. it must protect the interests of the states in which the resources are located and provide incentives for conservation of biological diversity without inhibiting growth or sustainable development.” (unep/bio.div.2/3, 1990) the ahwg further discussed the common heritage principle over gr and agreed that this principle did not mean the establishment of collective international rights to resources within national jurisdictions, nor did it infringe upon the permanent sovereignty of states over natural resources. the group underlined that free access did not mean access free of charge and accessibility should be based on mutual agreement and full respect for the permanent sovereignty of states over their natural resources. additionally, the ahwg agreed that those having jurisdiction and control over gr should receive preferential treatment for access to their germplasm and varieties developed from these resources. the second meeting of the ahwg discussed the two types of access and the compensation mechanisms for the provision of access and technology. the experts stated that biotechnology could assist in the conservation of gr which could be funded by enterprises that profit from the use of biotechnology. the ahwg suggested that this could be in the form of a tax to support conservation as well as biotechnology research in developing countries. additionally, the working group discussed that developers of biotechnology would require compensation for the provision of access to their technology. regarding the relationship of access to gr with to-be established financial mechanisms for the conservation of biological diversity, the report of the ahwg expressed that there was a consensus that “those who enjoy most the economic benefits of biological diversity should contribute equitably to its conservation and sustainable management.” some of the delegates argued for the potential of biotechnology to foster species conservation by means of adhering an immediate economic value to them and “if developing countries are given the capacity to develop and share the benefits of their biological diversity this will be a good incentive for natural resources conservation.” (unep/bio.div.2/3, 1990) the third meeting of the ahwg discussed the draft text of the cbd prepared by the international union for nature conservation (iucn) (unep/bio.div.3/12, 1990). the working group commissioned a study on the relationship between ip rights and access to gr. the report did not discuss what type of access measures could be implemented by provider countries, as it was presupposed that free access would remain the norm since the experts leaned on discouraging any measures including the amendment to the upov convention that would hinder free access to gr (unep/bio.div.3/6, 1990). under paragraph 5 of the report, the experts strongly emphasized the ‘undeniable’ importance of the principle of free access and argued that the iucn draft should not be allowed to result in a closing up of the system, for that would be against everyone’s interests. during the time of the second meeting of the ad hoc working group, discussions were ongoing on the revision of the upov convention. the draft revision of the upov convention introduced the concept of dependence, which meant that a variety ‘essentially derived’ from another variety protected by plant breeder’s rights cannot be used commercially without the permission of the breeder of the protected variety. at the same time, it introduced patent coverage over living matter under the agreements of the general agreement on tariffs and trade (gatt). during the meeting, the ahwg debated that the perception of the value of biological diversity was altering as the development of biotechnology enabled humanity to potentially create technological advancements out of any organism. therefore, the zones of biological diversity which were perceived to have no economic value, were presently considered to contain value that reflected the potential of gr contained in them (unep/bio.div.3/3, 1990). genetic resources (2022), 3 (6), 74–88 where abs comes from: a historical overview 81 the initial thought about sharing benefits had to do with technology transfer to improve the research capacity of developing countries. the study commissioned by ahwg on the relationship between ip rights and gr explicitly disapproved of monetary compensation for the costs of conservation (unep/bio.div.3/inf.4, 1990). it rather supported what was described as compensation mechanisms along the lines of technology-for-nature swaps. the study also argued that developments within upov on restricting access to varieties were “disturbing because free access (which she [the expert consultant] stressed did not mean free of charge) had been one of the essential factors in advances in genetics.” (unep/bio.div.3/12, 1990) it was recommended that the upov convention would harness ip rights with the aim of ensuring the more efficient use of biodiversity and also recommended a system of ‘paid open access’ yet warned that such a system could be endangered by the extension of patent law. another study on biotechnology commissioned during the third meeting argued that the ahwg should be wary of the fact that genetic material in private collections and information thereof will not be as easily available as the information on material in public sector genebanks and that the gr collected by seed companies were not likely to be freely exchanged and might be considered as trade secrets (unep/bio.div.3/7, 1990). the ahwg lastly commissioned a study on possible financial mechanisms for the conservation of biological diversity (unep/bio.div.3/5, 1990). as the study underlined that “the market prices of the genetic resources and functions do not reflect their real scarcity value or the ecological costs incurred by their use,” it also suggested the establishment of an international multilateral fund to enable their conservation and discourage their excessive use. from the wording of this report, it could be understood that grs were rather seen as physical, biological material whose excess use would result in negative ecological consequences. the working group suggested that contributions to the funding mechanism might be provided by the parties on an assessed basis. more specifically, “the scale of assessment could be related to a united nations scale, industrial and commercial exploitation of or trade in genetic resources or on some other equitable basis.” following the third meeting, a sub-working group on biotechnology (swg) gathered in november 1990. under annex 1 regarding possible additional elements for a biotechnology component in a global framework legal instrument on biological diversity, the swg considered the inclusion of the equitable sharing of the economic benefits derived from biotechnology with the country of origin of the biomaterials used (unep/bio.div/swgb.1/5/rev.1, 1990). hence, the discussions introduced monetary benefit-sharing arising from the utilization of gr for the first time, despite the previous recommendations discouraging them. the swg furthermore added that access to biological diversity should be based on agreements conforming with the sovereign right of states over their natural resources within their national jurisdiction. the swg stated that access to gr as well as access to technology would not be free of charge and should be based on mutual agreement (unep/bio.div/swgb.1/5/rev.1, 1990). in order to enhance the contribution of biotechnology to the conservation of biodiversity, it was urgently recommended to “increase the numbers of botanical gardens, seed banks and other ex-situ conservation facilities in various areas throughout the world, particularly in tropical areas, and to broaden the coverage of existing ones.” consequently, the swg has foreseen conservation via biotechnology through research and inventory on biodiversity and its conservation. it was not mentioned how shared economic benefits arising from gr utilization could enhance or how it would incentivize conservation. shortly after the meeting of the swg on biotechnology, the ahwg of legal and technical experts on biological diversity gathered for its first meeting, in november 1990 (unep/bio.div/wg.2/2/5, 1991). this meeting discussed the first cbd draft text, which was rather contested and heavily bracketed (lawson, 2015). the preamble of the text included the obligation of states to share in any increased knowledge as well as other benefits of the potential of biological diversity amongst bracketed suggestions for various wording such as “equitable sharing of benefits and conservation costs of biological diversity” or “the benefits derived from utilization and the cost of conservation of biological diversity should be shared” next to bracketed clarifications that free access does not mean free of charge. title vi of the draft text covered access to biological diversity under the same title as access to technology and information thereon. the commentary to this title discussed the details on which types of technology should be subject to access, and regarding the availability of both biological diversity and technology. the text also brought up the role of ip rights regarding these two types of access. title vii drafted the heavily bracketed obligation directed at developed countries to transfer technology – that supported biological diversity conservation and sustainable use – to developing countries on a non-commercial and preferential basis. the text also included options for research cooperation between developing and developed countries on scientific research and training, and joint ventures, taking into account the investments made by the private sector to develop these technologies as well as the possibility of establishing a mechanism to “ensure the acquisition of technology from the technology-rich states to the generich developing countries by providing funds to facilitate the necessary access to patents”. at his opening speech for the second meeting of the ahwg of legal and technical experts on biological diversity between 25 february and 6 march 1991, dr tolba, the executive director of unep, stated 82 sirakaya genetic resources (2022), 3 (6), 74–88 that access to biodiversity and the availability of biotechnology and other technology relevant to the rational use of biological resources were complementary and inseparable (unep/bio.div/wg.2/2/5, 1991). he continued that states should receive fair compensation for the provision of access and at the same time, the private sector should receive fair compensation for participating in technology transfer arrangements. another swg on biotechnology gathered during the second meeting to discuss issues related to access to gr. the swg discussed the possibility of introducing a prior informed consent mechanism to ensure that access to biological diversity would not endanger viable populations as well as to reflect the sovereignty of states over their gr. the swg added that access should not be regulated in a manner that resulted in blanket prevention of access. while there was a general consensus on the importance of access to technology for sustainable gr utilization, some delegates in the swg further requested the inclusion of gr utilization for other purposes such as pharmaceuticals in relation to transfer of technology to developing countries within the framework of the convention. some delegations requested further assessment of the transfer of both ‘hard’ (e.g. computers) and ‘soft’ (e.g. training) technologies and that technology transfer should not be specifically confined to biotechnology. additionally, some delegates argued that countries of origin of genetic material shall have equitable and/or preferential access to the benefits and profits arising from commercial exploitation thereof. regarding the question of how to financially incentivize all these activities, the swg could not come to an agreement. some delegates suggested the idea of a multilateral mechanism with a multitude of funding sources whereas some suggested bilateral settings. the swg, as later clarified in july 1991, defined access as “the right and/or means of acquiring biological resource or technology that can exploit the resource as well as relevant information and know-how, for scientific, commercial or other purposes on conditions agreed upon multilaterally or bilaterally.” (unep/bio.div./wg.2/3/6, 1991) access to biological diversity was defined to include both physical access to the genetic material and access to information about the genetic material. access to technology, on the other hand, was defined as access to know-how relevant to the conservation and sustainable use of biological diversity. during the same meeting, a multilateral trust fund was proposed to undertake the following activities: ”(a) to make money grants to habitat countries to enable these countries to undertake in situ or ex situ conservation of ecosystems and species; (b) to provide fair compensation to habitat countries for the use of their genetic resources; (c) to provide financial assistance to habitat countries to enable them to reach a technological, educational and training level that will facilitate national programmes for the conservation of biological diversity; (d) to provide financial assistance to habitat countries to enable them to conduct ecological surveys and to monitor technical assistance and strengthen relevant legal instruments for the conservation of biological diversity.” (unep/bio.div/wg.2/3/8, 1991) in between the second and third cbd negotiating sessions, the first bioprospecting agreement was signed between a provider country and an industrial user. in september 1991, costa rica’s national biodiversity institute (inbio), a private non-governmental entity, and merck & co., ltd, a pharmaceutical company based in the united states announced the freshly concluded bioprospecting agreement. according to the contract, inbio would provide merck with chemical extracts from wild plants, insects and microorganisms from costa rica’s conserved wildlands to be used for merck’s drugscreening programme in return for a two-year research and sampling budget of us$1,135,000 and royalties on any commercial products resulting from the use of the samples. inbio agreed to contribute 10% of the budget and 50% of any royalties to the government’s national park fund for the conservation of national parks in costa rica, and merck agreed to provide technical assistance and training to help establish drug research capacity in costa rica (aldhous, 1991). this was the first agreement serving the discussions on the economic value of biodiversity as well as its ability to demonstrate how companies can agree to return a portion of the benefits of commercial development to the developing country where gr were accessed (reid et al, 1993). following dr tolba’s recommendation on merging negotiations related to biodiversity and biotechnology, the ahwg was mandated to negotiate both matters and was renamed the intergovernmental negotiating committee (inc). after the third negotiating session/first meeting of the inc for a cbd, which lacked sufficient progress, the inc met for its fourth session between 23 september and 2 october 1991 (unep/bio.div/n4inc.2/5, 1991). the opening speech contained considerations on the negotiations related to the general agreement on tariffs and trade (gatt). this was followed up by cgiar centres arguing they should be allowed to freely sell their genetic material to the private sector without having to share profits with gr providers. likewise, it was contended by some members of the private sector that, if biotechnologies were transferred to developing countries, these developing countries would only be allowed to market their products locally which would constitute a disincentive to developing countries to acquire biotechnology. this was followed up by a speech by dr tolba, who shared the estimates of the global environmental fund (gef) on the financial cost of biodiversity conservation ranging from $500 million to $50 billion per year. he added that the countries with the richest biodiversity were also the ones least able to afford conservation measures and followed up by stating that the proposed economic system of access to resources increasingly depended on the activity of genetic resources (2022), 3 (6), 74–88 where abs comes from: a historical overview 83 access to biological diversity, yet the means of assessing the value of biodiversity were lacking. secondly, regarding access to technology, he contended that “. . . progress was measured in terms of development and use of sophisticated technologies, yet the way in which new technologies were regulated hindered their dissemination where they were most urgently needed. one hundred or perhaps more species were being made extinct every day as a result of human action. intensified scientific monitoring and assessment would help to fill in the gaps in knowledge, but it would take years if not decades.” (unep/bio.div/n4-inc.2/5, 1991) the inc adopted article 14bis on ‘traditional indigenous and local knowledge’ in addition to access to gr. the bracketed sentence requested contracting parties to acknowledge the contribution of this knowledge to biodiversity conservation and sustainable use and that they should endeavour to reflect the intrinsic economic value of this knowledge within national policies and legislative decisions. additionally, a less-bracketed version of article 15 on access to technology was adopted, which was complemented by article 16 on technology transfer and article 17 on scientific cooperation, both of which were still heavily bracketed (unep/bio.div/n4/inc.2, 1991). article 14 on access to gr was not further discussed in this session. at the fifth session of negotiations, which took place between 25 november and 4 december 1991, dr tolba informed the inc of a positive development, reading the statement recently made by the netherlands on behalf of the european community during the united nations general assembly. the representative indicated that “the industrialized countries, recognizing their responsibility towards the environment, should commit themselves to reducing the burden they imposed upon it, to the extent of their legitimate share.” (unep/bio.div/n5-inc.3/4, 1991) this session did not further discuss the abovementioned draft articles related to access to biological diversity and access to technology. the inc, however, released a document regarding the interpretation of the words fair and favourable, fair and most favourable, equitable, preferential and non-commercial, preferential, non-commercial at the relevant international fora (unep/bio.div/n5-inc3/3, 1991). during the sixth negotiating session, gathered from 6 to 15 february 1992, the inc prioritized the discussions related to financial resources, new and additional ones, mechanisms to review and manage those financial resources, access to genetic resources, fair distribution of benefits arising from the use of those resources, fair and favourable conditions for access to technology by developing countries, the question of biotechnology, the question of commitments by developed and developing countries, as well as national regulations and policies in dealing with biological resources at the national level. while the brackets from article 14 were largely removed and article 14bis was reformulated as article 7(j), article 15 on access to technology got merged with article 16 on technology transfer, which resulted largely in the removal of considerations related to ip rights restricting access to technology. at this point, article 16 did not contain as strong provisions on access to technology compared to the previous draft as it became less clear what access to technology or transfer of technology stood for. furthermore, the article read more as a mere recommendation than an obligation (unep/bio.div/n6-inc4/4, 1992). the seventh and final negotiating session held by the inc between 11 and 19 may 1992, renamed article 14 as article 16 and article 15 as article 17 (unep/bio.div/n7-inc5/2, 1992). after these drafts, the official documentation does not provide information on how these articles were renegotiated and what the reason behind removing the brackets and deleting certain sentences was. regarding the final negotiating environment during the united nations conference on environment and development (also known as the rio conference) in 1992, parson, haas and levy state the following: “the negotiations were plagued by the conflict over the financial mechanism, the sharing of benefits, and biotechnology regulation. france originally threatened not to sign the treaty because it did not include a list of global biodiversity-rich regions; japan threatened not to sign because it feared biotechnology regulation. at the last moment, both relented, and only the united states refused to sign the treaty because officials felt that the financial mechanism represented an openended commitment with insufficient oversight and control; that the benefitsharing provisions were incompatible with existing international regimes for intellectual property rights; and that the requirement to regulate the biotechnology industry would needlessly stifle innovation.” parson et al (1992) the rio conference adopted several international environmental treaties, including the cbd. in addition, the conference also adopted the first non-binding action plan of the united nations with regard to sustainable development, also known as agenda 21 (united nations conference on environment and development, 1992), which contained the following paragraph on sovereign rights of states over their gr: “governments should [. . . ] develop measures and arrangements to implement the rights of countries of origin of genetic resources or countries providing genetic resources, as defined in the cbd, particularly developing coun84 sirakaya genetic resources (2022), 3 (6), 74–88 tries, to benefit from the biotechnological development and the commercial utilization of products derived from such resources.” convention on biological diversity the provisions of the cbd (unep, 1992) originate from its three overarching objectives, which are: • conservation of biological diversity • sustainable use of the components of biodiversity • fair and equitable sharing of benefits arising from gr article 15 of the cbd reaffirms the states’ sovereign rights over their gr. this means that states have the right to regulate access to their gr, which includes the right to determine the conditions of such access and the fair and equitable benefit-sharing resulting from the utilization of gr (kamau and winter, 2013). article 15 paragraphs 3, 4 and 5 of the cbd stipulate that the access granted by a provider country shall be subject to prior informed consent (pic) and mutually agreed terms (mat) unless otherwise determined by the provider country. the cbd defined the key principles of a bilateral abs system between users and provider countries. article 15 of the cbd reaffirms the states’ sovereign rights over their gr. this means that states have the right to regulate access to their gr, which includes the right to determine the conditions of such access and the fair and equitable benefit-sharing resulting from the utilization of gr (kamau and winter, 2013). article 15 paragraphs 3, 4 and 5 of the cbd stipulate that the access granted by a provider country shall be subject to prior informed consent (pic) and mutually agreed terms (mat) unless otherwise determined by the provider country. the cbd defined the key principles of a bilateral abs system between users and provider countries. article 2 of the cbd defines gr as “genetic material of actual or potential value.” according to the same article, genetic material is defined as “any material of plant, animal, microbial or other origin containing functional units of heredity.” the right to determine the conditions of access and benefit-sharing of gr is given to the country of origin as well as the country providing gr. article 2 of the cbd defines the former as “the country which possesses those genetic resources in in-situ conditions” and the latter as “the country supplying genetic resources collected from insitu sources, including populations of both wild and domesticated species, or taken from ex-situ sources, which may or may not have originated in that country”. in relation to the latter definition, the cbd article 2 defines domesticated or cultivated species as “species in which the evolutionary process has been influenced by humans to meet their needs.” this definition is important due to the fact that the cbd also considers countries as providers of those gr that “have existed for some time away from their in-situ conditions and have become part of new natural and cultured ecosystems.” this article, therefore, refers to two situations under the definition of provider countries. kamau and winter (2013) consider this type of gr provider as first-level providers. the latter definition, however, also includes providers of gr from ex situ sources, meaning that these resources are kept and conserved outside of their natural habitat. kamau and winter consider this type of gr provider as second-level providers. the second-level providers become such by either rightfully obtaining gr from the country of origin (e.g. by entering into pic or mat when these are required by law) or by having obtained these resources before 29 december 1993 when the cbd came into force. article 8(j) of the cbd states that subject to their national legislation, states should promote the equitable sharing of benefits arising from the utilization of innovations and practices of indigenous and local communities. however, the cbd does not contain a definition and further description of traditional knowledge. article 15 and 8(j) are the two main provisions of the cbd relating to abs. several other articles of the cbd complement these provisions such as article 16 on access to and transfer of technology, article 17 on exchange of information, article 18 on technical and scientific cooperation, article 19/1 and 19/2 on biotechnology and distribution of its benefits, article 20 on financial resources and article 21 on a financial mechanism. article 16 is an important element in understanding the north-south debate that led to the insertion of the third objective of the cbd on fair and equitable sharing of benefits. as explained under the cbd negotiations section of this article access, in the early drafts of the cbd, was defined as access to biodiversity and technology. this definition did not find its place in the final text that got adopted. access to technology as an obligation was drafted as a separate article and perceived as crucial in establishing fairness and equity as access to gr would. according to this, governments would have to establish legislative measures to encourage the private sector to provide access to technology based on mutually agreed terms, and, in accordance with international obligations, refrain from imposing restrictions (such as ip rights). article 16 obliges contracting parties to provide and/or facilitate technologies relevant to the conservation of biological diversity and sustainable use of its components or technologies that make use of gr. as neither the act to “provide” nor to “facilitate” are defined, contracting parties have sizeable flexibility in implementing this obligation. according to glowka et al (1994), this could mean the provision of technologies within the public domain. regarding technology transfer, article 16(3) states that the contracting parties (be it developing or developed states) are obliged to create a framework permitting the transfer of technologies making use of gr. consequently, the obligation is not transferring technology yet merely creating the condigenetic resources (2022), 3 (6), 74–88 where abs comes from: a historical overview 85 tions enabling the transfer of technology, making what was an equal return for accessing gr in the beginning, a voluntary scheme in the end. discussion and conclusion the history behind the cbd negotiations demonstrates that the need to reinstate sovereign rights of states over their natural resources emanates from the global north vs global south debate on inequality resulting from the aftereffects of colonialism. the global north, or the developed countries, have been historically able to develop products, processes and technologies potentially beneficial to humanity as a whole, by utilizing the genetic resources the global south, or the developing countries had. in other words, the global south provided the resources, and the global north provided the technology for the development of the global society. nevertheless, the north traded the products developed with the gr of the global south, yet the global south had not participated in the benefits of these products. this resulted in the perception of inequality which paved the way for the first decision on state sovereignty on gr under the stockholm declaration. the mandate deriving from the stockholm declaration initiated the attempts of fao to establish cgiar centres and the first global abs instrument (though voluntary) under the iupgrfa. additionally, the north during the stockholm conference emphasized the need to conserve the environment, whereas the global south underlined that the poverty they were suffering would not enable them to make funds available for conservation as they had overarching priorities related to basic human needs. therefore, the need for a financing mechanism to allow the global south to conserve its resources became visible. moreover, biotechnology was seen as a key to overcoming food crises and poverty, and a solution to the global decline in biodiversity. the relief the iupgrfa provided as a multilateral benefit-sharing system operating under the common heritage principle soon lost its power due to the mistrust elevated by ip discussions under the upov convention regarding gr. the global south believed ip rights and privatization of gr through the storage thereof in private collections would deem the undertaking obsolete and undermine its free access principle. the global north, on the other hand, sustained its claims for the amendment of the upov convention for ip rights strengthening as their agricultural sector depended on the sales of seeds and other valueadded products developed with biotechnology. with this tension, the negotiations for an international instrument on biodiversity conservation began. separate from the attempts under the cbd, fao further developed the multilateral benefit-sharing system of pgrfa firstly by its amendments and later by adopting the itpgrfa to alleviate the concerns of the global south, especially risen after the amendment of the upov convention. during the negotiations on the cbd, the ahwg initially agreed that access should not be restricted, benefit-sharing should be based on technology-fornature swaps and that money as such would not make up for a benefit that would be mutually beneficial. the ahwg received pressure from the south on basing the cbd on the sovereign rights of states over their gr. the first cbd drafts reflected this demand and also the opinion of the ahwg on the importance of access to technology as well as to gr. therefore, access in the context of abs, at that stage, meant access to gr and access to technology. the north had concerns that this would overrule their ip rights on such technologies. the ahwg gathered experts to look into financing mechanisms that would serve as incentives for both access to gr and access to technology. the experts suggested the option of a multilateral benefit-sharing mechanism by which both the biodiversity-rich countries and owners of technology would be compensated for the provision of their assets. additionally, it was suggested that the fund would provide compensation for access to provider countries by means of funding conservation projects in those countries. on the contrary, some delegations supported a bilateral negotiation mechanism by means of acquiring pic and negotiating mat. both these options made it to the final text, however, the pic and mat appeared in the very article on access to gr, whereas the financing mechanism through a multilateral system was indirectly made an option through article 21. as for the definition and conceptualization of gr, it is visible from the early international documents that gr were perceived as a tangible, physical source that can potentially be subject to overexploitation. during the cbd negotiations, it was further stated that access to biodiversity meant access to gr and the information related to them. nonetheless, neither a definition of access, nor a definition of benefit-sharing have been included in the cbd final text. the overarching aim of the cbd is the conservation of biological diversity. however, the global south, starting from the preparations for the stockholm conference, called out the lack of ability to conserve biodiversity within their territories due to the continuing gap between development and technology as well as economic advancement. during the negotiations, next to restoring justice within access to resources vs technology equilibrium, several discussions took place on how sharing of benefits would allow incentivizing biodiversity conservation. the swg on biotechnology assumed that applications of biodiversity on gr such as research and creating inventories of gr would result in the conservation of biodiversity. it was however not clarified how sharing any other types of benefits would create incentives for biodiversity conservation. a very important note here, which surely has passed the test of time since the cbd negotiations, is that many countries of the global south still lack the ability, economic means and technology to be able to utilize and conserve gr. the cbd originally started as an equal exchange between gr and technology, yet the 86 sirakaya genetic resources (2022), 3 (6), 74–88 negotiations as well as concerns of the global north over the provenance of ip rights resulted in a final text unclear in its motivations, especially regarding the connection between articles 15 on access to genetic resources and articles 16 to 21 on transfer of technology and capacity building. arguably, the emphasis on article 15 in the abs realm resulted in an international bilateral abs framework under the cbd that is developed heavily around the concept of access and weaker around the concept of benefit-sharing. to this day, this reflects on the current discussions as the persisting lack of trust in the abs system since the benefits of the abs system are still blurry to many. in other words, the negotiations to the cbd aimed at introducing fairness and equity into innovation with biotechnology, as well as incentivizing conservation. however, it is doubtful whether the pic and mat mechanisms enabled provider countries to acquire the technology and know-how to become users of gr themselves, nor is it clear whether provider countries have been sufficiently financially incentivized with the abs mechanism the cbd introduced to the international legal realm. by analyzing the historical developments and negotiation documents that led to the cbd, this paper displays the narratives and needs of the global north and the global south with the hope of serving as guidance to the negotiations of further clarifications to the abs system. although many concepts have evolved throughout the two decades since the adoption of the abs system under the cbd, there are many lessons to be recalled regarding the tensions between the global north and the global south which persist today. for example, the visible demonstration of this tension often takes over the dsi debate. one of the more topical and lasting conclusions of this paper is that the current debates need to acknowledge the fact that the technological and economic prosperity gap between the global north and the global south, most likely on another level than in the late 1980s and early 1990s, continues to impact the prominence of the abs system and any novel concept which evolved with the current technological advancements, relevant to bioprospecting. without an effective solution addressing this underlying tension, the global north and the global south will continue to disagree on how to address the global biodiversity crisis and 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statement original article genetic resources (2024), 5 (10), 53–64 doi: 10.46265/genresj.epvo8349 https://www.genresj.org issn: 2708-3764 quantifying phenotypic relationships among arsi, bale and jemjem cattle breeds of ethiopia amine mustefa *,a, awoke melak a, hizkel kenfo b, seble sinke a, ahmed abdela c and abebe hailu a a ethiopian biodiversity institute, addis ababa, ethiopia b hawassa biodiversity center, hawassa, ethiopia c goba biodiversity center, goba, ethiopia abstract: nine morphometric and 16 morphological traits were used to characterize and quantify phenotypic relationships among arsi, bale and jemjem cattle breeds. a total of 441 randomly selected adult cattle (342 females and 99 males) from three purposively selected districts were used. univariate and multivariate analysis procedures of statistical analysis software (sas) were used to analyze the data. clear morphological and morphometric variations were not observed among the cattle breeds. the majority of the studied cattle possessed uniform coat colour pattern (78%), black coat colour (61%), forward-oriented horns (65.8%), widely spaced horns (71.4%) and curved horns (76%). they mostly had erected humps (96.2%), small humps (66.7), mainly located at the cervicothoracic position (77.8%) with a straight face (100%) and back profile (92%) while their rump was sloppy (100%). overall, 44.4% and 45.6% were mediumand long-tailed, respectively, while most (72.1%) of the cattle possessed medium dewlap width. most (57.6%) of the males had medium perpetual sheaths while naval flap was not observed on most (53.2%) of their female counterparts. in addition to the univariate analysis, the multivariate analysis also failed to show significant separation among the breeds, as indicated by the short mahalanobis distances and low eigenvalues. in conclusion, arsi, bale and jemjem cattle breeds were found to be phenotypically inseparable. however, the observed phenotypic similarities among these breeds do not necessarily mean that these cattle breeds are genetically the same. therefore, further molecular characterization is recommended to quantify the degree of genetic relationships among the studied breeds. keywords: cattle, characterization, ethiopia, indigenous breeds, morphology, morphometric, multivariate analysis citation: mustefa, a., melak, a., kenfo, h., sinke, s., abdela, a., hailu, a. (2024). quantifying phenotypic relationships among arsi, bale and jemjem cattle breeds of ethiopia. genetic resources 5 (10), 53–64. doi: 10.46265/genresj.epvo8349. © copyright 2024 the authors. this is an open access article distributed under the terms of the creative commons attribution license (cc by 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. introduction ethiopian indigenous cattle genetic resources contribute significantly both to farmers’ livelihoods and the country’s gross domestic product (gdp) (csa, 2021). cattle in ethiopia are primarily used for milk, meat and drought power. moreover, they are a source of income and manure, as well as provide social and cultural values (zerabruk and vangen, 2005; genzebu et al, 2012; yimamu, 2014; kebede et al, 2017; getachew et al, 2020). ethiopia has about 70.3 million heads of cattle (csa, 2021), making them the most populous ∗corresponding author: amine mustefa (amine.mustefa@ebi.gov.et) livestock species in the country. furthermore, according to statista (2020), ethiopia has the largest cattle population in africa. to ensure that cattle production contributes sustainably to the country’s food and nutrition security, proper management of the diversity of indigenous breeds is essential (fao, 2007). to achieve this goal, ethiopia has adopted the global plan of action (gpa) for animal genetic resources which has four strategic priority areas (spas) and 23 strategic priorities (sp) (ebi, 2016). the first spa – characterization, inventory and risk monitoring – aims to produce sufficient and accurate information for enhanced management of animal genetic resources (angr). outputs from this spa include knowledge of the genetic diversity, population structure received: 16.05.2024 accepted: 11.09.2024 published online: 01.10.2024 https://www.genresj.org https://www.doi.org/10.46265/genresj.epvo8349 https://www.genresj.org https://www.doi.org/10.46265/genresj.epvo8349 mailto:amine.mustefa@ebi.gov.et 54 mustefa et al genetic resources (2024), 5 (10), 53–64 and population differentiation of indigenous breeds. to achieve these outputs, phenotypic and genetic characterization studies are required (fao, 2012; ajmone-marsan et al, 2023). several cattle phenotypic and genetic characterization studies have been carried out in ethiopia in the past three decades, leading to the registration of 28 indigenous breeds (ebi, 2016; mustefa, 2023). the phenotypic characterization studies provided a list of the breeds believed to exist in the country, the breeds’ distribution areas and characteristics, and their linear body measurements. similarly, molecular characterization studies assessed the withinand among-breed genetic diversity and differentiation. however, the phenotypic and molecular characterization studies carried out so far have not been comprehensive, particularly in terms of breed differentiation and registration. the phenotypic studies were limited by narrow geographic coverage, inconsistent naming and varying methodologies. molecular studies were contradictory, and showed discrepancies between phenotypic characteristics and geographical distances among the breeds (mustefa, 2023). moreover, some cattle breeds including adwa, ambo, bale, hamer, jemjem, jigjiga, and smada were registered without adequate phenotypic characterization studies. addressing these gaps is essential to provide a complete and country-wide picture, which in turn will inform the design of breed-specific genetic improvement and conservation programmes. the current study targeted three registered cattle breeds: the arsi, bale, and jemjem (ebi, 2016; assefa and hailu, 2018). two of them, bale and jemjem, were not studied before while arsi was studied by yimamu (2014), which revealed some of the unique characteristics and distribution areas of this cattle. the breed has a compact body with a uniformly patterned black coat colour. it is reported to have originated in the arsi highlands, with a distribution up to bale and sidama highlands (assefa and hailu, 2018). these zones were also identified as the home to other cattle breeds: bale highland is the breeding tract of bale cattle (assefa and hailu, 2018), and sidama highland is the breeding tract of jemjem (sidama highland) cattle (assefa and hailu, 2018; legesse and zeleke, 2021). the study by legesse and zeleke (2021) on sidama highland cattle showed some phenotypic resemblances with arsi. furthermore, legesse and zeleke (2021) reported the neighbouring arsi and bale highland areas as the origin of sidama highland cattle. therefore, the breeds that exist in the arsi, bale, and sidama highlands seem to be the same breeds with different names. therefore, an inclusive study taking representative samples from these areas is required to quantify the level of relationships among these breeds. the current study aimed to phenotypically characterize arsi, bale, and jemjem cattle breeds and quantify the level of phenotypic relationships using multivariate analysis. materials and methods study areas the study was conducted in two regions, oromia and sidama. three districts – diksis district of arsi zone and goba district of bale zone in oromia, and hula district in sidama (figure 1) – were covered. some parameters of the sampled districts including weather conditions and agroecology are presented in table 1. site and animal selection representative samples of arsi, bale and jemjem cattle breeds were selected from their respective breeding tract. information on their breeding tract and distribution areas were identified from previous studies (rege and tawa, 1999; yimamu, 2014; assefa and hailu, 2018; legesse and zeleke, 2021). accordingly, diksis district was randomly selected from the highland districts of the arsi zone to represent arsi cattle, goba district was randomly selected from the highland districts of the bale zone to represent bale cattle, while hula district was randomly selected from the highland districts of the sidama region to represent jemjem cattle. two sampling sites (kebeles) were randomly selected from each district. forty households that reared cattle were randomly selected from each sampling site (kebele). within each household, the adult cattle aged four years and above were first separated from the young ones to avoid age bias. genetically unrelated animals were separated to make the sampling representative. then, two animals were selected randomly for the morphometric and morphological recording to avoid sampling bias. selected animals were controlled carefully by their owners and trained labourers. aggressive animals that could not properly stand on the flat ground were not recorded to avoid measurement bias. data collection data on morphometric (quantitative linear body measurements) and morphological (qualitative characteristics) traits were collected based on the data collection procedures described in the un’s food and agriculture organization (fao) guidelines (fao, 2012). data collection was performed in the morning to avoid errors regarding feeding and watering. five researchers were involved in the data collection procedure: three handled the quantitative data while the remaining two took care of the qualitative data decision-making and recording. to reduce bias, morphometric data recording was performed by the same researcher throughout the study. animals were measured using a centimeterunit textile measurement tape. a total of 441 cattle (342 females and 99 males) were subjected to nine morphometric measurements (table 2) and 16 morphological/qualitative traits (table 3). genetic resources (2024), 5 (10), 53–64 phenotypic relationships among ethiopian cattle breeds 55 figure 1. map of the study areas table 1. weather and agroecology-related information of the selected districts. source: (yimamu, 2014; tiki et al, 2016; teshale et al, 2017). parameters districts diksis goba hula altitude of the district (m.a.s.l.) 2,200–2,800 1,500–4,377 1,501–3,500 altitude of the sampled locations (m.a.s.l.) 2,710–2,721 2,588–2,596 2,709–2,718 temperature(oc) 18 0–23 12–22.5 rainfall (mm) 700–1,300 1,033–1,112 1,200–1,600 area (km2) 283 270 cattle population 139,568 124,472 human population 215,337 165,712 161,214 ethnicity oromo oromo sidama table 2. list of the linear body measurements with their definitions. these measurements were carried out using a centimetre (cm) unit measuring tape. source: fao (2012). no. morphometric traits definitions 1 body length distance from shoulder point to pin bone 2 heart girth chest circumference right behind the two front legs 3 height at withers distance from ground to withers of the front foot 4 pelvic width distance between the two ends of the pelvic bone 5 muzzle circumference perimeter of the mouth 6 ear length distance from the root to the tip of the back side of the ear 7 horn length outer side distance between root and tip of the horn 8 cannon bone length distance between the fetlock joint (ankle) and the knee 9 hock circumference perimeter of the hock bone 56 mustefa et al genetic resources (2024), 5 (10), 53–64 data analysis a microsoft office excel worksheet was used to enter and manage data, while the overall data analysis was carried out using various procedures of the statistical analysis system (sas) software 9.0 (sas, 2002). univariate analysis univariate procedure of sas (sas, 2002) was used for data normality test, the frequency procedure (chisquare test) was used for morphological (qualitative) data analysis, and the general linear model (glm) procedure was used for morphometric (quantitative) data analysis. the following statistical analysis model was used to analyze the morphological data: yij = µ + si + bj + eij where yij is an observation, µ is the overall mean, si is the fixed effect of sex (i = male, female), bj is the fixed effect of breed (j = arsi, bale, jemjem) and eij is the random error. quantitative data were analyzed separately for each sex by fitting breed as a class variable. means (lsm) were separated using the adjusted tukey-kramer test (tukey, 1953; kramer, 1956). multivariate analysis stepwise discriminant analysis (sas, 2016) was used to detect morphometric traits that better discriminate the cattle breeds, while discriminant analysis was applied to allocate individuals to known breeds and assess possibilities of misclassifications. canonical discriminant analysis was also employed to deliver maximal separations between breeds (sas, 2002). graphic interpretation of breed differences was plotted using the scored canonical variables. pairwise mahalanobis distances between breeds were computed as d2 (i|j) = (xi − xj) ′ cov−1 (xi − xj). where d2 (i|j) is the distance between breeds i and j, cov−1 is the inverse of the covariance matrix of measured variables, xi and xj are the means of variables in the ith and jth breeds. results qualitative characteristics figure 2 shows the coat colour distribution across the two sexes and three breeds studied. while coat colour was not significantly influenced by the animals’ sex (chisquare value 4.5, p = 0.3480), it did vary by breed (chi-square value 37.9, p < 0.0001). black coat colour was predominant across all three breeds, while black + white coat animals were observed more frequently in jemjem cattle. black + white coloured animals are those with predominantly black coat colour with some white patches, spots or shades. their coat colour pattern can be also indicated as pied, spotty or shaded. the same applied to the red + white coloured animals. the effects of breed and sex on the qualitative characteristics of arsi, bale and jemjem breeds are presented in table 3 along with the respective chisquare values and levels of significance. sex affected 6 out of the 13 traits while breed significantly affected 7 out of the 15 traits. the majority of the studied cattle had forward-oriented (65.8%), widely spaced (71.4%) and curved horns (76%). they also mainly had small (66.7%), erected (96.2%) humps located at the cervicothoracic position (77.8%). all (100%) of the studied cattle had a straight face and a back profile as well as a sloppy rump. medium (44.4%) or long (45.6%) tails were equally common and medium dewlap width were observed on most (72.1%) of the cattle. on the other hand, 57.6% of the males had medium perpetual sheath while naval flap was not observed on 53.2% of their female counterparts. a uniform coat colour pattern was observed on most (78%) of the cattle while all of them (100%) had straight-edged ears (figure 3). comparing sexes, laterally oriented straight horns were more frequently observed in males than females. the majority of females had an erect hump while some males had a dropping hump. males also had larger humps located at the thoracic position while females possessed small humps located at the cervicothoracic position. comparing breeds, a higher proportion of narrow horn spacing was observed in jemjem cattle. however, no significant differences were observed among the cattle breeds in terms of most of the qualitative characteristics. morphometric traits means (least squares), standard errors and pairwise comparisons showing the effect of breed on the morphometric traits of the studied male and female cattle populations are presented in table 4. relative differences among breeds were observed more in females than males. within females, bale cows had the largest body length (101.6cm), heart girth (139.4cm) and hock circumference (28.8cm). the arsi cows had the smallest body length (97.1cm), pelvic width (29.5cm) and muzzle circumference (36.1cm) while their horns were the longest (22.6cm). the jemjem cows had relatively intermediate measurements for most of the traits including body length (99.7cm), pelvic width (30.5cm) and hock circumference (27.6cm). similarly, the bale males had the largest heart girth (150.5cm) and hock circumference (30.7cm). multivariate analysis stepwise discriminant analysis all nine morphometric traits were used in discriminating the females while only six morphometric traits were used to discriminate the males. the three most important morphometric variables used in discriminating the cattle breeds were heart girth, muzzle circumference and horn length among females, and heart girth, horn length and pelvic width among males (table 5). however, low partial r-square and f-values were observed. genetic resources (2024), 5 (10), 53–64 57 figure 2. distribution of coat colour among three cattle breeds and separated by sex. figure 3. a, arsi cattle; b, bale cattle; c, jemjem cattle. female individuals on the left, male individuals on the right. phenotypic relationships among ethiopian cattle breeds 58 mustefa et al genetic resources (2024), 5 (10), 53–64 table 3. percentage distributions of qualitative characteristics of cattle populations by sex and breed. χ2 , chi-square; p, chi-square probabilities; ns, not significant; na, not available; *, p < 0.05; **, p < 0.01; ***, p < 0.0001 qualitative traits breed sex overall meanarsi bale jemjem χ2 value p male female χ2 value p number of animals 152 137 152 99 342 horn spacing narrow 13.2 24.1 48.0 47.2 *** 29.3 28.4 0.03 ns 28.6 wide 86.8 75.9 52.0 70.7 71.6 71.4 horn shape straight 17.1 32.1 23.7 8.9 * 47.5 17.3 38.4 *** 24.0 curved 82.9 67.9 76.3 52.5 82.7 76.0 horn orientation lateral 17.1 29.9 22.4 27.0 ** 45.5 16.4 44.2 *** 22.9 upright 19.1 5.1 5.9 14.1 9.0 10.2 forward 63.8 62.8 70.4 40.4 73.1 65.8 dropping 0 2.2 1.3 0 1.5 1.1 colour pattern uniform 82.9 85.4 66.5 29.6 *** 81.8 76.9 1.3 ns 78.0 spotty 1.3 0.7 7.2 2.0 3.5 3.2 pied 11.8 8.8 23.7 13.2 15.5 15.0 shaded 4.0 5.1 2.6 3.0 4.1 3.8 coat colour black 63.2 61.3 58.5 37.9 *** 61.6 60.8 4.5 ns 61.0 red 21.0 24.1 8.6 21.2 16.7 17.7 black + white 9.2 11.7 29.6 16.2 17.3 17.0 grey 2.6 2.2 2 1.0 2.6 2.3 red + white 4 0.7 1.3 0 2.6 2 ear shape straight edged 100 100 100 na ns 100 100 na ns 100 hump shape erect 94.7 96.3 97.4 1.4 ns 82.8 100 61.1 *** 96.2 dropping 5.3 3.7 2.6 17.2 0 3.8 hump size small 72.4 59.8 67.1 12.9 * 9.1 83.3 224.4 *** 66.7 medium 19.7 29.2 30.3 59.6 16.7 26.3 large 7.9 11.0 2.6 31.3 0 7.0 hump position thoracic 25.7 24.8 16.5 4.5 ns 80.8 5.3 253.5 *** 22.2 cervico-thoracic 74.3 75.2 83.5 19.2 94.7 77.8 face profile straight 100 100 100 na ns 100 100 na ns 100 back profile straight 92.8 92.0 91.5 0.18 ns 91.9 92.1 0.004 ns 92.0 curved 7.2 8.0 8.5 8.1 7.9 8.0 rump profile sloppy 100 100 100 na ns 100 100 na ns 100 tail length short 13.2 5.1 11.2 9.6 * 9.1 10.2 0.85 ns 10.0 medium 45.4 40.2 47.4 48.5 43.3 44.4 long 41.4 54.7 41.4 42.4 46.5 45.6 dewlap width small 5.9 2.9 5.2 16.0 ** 0 6.1 82.3 *** 4.8 medium 69.1 65.0 81.6 43.4 80.4 72.1 large 25.0 32.1 13.2 56.6 13.5 23.1 naval flap width absent 58.9 53.8 48.2 7.3 ns 53.2 na na 53.2 small 28.6 33.3 36.5 33.1 33.1 medium 11.6 7.5 12.4 10.8 10.8 large 0.9 5.4 2.9 2.9 2.9 perpetual sheath small 25.0 29.6 26.7 4.8 ns 27.3 na na 27.3 medium 52.5 56.8 73.3 57.6 57.6 large 22.5 13.6 0 15.1 15.1 genetic resources (2024), 5 (10), 53–64 59 table 4. the effect of breed of the cattle on their morphometric measurements by sex. *, p < 0.05; **, p < 0.01; ***, p < 0.0001; ns, not significant. means within a row bearing different superscripts are significantly different; a is given to the highest value. traits females arsi bale jemjem p number 112 93 137 body length 97.1±0.56c 101.6±0.61a 99.7±0.52b *** heart girth 132.1±0.63b 139.4±0.68a 132.0±0.58b *** height at withers 108.9±0.51ab 109.4±0.55a 107.4±0.47b * pelvic width 29.5±0.19b 30.6±0.21a 30.5±0.18a *** muzzle circumference 36.1±0.19b 37.4±0.20a 37.8±0.17a *** ear length 16.2±0.15 15.8±0.16 15.9±0.13 ns horn length 22.6±0.55a 19.3±0.60b 16.7±0.50c *** cannon bone length 16.9±0.13ab 16.5±0.15b 17.1±0.12a ** hock circumference 27.4±0.16b 28.8±0.18a 27.6±0.15b *** males arsi bale jemjem p number 40 44 15 body length 105.2±1.03 108.1±1.04 105.1±1.70 ns heart girth 144.7±1.25b 150.5±1.26a 140.7±2.04b *** height at withers 115.3±0.98a 115.6±0.99a 110.7±1.61b * pelvic width 30.0±0.37 30.3±0.37 29.7±0.60 ns muzzle circumference 39.2±0.33b 40.3±0.34a 39.6±0.55ab * ear length 16.4±0.22a 15.8±0.22b 15.8±0.36ab * horn length 23.8±1.25a 24.4±1.26a 15.3±2.05b ** cannon bone length 17.5±0.24a 16.7±0.24b 17.4±0.39ab * hock circumference 29.6±0.30b 30.7±0.31a 28.8±0.50b ** table 5. order of traits used in discriminating the cattle populations from different breeds. sex step variables entered partial r-square f value pr > f wilks’ lambda pr < lambda females 1 heart girth 0.1832 38.01 < 0.0001 0.8168 < 0.0001 2 muzzle circumference 0.1394 27.31 < 0.0001 0.7029 < 0.0001 3 horn length 0.1928 40.25 < 0.0001 0.5674 < 0.0001 4 pelvic width 0.0752 13.66 < 0.0001 0.5247 < 0.0001 5 canon bone length 0.0583 10.37 < 0.0001 0.4941 < 0.0001 6 ear length 0.0307 5.29 0.0055 0.4789 < 0.0001 7 hock circumference 0.0278 4.76 0.0091 0.4656 < 0.0001 8 body length 0.0201 3.41 0.0341 0.4562 < 0.0001 9 height at withers 0.0189 3.19 0.0425 0.4476 < 0.0001 males 1 heart girth 0.2543 16.37 < 0.0001 0.7456 < 0.0001 2 horn length 0.1577 8.90 0.0003 0.6280 < 0.0001 3 pelvic width 0.0900 4.65 0.0119 0.5715 < 0.0001 4 canon bone length 0.0974 5.02 0.0085 0.5158 < 0.0001 5 height at withers 0.0568 2.77 0.0677 0.4865 < 0.0001 6 ear length 0.0422 2.01 0.1404 0.4659 < 0.0001 phenotypic relationships among ethiopian cattle breeds 60 mustefa et al genetic resources (2024), 5 (10), 53–64 discriminant analysis results of the discriminant analysis show moderate classification (65.83%) of individual animals into their corresponding breed with an error rate of 34.17% (table 6). the highest classification into their respective breed was observed in arsi cows while the lowest classification was observed in arsi males. o canonical discriminant analysis multivariate statistics including eigenvalues using the first and the second canonical structures (can 1 and can 2) are shown in table 7. in classifying the cattle breeds, can 1 had a higher proportion for females (0.6066) and males (0.7876) than can 2. however, the lowest eigenvalues were observed for both canonical structures under both sexes. pairwise mahalanobis distances between the breeds studied are presented in table 8. the shortest and the longest distances were observed among males. the shortest distance (1.77) was observed between arsi and bale males while bale and jemjem oxen were related distantly (7.31). the overall results showed the lowest and non-significant distances among arsi, bale and jemjem cattle breeds. a plot of can 1 and can 2 showing the maximum separation among the cattle breeds is presented in figure 4. in line with the result of the mahalanobis distances, females were separated less than males. accordingly, arsi, bale and jemjem cows were inseparable and categorized in the same group while relative separation was observed between arsi and jemjem cows. similarly, a relative separation between bale and jemjem oxen was also observed. discussion qualitative characteristics due to their easily observable nature, unique qualitative characteristics can be used for breed differentiation. alongside other morphometric and morphological traits, similarities in coat colour and coat colour pattern among breeds may indicated genetic similarity (getachew et al, 2014; mustefa et al, 2024). according to getachew et al (2014), the majority (73.62%) of ogaden cattle exhibited a uniform body colour pattern, with most (69.33%) having a grey coat colour. similarly, mustefa et al (2024) suggested that the guraghe and jimma cattle populations might belong to the same breed based on their phenotypic similarities. they reported that 66% of guraghe and 77% of jimma cattle populations had a uniform coat colour pattern with 55% of guraghe and 65% of jimma cattle populations having a red coat (mustefa et al, 2024). in line with these results, the cattle breeds examined in the current study – arsi, bale and jemjem – shared similarities in both coat colour and coat colour patterns. these phenotypic similarities suggest a potential genetic link between these breeds. however, contrasting reports from mustefa et al (2023) on harar cattle, which displayed a diverse range of coat colours and patterns, highlight the need for further molecular characterization to confirm the results of the phenotypic study. the dominantly black coat colour and uniform body colour pattern observed in this study are in line with the results of yimamu (2014) on arsi cattle. the dominance of dark colours over light colours might be associated with the highland environment (titto et al, 2016), since animals with darker coats are better adapted to cold conditions by absorbing more heat than lighter colour coats (titto et al, 2016). moreover, the dominantly observed black coat colour might also be associated with farmers’ preferences and selection criteria as black was favoured in the studied areas. beyond coat colour, similarities in other qualitative characteristics were also observed. the resemblance in horn, hump, tail, dewlap, naval flap and perpetual sheath besides their perfect match in the face, back and rump profiles among arsi, bale and jemjem challenge their classifications as different breeds. the slight differences noted can be taken as within-breed differences. such differences in cattle sampled from different locations were reported by terefe et al (2015) in mursi cattle and mustefa et al (2021) in raya cattle. morphometric traits results of morphometric traits, alongside qualitative traits, can provide reliable information for quantifying the degree of relationships among breeds. in this study, the observed qualitative similarities among arsi, bale and jemjem were also supported by quantitative measurements. significant differences in morphometric measurements that would indicate distinct breeds were not observed. this was also in line with mustefa et al (2024), who reported comparable morphometric measurements between guraghe and jimma cattle populations suggesting they belong to the same breed. as noted in the qualitative analysis, the differences observed among the three cattle breeds might be due to within-breed variation (mustefa et al, 2024). bale cows seem to be the largest, with higher measurement values for body length, heart girth and hock circumference. intermediate measurement values were observed in jemjem cows, while arsi cows were the smallest, with lower values for body length, pelvic width and muzzle circumference although they possessed the longest horns. however, yimamu (2014) reported relatively higher measurements for body length, heart girth and height at withers for arsi cattle in the same study area. when compared to other ethiopian breeds, the morphometric values of arsi, bale and jemjem cattle were lower than afar cattle (tadesse et al, 2008), begait cattle (ftiwi, 2015), begaria cattle (getachew et al, 2020), fogera cattle (girma et al, 2016), gojjam highland cattle (getachew and ayalew, 2014), harar cattle (mustefa, 2023), kereyu cattle (nigatu and tadesse, 2020), nuer cattle (minuye et al, 2018), ogaden cattle (mustefa et al, 2023) and raya cattle (mustefa et al, 2021). on the other genetic resources (2024), 5 (10), 53–64 61 table 6. number and (percentage) of observations classified into breed based on discriminant analysis of morphometric traits. the diagonal bold values indicate the correct classifications of sampled animals into their respective breed. sex from breed arsi bale jemjem total females arsi 77 (68.75) 20 (17.86) 15 (13.39) 112 (100) bale 18 (19.35) 60 (64.52) 15 (16.13) 93 (100) jemjem 24 (17.52) 25 (18.25) 88 (64.23) 137 (100) error rate 0.3125 0.3548 0.3577 0.3417 males arsi 23 (57.50) 9 (22.50) 8 (20.00) 40 (100) bale 11 (25.00) 30 (68.18) 3 (6.82) 44 (100) jemjem 2 (13.33) 3 (20.00) 10 (66.67) 15 (100) error rate 0.4250 0.3182 0.3333 0.3588 figure 4. plots of canonical discriminant analysis of ethiopian cattle based on morphometric traits. a, females; b, males. breed is indicated by numbers: 1, arsi; 2, bale; 3, jemjem. phenotypic relationships among ethiopian cattle breeds 62 mustefa et al genetic resources (2024), 5 (10), 53–64 table 7. multivariate statistics outputs from the two canonical structures. can, canonical structure. females males multivariate statistics can 1 can 2 can 1 can 2 canonical correlation 0.6138 0.5307 0.6814 0.4353 proportion 0.6066 0.3934 0.7876 0.2124 eigenvalue 0.6047 0.3921 0.8670 0.2338 table 8. pairwise squared distances between breeds. females above diagonal, males below diagonal. from breed arsi bale jemjem arsi 0 2.88 3.32 bale 1.77 0 2.72 jemjem 4.21 7.31 0 hand, the morphometric values of abergelle and irob cattle (zegeye et al, 2021) were lower than the arsi, bale and jemjem. comparable morphometric values were also reported in arado cattle (genzebu et al, 2012), gofa cattle (kebede et al, 2017), horro cattle (bekele, 2015) and mursi cattle (terefe et al, 2015). effect of sex in most morphometric traits, males were observed to be larger than females. such differences might be attributed to the secretion of testosterone in males, which promotes skeletal development and muscle mass growth (baneh and hafezian, 2009). the endocrine system plays a significant role in differentiating the two sexes, with the growth-limiting effects of estrogen being more prominent in females (chriha and ghadri, 2001; baneh and hafezian, 2009). the findings of this study are in line with the reports of mustefa et al (2023) on harar and ogaden cattle, mustefa et al (2021) on raya cattle, and terefe et al (2015) on mursi cattle. multivariate analysis morphometric traits were identified and ranked based on their ability to differentiate between the cattle breeds. in line with the results of mustefa et al (2024) on guraghe cattle, lower partial r-square and f-values (table 5) were observed in the stepwise analysis, showing that morphometric traits have limited potential to discriminate the breeds into different categories. the higher the r-square and f-values the higher the potential of the traits in differentiating the cattle breeds (mustefa et al, 2023). the higher error rate (table 6) suggests greater shared similarities among the breeds, which reduces the chances of clearly categorizing the breeds into different clusters. on the other hand, the lower the error rate, the lower the similarities shared among the breeds. this highlights the uniqueness of each breed. the moderate classification with a considerably higher error rate (34.17%) observed in the current study, showed the presence of shared similarities among the breeds. an error rate of 1% was reported in classifying the phenotypically unrelated harar and ogaden cattle breeds (mustefa et al, 2023). the low eigenvalues reported for both canonical structures in both sexes (table 7) do not support the classification of the animals into different breeds. an eigenvalue higher than 1 is accepted to approve the discrimination analysis. if the value is lower than 1, the discrimination of the studied animals into different breeds is not significant. in this study, the observed low eigenvalue disproved the presence of three breeds in the study area. similarly, the higher the mahalanobis distances between breeds (table 8) the higher the possibility of classification into different clusters. however, the mahalanobis distances in the current study were low although jemjem males showed relatively higher distances. this could be due to the small sample size of jemjem oxen. the accuracy of the analysis increases with larger sample sizes. due to the low eigenvalue (< 1) in the multivariate analysis, the distances observed were not significant, supporting the conclusion that the studied cattle breeds are phenotypically inseparable. conclusion according to the univariate (morphometric measurements and qualitative characteristics) as well as multivariate analysis results, the arsi, bale and jemjem cattle breeds were found to be phenotypically inseparable. however, the observed phenotypic similarities among these breeds do not necessarily mean that they are genetically the same. therefore, further molecular characterization is recommended to quantify the degree of genetic relationships among these breeds. acknowledgments the authors are highly indebted to the ethiopian biodiversity institute (ebi) for covering all the budget needs for this work. our special appreciation also goes to the smallholder farmers/breeders for providing their animals for this work for free. we also take this opportunity to thank the animal science experts and development agents for their endless help during the data collection. a special word also goes to our friend and work partner mr tadesse hunduma for mapping the study area. author contributions all authors contributed to the study’s conception and design. material preparation and data collection were performed by amine mustefa, awoke melak, hizkel kenfo, seble sinke and ahmed abdela. data analysis and writing the first manuscript draft were performed by amine mustefa. abebe hailu reviewed the manuscript. all authors commented on the various versions of the manuscript, and read and approved the final manuscript. genetic resources (2024), 5 (10), 53–64 63 data availability statement the datasets generated and/or analyzed during the current study are not publicly available due to data confidentiality but are available from the corresponding author upon reasonable request. conflicts of interest the authors declare that they have no conflicts of interest. references ajmone-marsan, p., boettcher, p. j., colli, l., ginja, c., kantanen, j., and lenstra, j. a. 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morphometric traits multivariate analysis stepwise discriminant analysis discriminant analysis canonical discriminant analysis discussion qualitative characteristics morphometric traits effect of sex multivariate analysis conclusion acknowledgments author contributions data availability statement conflicts of interest original article genetic resources (2025), 6 (11), 71–81 doi: 10.46265/genresj.sclc1551 https://www.genresj.org issn: 2708-3764 phenotypic characterization of cattle breeds in southern ethiopia: implications for breed differentiation and conservation bergene banjaw a, habtamu lemma didanna a and amine mustefa *,b a college of agriculture, wolaita sodo university, wolaita, ethiopia b ethiopian biodiversity institute, addis ababa, ethiopia abstract: this study aimed to characterize and quantify the phenotypic relationship between gamo and gofa cattle breeds using nine morphometric measurements and 11 morphological traits. a total of 600 adult cattle (486 females and 114 males) were randomly selected from six purposively chosen districts. univariate and multivariate analyses were conducted using statistical analysis software. the univariate analysis revealed the morphometric values and morphological characteristics of both cattle breeds but did not show significant variations between them. the majority of the cattle exhibited uniformly patterned coat colour, upward-oriented, straight-shaped horns with black colour, laterally oriented ears with rounded edges, straight face profiles, small hump sizes, short coat hair, and medium tail length. in accordance with the phenotypic similarities observed in the univariate analysis, multivariate analysis also failed to identify significant differences between the two breeds. these results suggest that the two cattle breeds are phenotypically inseparable. however, these phenotypic similarities do not necessarily indicate genetic similarities. therefore, further genetic characterization is recommended to assess the degree of genetic relationship between the breeds. in the meantime, it is advised to design breed-specific in situ conservation and genetic improvement programmes without separating the cattle breeds. keywords: gamo-gofa, morphological traits, phenotypic characterization, univariate analysis citation: banjaw, b., didanna, h. l., mustefa, a. (2025). phenotypic characterization of cattle breeds in southern ethiopia: implications for breed differentiation and conservation. genetic resources 6 (11), 71–81. doi: 10.46265/genresj.sclc1551. © copyright 2025 the authors. this is an open access article distributed under the terms of the creative commons attribution license (cc by 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. introduction ethiopian indigenous cattle are vital to the livelihoods of smallholder farmers and significantly contribute to the nation’s economy, particularly through their role in the agricultural gdp (csa, 2022). these cattle are primarily valued for milk, meat and draught power, while also serving as sources of income, manure and cultural capital (zerabruk and vangen, 2005; genzebu et al, 2012; yimamu, 2014; kebede et al, 2017; getachew et al, 2020). with an estimated population of 70.3 million cattle, ethiopia hosts the largest cattle herd in africa (csa, 2022; statista, 2024), underscoring their prominence within the livestock sector. ∗corresponding author: amine mustefa (aminemustefa32@gmail.com) genetic diversity, both within and among breeds, is a critical foundation for conservation and genetic improvement strategies. indigenous breeds dominate ethiopia’s cattle population, comprising 28 officially registered breeds (ebi, 2016; mustefa, 2023). however, several gaps exist in breeds’ documentation and characterization. for instance, several phenotypically studied breeds – including bonga, fellata, gamo and qocherie – remain unregistered, while others (e.g. adwa, hamer and smada) lack comprehensive phenotypic data despite formal registration (mustefa, 2023). addressing these inconsistencies is essential to establish a nationwide framework for breed-specific conservation and genetic improvement programmes. this study focused on two cattle breeds, gamo and gofa. the gofa cattle breed, which was first studied by rege and tawa (1999), is officially registered in the ethiopian indigenous cattle breeds database (ebi, received: 06.02.2025 accepted: 30.04.2025 published online: 10.06.2025 https://www.genresj.org https://www.doi.org/10.46265/genresj.sclc1551 https://www.genresj.org https://www.doi.org/10.46265/genresj.sclc1551 mailto:aminemustefa32@gmail.com 72 banjaw et al genetic resources (2025), 6 (11), 71–81 2016). gofa cattle, primarily used for work, milk and meat, were categorized as small east african zebu (seaz) (rege, 1999). according to rege and tawa (1999), gofa cattle are one of the smallest strains not only among the abyssinian zebu but also among all ethiopian cattle. small hump, small to medium horns and dominantly red colour are some of their qualitative characteristics (rege and tawa, 1999). the study by kebede et al (2017) on gofa cattle has also reported the existence of diverse coat colours, patterns and qualitative traits. the name gofa cattle was first introduced by rege and tawa (1999) and then by kebede et al (2017) after the ‘gofa’ ethnic community which raised them. on the other hand, gamo cattle, first studied by chebo et al (2013), were not officially registered in the ethiopian indigenous cattle breeds database (ebi, 2016). however, the study by chebo et al (2013) showed the existence of potential cattle breeds in the area. according to chebo et al (2013), the gamo cattle were further divided into two subpopulations: the gamo highland and the gamo lowland. the gamo highland were relatively smaller with compact bodies compared to the medium-to-large-bodied gamo lowland subpopulations. the name gamo cattle was first introduced by chebo et al (2013) after the ‘gamo’ ethnic community which raised them. the production system of both gamo and gofa cattle breeds was reported to be similar, with cattle owners practising comparable husbandry methods. own and communal grazing lands were the source of feed, while natural and controlled breeding were the common breeding systems among the owners of both cattle breeds (chebo et al, 2013; kebede et al, 2017; zeleke et al, 2017). however, as mentioned above, the two cattle breeds were studied separately and at different times. this hindered the comparison of the two breeds, which further affected the breed registration as well as the development of breedspecific breeding programmes. therefore, an inclusive phenotypic characterization study was mandatory to understand the relationships between the breeds. thus, the current study aimed to conduct an on-farm phenotypic characterization of gamo and gofa cattle, assess their morphological diversity, and quantify the degree of phenotypic divergence between them. materials and methods study areas the study was carried out in the gamo and the gofa zones. three districts were selected from each zone: kucha, daramalo and dita districts from gamo zone, as well as zala, denbagofa and oyda districts from gofa zone (figure 1). weather and agroecology-related information of the selected districts are presented in table 1. site and animal selection representative samples of gamo and gofa cattle breeds were selected from their respective breeding areas. information on their breeding regions and distribution zones was gathered using secondary sources. the gamo cattle breed is reported to be native to the gamo zone, with its distribution extending into the neighbouring gofa zone (rege and tawa, 1999; chebo et al, 2013). thus, three districts – kucha, daramalo and dita – were randomly chosen from gamo zone to represent the indigenous gamo cattle. similarly, gofa cattle are reported to be primarily found in gofa zone, with their distribution reaching into the neighbouring gamo zone (rege and tawa, 1999; kebede et al, 2017). accordingly, three districts – zala, denbagofa, and oyda – were randomly selected from gofa zone to represent the indigenous gofa cattle. from each district, two sampling sites (known as ‘kebeles,’ the smallest administrative units) were randomly chosen. twenty-five households-raising cattle were then randomly selected from each sampling site. from each household, two unrelated adult cattle, aged four years and older, were randomly chosen. one male cattle was sampled every two households within each kebele. these animals were carefully monitored by their owners and trained labourers. aggressive cattle that were unable to stand properly on flat ground were excluded from measurements. data collection morphometric and morphological data were collected following the fao (2012) guidelines. data collection was conducted in the morning to minimize the effects of feeding and watering on the measurements. three researchers were involved in the data collection process: two handled the morphometric data, while the third recorded the morphological data. to minimize bias, the same researchers performed the data collection in all sites throughout the study. the animals were measured using a textile measuring tape in centimetres. a total of 600 cattle (486 females and 114 males) were subjected to nine morphometric measurements (table 2) and 11 qualitative (morphological) traits (figure 2, table 3). for data analysis, the cattle were grouped into three age categories based on the classification by tatum (2011): group one (3–5 years), group two (6–7 years) and group three (8 years and older). data analysis the overall data analysis was carried out using the statistical analysis system (sas) software 9.0 (sas, 2002). univariate procedure for data normality test, the frequency procedure for morphological (qualitative) data analysis, and the general linear model (glm) procedure for morphometric (quantitative) data analysis were used. data analysis was carried out using the following model: yijk = µ + xi +yj +zk + eijk where genetic resources (2025), 6 (11), 71–81 characterization of gamo and gofa cattle in ethiopia 73 figure 1. map of the studied areas table 1. weather and agroecology-related information of the selected districts (gegnaw and hadado, 2014; leulalem et al, 2016; kebede et al, 2017; cholo et al, 2018; chankalo, 2022; csa, 2022; kassa et al, 2022). parameters gamo zone gofa zone kucha daramalo dita zala dembagofa oyda human population 163,832 110,815 111,283 105,949 114,382 51,784 cattle population 211,574 219,452 157,300 303,095 289,097 121,431 temperature (ºc) 20–25 19–22 10–23 18–32 18–28 15–25 rain fall (mm) 1,100–1,600 1,300–1,900 2,500–3,500 500–900 900–1,100 1,000–2,000 altitude 800–2,250 1,217–2,700 1,800–3,500 1,194–1,484 800– 2,860 1,000–3,200 agroecology (%) lowland 49.4 29.2 90 75 27 midland 50.6 33.3 40 10 15 40 highland 37.5 60 10 33 table 2. listof morphometric traits with their respective definitions. measurements were conducted in centimetres (fao, 2012). no. morphometric traits definitions 1 body length distance from shoulder point to pin bone 2 heart girth chest circumference right behind its front two legs 3 height at withers distance from ground to withers of the front foot 4 pelvic width distance between the two ends of the pelvic bone 5 muzzle circumference perimeter of the mouth 6 ear length distance from the root to the tip of the back side of the ear 7 horn length outer side distance between root and tip of the horn 8 cannon bone length distance between the fetlock joint (ankle) and the knee 9 hock circumference perimeter of the hock bone 74 banjaw et al genetic resources (2025), 6 (11), 71–81 yijk is an observation, µ is the overall mean, xi is the fixed effect of breed (i = gamo, gofa), yj is the fixed effect of sex (j = male, female), zk is the fixed effect of age (k = 3–5, 6–7, ≥8 years) and eijk is the random error. however, the effect of age on all qualitative traits was found to be not significant; hence, it is omitted from the model. additionally, the quantitative data were analyzed separately for each sex by fitting breed as a class variable. means (lsm) were separated using the adjusted tukey-kramer (tukey, 1953; kramer, 1956). multivariate analysis was carried out separately for each sex using both the morphometric and morphological traits at the same time. prior to the analysis, the morphological traits were coded using discrete values. stepwise discriminant analysis to detect morphometric traits that could better classify the cattle breeds, discriminant analysis to allocate individuals to known breeds and assess possibilities of misclassifications, and canonical discriminant analysis to deliver maximal separations between breeds were used. graphic interpretation of breed differences was plotted using the scored canonical variables. pairwise mahalanobis distances between breeds were computed as d2 (i|j) = (xi − xj) ′ cov−1 (xi − xj). where d2 (i|j) is the distance between breeds i and j, cov−1 is the inverse of the covariance matrix of measured variables, xi and xj are the means of variables in the ith and jth breeds. results qualitative characteristics the coat colour of the two studied breeds and sexes is presented in figure 2. the coat colour was not significantly affected by breed, but it was affected by their sex. the majority of the studied cattle possessed red and light red coat colour. sex-wise results revealed a high proportion of red-coloured females, while males possessed a light-red coat colour. the effect of breed and sex on the qualitative characteristics of gamo and gofa cattle breeds is presented in table 3 along with the respective chisquare values and levels of significance. relatively, breed affected more morphological traits than sex: three out of the ten morphological traits were affected significantly by the breed of the animals, while sex affected only one trait. accordingly, the majority of the studied cattle populations possessed uniformlypatterned coat colour, upward-oriented straight-shaped horns with black colour, laterally-oriented round edge ears, straight face profile, small hump size, short coat hair size, and medium tail length. morphometric traits tables 4 and 5 present the least square means, standard errors and pairwise comparisons showing the effect of breed, sex and age on the morphometric traits of the studied cattle populations. breed affected two out of the nine morphometric traits, with gofa cattle exhibiting greater pelvic width and horn length measurements compared to gamo cattle. however, the effect of breed on the morphometric measurements was found to be sex-dependent. breed significantly affected pelvic width and horn length measurements of the females, with values higher in gofa females. however, these measurements of the male cattle populations were not significantly affected by breed differences. on the other hand, the heart girth measurement of the males was significantly affected by breed, with gamo males having higher measurements than their counterparts from gofa. however, the heart girth measurement of the females was not significantly affected by breed. similarly, sex affected seven out of the nine morphometric traits, with males exhibiting larger measurements than females in most of the traits except for ear and horn length. age affected five of the nine morphometric traits, with most measurements increasing as the animal grew older. body length and heart girth measurements were found to be stable after the age of six years. however, muzzle circumference and horn length measurements of middle-aged animals were found to be the highest among the compared age groups. multivariate analysis stepwise discriminant analysis ten out of the 20 morphometric and morphological traits were used to discriminate the female cattle populations while six traits were used to discriminate the males. the three most important morphometric variables used in discriminating the cattle breeds were horn length, pelvic width and coat colour pattern among females, and horn shape and horn length among males (table 6). the overall results show the existence of low partial rsquare and f-values. discriminant analysis results of the discriminant analysis showed moderate classification of individual animals into their corresponding breed (table 7). the highest classification into their respective breed was observed in gamo males, while the lowest classification was observed in gofa females. canonical discriminant analysis canonical correlations and eigenvalues for both male and female cattle populations are shown in table 8. in line with the low partial r-square and f-value outputs in table 6, the eigenvalues were also small enough to discriminate between the two cattle breeds in both sexes. however, relatively higher eigenvalues were observed for males than females. similarly, the canonical correlation, which was used to build the canonical variate 1 (can 1) from the used traits, was also low. however, a relatively higher canonical correlation was observed for males than females. pairwise squared mahalanobis distances between the breeds were calculated as 1.43 for females and genetic resources (2025), 6 (11), 71–81 characterization of gamo and gofa cattle in ethiopia 75 table 3. percentages of qualitative characteristics of cattle populations by sex and breed. qualitative traits cattle breed sex gamo (300) gofa (300) χ 2 value p males (114) females (486) χ2 value p coat colour pattern uniform 85.3 78.0 5.4 ns 82.5 81.5 0.27 ns patchy 9.7 14.0 10.5 12.1 spotted 5.0 8.0 7.0 6.4 horn shape straight 69.3 59.0 7.0 ** 65.8 63.8 0.16 ns curved 30.7 41.0 34.2 36.2 horn orientation lateral 8.7 14.7 7.3 ns 15.8 10.7 13.2 * upward 69.7 68.7 63.2 70.6 downward 10.0 7.7 14.0 7.6 forward 10.0 8.3 4.4 10.3 backward 1.6 0.6 2.6 0.8 horn colour black 59.3 59.3 5.2 ns 50.9 60.7 4.6 ns brown 9.0 6.3 9.6 7.2 white 28.0 33.7 35.1 29.8 black + white 3.7 1.7 4.4 2.3 ear shape round edged 93.0 94.7 0.72 ns 91.2 94.4 1.7 ns straight edged 7.0 5.3 8.8 5.6 ear orientation erect 16.3 20.0 10.5 ** 18.4 18.1 2.0 ns lateral 78.7 68.7 70.2 74.5 dropping 5.0 11.3 11.4 7.4 face profile straight 83.7 90.7 7.0 * 89.5 86.6 1.5 ns concave 13.0 8.0 9.6 10.7 convex 3.3 1.3 0.9 2.7 hump size absent 5.3 3.3 2.7 ns 7.9 3.5 0.27 ns small 61.3 59.7 54.4 61.9 medium 32.3 35.0 35.1 33.3 large 1.1 2.0 2.6 1.3 coat hair length short 93.7 94.0 0.03 ns 93.9 93.8 1.6 ns medium 5.3 5.0 6.1 5.0 long 1.0 1.0 0 1.2 tail length short 4.3 6.0 0.86 ns 2.6 5.8 1.9 ns medium 74.0 73.0 76.3 72.8 long 21.7 21.0 21.1 21.4 table 4. the effect of breed on the cattle morphometric measurements by sex. measurements are in centimetres. n, number of animals sampled; bl, body length; hg, heart girth; hw, height at withers; pw, pelvic width; mc, muzzle circumference; el, ear length; hl, horn length; cbl, cannon bone length; hc, hock circumference; *, p < 0.05; **, p < 0.01; ***, p < 0.0001; ns, not significant. traits aggregate sex females males gamo gofa sig. gamo gofa sig. gamo gofa sig. n 300 300 249 237 51 63 bl 109.1±0.37 109.4±0.35 ns 106.9±0.33 107.3±0.34 ns 111.4±0.87 111.1±0.81 ns hg 137.3±0.40 137.1±0.39 ns 134.1±0.36 135.4±0.37 ns 142.9±0.86 136.0±0.80 *** hw 108.9±0.43 108.8±0.41 ns 106.2±0.38 106.2±0.39 ns 112.2±1.13 111.4±1.05 ns pw 31.3±0.21 32.9±0.21 *** 30.7±0.18 32.4±0.18 *** 32.0±0.61 33.0±0.57 ns mc 36.9±0.15 36.8±0.14 ns 36.4±0.14 36.3±0.14 ns 37.2±0.34 37.1±0.31 ns el 17.0±0.11 17.3±0.11 ns 17.3±0.10 17.7±0.11 ns 16.9±0.22 16.6±0.20 ns hl 16.5±0.21 19.0±0.21 *** 17.2±0.20 20.1±0.20 *** 16.6±0.41 17.6±0.38 ns cbl 25.8±0.15 25.7±0.14 ns 25.7±0.14 25.6±0.14 ns 25.7±0.31 25.8±0.29 ns hc 30.4±0.17 30.6±0.17 ns 30.2±0.16 30.4±0.17 ns 30.8±0.37 30.9±0.35 ns 76 banjaw et al genetic resources (2025), 6 (11), 71–81 table 5. the effect of sex and age on the cattle morphometric measurements. measurements are in centimetres. n, number of animals sampled; bl, body length; hg, heart girth; hw, height at withers; pw, pelvic width; mc, muzzle circumference; el, ear length; hl, horn length; cbl, cannon bone length; hc, hock circumference; *, p < 0.05; **, p < 0.01; ***, p < 0.0001; ns, not significant. traits sex age males females sig. < 6 6–7 > 7 sig. n 114 486 169 234 197 bl 111.3±0.51 107.1±0.25 *** 107.8±0.44b 109.7±0.38a 110.1±0.45a ** hg 139.6±0.56 134.8±0.27 *** 135.9±0.48b 137.8±0.42a 137.8±0.50a ** hw 111.4±0.60 106.3±0.29 *** 107.5±0.51b 108.7±0.45b 110.4±0.53a *** pw 32.6±0.30 31.6±0.14 ** 31.7±0.26 32.4±0.22 32.3±0.26 ns mc 37.3±0.21 36.3±0.10 *** 36.3±0.17b 37.4±0.15a 36.7±0.18b *** el 16.9±0.15 17.5±0.07 ** 17.2±0.13 17.1±0.12 17.2±0.14 ns hl 16.9±0.30 18.6±0.14 *** 16.4±0.26c 19.0±0.22a 17.8±0.26b *** cbl 25.9±0.20 25.7±0.10 ns 25.7±0.17 25.9±0.15 25.7±0.18 ns hc 30.7±0.24 30.2±0.12 ns 30.5±0.21 30.4±0.18 30.5±0.21 ns table 6. order of traits used in discriminating between the two cattle populations using a stepwise discriminant analysis (stepdisc). sex step variables entered partial r-square f value pr > f wilks’ lambda pr < lambda females 1 horn length 0.1630 94.25 < 0.0001 0.8370 < 0.0001 2 pelvic width 0.0522 26.60 < 0.0001 0.7933 < 0.0001 3 coat colour pattern 0.0121 5.91 0.0154 0.7837 < 0.0001 4 canon bone length 0.0099 4.79 0.0291 0.7760 < 0.0001 5 face profile 0.0085 4.12 0.0428 0.7694 < 0.0001 6 body length 0.0096 4.63 0.0319 0.7620 < 0.0001 7 horn shape 0.0076 3.64 0.0570 0.7562 < 0.0001 8 muzzle circumference 0.0074 3.55 0.0600 0.7506 < 0.0001 9 hair length 0.0065 3.10 0.0792 0.7458 < 0.0001 10 horn orientation 0.0047 2.22 0.1368 0.7423 < 0.0001 males 1 heart girth 0.2461 35.25 < 0.0001 0.7539 < 0.0001 2 horn shape 0.1164 14.10 0.0003 0.6661 < 0.0001 3 horn length 0.0585 6.58 0.0117 0.6272 < 0.0001 4 hump size 0.0497 5.49 0.0210 0.5960 < 0.0001 5 pelvic width 0.0388 4.19 0.0431 0.5729 < 0.0001 6 hock circumference 0.0315 3.35 0.0701 0.5548 < 0.0001 table 7. number and (percentage) of observations classified into breed. sex breed gamo gofa total females gamo 187 (75.10) 62 (24.90) 249 (100) gofa 80 (33.76) 157 (66.24) 237 (100) error rate 0.2490 0.3376 0.2933 males gamo 43 (86.00) 7 (14.00) 50 (100) gofa 16 (26.67) 44 (73.33) 60 (100) error rate 0.1400 0.2667 0.2033 genetic resources (2025), 6 (11), 71–81 characterization of gamo and gofa cattle in ethiopia 77 figure 2. effect of breed on coat colour (chi-square value 11.2, p = 0.0836), and effect of sex on coat colour (chi-square value 15.9, p = 0.0142). 3.68 for male cattle, indicating that males were more distantly related than females. however, both distances were small and insufficient to indicate a significant distance between the breeds. the overall multivariate analysis results showed low and nonsignificant distances between gamo and gofa cattle breeds. plots of the first two canonical variables to discriminate the cattle breeds are presented in figure 3. in line with the result of the mahalanobis distances, the studied gamo and gofa breeds were found to be inseparable and categorized in the same group, while a relative separation was observed between the males. discussion qualitative characteristics qualitative traits, due to their easily observable nature, are valuable for distinguishing between cattle breeds. coat colour and coat colour patterns are among the most easily observed traits used to differentiate breeds. however, in this study, these traits did not distinguish the gamo and gofa cattle breeds, as most animals from both breeds exhibited uniformly patterned red and light red coat colours. the similarities in coat colour and pattern, along with other morphological and morphometric similarities between the breeds, may suggest genetic relatedness (mustefa et al (2021) for table 8. multivariate statistics outputs of the canonical structures. multivariate statistics females males canonical correlation 0.5140 0.7087 eigen value 0.3591 0.9060 raya cattle; getachew et al (2014) and mustefa et al (2023) for ogaden cattle). therefore, the observed similarities in coat colour and pattern between gamo and gofa cattle imply phenotypic resemblance, which may reflect underlying genetic similarities. these findings, however, should be validated through genetic analysis. the red-dominant coat colour observed in this study aligns with the findings of rege and tawa (1999), who identified red as the primary coat colour in gofa cattle. similarly, kebede et al (2017) also reported that red and white were the most common coat colours in gofa cattle. in agreement with these findings, chebo et al (2013) reported that dark and light red were the predominant coat colours in gamo cattle. the farmers’ preference for red coat colour in both breeds may be linked to farmers’ selection criteria, as red is a preferred colour in the studied areas. according to kebede et al (2017), coat colour was a significant selection criterion for farmers, following milk yield. similarities in other qualitative traits, such as horn, ear, hump, face, hair, and tail lengths, were also observed between the gamo and gofa cattle breeds, which challenges their classification as distinct breeds. the minor differences observed could be attributed to variations within the breeds. such intra-breed variations across different sampling locations were also reported by terefe et al (2015) in mursi cattle, mustefa et al (2021) in raya cattle, and mustefa (2023) in harar cattle. morphometric traits morphometric measurements, in conjunction with qualitative traits, provide reliable information for assessing the degree of relationship between breeds. most of the 78 banjaw et al genetic resources (2025), 6 (11), 71–81 figure 3. plots of canonical discriminant analysis based on morphometric traits. a, females; b, males. breed is indicated by numbers: 1, gamo; 2, gofa. morphometric measurements were similar between the two cattle breeds, which supports the observed qualitative similarities. consequently, no significant differences were found between the gamo and gofa cattle breeds that could serve to differentiate them. as mentioned in the qualitative section, the slight differences observed may reflect within-breed variations. these variations are crucial for designing conservation and genetic improvement programmes. terefe et al (2015) in mursi cattle, mustefa et al (2021) in raya cattle, and mustefa (2023) in harar cattle also reported morphometric variations within the same breed across different locations. in comparison with the previous study on gofa cattle by kebede et al (2017), the gofa females in this study showed similar body length, heart girth and wither height. however, they had larger muzzle and hock circumferences, and lower ear and horn lengths. in contrast, the gofa males in this study exhibited comparable body length but lower values for other morphometric measurements. these findings suggest a reduction in body size of gofa males over the past seven years, possibly due to negative selection practices by farmers. similarly, most of the morphometric measurements for the cattle breeds in this study were lower than those reported by chebo et al (2013) for gamo cattle, but comparable to those reported by zeleke et al (2017). the gamo and gofa cattle breeds were found to be smaller than many other ethiopian indigenous cattle breeds. this observation is consistent with rege and tawa (1999) description of gofa cattle as the smallest strain of ethiopian cattle. their morphometric measurements were smaller than those of breeds such as afar (tadesse et al, 2008), begait (ftiwi, 2015), begaria (getachew et al, 2020), fogera (girma et al, 2016), gojjam highland (getachew and ayalew, 2014), harar (mustefa, 2023), kereyu (nigatu and tadesse, 2020), mursi (terefe et al, 2015), nuer (minuye et al, 2018), ogaden (mustefa, 2023) and raya cattle (mustefa et al, 2021). however, their measurements were larger than those of abergelle and irob cattle breeds (zegeye et al, 2021). similar morphometric traits genetic resources (2025), 6 (11), 71–81 characterization of gamo and gofa cattle in ethiopia 79 were also observed in arado (genzebu et al, 2012) and horro cattle (bekele, 2015). multivariate analysis high partial r-square and f-values are necessary to demonstrate significant discrimination between populations. additionally, low error rates are essential to show the distinctiveness of separate breeds. however, the results from the stepwise analysis (table 6) exhibited low partial r-square and f-values, indicating weak discriminatory potential of the morphometric traits. furthermore, high error rates (table 7) were observed, indicating greater similarities between the cattle populations, which lowers the likelihood of classifying the breeds into separate clusters. in contrast, lower error rates would indicate distinct differences between the breeds. for example, an error rate of 1% was reported in the classification of phenotypically unrelated harar and ogaden cattle breeds (mustefa, 2023). an eigenvalue greater than 1 is required for breed discrimination. if the value falls below 1, discrimination between the studied animals is not significant. the lowest eigenvalues observed in both sexes (table 8) failed to distinguish the cattle populations into separate clusters, suggesting the absence of two distinct breeds. similarly, high mahalanobis distances between breeds are needed for clear cluster separation, but the mahalanobis distance results in this study were low, with males showing slightly higher distances. this could be attributed to the smaller sample size of oxen. the accuracy of the analysis improves with larger sample sizes. due to the low eigenvalue (< 1) and short mahalanobis distances in the multivariate analysis, the studied cattle breeds were found to be phenotypically inseparable. however, phenotypic similarities do not necessarily imply genetic similarities between breeds (zechner et al, 2001). effect of sex and age sex and age had minimal effects on the qualitative traits of the studied cattle populations since qualitative traits are typically controlled by fewer genes (falconer, 1989). therefore, successive planned selection activities are required to bring changes to the qualitative traits. on the other hand, the morphometric traits were influenced by both sex and age, because quantitative traits are influenced by a greater number of genes (falconer, 1989). this means a few natural or artificial selection activities can produce significant changes to the quantitative traits. accordingly, males were generally larger than females in most morphometric traits of both breeds, aligning with rensch’s rule (rensch, 1950), which suggests that females of a species are usually smaller than males. these differences could be attributed to testosterone, which promotes the development of skeletal and muscle mass in males (baneh and hafezian, 2009). the effect of the endocrine system on growth was also significant, with estrogen’s influence on growth being more limited in females (chriha and ghadri, 2001; baneh and hafezian, 2009). similar findings, showing male dominance in size, were reported by mustefa (2023) for harar and ogaden cattle breeds, mustefa et al (2021) for raya cattle, and terefe et al (2015) for mursi cattle. age significantly affected five morphometric traits. body length and heart girth measurements showed stable body development from the middle-aged group, while muzzle circumference and horn length measurements indicated the middle-aged group as optimal for these traits. significantly different breeds need to be registered separately, while the same breed should be registered only once. this is because our next step as a country is to design breeding programmes that include both conservation and genetic improvement activities for each breed individually. therefore, conducting these programmes separately for breeds without significant differences would be inappropriate. the currently observed differences among these breeds can be considered as within-breed variation; however, this needs to be supported by further genetic characterization studies. conclusion in accordance to the observed similarities in morphological and morphometric traits between gamo and gofa cattle breeds, multivariate analysis failed to identify significant differences, suggesting that the two breeds are inseparable. however, phenotypic similarities do not necessarily indicate genetic similarity. therefore, further genetic characterization is recommended to assess the genetic relationship between these breeds. in the meantime, the studied cattle populations should not be regarded as separate breeds. breed-specific in situ conservation and genetic improvement programmes should consider the cattle populations as a single entity. additionally, a unified breed name that can represent both populations is recommended for consideration by the country’s national advisory steering committee for animal genetic resources. acknowledgments the authors are highly indebted to the ethiopian biodiversity institute (ebi) and wolita sodo university for covering some 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areas site and animal selection data collection data analysis results qualitative characteristics morphometric traits multivariate analysis stepwise discriminant analysis discriminant analysis canonical discriminant analysis discussion qualitative characteristics morphometric traits multivariate analysis effect of sex and age conclusion original article genetic resources (2024), 5 (10), 94–106 doi: 10.46265/genresj.lyoq7265 https://www.genresj.org issn: 2708-3764 genetic improvement of indigenous cattle breeds in ethiopia: a systematic review of the fogera cattle open nucleus breeding scheme assemu tesfa *,a,b, kefyalew alemayehu a,c, mengistie taye a,d and demelash kassahun b a college of agriculture and environmental sciences, bahir dar university, bahir dar, ethiopia b andassa livestock research center, bahir dar, ethiopia c ethiopian agricultural transformation institution (ati), amhara agricultural transformation center (aatc), bahir dar, ethiopia d institute of biotechnology, bahir dar university, bahir dar, ethiopia abstract: fogera cattle are one of the valuable indigenous milk-type local breeds of ethiopia, widely adapted to the area around lake tana in the amhara region. the objective of this systematic review was to evaluate the performance of the fogera cattle breed under an open nucleus breeding scheme. the review was done systematically by collecting published and unpublished data sources on the breed. the overall milk yield of the nucleus fogera cattle herd was 2.26±0.794l/day. from the total herd, the top 10% and 25% of them produced daily milk yields of 3.31 and 2.87l, respectively, and some elite cows gave an average of 5.45±0.73l/day with a maximum yield of 8l/day. the predicted 305-day milk yield for the top 10% and 25% of the total herd was 883.64 and 772.83l, respectively. the average lactation milk yield and lactation length were reported to be 489±184l and 243±72.79 days, respectively. the respective heritability estimates for the aforementioned traits were 0.20±0.23 and 0.27±0.001. the birth and weaning weights (at 8 months of age) of village fogera cattle born from community-based breeding programmes (cbbp) were 23.77±.21 and 85.89±1.07kg, respectively. the average weaning age for the cbbp herds was reduced to 8 months. the overall calf mortality in the nucleus herd was 3%. the cbbp demonstrated that it could act as a significant entry point for ensuring the conservation and restocking efforts of this breed as a country asset. keywords: community-based breeding programme, ethiopia, growth traits, milk yield, onbs, reproductive traits citation: tesfa, a., alemayehu, k., taye, m., kassahun, d. (2024). genetic improvement of indigenous cattle breeds in ethiopia: a systematic review of the fogera cattle open nucleus breeding scheme. genetic resources 5 (10), 94–106. doi: 10.46265/genresj.lyoq7265. © copyright 2024 the authors. this is an open access article distributed under the terms of the creative commons attribution license (cc by 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. introduction indigenous animal genetic resources are believed to preserve much of the current global genetic diversity, with millions of people directly depending on them (fao, 2023). indigenous cattle breeds constitute an important reservoir of genetic material, for which developing nations have failed to provide adequate recognition through their sustainable use and conservation, which ∗corresponding author: assemu tesfa (assemu546@gmail.com) puts them at risk of extinction. for example, sheko cattle in ethiopia are highly threatened because of crossbreeding with other local cattle, and others like fogera, begayit, ogaden and borena cattle breeds in ethiopia are also facing various degrees of threat that challenge their existence as a breed (ibc, 2004). these breeds are decreasing and deteriorating in terms of both population size and genetic diversity due to paradigm shifts in the existing farming system and production system and farm size dynamics of the native habitat, leading to a subsequent genetic dilution (kebede et al, 2013; tesfa et al, 2017; adisu et al, 2021). received: 18.10.2023 accepted: 23.09.2024 published online: 05.11.2024 https://www.genresj.org https://www.doi.org/10.46265/genresj.lyoq7265 https://www.genresj.org https://www.doi.org/10.46265/genresj.lyoq7265 mailto:assemu546@gmail.com genetic resources (2024), 5 (10), 94–106 genetic improvement of fogera cattle in ethiopia 95 the increasing demand for dairy products because of high population growth, urbanization and improved standards of living is pushing ethiopia’s livestock keepers to increase the productivity of their animals (shapiro et al, 2015). shapiro et al (2017) indicated that, to realize genetic improvement programmes in ethiopia, the best contributors are a range of agro-climates and production systems, feed and water availability, and small to large-scale farms. since the 1960s, selective breeding and crossbreeding have been implemented for the genetic improvement of ethiopian cattle (chebo and alemayehu, 2012). however, the anticipated improvement in milk production and productivity has not yet been achieved, even with crossbreeding. the reasons for this, as indicated by shapiro et al (2015), include separate implementation interests between research and development officials, the absence of a clear and implementable strategy that targets the improvement scenario of indigenous breeds, and the absence of sustained support from different stakeholders. fogera cattle are one of the promising breeds of ethiopia that are widely adapted to the plain of lake tana in the amhara region. the fogera cattle breed is known for its relatively higher milk yield, larger body frame and traction power, better resistance to internal parasite infestation, and sound adaptability to waterlogged fogera plains, which is attributed to its long legs (tesfa, 2015). the milk production potential and draught power of the breed are farmers’ preferred traits (zewdu, 2004; bitew et al, 2007). in its production environment, the breed is used as a dam line for milk yield improvement (with holstein frisian semen) while the bulls are used in crossbreeding with local highland zebu cattle, which perform poorly under smallholder production system, to improve milk yield, growth rate and draught power (tesfa et al, 2017, 2022). the population size of fogera cattle is declining at an alarming rate, therefore urgently requiring conservation efforts for future potential use of the breed. the andassa livestock research center (alrc) has been implementing selective breeding and conservation efforts both on station and on farm to safeguard the fogera breed from extinction and increase its productivity. the center was established in 1964 as the imperial fogera cattle conservation centre, with 57 fogera cows and three bulls purchased from the local area. it was then re-organized as a farm in 1980-1981 and started its operation with the main objective of conserving fogera cattle and producing f1 holstein × fogera crossbreds for distribution to farmers to increase milk production. it was upgraded into a livestock research centre in 2000 (alrc, 2017). however, in its 40-year journey, it did not provide any visible, significant and sustainable positive change to farmers’ lives. among the reasons is the inability to design and formulate an effective breeding programme. this is a key problem not only for alrc case but also for all farms, ranches and multiplication centres found in the country. in 2007, an open nucleus breeding scheme linking the alrc nucleus and the village herds was designed for fogera cattle improvement, and the programme was also used as a model for other breeds under different research centres, farms and multiplication centres. the ultimate objective of the breeding scheme was to restock the declining village fogera cattle populations and improve the livelihood of the farmers. this manuscript summarizes the productive performances, past achievements, shortcomings and lessons learned under the open nucleus breeding scheme and community-based breed programme (cbbp) and indicates gaps and future directions for the sustainability of breeding initiatives. material and methods data sources various researchers, professionals and students (phd and msc) involved in the fogera cattle breeding programme published articles, proceedings and case studies at the level of the two nucleus herds (alrc and ccbir) as well as on the community herds. from the summary, above 10 mscs, 2 phd dissertations, 42 published journal articles and above eigh national, regional, and societal proceedings had information regarding fogera cattle production and productivity. besides, more than ten articles have used the genetic potential of fogera cattle in comparison with other national and internationally recognized breeds for genetic diversity, selection signatures and molecular conservation studies. for this case study, data from more than 17 papers and thesis works with full information on the breed done under the open nucleus breeding scheme were analyzed and compared, while research articles with little information on the fogera cattle were used to discuss and supplement the main findings. the collected data were analyzed by sas (2002) and ms excel was used to develop graphs and trend lines. besides, articles published online on different ethiopian breeds were used to discuss the main findings of the case study (table 1). data analysis daily milk yield (l), lactation milk yield (l), birth weight (kg) and weaning weight (kg) performance were the dependent variables, while breeding period (year), sex, season and district were used as independent variables for the analysis of quantitative traits. the fixed factors were analyzed by the glm procedure of sas (2002) software. genetic parameter estimation for pre-weaning growth performance and milk-related traits was done by the statistical procedure of wombat software (meyer, 2007) in the case of kassahun et al (2020) and asreml (gilmour et al, 2015) in the case of tesfa (2015). heritability, repeatability, and genetic and phenotypic correlations were estimated. survival of calves at the nucleus herd was done with the cox proportional hazards model (cox, 1972) of stata software (se 14) (1996–2021) in the case of gessesse 96 tesfa et al genetic resources (2024), 5 (10), 94–106 table 1. data sources used for the systematic review. alrc, andassa livestock research center; ccbir, chagni cattle breeding and improvement ranch. publication types publication/ releasing years articles’ focus working sites study topics no. of publications msc thesis 1992 to 2019 fogera cattle breed alrc, ccbir and on farm growth, milk, reproduction, survival 6 scientific articles 2005 to 2023 fogera cattle breed alrc, ccbir and on farm growth, milk, reproduction, survival, population number 15 1982 to 2023 ethiopian indigenous cattle breeds different sites in ethiopia production system, growth, milk, reproduction 10 proceedings 2007 to 2019 fogera cattle breed alrc, ccbir and on farm production system, growth, milk, reproduction, survival 5 reports and working documents 2004 to 2023 fogera and other ethiopian indigenous cattle breeds different sites in ethiopia production system, breeding design, strategic plan 8 et al (2021a), and the chi-square test of spss (version 22) was used in the case of mola et al (2019). results description, distribution and adaptive potential of fogera cattle the fogera breed is characterized and well known by its pied coat of black-and-white or black-and-grey; short, stumpy, pointed horns; hump ranging from thoracic to cervico-thoracic; folded dewlap, of moderate to large size; docile temperament; and is used for draught, milk and meat (rege and tawah, 1999; dagris, 2007). it is highly tolerant or resistant to heat stress and solar radiation, which could be due to its dominantly white coat colour with short hair. additionally, the breed is known for its adaptation to high altitudes, tolerance to parasite and disease infestation, fly burden, wet soils or swampy areas, low-quality feed and other unfavourable environmental conditions (alberro and haile-mariam, 1982). farmers keeping fogera cattle reported the breed is known for its drought tolerance, better milk yield and growth rate (tesfa et al, 2022), and its environmental adaptation and meat production potential (kassahun, 2019). the breed is reared in districts surrounding lake tana and is one of the most populous and productive breeds in the amhara region (tesfa, 2015) and the country (ibc, 2004). additionally, the breed is found and conserved at alrc, chagni cattle breeding and improvement ranch (ccbir), and their surrounding kebeles (a small administrative unit in ethiopia). figure 1 shows the fogera cattle distribution districts and working sites. description of the breeding strategy the open nucleus breeding scheme (figure 2) was the strategy employed since 2007 to conserve, improve and restock the declining population of fogera cattle. the nucleus was established in early 1964 by the emperor’s regime (alrc, 2017). the scheme was implemented in selected districts where the pure line breed was intended to be produced, namely wagetera kebele of fogera district and metrha abawarka kebele of gondar zuria district. however, due to high levels of admixture observed in the village herd, the breeding scheme has been closed since 2015. this has prevented the introduction of heifers from the village herd to the nucleus herd to safeguard the latter from genetic dilution (figure 3; tesfa et al (2017)). the districts involved in the open nucleus breeding scheme study were selected through the participation of researchers and experts, with criteria such as the presence of true-to-type fogera cattle (50%), accessibility and presence of knowledgeable farmers (25%), and others like the willingness of farmers, availability of communal grazing land, and enough land for feed development (25%). after the selection, a discussion was done with the community on points like the importance and productivity of the breed, its value for them, and the need for conservation and improvement strategies. after a consensus was built with the community, farmers were selected to hold the breeding bulls and serve the community. those farmers were selected based on wealth status, cattle management practices and the presence of a better educational background for record keeping. the bulls were provided under a written contractual agreement for four years of service to avoid inbreeding, after which the bulls became the property of the recipients. the participating farmers were then arranged as a community-based breeding programme, having their own committee to manage and decide on the activity with the researchers leading the programme (tesfa et al, 2019). the meta-analysis was done for production traits like daily milk yield, lactation milk yield, lactation length; genetic resources (2024), 5 (10), 94–106 genetic improvement of fogera cattle in ethiopia 97 figure 1. a) distribution of fogera cattle in the amhara region, and andassa livestock research center (alrc) and chagni cattle breeding and improvement ranch (ccbir) nucleus sites (tesfa et al, 2022); b) districts suitable for the production of fogera cattle breed and community-based breeding programmes (cbbp) working sites (tesfa et al, 2017) growth-related traits for birth weight and weaning weight; reproductive performance traits for age at first calving, calving interval, and days open; and calf survival in fogera cattle kept under an open nucleus breeding scheme. productive and reproductive performances milk yield performance of fogera cattle according to the report of tesfa et al (2019), the milk yield of fogera cattle at alrc showed an increasing trend from 2002 to 2017, with a lower yield between 2005 and 2007. the inbreeding rate during this time, according to the author, contributed to this outcome. removing the inbred individuals increased the output from 1.92 to 2.43l per day. from the total herd, the top 10% and 25% had an average milk yield of 3.31l and 2.87l per day, respectively. at alrc, there were four groups of pure nucleus herds with an average of 45 cows and one mating bull. these were selected and grouped based on their estimated breeding value (ebv) for milk yield and family relationships to minimize inbreeding; the top 45 individuals with higher ebv were grouped as group i, and next 45 individuals with group ii and the like for group iii and iv (table 2). 98 tesfa et al genetic resources (2024), 5 (10), 94–106 figure 2. open nucleus breeding scheme (onbs) (tesfa et al, 2019) as indicated in table 2, the daily milk yield per cow had a big variation from 0.4 to 7.2l per day, which still allows for improvement within breed selection. the predicted 305-day milk yield also indicated a big variation among breeds and ranged from 274 to 1,194l of milk with an overall average of 578.26l. the top 10% and 25% of the total herd had a 305-day milk yield of 883.64 and 772.83l, respectively (tesfa et al, 2019). according to the experiment by bitew et al (2021) on the potential exploitation of the breed for milk yield, cows were divided into two groups: group one received concentrate feed adjusted based on their daily milk yield in addition to grazing, while group two relied on grazing alone (control). the cows in group one gave 4.69l of milk per day compared to 2.21l in the control group. this indicated that the breed’s genetic potential may have been masked by poor environmental conditions, which need to be improved through delivering additional feeds and husbandry practices. according to kassahun et al (2020), the average daily milk yield (dmy) and lactation length (ll) of the breed were 1.98±0.60l and 243±72.79 days, respectively, and the lactation milk yield (lmy), which is calculated by multiplying dmy by ll, was 489±184l. as shown in the trend (figure 4), the results for these parameters exhibited significant variability and inconsistency over the reported years, which was due to the lower selection intensity and number of animals considered in the selection procedure (kassahun et al, 2020). according to the genetic parameters estimate, heritability for dmy, ll and lmy was 0.33±0.27, 0.20±0.23, and 0.27±0.001, respectively. besides, the repeatability of the respective traits of interest was 0.33, 0.48, and 0.55 (kassahun et al, 2020). genetic resources (2024), 5 (10), 94–106 genetic improvement of fogera cattle in ethiopia 99 figure 3. closed breeding scheme (tesfa et al, 2017) table 2. milk yield performance of the four pure nucleus herds at the andassa livestock research center (alrc). source: tesfa et al (2019) herd type milk yield (l/day) mean sd minimum maximum overall 2.26 0.794 0.4 7.2 fogera group i 2.24 0.731 0.7 5.5 fogera group ii 2.52 0.863 0.6 6.1 fogera group iii 2.05 0.814 0.4 7.2 fogera group iv 2.18 0.707 0.6 4.5 100 tesfa et al genetic resources (2024), 5 (10), 94–106 figure 4. lactation length (ll), lactation milk yield (lmy) and daily milk yield (dmy) of fogera cattle across selection years (kassahun et al, 2020) growth performances performance under nucleus herd. the birth and weaning weight of fogera calves in the two nucleus herd populations is presented in figure 5. according to the trend across selection years, both weaning and birth weight showed variability in both sites. the main reasons for this inconsistency were variations in the management of pregnant cows and calves, climate variability exerting an effect on feed availability, poor recording systems, and differences among individual animals included in the selection process during different years (bekele, 2012; tesfa et al, 2016; kassahun et al, 2022). besides, the observed inbreeding in the nucleus herd during 2006-07 (mekuriaw and bitew, 2006) and varying management aspects (kassahun et al, 2022) at alrc contributed to the declining trend of birth and weaning weight of fogera calves. the heritability estimate for the pre-weaning growth rate of fogera cattle at the two nucleus herds is summarized in table 3. it was observed that the heritability for birth weight had declined with the advancement of years at alrc. differences across years might be due to management differences that influence the environmental part of the estimated genetic parameter, the number of data points, and differences in methods of estimation among the authors. the heritability estimate at ccbir was lower compared to alrc; even the estimate for the f1 crossbreed between the fogera and holstein friesian was lower for birth weight and pre-weaning average daily gain (pwadg) and comparable for weaning weight (table 3). the lower estimate at ccbir for pre-weaning growth parameters was attributed to the presence of management variations, poor nutritional status of the animals, high environmental stress and data record quality (bekele, 2012; zeleke, 2014). performances under community-based breeding programme (cbbp) . as an open nucleus breeding scheme, alrc distributed about 17 pure improved fogera bulls selected based on their estimated breeding value and physical soundness in two kebeles to conduct a community-based breeding programme since 2012. trained enumerators collected data for 8 conscutive years on birth and weaning weight from 2,180 calves born by distributed improved bulls and village local bulls for performance comparison. the birth and weaning weights (at 8 months of age) of the village herds born from improved bulls were 23.77±0.21 and 85.89±1.07kg, respectively (tesfa et al, 2019). the average weaning age was lowered from one year to 8 months. based on the monitoring data at cbbp villages, the average birth and weaning weight of calves born from village local bulls were 20.21 and 85.14kg (at 1 year of age), respectively. this indicated that attempts made through the open nucleus breeding programme, beyond the conservation and restocking efforts, can achieve the genetic improvement of the indigenous fogera cattle breed (tesfa et al, 2019). strategic deworming of internal parasites and scheduled vaccination against known diseases (anthrax, lumpy skin disease, foot and mouth disease and bovine pasteurellosis) were implemented as part of the flock health monitoring. additionally, grazing land management, the introduction of waterlogged tolerant grass varieties and fattening technology for castrated village local bulls were introduced to cbbp villages to improve the production environment and create an income source for producers. according to (tesfa et al, 2019), under the cbbp, the age at first calving (afc) was reported to be 36 months, and compared with the genetic resources (2024), 5 (10), 94–106 genetic improvement of fogera cattle in ethiopia 101 figure 5. birth weight and weaning weight at the two nucleus herds. alrc bwt, birth weight at the andassa livestock research center (alrc); ccbir bwt, birth weight at the chagni cattle breeding and improvement ranch (ccbir); alrc wwt, weaning weight at alrc; ccbir wwt, adjusted weaning weight at ccbir (bekele, 2012; tesfa et al, 2016; kassahun et al, 2022). table 3. direct heritability estimate for pre-weaning growth traits at the two nucleus herds. bwt, birth weight; wwt, weaning weight; pwagd, pre-weaning average daily gain; *, estimates were done for fogera × holstein friesian (f1). parameters bwt wwt pwadg references alrc 0.38±0.32 0.22±0.25 sewalem (1992) 0.24±0.09 0.18±0.05 tesfa et al (2019) 0.21±0.07 0.26±0.01 0.55±0.19 kassahun et al (2022) ccbir 0.03±0.02 0.06±0.03 0.05±0.03 bekele (2012) 0.13±0.04 0.24±0.08 0.16±0.07 zeleke (2014)* actual results before the programme, there was a oneyear shortening of afc. this shows how pure fogera bulls contributed to upgrading the genetic makeup of the village herds. as traction power is a selective trait of the farmers, calves born through the breeding programme started ploughing at 31 months of age, while the farmers’ local bulls did the same work at 41 months tesfa et al (2019). the positive contribution of the open nucleus breeding programme at the on-farm level to the improvement of the genetics and environment was appreciated by participant farmers (kassahun, 2019). reproductive performances of nucleus herd the average reproductive performance of fogera cattle at alrc and ccbir is presented in figure 6. the overall average age at first calving (afc) of 52.00±3.27 months and 52.17±3.17 months; calving interval (ci) of 19.86±2.15 months and 18.65±1.12 months; and days open (do) of 341.62±90.89 and 280.27±53.86 days were reported at alrc and ccbir nucleus herds, respectively. the afc (month) and do (day) were lower at alrc nucleus herds compared with ccbir’s while ci (month) was shorter in the ccbir nucleus herd compared with the alrc’s (figure 6). variations between these two nucleus breeding herds might be attributed to the presence of agroecological differences among the sites. due to the objectives of the breeding scheme, the improvement in reproductive performance was attained through indirect selection with daily and lactation milk yield. a comparable result for do (9.5 months), higher ci (19.56 months), and lower afc (50.8 months) was reported for the nucleus herd at ccbir (melaku et al, 2011) and a slightly lowered do (10.17 months) (gebeyehu et al, 2005), afc (51.76 months), and ci (19.53 months) was reported for nucleus herd at alrc (tesfa et al, 2016). as reported by sendeku et al (2016), the afc and ci for on-farm fogera cattle was 51.4±0.05 and 21.18±0.70 months, respectively. calf mortality rate the overall calf mortality at alrc and ccbir was 3% (gessesse et al, 2021b), which is comparable with the minimum standard (3–5%) set for the calf mortality rate (heinrichs and radostits, 2001). gessesse et al (2021a) reported that season, breed and birth weight had a significant (p < 0.05) association with the incidence of calf mortality in both fogera cattle nucleus herds, with a respective hazard ratio (hr) of 102 tesfa et al genetic resources (2024), 5 (10), 94–106 figure 6. reproductive performance of fogera cattle at the two nucleus herds. alrc afc, age at first calving at the andassa livestock research center (alrc); ccbir afc, age at first calving at the chagni cattle breeding and improvement ranch (ccbir); alrc ci, calving interval at alrc; ccbir ci, calving interval at ccbir; alrc do, days open at alrc; ccbir do, days open at ccbir (bekele, 2012; tesfa, 2015; kassahun, 2019) 1.6, 0.55, and 0.88. conversely, other potential risk factors such as calf sex, dam parity, year of birth and location did not show a significant effect on calf mortality rates at an early age of the calves at both alrc and ccbir (gessesse et al, 2021b). the author indicated that among the significant risk factors, birth weight (hr = 0.88, p = 0.000) was found to be a very important determinant of calf mortality. based on the health monitoring data, the overall morbidity and mortality rate reported for fogera calves at alrc were 12.96% and 7.5%, respectively (figure 7) (mola et al, 2019), and the diseases contributing to calf mortality were diarrhea, systemic infection, coccidiosis, and gastrointestinal parasites, in order of importance. on the other hand, the work done at alrc (kassahun et al, 2023) indicated that the pre-weaning mortality rate of male calves (21.3%) was higher than that of female calves (13.4%). based on the factors considered, the dry season and lightweight calves contributed more to calf mortality than the wet season and heavyweight calves, respectively. the main reasons for the contribution of these factors were the variability of feedstuffs across seasons and the ability to resist the new environment with a heavier weight at birth, respectively. the heritability estimates done for calf survival at 1, 4 and 8 months of age were 0.26, 0.22 and 0.38, respectively (kassahun et al, 2023), which is categorized as a medium level of heritability. population status of fogera cattle the estimated population size of the fogera cattle breed is declining progressively from about 800,000 in the 1980s (alberro and haile-mariam, 1982) to 55,646 heads in 2017 (tesfa et al, 2022), even though girma et al (2016) argued for the presence of satisfactory genetic diversity in fogera cattle. according to the breed keepers, the population of the fogera cattle has decreased (40%), increased (13%), is stable (6%) or is not known (41%) (tesfa et al, 2022), while respondents who replied to kassahun (2019) indicated the population had increased (51.81%), decreased (31.81%), is constant (4.54%) or is not known (11.84%). the difference between the population trends of the two studies is that the respondents to kassahun (2019) were participants in the open nucleus breeding scheme done by alrc. tesfa et al (2022) indicated the households in the production track of the breed had kept relatively pure fogera (41%), fogera-zebu mix (35%), and highland zebu (24%). the fogera breed appeared to be threatened due to changes in the agricultural production system in the area and genetic dilution by interbreeding with other adjacent indigenous cattle breeds and by indiscriminate crossbreeding with exotic dairy cattle breeds. population viability analysis indicated that the pure fogera cattle are not viable, and population growth is decreasing due to feed shortages, interbreeding with other indigenous breeds, disease and parasites (alemayehu et al, 2015). genetic resources (2024), 5 (10), 94–106 genetic improvement of fogera cattle in ethiopia 103 figure 7. morbidity and mortality percentage of fogera cattle at the andassa livestock research center (alrc) (adapted from mola et al., 2019) discussion the daily milk yield of the fogera cattle at the onstation level indicated a wider variation across selection years, ranging from 0.4 to 7.2l per day, with an average of 2.26l. the reported average daily milk yield (dmy) and lactation milk yield (lmy) were higher than the 1.65±0.03l and 475.85l, respectively, reported for horro cattle at on-farm conditions (mekonnen et al, 2012) and lmy (425.34±24.06l) at bako agricultural research centre, ethiopia (dabi, 2020). relatively lower respective values of dmy, lmy and lactation length (ll) of 1.5±0.01l, 419.8±4.45l, and 284.1±0.15 days were reported for horro cattle at the on-fam level (mekonnen et al, 2021). similarly, mekonnen et al (2021) reported that the top 10% and 25% of the horro cattle population produced 2.01 and 1.86l of milk/head/day, respectively, which is lower than that reported for fogera cattle (tesfa et al., 2019). lower average dmy (1.7l) and higher lmy (507l) were also reported for boran cattle (haile et al, 2011). mezgebe et al (2018) reported a higher average dmy (4.04kg) and average lmy (936kg) for begait cattle in northern ethiopia. the reproductive performance of fogera cattle observed under both open nucleus and communitybased approaches at village herds was comparable with those of other ethiopian indigenous cattle (mohammed, 2020; adisu et al, 2021). according to tenagne et al (2023), the performance for calving interval (ci) and age at first calving (afc) for indigenous cattle in northwest ethiopia was 20.4±6.1 and 52.5±6.8 months, respectively. a slightly lower result for afc and ci for ethiopian boran cattle at on-station set-up was 48.39±1.41 and 17.91±1.01 months, respectively (hordofa and melua, 2021), while a higher value for afc and ci for the same breed at on-farm level was 53.0 and 18.0 months, respectively (wario et al, 2016). lower results for afc (41.2±0.28 months), ci (13.9±0.3 months), and days open (100.5±4.5 days) were reported for horro cattle in on-station conditions (jalata et al, 2023). the observed variations in the reproductive performance traits between the ethiopian indigenous cattle breeds might be due to differences in the adaptation of the various local breeds that make them useful for husbandry in different areas and their genetic diversity should therefore be conserved through appropriate programmes. conclusion and ways forward this systematic review summarizes the efforts towards genetic improvement and conservation of fogera cattle under the open nucleus breeding scheme at alrc and ccbir as a nucleus herd and at the on-farm level under cbbp. the results indicate that there was an improvement in milk and growth-related traits, although the trend was slow and decreasing. the presence of variation among individuals in traits of interest suggested there is room for improvement of the breed through selection. one can also assume that the breeding programme was successful, but there are still different problems masking the genetic potential, such as feed availability, management system of dams, data recording, and transfer of data from one researcher to the other. the closed breeding scheme, which the centre currently follows, has been strengthened with the full involvement of farmers, other stakeholders, researchers and experts at different levels. on top of this, the community-based breed productivity improvement programme acts as a big entry point to assure the conservation and restocking efforts of the breed as a country asset, and the programme is better supported with forage, health and extension works to assure sustainability. with these conclusions, the following recommendations are 104 tesfa et al genetic resources (2024), 5 (10), 94–106 suggested for sustainable breeding programmes in the future: • consistent data records are important for an accurate estimation of genetic and non-genetic parameters for better selection. thus, a standard record-keeping practice should be adopted. • the researchers working on the community-based breeding programme should estimate the breeding values of distributed bulls from the nucleus and their daughters for milk and growth-related traits in order to ensure the contribution of a real genetic gain from the distributed bulls. • the breeding objective developed for milk-related traits should be revised to incorporate the potential of the breed for meat-related traits. • as heritability for calf survival has been categorized as medium, it is advisable to include it in the selection index under the breeding programme. • it is recommended to develop a reproductive biotechnology unit to speed up the genetic gain and multiply elite high-yielding animals, which should be established at one of the nucleus sites. author contributions assemu tesfa and kefyalew alemayehu contributed to the study’s conception and design. data collection and meta-anlysis were done by assemu tesfa, mengistie taye and demelash kassahun. data analysis and writing of the first manuscript draft were performed by assemu tesfa. all authors commented on the various versions of the manuscript, and read and approved the final manuscript. conflict of interest the authors declare that they have no conflicts of interest. references adisu, a., zewdu, w., and moreda, t. 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(2004). indigenous cattle genetic resources, their husbandry practices and breeding objectives in north-western ethiopia. msc thesis, alemaya university of agriculture, dire dawa, ethiopia. https://doi.org/10.1007/s11250-018-1560-4 https://doi.org/10.1007/s11250-018-1560-4 https://www.iiste.org/journals/index.php/jnsr/article/view/54106 https://www.iiste.org/journals/index.php/jnsr/article/view/54106 https://doi.org/10.1017/s1014233900001152 https://doi.org/10.1017/s1014233900001152 https://www.ijpmbs.com/papers/9-a0223.pdf https://faolex.fao.org/docs/pdf/eth191493.pdf https://faolex.fao.org/docs/pdf/eth191493.pdf https://cgspace.cgiar.org/server/api/core/bitstreams/dabb7069-888d-4caa-900a-d37dca144fbc/content https://cgspace.cgiar.org/server/api/core/bitstreams/dabb7069-888d-4caa-900a-d37dca144fbc/content https://cgspace.cgiar.org/server/api/core/bitstreams/dabb7069-888d-4caa-900a-d37dca144fbc/content https://doi.org/10.1080/27685241.2023.2211533 https://doi.org/10.1080/27685241.2023.2211533 https://www.amazon.com/genetic-parameters-estimate-performance-traits/dp/3659762474 https://www.amazon.com/genetic-parameters-estimate-performance-traits/dp/3659762474 https://doi.org/10.3389/fanim.2022.998628 https://doi.org/10.3389/fanim.2022.998628 https://doi.org/10.1155/2017/2149452 https://www.lrrd.cipav.org.co/lrrd28/1/tesf28004.htm https://www.lrrd.cipav.org.co/lrrd28/1/tesf28004.htm https://doi.org/10.1071/an15215 introduction material and methods data sources data analysis results description, distribution and adaptive potential of fogera cattle description of the breeding strategy productive and reproductive performances milk yield performance of fogera cattle growth performances reproductive performances of nucleus herd calf mortality rate population status of fogera cattle discussion conclusion and ways forward author contributions conflict of interest original article genetic resources (2023), 4 (7), 32–45 doi: 10.46265/genresj.qgsb7051 https://www.genresj.org issn: 2708-3764 trends, challenges and opportunities in the in situ conservation of cereal landraces in scottish islands peter martin a,*, olivia shoemark b, maria scholten c, john wishart a, adam g druckerd and nigel maxted b a agronomy institute, orkney college uhi, kw15 1lx, kirkwall, orkney, scotland b school of biosciences, university of birmingham, b15 2tt, birmingham, uk c independent researcher, iv2 3an, inverness, scotland d alliance bioversity international ciat, via di san domenico, 1, 00153, rome, italy abstract: landraces are traditional crop varieties that often have special adaptations to the farming environment in which they have evolved and are therefore a valuable source of useful traits for plant breeders. in most agriculturally advanced countries, landraces of the main crops have generally been superseded by modern varieties. an exception to this in the united kingdom is the cultivation on the scottish archipelagos of orkney, shetland and the outer hebrides of three cereal landraces: bere, a 6-row barley (hordeum vulgare), small oat (avena strigosa) and hebridean rye (secale cereale). our study focused on trends in their cultivation and use over the past 20 years. in the outer hebrides, a mixture of all three has continued to be grown on more than 200ha for feed because of its tolerance of nutrient-deficient sandy soils. future cultivation is threatened, however, by damage from geese and deer, especially to fields used for seed production. in orkney and shetland, only bere and small oat are grown, and always as sole crops. the area of bere has increased in orkney, from about 10ha in 2004 to almost 75ha in 2020 and has been driven by two supply chains producing bere for milling and malting. however, small oat in orkney, and both small oat and, especially bere, in shetland have been grown by very few farmers since 2018 and are at serious risk of being lost from cultivation. we discuss these results in the context of measures to support greater on-farm cultivation of these landraces. keywords: in situ, on farm, conservation, bere, small oat, hebridean rye citation: martin, p., shoemark, o., scholten, m., wishart, j., drucker, a. g., maxted, n. (2023). trends, challenges and opportunities in the in situ conservation of cereal landraces in scottish islands. genetic resources 4 (7), 32–45. doi: 10.46265/genresj.qgsb7051. © copyright 2023 the authors. this is an open access article distributed under the terms of the creative commons attribution license (cc by 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. introduction background food security is of growing global concern due to the rapidly increasing human population and the adverse impact of climate change on food production (godfray et al, 2010; fao, ifad, unicef, wfp and who, 2017). research into food security should focus on all aspects of the human food chain but logically should start with ensuring stable production of highly nutritious and diverse crops (dhankher and foyer, 2018). however, in recent years industrial agriculture has focused on the cultivation of fewer genetically homogeneous crop varieties leading to a reduction in crop diversity (fao, 2012) caused by fewer crops in cultivation, fewer cultivars of each crop and reduced genetic diversity within each cultivar (negri et al, 2009). there are about 31,128 plant species which currently have documented uses, of which 5,538 are associated with direct food production and a further 5,338 have potential for adaptive allele donation (rbg kew, 2016). however, basic global human nutrition comes from just 30 species, and three major crops – rice (oryza sativa), wheat (triticum aestivum) and maize (zea mays) – provide 50% of calories consumed globally. food production is increasingly focused on these few crops while the bulk of semi-domesticated and wild edible species are underutilized (fao, ifad, unicef, wfp and who, 2017; padulosi et al, 2013). furthermore, industrial *corresponding author: peter martin received: 27.09.2022 accepted: 07.02.2023 published online: 03.04.2023 (peter.martin@uhi.ac.uk) https://www.genresj.org https://www.dx.doi.org/10.46265/genresj.qgsb7051 https://www.genresj.org https://www.dx.doi.org/10.46265/genresj.qgsb7051 genetic resources (2023), 4 (7), 32–45 33 production and consumer demand are often focused on uniformity and achieving maximum production and consumer shelf appeal (kilcast and subramamiam, 2011). to achieve this, modern cultivars are genetically homogeneous, lacking the geographically localized infra-varietal diversity found in traditional varieties. these traditional varieties are produced via generational cycles of farmer seed selection, which results in adaptations to local environment and higher levels of genetic diversity than occur in modern varieties. cultivar uniformity increased production overall but the switch from traditional genetically variable crop landraces significantly depleted the gene pool of diversity available to breeders, which is required to sustain crop improvement in the future (hawkes et al, 2000; van de wouw et al, 2009; maxted et al, 2020). the price paid for crop uniformity is that these homogeneous cultivars are vulnerable to new strains of diseases or pests, or extreme changes in the growing environment (committee on managing global resources, 1993; fao, ifad, unicef, wfp and who, 2017). the threat to overall crop diversity by the replacement of landraces by higher-yielding but genetically uniform cultivars is well established at a global level (negri et al, 2009; fao, 2010). while modern varieties usually outperform landraces under optimal conditions, landraces may still be competitive in marginal environments where modern cultivars lose their advantage or where there is an associated niche market to sustain production (yahiaoui et al, 2014; marone et al, 2021). the intrinsic genetic diversity present in landraces means they will often produce some yield even in the face of new strains of diseases or pests, or sudden extreme changes in the growing environment. conserving the maximum pool of diversity in landraces is a priority to sustain future agricultural production (fao, 2012), and landraces grown in regions subject to very variable conditions are likely to have evolved traits that allow them to tolerate such changes. these traits are likely to be particularly valuable to breeders to mitigate the impact of conditions like climate change (hawtin et al, 1997; maxted et al, 2020). within the united kingdom (uk), the agricultural regions where landrace cultivation and resulting diversity are greatest, are the scottish archipelagos of orkney and shetland (the northern isles) and the outer hebrides (kell et al, 2009) (figure 1). the main landraces grown are three cereals: bere,1 an ancient type 1 under current terminology the name ‘bere’ is used to describe a distinct landrace population of lax-eared, 6-row, hulled spring barley that has been grown in scotland and the north of britain for over 1,000 years (drosou et al, 2022). in the past, several synonyms appear to have been applied to bere including ‘beare’, ‘bygg’ or ‘bigg’ (jarman, 1996; wallace et al, 2019). in most cases, these names appear to refer to a similar type of barley to today’s bere, but sometimes they were used to refer to any 6-row barley (jarman, 1996). phenotypically, bere from the different scottish archipelagos as well as most of 35 accessions in a germplasm collection held by the james hutton institute, are similar although differences occur in some characteristics like days to heading and tolerance to manganeseof 6-row barley (hordeum vulgare l.), small oat2 (avena strigosa schreb.) and hebridean rye3 (secale cereale l.). while bere and small oat are grown in all archipelagos, hebridean rye is only grown in the outer hebrides. there are fundamental differences in the manner of cultivation of the landraces between the archipelagos. in the outer hebrides, the most common practice is to grow all three together as a mixture (scholten et al, 2009) for animal forage. in contrast, in orkney and shetland, bere and small oat are grown as sole crops, with bere being used in orkney for milling and malting. in shetland, small oat is usually grown for straw used in basketry, making traditional straw-backed chairs or for thatching. in the outer hebrides and shetland, and to a lesser extent in orkney, cultivation of the cereal landraces is usually associated with crofting (see supplemental note 2), a small-scale, low-input traditional type of farming. two other landraces that survive in shetland are shetland cabbage or kale (brassica oleracea l.) and shetland black potato (solanum tuberosum l.). livestock landraces have also been preserved in the northern isles, with the multipurpose shetland cow in shetland and seaweed-eating north ronaldsay sheep in orkney. locational and environmental context undoubtedly, the location and history of these archipelagos are important factors contributing to the survival of landraces in these areas. they are all peripheral areas, geographically isolated from mainland scotland, which have always experienced difficulties, or high costs, in importing goods, resulting in a requirement for self-sufficiency. climatically, they are hyper-oceanic with high rainfall, frequent gales and cropping seasons that are cooler and shorter than in other parts of britain (chappell et al, 2017). other challenges for cropping include relatively poor soils and limited availability of machinery and equipment. the three archipelagos also have unique cultural identities and traditions. although all were settled by viking invaders during the 9th century, norse control of the outer hebrides ended earlier, in the 13th century, and was superseded by a gaelic culture which includes its own language still deficient soils. genotypically, these beres are also closely related to each other although cluster analysis has shown that the accessions separate into three distinct groups according to their archipelago of origin (schmidt et al, 2019). in spite of these phenotypic and genotypic differences, different types of bere are not currently recognized by growers in scotland although in the 19th century named selections were available (martin et al, 2009). 2 we have used the name small oat for the scottish a. strigosa landrace population which is the terminology used by sasa’s (science and advice for scottish agriculture) scottish landrace protection scheme (scottish landrace protection scheme (slps) — sasa (science & advice for scottish agriculture)). on orkney it is referred to as black oats and on shetland as shetland aets (supplemental table 1). although there are phenotypic and genetic differences amongst accessions of small oat, growers in the island groups do not identify different types (scholten, 2012). 3 we again follow the terminology of the slps for the scottish s. cereale landrace population. this is represented by relatively few accessions in the slps collection and has been much less studied than bere or small oat. in situ conservation of cereal landraces in scottish islands 34 martin et al genetic resources (2023), 4 (7), 32–45 figure 1. map of northern scotland with red arrows showing the location from north to south of the shetland isles, orkney isles and outer hebrides. the image was sourced from scotland topographic map-en.jpg (2400×3450) (wikimedia.org) and originally produced by eric gaba; supplemental note 1 provides the complete attribution. spoken by most islanders. orkney and shetland only became part of scotland in the late 15th century and many traditions and aspects of culture in the northern isles reflect the earlier scandinavian links, especially in shetland. in all the archipelagos, but particularly the outer hebrides, a significant proportion of arable land is on sandy, coastal soils which have a high ph and low availability of trace elements, especially manganese and copper, forming a special habitat called ‘machair’ (scottish natural heritage, 2003). locally adapted types of bere have a remarkable ability to tolerate these deficient soils and produce reasonable grain yields while modern barley varieties lack this trait and yield very poorly or not at all (schmidt et al, 2019). the hebridean rye and small oat landraces are also tolerant of these soil conditions and this trait is likely to have been an important factor explaining the survival of all three landraces in cultivation down to current times. the machair is a particularly harsh agricultural environment because of its soil properties but also because of its exposure to gales, salt spray and periodic droughts; drifting of sand and soil erosion also occur (scottish natural heritage, 2003). over centuries, landraces have adapted to these conditions and on the outer hebrides the bere, small oat and hebridean rye mix is able to produce a reasonable yield with minimal inputs, apart from fertilizer, for which local stranded seaweed and animal manure are often used (brown et al, 2020). traditional cropping of the cereal landraces involves leaving land fallow as part of the rotation, resulting in the development of a unique coastal grassland with a rich and distinctive flora and fauna. its importance is recognized by its inclusion in annex 1 of the eu habitats directive (walton and mackenzie, 2009), and several machair areas are included amongst natura 2000 sites (scottish natural heritage, 2018). continued low-input cultivation of these cereal landraces in combination with the application of organic fertilizers and fallow years is key to machair conservation (scottish natural heritage, 2003), and so local biodiversity and cultural interests in this rare habitat both favour continued landrace use. in orkney and shetland, cropping of the machair is less formalized and, especially in orkney, there are larger areas of better quality agricultural land for cropping. some bere accessions collected from orkney, which do not originate from machair areas, lack a marked ability to tolerate its manganese-deficient soils (schmidt et al, 2019). although the three cereal landraces are welladapted to the scottish island environment, compared with modern varieties, they have several agronomic shortcomings (martin et al, 2010), which limit their attraction to farmers. in particular, their grain yields are low and their straw is long, making crops very susceptible to lodging close to harvest. lodging is aggravated on fertile land or with high levels of nitrogen and is sometimes accompanied by premature grain germination because the heads of lodged crops remain wet for long periods after rain. this is of concern where crops are grown for malting, milling or seed. bere has relatively long awns and in some years these do not completely break off the grain during combine harvesting and this can result in timeconsuming blockages in machinery. historical context bere was probably the earliest of the three cereal landraces to be introduced to scotland, most likely during the bronze age or possibly in the neolithic (drosou et al, 2022). small oat and hebridean rye were probably more recent introductions, with oats first appearing at archaeological sites during the iron age (bond, 1998) and rye during viking times when there was an intensification of cereal production in the scottish isles (smith, 2005). there may also have been more recent seed introductions of both hebridean rye and small oat to the outer hebrides (scholten et al, 2009). bere was widely grown throughout scotland until the 19th and 20th centuries. it was mostly cultivated as a sole crop, even in the outer hebrides, and was important for in-kind and land rental payments and for milling into beremeal and making into malt, which was used for brewing and distilling. the latter allowed significant value to be added to the crop (devine, 1994). given the short cool growing season of all the archipelagos, a major advantage of bere was its ability to produce a crop of grain in a short time. historically, this allowed bere to be sown after oats (both a. sativa l. and a. strigosa), which had a longer period to maturity, but to be still harvested before them in late august or september (dodgshon, 2004), usually before the weather deteriorated and spoilt the crop or prevented harvesting. consequently, bere was more likely to be harvested successfully than oats and was therefore important for food security. in all the islands, the importance of local cereals for food and malt declined as transportation links improved in the 19th century, allowing the import of cheap wheat flour to the area and providing distilleries with access to barley with better malting characteristics than bere (martin et al, 2009). the introduction of potatoes during the 18th century and their widespread adoption for food also reduced the importance of cereal landraces as food staples. during the 20th century, mechanization effectively lengthened the cropping season in the study area because it reduced the time required for cultivation and harvesting. this helped growers to adopt later-maturing, higher-yielding varieties than bere. in orkney, the conservation of bere during the latter part of the 20th century can largely be attributed to the presence of a functioning water mill (barony mill) producing beremeal for local bakeries which provided a small market for locally grown bere. since 1998, the mill has been run by birsay heritage trust (bht), which maintains its own line of bere and organizes its growing in the birsay area with a local agricultural contractor, mostly leasing land from farmers. from 2004, the agronomy institute (ai) started to develop new brewing and distilling markets for bere and established its own supply chain with a small number of local growers (3-4 per year). cereal cultivation in shetland has always been more difficult than in orkney because of its more challenging climate and much smaller area of suitable arable land (martin, 2015). during the 20th century, the area of bere grown on shetland declined from about 419ha in 1912 (board of agriculture for scotland, 1913) to a few hectares by the end of the century and there were estimated to be only two to three growers of bere in 2002 (shetland organic producers group, 2003). crucially, unlike orkney, no large water mill survived in operation on shetland into the 21st century and there was therefore no milling market for bere. it is not known when the practice of growing bere, small oat and hebridean rye in a mixture started in the outer hebrides, but it may be relatively recent as historical accounts from the early 20th century do not mention this practice (macdonald, 1919). it likely became possible as the importance of local cereals for food and malt declined, as hance (1952) reported that the decline in barley cultivation in the uists from about 1,797ha in 1925 to about 335ha in 1946 had been genetic resources (2023), 4 (7), 32–45 in situ conservation of cereal landraces in scottish islands 35 36 martin et al genetic resources (2023), 4 (7), 32–45 offset by an increase in rye and mixed grains. it has been suggested that a major advantage of the landrace mixture, apart from its tolerance to low levels of soil trace elements, is that it provides production resilience under the very variable soil moisture conditions of coastal, sandy soils (smith, 1994). this can vary from drought to waterlogging depending on rainfall and the drainage characteristics and topography of individual fields. within the mixture, hebridean rye is the most drought-tolerant species while bere and small oat perform better on wetter land and bere is also valued for its nutritional contribution (smith, 1994). bere matures earlier than the other two species, however, and often starts to shed seed before harvest; the other two species, therefore, tend to dominate the mixture. study aims an initial survey in 2003 of uk landraces (scholten et al, 2011) identified the three scottish island archipelagos as regions where the three cereal landraces as well as shetland cabbage (scholten et al, 2008) were still prized and maintained. the aim of the present paper is to update the results of the first assessment with more recent information on the landraces grown, grower numbers, area under cultivation and to identify interventions which could encourage wider growing of these landraces. these results are presented within the context of other factors which have affected the cultivation of the cereal landraces on these archipelagos. this update includes data collected during a survey undertaken in 2018 as part of the ec-funded farmer’s pride project (farmer’s pride, 2019), one element of which was to study the economic feasibility of landrace cultivation. although the scope was inclusive, the main focus was on cereal landraces and the results reported are mainly for these. materials and methods area of study the area of study included the three scottish archipelagos of orkney, shetland and the southern part of the outer hebrides (the ‘uists’) comprising north uist, benbecula, south uist and barra (figure 1). the latitudinal range of these archipelagos is from approximately 57on to 61on. assessing area of landraces and grower numbers 2002–2020 for orkney and shetland, the number of landrace growers and area grown was small and we provide estimates of this based on our knowledge and information from key informants (for example, shetland organic producers group, the agronomy institute (ai) at orkney college uhi and birsay heritage trust). with the much larger number of growers in the outer hebrides, we can only provide an estimate of grower numbers and the area grown and consider this to be most reliably based upon scottish government annual census data from 2002 to 2019 for the area of crops grown by region. for most years, data for the outer hebrides (na h-eileanan siar) include the area of oats and mixed grains grown on agricultural holdings. several local key informants, as well as extensive field surveying in 2006 (scholten et al, 2008) indicated that most oat fields were small oats, and the mixed grain crops were the mixture of landraces – mainly small oat and hebridean rye, but often also included some bere. another strong reason for assuming that the census data for these crops reflect landraces is that the arable areas are mostly on the machair, and landraces are much better suited to these soil conditions than modern varieties. in some years the census provides the same data for the barley crop and, for the same reasons, this is most likely to be bere. however, we stress that for crofting communities, where crofts are often extremely small and holdings can contain several crofts, the use of the census data is likely to underestimate the area and, especially, grower numbers. given the uncertainties in the data, we provide both the agricultural census data and estimates derived from consulting key informants. design of the 2018 survey and questionnaire in 2018, the farmer’s pride project provided an opportunity to reassess the landrace situation in the scottish islands. a landrace grower questionnaire (supplemental note 3) was developed to update and further our understanding of the diversity of landrace cultivation in the uk and continental europe (austria, hungary and turkey). the questionnaire was used in face-to-face interviews with current or former landrace growers on the three island archipelagos. interviews started with an introduction to the farmers pride project and the reasons for the interview. during the interview, background information was requested on the farmer’s household and land use (sections a, b and f) and participation in agri-environmental schemes (section d). specific information on landraces was requested in the following sections: section b (landraces grown and the reasons for growing them or stopping this); section c (the interviewee’s intentions about future landrace cultivation); section e (the interviewee’s response to a hypothetical support scheme for growing landraces); section g (the extent of interviewee’s agreement/disagreement with various statements about the situation of landrace conservation and support measures). crofters and farmers cultivating landraces were interviewed in july and august 2018. interviewees were each questioned face-to-face, and answers were recorded in a microsoft excel table directly by the interviewer. crofter and farmer selection in 2018 in each of the island groups, key informants were used to identify an initial target group of farmers known to have recently grown landraces. additional farmers were added to the survey based on the recommendations of interviewees. a short public awareness article was published in the crofter (maxted et al, 2018) and local radio interviews were undertaken in orkney and shetland to increase visibility among the target community. by the end of the 2-month surveying period, a total of 42 farmers and crofters had been interviewed. travelling was time-consuming in all the archipelagos in the study and it was not possible to reach the remoter islands. in the outer hebrides, the study focused on growers in the uists. due to the scale of cultivation on the uists, data were also collected from local scottish government offices and local observers on north uist who had been consulted during the first field survey in 2006. results landrace diversity, area grown and grower numbers except for bere on shetland, the 2018 landrace survey suggested relative stability in crop diversity (the number of landrace species cultivated) and also in their uses compared with the 2003 study (table 1). on shetland from 2004 to 2006, an attempt was made to revive the growing of bere and small oat by shetland organic producers group. although this resulted in a brief increase in growers (table 2 ) to about ten of bere and five of small oat (3.3ha of bere and 0.7ha of small oat in 2004), several growers only planted extremely small areas and most failed to maintain their own supply of seed. crucially, no new markets were developed for either crop. we estimate that from at least 2018, there has only been one grower of bere and just a few growers of small oat on shetland. between 2003 and 2006 on orkney, the area of bere grown by the ai and bht supply chains was about 9–12ha and most of the growers included in the 2003 desktop survey were associated with these supply chains (table 2). subsequently, bht successfully expanded the market for beremeal, exporting it to other parts of scotland, and there was a considerable increase in interest in bere for both brewing and distilling following the release in 2012 of single malt whiskies made with bere (martin and wishart, 2015). as a result, the combined area of bere grown by the ai and bht supply chains increased to about 51ha in 2018 and 69ha in 2020 when about 200t of bere grain were supplied for producing specialist whiskies. for the outer hebrides, scottish government census data indicate a very slight decline in the area of oats and mixed grains grown between 2005 and 2018 (table 2) and a more marked decline in the number of holdings growing these crops. it is possible that this reflects a consolidation of growing on the larger holdings. census data for the outer hebrides only include barley from 2002 to 2008 and over this period the average area was 28ha on eight holdings; it is likely that most of this was bere. a later study (scholten et al, 2009) estimated about 12 growers of sole crop bere on the uists in 2008. landrace cultivation by growers in the 2018 survey in 2018, amongst the 42 landrace growers interviewed, 29 were still growing at least one landrace (table 3). the number of interviewees growing landraces in each archipelago (table 3) broadly reflected the estimated grower numbers (table 2) and was highest on the uists (15), followed by orkney (9) and shetland (5). amongst the growers of cereal landraces, most on the uists (12 out of 14) were growing two or three species in mixtures while in shetland there were only two growers of small oat and in orkney, there were two growers of small oat and five of bere. average arable land use per croft or farm varied between the island archipelagos (table 3), with orkney growers having the highest proportion of their land in arable cultivation (70% arable, compared with 30% pasture) and the highest average area of arable land per farm (49.7ha). growers in the uists had the second-highest average area of arable land per farm (13.5ha), but this was only 8% of their land, with 92% being pasture. shetland had the lowest average area of arable land per farm (1.2ha). these figures broadly reflect more comprehensive agricultural census data for these areas (supplemental note 2). reasons for growing, or ceasing to grow, landraces growers were asked their reasons for growing landraces and given the option of multiple responses (table 4). the most consistent replies came from uist growers who unanimously listed the adaptation of the landraces to local soil conditions as the main reason for growing them (15 out of 15), followed by tradition (12) and low management requirements (9), with market demand figuring quite low (3). tradition was also the most common reason given by respondents in shetland (5 out of 5) and orkney (5 out of 9). the number of interviewees who had ceased growing landraces was largest on orkney (7 out of 16) and the reasons mentioned included: lodging, weak straw, inability to apply fertilizer, lack of a market for bere, problems during combine harvesting, disease, loss of traditional knowledge, unfavourable weather and a high workload. on shetland, 3 out of 8 respondents had stopped, and the reasons provided were: damage from sparrows, low yield, difficult to grow and problems with machinery. on the outer hebrides, 3 out of 18 had stopped and mentioned damage from geese, machinery problems and costs and adverse weather as the main reasons. participation in financial support schemes although several growers, especially on the uists, preferred not to say what percentage of annual household income was derived from their croft/farm, genetic resources (2023), 4 (7), 32–45 in situ conservation of cereal landraces in scottish islands 37 38 martin et al genetic resources (2023), 4 (7), 32–45 table 1. landrace species and their uses documented between 2003 and 2020 in the different scottish archipelagos. archipelago landrace 2003 2018 2019-2020 (scholten et al, 2011) (source: farmer’s pride survey) (sources: agronomy institute; cdp! project1) shetland cabbage winter fodder winter fodder winter fodder bere potential food and drink none grown maintaining a local seed supply small oat animal feed; straw for basketry, chairs and thatch animal feed; straw for basketry, chairs and thatch animal feed; straw for basketry, chairs and thatch orkney bere food and drink food, drink and animal feed food, drink and animal feed small oat heritage crafts heritage crafts heritage crafts uists small oat mixtures with hebridean rye and /or bere winter animal feed winter animal feed winter animal feed; exploring use of bere and rye for whisky 1cdp! project (crofters’ diversity pays!, 2020) table 2. approximate area of landraces grown and estimated grower numbers in the scottish archipelagos. archipelago landrace 2002–2005 2018 survey 2018–2020 shetland1 shetland cabbage data not available data not available data not available bere 4ha; 10 growers none grown < 0.1ha; 1 grower small oat 1–2ha; 5 growers 3ha; 2–5 growers 1–2 ha; 2–5 growers orkney1 bere 9–12ha; 3–7 growers 59ha; 5–8 growers 73ha; 5–8 growers small oat 1ha; 2 growers < 1ha; 2 growers < 1ha; 2 growers outer hebrides2 small oat mixtures with hebridean rye and/or bere i) 275–317ha of oats and mixed grains; 215–232 holdings; (scottish executive, 2005) ii) 300–600ha; 200–400 growers estimated by scholten et al (2008) i) 269 ha of oats and mixed grains; 147 holdings; (scottish government, 2018) ii) key informants estimated the areas to be similar to those given by scholten et al (2008) i) 260ha of oats and mixed grains; 139 holdings; (scottish government, 2020) ii) key informants estimated the areas to be similar to those given by scholten et al (2008) 1 the areas and numbers of growers of landraces in orkney and shetland are based upon our own knowledge and information provided by key informants (for example, shetland organic producers group, the agronomy institute (ai) at orkney college uhi and birsay heritage trust) 2 two estimates are provided for the areas and numbers of holdings of small oat and small oat mixtures in the outer hebrides: i) derived from the areas for oats and mixed grains given in scottish government census data and ii) derived from local key informants. table 3. summary of respondent characteristics in the 2018 survey by scottish isle archipelago. archipelago number of interviewees; number growing landraces in brackets range of arable area (ha); averages in brackets number of landrace growers in agri-environment schemes number of landrace growers with more than 50% of income from croft/farm; number of responses in brackets shetland 8 (5) 0–3 (1.2) 4 1 (5) orkney 16 (9) 1–324 (49.7) 5 4 (8) uists 18 (15) 1–30 (13.5) 9 3 (4) table 4. summary, by archipelago, of reasons for farmers growing landraces. archipelago number of respondents reasons for growing landraces (number of respondents who gave each answer in brackets) shetland 5 tradition (5); not available elsewhere (2); market demand (3); adaptation to soils (3); disease resistance (2); quality of products (1); low management requirements (1) orkney 9 low management requirements (5); tradition (5); market demand (3); adaptation to soils (3); disease resistance (2); quality of products (2) uists 15 adaptation to soils (15); tradition (12); low management requirements (9); market demand (3); good feed for cows (2); yield (2); tolerance to extreme temperatures (1); quality of product (1). this appeared to be greater on orkney and the uists than on shetland with 50%, 75% and 20% of those replying to this question on orkney, the uists and shetland, respectively, reporting that it contributed 50% or more of household income (table 3). half of those interviewed mentioned that they were currently, or had in recent years, taken part in an agri-environment scheme (table 3). amongst these, 67% were currently taking part in one, and 33% had done so previously. accurate collection of data on scheme participation was not possible as not all growers had kept or had access to the relevant paperwork, and not all could recall which schemes they had taken part in or when. amongst those who could recall, the following schemes were mentioned: the agri-environment climate scheme (9 growers), the scottish rural development programme (srdp; 4), the basic payment scheme (4), the environmentally sensitive area scheme (3), the habitat scheme (1), the organic maintenance scheme (1), the rural stewardship scheme (1). hypothetical bere barley support measures none of the growers interviewed were currently involved in any specific support programme for bere. nevertheless, 48% said that they would be interested in a support scheme which rewarded growers for each hectare of bere grown as a pure stand with the intention of saving seed each year. when asked how much monetary support they would require for this, responses ranged from £110 to £1,000 per ha with a mean of £338 per ha, which compares with previous support schemes such as srdp payments which had a maximum of £500 per ha and the agri-environment scheme for machair soils of £230 per ha. planned croft or farm future growers were asked what they intended to do with the croft or farm once they stopped working it. the majority (61% of those asked) planned to hand them on to younger family members, while just over a quarter (27%) planned to sell them outside of the family. those who were tenants (7%) would return the crofts to the owner upon retirement and the remaining 5% were not sure what they would do with the croft. on both orkney and shetland, the majority of growers planned to sell them outside the family. the uists had the highest number of younger crofters and all of those interviewed intended the croft to continue to be worked upon retirement, with the majority planning to pass it on to a relative. discussion landrace maintenance in orkney since 2002, there has been a very significant increase in the area of bere grown in orkney from about 10 to 73ha, although this has not been matched by a large increase in growers (table 2). this is explained by the role of bht and the ai in managing and expanding supply chains for bere in which growing tends to be carried out by just a few growers and on leased land. to put the area of bere grown in orkney into perspective, over the 20th century this declined from about 1,600ha in 1912 (board of agriculture for scotland, 1913) to about 5ha at the end of the century. despite the recent increase in the area of bere, there are essentially only two maintainers of bere seed – the ai and bht – and these organizations provide seed to their own growers. the focus in orkney on grain production for higher value off-farm use is facilitated by the larger arable area on orkney farms (see table 3 and supplemental table 2), a high level of mechanization and the availability of several batch grain dryers. in spite of the success of the ai and bht supply chains, the 2018 survey showed that several farmers had stopped growing landraces (mostly bere) because of their agronomic shortcomings. these would be very obvious to orkney farmers because of the large area (over 4,000ha) of modern 2-row barley grown on the island; this is higher yielding and less susceptible to lodging than bere. nevertheless, other factors like tradition and bere’s low management requirement are also important to some growers, as found by both the 2018 survey and an earlier study (mahon et al, 2016). here, management includes aspects like the use of inputs (fertilizer, herbicide, growth regulator and fungicide) and the quality of land required by the crop. although not mentioned during the survey, straw is an important byproduct of bere grown for grain and is readily sold if farmers do not require it themselves. since bere is usually harvested before the weather deteriorates too badly, the quality of its straw is normally very good and is used for feeding as well as bedding. on orkney, the main driver for continued growing of bere has been the demand for grain processed genetic resources (2023), 4 (7), 32–45 in situ conservation of cereal landraces in scottish islands 39 40 martin et al genetic resources (2023), 4 (7), 32–45 locally into beremeal or sent to mainland scotland for malting. these are then used to produce niche market, high-provenance food and drink products for both the local and export market and the tourism sector, including traditional bere bannocks (a type of flat bread), biscuits and bread made from beremeal and beers, whiskies and a craft vinegar made from bere malt. long-term collaborations between the bere supply chains and distilleries have been important in building up a consistent market for bere, and several growers in the 2018 survey commented that they would be unlikely to continue cultivating bere without an assured buyer. apart from being the only mill producing beremeal in scotland, barony mill is also a visitor attraction which promotes bere, beremeal and other bere products. although the conservation status of orkney bere appears strong at present, it is dependent on demand from a relatively small number of end-users and would benefit from further market diversification and on-farm use. increased interest in using bere for both brewing and distilling has resulted in small areas of the crop being grown for malting in parts of mainland scotland and the north of england since about 2020. the success of bere has also encouraged farmers, craft maltsters and distilleries to start experimenting with other scottish barley landraces like scotch common and scotch annat. in contrast to bere, the growing of small oat in orkney has virtually ceased. its main potential value would likely be as a feed crop on orkney’s coastal sandy land where it was once commonly grown because of its tolerance to manganese deficiency. its straw might also be of value for straw products and was once used for making traditional orkney straw-back chairs. a major constraint on the wider growing of small oat in orkney is a lack of seed. there is also a reluctance to grow it because of its ability to contaminate other crops and fields through volunteer plants or seed remaining in machinery. landrace maintenance in shetland the study showed that the situation of cereal landraces is most precarious in shetland, both in terms of the number of growers and the areas of crops grown and the most cited constraint there was the lack of market demand for landrace products. by 2020 it is thought that there was only one grower of bere on shetland who grew it on a very small scale in a net tunnel to protect it from damage by sparrows and about three growers of small oat who were growing this for straw. the difficulties of commercializing bere in shetland have included a scarcity of machinery and equipment for growing the crop and drying grain on a larger scale, a lack of processing equipment for local milling or malting and high transportation costs, which make it expensive to ship elsewhere for processing. potentially, in shetland, the growing of small oat for straw for basketry, straw-backed chair or thatching could be expanded, but these markets are very small. additionally, farmers growing it for straw cannot use combines for harvesting as these crush the straw and they must either harvest using scythes or old reaper binders, which often have maintenance issues. landrace maintenance in the uists in the uists, our study showed that the cereal landrace mixture is still a very valuable component of the machair crofting system and is economically important as a well-adapted, low-input crop for winter feed which allows growers to avoid the expense of importing feed (estimated to be £32–38 per tonne in 2018 (jones, 2018)) and its associated carbon footprint. the continued use of seaweed and animal manure as a fertilizer for the mixture by some growers also contributes to its sustainability. a crucial trait underpinning the value of the mixture is the tolerance of all its component species to high-ph soils deficient in manganese and copper. this was reflected by replies in the 2018 survey where all respondents included the ability of the landrace mixture to grow on poor soil as a reason for growing the crop. for continuity of cultivation, it is vital to attract a new generation of stakeholders to adopt and use landraces (raggi et al, 2021). at the initial assessment in 2003 most of the uist crofters were in their fifties or sixties, but during the 2018 field work it was apparent that there are now many younger crofters in north uist involved in growing landraces and these showed a keen awareness of the uniqueness of these crops and, specifically, of the potential of bere. one example of this is the interest from a new distillery in the baleshare crofting community, in sourcing both hebridean rye (blackley et al, 2022) and bere from local growers. local seed production is crucial for growing the landrace mixture and while some growers obtain seed from others, many maintain their own seed (scholten et al, 2009). the composition of species and proportion of each is dynamic and varies with the grower and how weather and field conditions affect the crop. a major threat to the island seed system is damage from geese and deer. data for greylag (anser anser l.) and barnacle (branta leucopsis bechstein) geese numbers (mitchell and hall, 2020; wwt, 2022) indicate increases for the two species on the uists from about 7,400 in 2003 to around 11,800 in 2018. at the time of the 2018 survey, there were at least four areas, one on north uist and three on south uist, where seed production had ceased because of the geese threat. this potentially threatens local landrace maintenance. almost two out of three crofters in the survey mentioned geese as the main constraint on growing landraces, and this was included as a reason for stopping, or for not resuming, growing landraces. while some government funding has been available for the management of geese numbers, local crofting stakeholders consider this inadequate for the scale of the problem (scottish crofting federation, 2022). it has also been reported that some growers are using farmer contacts in other parts of scotland to grow the landrace mix to provide seed for them. tradition across all archipelagos, tradition was an important factor cited by many growers (22 out of 29) driving their current use of landraces. on orkney, this may be due to the rise in demand for traditional products made from bere, but is not the case on the other archipelagos, indicating that growers enjoy the tradition for the connection that growing the crops provides with their heritage. many commented to this effect during the survey and it was also identified as an important consideration in an earlier study (mahon et al, 2016). another possible explanation is that tradition is linked to the less intensive approach to agriculture which crofters and small farmers often adopt, including aspects like fewer inputs, small-scale production on more marginal land and use of less costly or technically advanced machinery and equipment. despite the importance of tradition, crofters and farmers have not restricted themselves to traditional methods of cultivating them. thus, over the last 40 years on the uists, most crofts and farms have transitioned from harvesting the landrace mix with reaper binders and putting sheaves into stacks for later threshing, to using combine harvesters and, most recently, to harvesting the crop earlier and preserving it in wrapped bales. the latter, however, reduces the biodiversity value of the crop as it is harvested before wildflowers within it can set seed. as well as tradition, adaptation to local soils and low management requirements were also important reasons for continuing to grow landrace crops on all archipelagos. while all uist crofters gave this reason, many also commented that if they could grow modern higher-yielding varieties, they would. grower satisfaction with current support measures half of the growers interviewed had taken part either currently or in the past in agri-environmental schemes (table 3), of which seven different schemes were mentioned. this diverse range of grower support indicates an awareness that growers need support but also suggests that some rationalization could simplify the system for the growers and aid implementation. a major criticism of past schemes was that some, which were designed to protect wildlife (primarily birds), did not allow early harvesting of cereals. consequently, harvesting was later than desirable and often resulted in crop lodging because of unfavourable weather. another major complaint was that migrating geese arrived at harvest time and ate or damaged the crop before it could be harvested, compounding the problem of the specified harvesting date and adding to the uncertainty of a successful harvest. the consensus amongst crofters was that if there was more flexibility, they would be more likely to join agri-environment schemes. most of those interviewed considered that resolving the problem of the geese outweighed the importance of financial support. another issue raised was the lack of a market for landrace products, suggesting that stimulating this market would increase landrace conservation. therefore, the solution is not simple and possibly a mix of financial support and appropriate policy actions would best support landrace maintenance long-term. recently a new post-brexit uk agriculture bill has received royal assent. part of the bill includes a clause allowing the secretary of state to: “give financial assistance for or in connection with any one or more of the following purposes . . . (i) conserving plants grown or used in carrying on an agricultural, horticultural or forestry activity, their wild relatives or genetic resources relating to any such plant” (uk parliament, 2020). through the implementation of this clause, the secretary of state can provide long-term funding for in situ/onfarm conservation of uk crop wild relatives and landrace genetic diversity conservation which could significantly support landrace maintenance and help underpin future uk food security. the practical application of this new law in england has yet to be formulated but such consideration as the flexibility of harvest time could be accommodated to ensure a desirable outcome for landrace maintenance. within the uk, agriculture is a devolved policy area, however, and this clause has not yet been included in scottish law. heightened profile of scottish cereal landraces since 2002 the development and marketing of a diverse range of new commercial products from bere have helped considerably to raise its profile and that of other scottish cereal landraces and have demonstrated the potential for realizing significant economic benefits from their conservation. notable amongst bere products have been several single malt whiskies. the use of bere in such a high-value, iconic scottish product has introduced a new clientele and audience to both the crop and discussions about the importance of landraces through the marketing and social media activities of distilleries with a global reach. this demonstrates the potential for commercialization to assist the promotion of landraces as well as add value to them (raggi et al, 2021). commercialization of bere has also helped lever funds for scientific research on scottish landraces which in turn has stimulated further interest in these crops. for example, research on bere has encompassed a diverse range of topics including: the nutritional properties of its grain and beremeal (chappell et al, 2017; theobald et al, 2006); agronomy (martin et al, 2010; brown et al, 2020); sustainability traits (schmidt et al, 2019; cope et al, 2021); and the origins of bere (drosou et al, 2022; wallace et al, 2019). much of this research has been possible because of the collection of bere accessions that has been built up at the james hutton institute (jhi), and progress with genotyping the collection will allow genetic diversity to be taken into account as in genetic resources (2023), 4 (7), 32–45 in situ conservation of cereal landraces in scottish islands 41 42 martin et al genetic resources (2023), 4 (7), 32–45 situ conservation strategies are developed. a spin-off of this research has been the multiplication of bere seed and the distribution of scottish island landraces to the mainland by the jhi working with the seed sovereignty programme in 2020 and 2021. the need for an in situ on-farm conservation strategy given the threats to continued in situ growing of cereal landraces in the scottish islands and the relatively good knowledge about their cultivation, utilization and genetic variation, it is an appropriate time to develop an in situ on-farm conservation strategy for them. ideally, this would identify desirable conservation targets in terms of numbers of growers and seed maintainers and the area of crops required for this on each archipelago. it should also include measures for conserving both their genetic diversity and the underpinning traditional knowledge about these crops and support ways of passing this on to new, younger growers. results from recent and ongoing genotyping and phenotyping (schmidt et al, 2019; scholten, 2012; hagenblad et al, 2016) will be important for ensuring efficient conservation of genetic variation and providing these crops with the greatest possibility of being able to adapt to future changes in growing conditions. the strategy could be included in scotland’s evolving postbrexit agricultural policy. conclusions implications for future in situ conservation of scottish landraces this study has shown that both the cereal landrace mix grown in the outer hebrides and bere grown in orkney continue to contribute value to their respective farming communities and, for bere, there are considerable added value benefits to others in the food and drink value chains. while the conservation status of these crops in these locations seems promising, and for bere in orkney, is much better than it was in 2003, there are still causes for concern. in particular, seed production of the landrace mix is threatened by geese in the outer hebrides and, with most orkney bere being grown for distilling, it is vulnerable to changes in market trends. also, there are only two organizations maintaining bere seed in orkney and they both grow very similar lines which probably do not include the range of genetic variation found amongst orkney bere within germplasm collections (schmidt et al, 2019). therefore, a review of the regeneration strategy to ensure a broader coverage of the diversity that exists would be beneficial. there are now very few growers of small oat and, particularly, bere in shetland and of small oat in orkney and these crops are under serious threat of being abandoned and lost in these locations. it is important that accessions of this material continue to be incorporated into ex situ collections at the jhi and sasa’s scottish landrace protection scheme (green et al, 2009) to provide a foundation for any potential future rejuvenation schemes. the situation demonstrates the difficulty of in situ conservation of crops when they do not generate value (not necessarily financial) for growers. in both orkney and shetland, there is potential for wider use of both small oat and bere, alone or in mixtures, for animal feed on coastal, sandy soils as they are used in the outer hebrides. a key requirement for this, however, would be the availability of seed and this could become a commercialization opportunity for a grower or group of growers. the food and drink value chains developed for orkney bere might be replicated in the outer hebrides and shetland to promote greater growing of bere and hebridean rye but this would require suitable processing facilities, collaboration between growers and commercial end-users and probably support to growers to acquire appropriate machinery. these value chains also provide successful examples of landrace maintenance of much wider, even global, relevance. within the uk, the development, post-brexit, of new devolved agricultural policies provides the scottish government with an important opportunity to develop an in situ on-farm conservation strategy for cereal landraces on the scottish islands. acknowledgements the 2018 survey work was undertaken as part of the farmer’s pride project (farmer’s pride, 2019) which was supported by the horizon 2020 framework programme of the european union under grant agreement no. 774271. the contribution of pm and jw was funded by the rural and environment science and analytical services division of the scottish government. author contributions os carried out the farmer survey with assistance from the other authors. all authors contributed to writing the manuscript, which was finalized by pm. conflict of interest statement the authors declare that they have no financial or competing interests. supplemental data • supplemental note 1. figure 1 • supplemental note 2. crofting • supplemental table 1. linguistic diversity in scottish cereal landraces • supplemental table 2. main differences and similarities in cropping land use and crofting between the archipelagos in this study • supplemental note 3. survey questionnaire for landraces on northern and western scottish islands 2018 https://www.genresj.org/index.php/grj/article/view/genresj.qgsb7051/suppdata107 https://www.genresj.org/index.php/grj/article/view/genresj.qgsb7051/suppdata107 https://www.genresj.org/index.php/grj/article/view/genresj.qgsb7051/suppdata107 https://www.genresj.org/index.php/grj/article/view/genresj.qgsb7051/suppdata107 https://www.genresj.org/index.php/grj/article/view/genresj.qgsb7051/suppdata107 https://www.genresj.org/index.php/grj/article/view/genresj.qgsb7051/suppdata107 references blackley, s., mcvey, d., scholten, m., and veitch, a. 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(2014). spanish barley landraces outperform modern cultivars at low-productivity sites. plant breed 133, 218–226. doi: https://doi.org/10.1111/pbr.12148 genetic resources (2023), 4 (7), 32–45 in situ conservation of cereal landraces in scottish islands 45 https://doi.org/10.1111/j.1467-3010.2006.00528.x https://commonslibrary.parliament.uk/research-briefings/cbp-8702/ https://commonslibrary.parliament.uk/research-briefings/cbp-8702/ https://doi.org/10.1017/s1479262109990062 https://doi.org/10.1017/s1479262109990062 https://doi.org/10.1007/s10816-018-9402-2 https://doi.org/10.1007/s10816-018-9402-2 https://www.glasgownaturalhistory.org.uk/machair/conservation.pdf https://www.glasgownaturalhistory.org.uk/machair/conservation.pdf https://monitoring.wwt.org.uk/our-work/goose-swan-monitoring-programme/species-accounts/british-greylag-goose/ https://monitoring.wwt.org.uk/our-work/goose-swan-monitoring-programme/species-accounts/british-greylag-goose/ https://monitoring.wwt.org.uk/our-work/goose-swan-monitoring-programme/species-accounts/british-greylag-goose/ https://doi.org/10.1111/pbr.12148 introduction background locational and environmental context historical context study aims materials and methods area of study assessing area of landraces and grower numbers 2002–2020 design of the 2018 survey and questionnaire crofter and farmer selection in 2018 results landrace diversity, area grown and grower numbers landrace cultivation by growers in the 2018 survey reasons for growing, or ceasing to grow, landraces participation in financial support schemes hypothetical bere barley support measures planned croft or farm future discussion landrace maintenance in orkney landrace maintenance in shetland landrace maintenance in the uists tradition grower satisfaction with current support measures heightened profile of scottish cereal landraces since 2002 the need for an in situ on-farm conservation strategy conclusions implications for future in situ conservation of scottish landraces acknowledgements author contributions conflict of interest statement supplemental data the versailles arabidopsis stock center (vasc): original genetic resources exploiting both induced and natural diversity to investigate gene functions and analyze the impact of variation on plant biology the versailles arabidopsis stock center (vasc): original genetic resources exploiting both induced and natural diversity to investigate gene functions and analyze the impact of variation on plant biology anthony ricou 1 , carine géry 1 , christine horlow 1 , olivier loudet 1 , christine camilleri ​✉ 1 1 université paris-saclay, inrae, agroparistech, institute jean-pierre bourgin for plant sciences (ijpb), versailles, 78000, france abstract arabidopsis thaliana is a powerful plant model for functional biology, genetics and, more recently, population genomics. the versailles arabidopsis stock center collects, produces, preserves, characterizes and distributes various arabidopsis biological resources. besides large collections of mutants, including homozygous mutant lines, this stock centre offers numerous natural genotypes collected worldwide, as well as resources resulting from crosses between these variants. most of the resources are unique and can be useful to a wide range of users, ensuring cumulative characterization of the same material over time. they are accompanied by molecular characterization, genotyping or sequencing data, enabling the analysis of diversity’s impact, particularly on complex plant traits. the collections are made easily and reliably available through an information system comprising a database and a web portal for description and distribution (https://publiclines.versailles.inrae.fr/). several thousand seed lots are provided each year to the international scientific community. keywords plant genetic resources, arabidopsis thaliana, natural variation, mutants introduction arabidopsis thaliana (l.) heynh. is a small wild plant belonging to the brassicaceae family, like rapeseed, cabbages, radish or mustard. it is easy to grow, has a short life cycle in greenhouse conditions, is mainly self-pollinating but can undergo crosses, and produces many seeds. thanks to these biological characteristics, it became a plant model species in the 1980s (meinke, cherry, dean, rounsley, & koornneef, 1998). in 2000, it was the first plant whose genome was completely sequenced (arabidopsis genome initiative, 2000). as the international scientific community working on arabidopsis has grown, numerous shared molecular tools, data and genetic resources have emerged and developed, making arabidopsis the model system of choice in plant functional biology. in addition to enabling the understanding of many biological questions in this species, findings or biotechnological methods developed in arabidopsis have also been transposed into crops or other organisms, and to more applied scientific fields such as plant breeding or even medicine (yaschenko, alonso, & stepanova, 2024). community-driven databases and stock centres have been created and have played a major role in the advancement of many research programmes. the arabidopsis information resource (reiser et al., 2024) maintains an extensive database, with links to other arabidopsis resources. besides the historical stock centres – the arabidopsis biological resource center (abrc), ohio, usa and nottingham arabidopsis stock centre (nasc), uk, the versailles arabidopsis stock center (vasc) was developed in the early 1990s in versailles (france), at the institute jean-pierre bourgin for plant sciences (ijpb) of the national research institute for agriculture, food and environment (inrae), with a first collection of t-dna insertion mutants to explore gene function. since then, vasc has produced many specific resources exploiting both induced and natural diversity. except for natural genotypes collected worldwide, these resources are unique, they are not distributed elsewhere, so vasc is complementary to other existing arabidopsis stock centers. in addition to t-dna insertion mutant lines and homozygous ems mutant lines, the collections include worldwide natural genotypes and segregating populations or cytolines derived from crosses between these genotypes, to analyze the impact of natural diversity particularly on complex plant traits such as growth, development, reproduction or stress tolerance. the resources are molecularly characterized and provided to the arabidopsis community all over the world. mutant collections t-dna insertion lines the earliest collection was a set of 55,000 t-dna insertion mutant lines, generated in the ws (wassilewskija) background (bechtold, ellis, & pelletier, 1993), in which t-dna was inserted randomly in the genome. this collection has been extensively used in numerous studies of forward genetics, based on the screening of mutated lines affected in diverse phenotypes, and the subsequent cloning of the tagged genes. then, genomic sequences flanking the t-dna insertions (flanking sequence tags, fst) have been determined for all the t-dna lines. a total of 46,236 fst have been systematically sequenced (balzergue et al., 2001). they are available in the databases signal (http://signal.salk.edu/cgi-bin/tdnaexpress) and tair (https://www.arabidopsis.org/), allowing reverse genetics approaches which consist in looking for a line with an insertion in a candidate gene and then analyzing the mutant phenotype. genetic screens have played a major role in deciphering the genetic basis of many biological processes. both forward and reverse genetics have been used for example to get insight into plant meiosis (mercier, grelon, vezon, horlow, & pelletier, 2001). many genes involved in meiosis were identified in a. thaliana by using a phenotypic screen on reduced fertility in the greenhouse, and, in parallel, by searching mutants in homologs of genes that play a role in meiosis in non-plant organisms, for example saccharomyces cerevisiae (couteau et al., 1999; gallego et al., 2001). today, these t-dna insertion mutants are still used to validate candidate genes involved in numerous biological processes. homozygous ems mutant lines (hems) after a while, forward genetic screens had identified most of the meiotic genes which, when mutated, cause a dramatic reduction in fertility in a. thaliana. however, an increasing number of genes that play a role in meiosis without causing marked phenotypes when mutated were being identified by reverse genetics, suggesting that many genes with a meiotic function remained to be discovered. to this end, vasc, together with the ijpb team working on meiosis, produced about 900 lines randomly mutagenized by ems (ethyl methyl sulfonate), which were then made homozygous or nearly-homozygous through either haplodiploidization or four generations of selfing by single seed descent (capilla-perez et al., 2018). in both cases, each line is composed of identical or nearly identical plants. in addition to mutations in promoters and untranslated regions (utrs) that can impact gene expression, each line contains between 100 and 500 homozygous mutations that affect the sequence of protein-coding genes (e.g. amino-acid change, stop codon, loss of splicing sites). these resources can be used for forward genetic screening, examining either a single plant per line, or several plants to observe a more quantitative phenotype, and enable subtle and repeated phenotyping. in the hem collection, 43 lines with meiotic defects were phenotypically identified, of which 21 lines had a mutation in a gene whose role in meiosis had already been demonstrated in another organism. for six of these genes, this was the first time they were identified in a direct screen in arabidopsis (capilla-perez et al., 2018). these results show the value of the hem population and illustrate its potential to screen for any qualitative or quantitative phenotype. in addition, the whole-genome sequences of all the hem lines were recently made available (carrère et al., 2024), enabling reverse genetics approaches. on average, three mutations affecting protein sequences are found per gene in the collection. the athem web interface (https://lipm-browsers.toulouse.inra.fr/pub/athem/) provides the community with the raw sequences, snp calling results, and an interface to search for snps in given hem lines or genes. reverse genetic screens for various functions show the power of this resource to obtain different types of mutant alleles (carrère et al., 2024). in addition, the knowledge of mutations greatly accelerates the search for causal genes in forward genetic screens. since 2020, this resource has been the most widely distributed by vasc. collections exploiting natural diversity natural variants (accessions) arabidopsis grows naturally throughout the northern hemisphere, in a wide variety of ecological conditions. this makes it an excellent model for studying natural diversity and adaptation, either directly in association studies using natural genotypes, or through segregating populations (bazakos, hanemian, trontin, jiménez-gómez, & loudet, 2017). at present, over 600 natural accessions – individuals collected worldwide in diverse environments – are available at vasc. most of these genotypes exist in other stock centres or laboratories under the same name, but correspond to different batches of seeds. due to possible mislabeling or sequence divergence across time between lineages, these seed batches can be genetically different and should not be mixed to avoid affecting genetic analyses. each seed batch of the vasc accessions was identified by genotyping with a set of 384 snp markers (simon et al., 2012), and the genotyping data are available on the dedicated web interface anatool (https://www.versailles.inra.fr/ijpb/crb/anatool/index.html). this interface also provides tools that offer a simple and efficient means to verify or determine the identity of the accessions in any laboratory, without the need for any specific or expensive technology. recently, chromosome-level genome assemblies were generated from long-read de novo sequencing for 69 natural accessions, using the dna of plants issued from the vasc seed batches (lian et al., 2024; simon et al., 2022). these data provide insight into the overall genetic variation of the species and add value to our collection of natural accessions. all the parental lines of the vasc recombinant inbred lines (rils), heterogeneous inbred families (hifs), and cytolines (see below) are part of these 66 sequenced accessions. mapping populations: f2s, rils and hifs the most important plant traits are quantitative traits, controlled by several genes at different loci and their interactions. to characterize the genetic architecture and identify the molecular basis of such traits, segregating populations dedicated to quantitative trait loci (qtl) analyses have been developed. the vasc generated 262 f2 families and 16 ril populations from crosses between natural accessions. rils are particularly interesting because they are nearly homozygous and can be propagated as genetically identical individuals, enabling the phenotyping of many traits on the same material genotyped only once. the vasc ril populations (simon et al., 2008) have been generated from genetically and phenotypically distant accessions, covering a wide range of diversity (mckhann et al., 2004). they are composed of large numbers of individuals (343 on average per population) to enhance the statistical power of qtl detection. in addition, an optimal subset of 164 lines (core population) was determined for each ril population, allowing users to phenotype a reduced number of lines with limited qtl detection power loss. the genetic maps rely on common markers, enabling the localization of qtls mapped with different ril populations to be compared. a very large number of studies have been published using this resource to decipher the genetic basis of various traits (for example brachi et al. (2010); brock, rubin, dellapenna, and weinig (2020); gravot et al. (2011); hanemian et al. (2020); poque et al. (2015); shahzad et al. (2016); wuest and niklaus (2018)). hifs are nearly isogenic lines used as a complement to the ril populations to confirm qtls (loudet, gaudon, trubuil, & daniel-vedele, 2005). they were selected in the progeny of rils that show a single residual heterozygous region. three complete hif populations covering the whole genome are currently available. cytolines because the functioning of organelles (mitochondria and plastids) involves the interaction of proteins encoded by the nuclear and cytoplasmic genomes, these genomes are coadapted at the species level. to assess the impact of cytoplasmic variation and nucleo-cytoplasmic interactions on plant phenotypes, we created a unique series of 56 cytolines, whose cytoplasmic and nuclear genomes come from two different natural accessions (roux et al., 2016). the cytolines were generated from reciprocal crosses between eight natural accessions representative of the species diversity, followed by recurrent backcrossing with the nuclear genome donor. cytonuclear interactions were shown to affect several phenotypic traits, 1) indicating that cytoplasmic and nuclear genomes can interact to shape integrative traits that contribute to adaptation, and 2) highlighting a possible role for these interactions in the evolutionary dynamics of the species (roux et al., 2016). epigenetic recombinant inbred lines in addition to genetic variation, epigenetic variation can affect plant phenotype. epigenetic modifications, such as dna methylation, do not alter the dna sequence but can be transmitted from one generation to the next. dna methylation is a source of heritable phenotypic variation notably because it can affect gene expression. a set of 500 epigenetic recombinant inbred lines (epirils) was generated to study the impact of dna methylation on phenotypic variation (johannes et al., 2009). these epirils are derived from two closely related parents that have few dna sequence differences but contrasting dna methylation profiles. one parent is the accession col-0, and the other is a homozygous mutant in col-0 for the ddm1 gene, involved in the maintenance of dna methylation (vongs, kakutani, martienssen, & richards, 1993). these epirils enable the analysis of epigenetic variation and the mapping of epigenetic qtl associating epialleles with phenotypic traits (petitpas et al., 2024; zhang et al., 2021). management staff and partnership vasc is run by two permanent inrae staff members, a scientific manager (research engineer) and an operating manager (technician), for its scientific and technical activities. since 2022, these two members have been supported by two additional staff, each for 20% of their time, in charge of the quality and certification procedures. governance includes a steering committee comprising these four persons plus the head of the ijpb, a user committee comprising the steering committee plus ijpb researchers and an external scientist, and a scientific advisory board made up of the user committee plus a foreign scientist. despite its limited staff, vasc manages to continue the development and characterization of new genetic resources, such as the hem collection recently, through projects carried out in partnership with other research teams, at ijpb or more widely. vasc is always open to developing new collaborations. we can provide our expertise in producing resources dedicated to specific approaches that can subsequently be useful to a wide audience. we can also maintain, host and distribute resources collected or generated by other laboratories. to this end, vasc benefits from ijpb's infrastructures (large-scale greenhouses, growth chambers, seed conservatories) and a skilled workforce for plant growing. within the ijpb plant observatory, vasc also interacts closely with the phenoscope high-throughput phenotyping platform (tisné et al., 2013). a large proportion of our genetic resources (accessions, ril, hif, cytolines) have been phenotyped using this tool under homogeneous and highly controlled conditions (for example, marchadier et al. (2019)). vasc is part of the national research infrastructure of agronomic biological resource centers rare (agronomic resources for research). this enables us to share experiences with other resource centres, particularly in terms of management, regulation and quality. information system, distribution and funding model vasc has established its own information system comprising a database and a web portal for data and distribution (https://publiclines.versailles.inrae.fr/). the online catalogue presents all resources and their descriptions. collections are systematically characterized and molecular data are made easily available to the scientific community via downloadable files or hypertext links. seeds can be ordered directly from the catalogue pages of the website. the price of orders is calculated automatically, and seeds are paid for online at the time of ordering. an invoice is issued and sent automatically to the client. an e-mail is automatically sent when the seeds are shipped, on average within four working days. the website enables the vasc staff to track all orders and clients. over the past five years, an average of more than 5,000 seed samples were distributed annually. more than 200 customers, from 26 countries, have placed orders, of which around one-third in france and two-thirds abroad. the most represented foreign countries were germany, the usa, the netherlands, belgium, the united kingdom, italy, switzerland and china. the most widely distributed resources are always the most recent. the ems collection, which is the most recent, has been the most widely distributed since 2020. this motivates us to acquire new resources. seed sales represent a total income of about €20,000 per year. vasc is part of ijpb and has no funding of its own: vasc revenues are pooled at the institute level and operating costs are covered by ijpb funds. quality multiplication of seed stocks is conducted according to defined protocols designed especially to avoid seed contamination. seeds are kept in a seed conservatory under controlled conditions at a low temperature (4°c) with 12% hygrometry. security duplicates are maintained at -20°c to ensure preservation of the resources in the long term. germination rates are regularly evaluated on samples from the different collections, testing 100 seeds per sample. to regenerate seed stocks, propagation is carried out by self-fertilization in insect-proof greenhouses. an identification number is assigned to each seed batch and is associated with a barcode that enables computerized tracking from sowing to harvesting and distribution. these procedures guarantee traceability and reliability during the production and distribution of the resources. under these high-quality standards, vasc obtained the ibisa 1 label in 2023. it has also implemented a quality management system based on the iso9001:2015 standard, and achieved certification in 2024. our efforts in the production, conservation and characterization of resources, as well as in the establishment of an efficient information and distribution system, have already earned us worldwide recognition for the interest and quality of our collections, our prompt distribution and the support we provide to our customers. past and present research projects the resources produced have always been exploited by the vasc team in research projects. this enables us to anticipate the needs in terms of genetic resources, to obtain funding and gain recognition. our research focuses on genomes, both their expression and their evolution, particularly from the point of view of genomic conflicts that can lead to the establishment of reproductive barriers. a transcriptome study of two ril populations has revealed, in each population, several thousands of expression qtls (eqtls;cubillos et al. (2012)) providing a basis for identifying the gene networks involved in different pathways (xue et al., 2024). we have observed genetic incompatibilities in the progenies of certain crosses, where particular combinations of alleles at different loci lead to lethality (e.g. at the embryonic stage) or to total or partial sterility. we have found in our ril populations several different pairs of loci that lead to this type of situation, and we have identified the partner genes and elucidated the mechanisms involved, some of which are epigenetic in origin (agorio et al., 2017; bikard et al., 2009; durand, bouché, strand, loudet, & camilleri, 2012; jiao et al., 2021). these phenomena can explain the lethality observed in hybridizations between varieties or species, which can have major implications for plant breeding and introgression programmes. studying the reproductive barriers they create can also help us understand the mechanisms that lead to the formation of new species, an overarching goal in biology. we uncovered a cryptic cytoplasmic male sterility (cms) in a. thaliana. cms, which is a source of reproductive polymorphism in angiosperms and of major relevance in hybrid breeding, is genetically determined by both mitochondrial and nuclear factors. a new mitochondrial gene causing sterility (gobron et al., 2013) as well as a nuclear gene restorer of fertility (durand et al., 2021) were identified, and the process of pollen abortion in this cms system was characterized (dehaene et al., 2024). this cms participates in the hybrid sterility phenotypes observed in some crosses, together with segregation distorter loci responsible for pollen lethality (simon et al., 2016). we characterized one of these pollen killers, identifying three genes involved in its functioning and exploring the high locus diversity at the species level (ricou et al., 2025; simon et al., 2022). we found both sensitive and killer plants coexisting in local french populations, which constitutes an invaluable resource for studying pollen killer evolution in the wild. indeed, understanding how gamete killers appear and propagate in populations remains a major issue in evolutionary biology, and arabidopsis proved to be a powerful model for investigating evolutionary dynamics at complementary geographical scales. conclusion and perspectives we are determined to continue our commitment to proposing high-quality genetic resources, guaranteeing their long-term conservation, and generating knowledge on these resources to increase their value for research. our recent results (ricou et al., 2025) underline that arabidopsis, originally mainly a functional biology model, is also a valuable model for conducting studies in population biology, thanks to tens or hundreds of genotypes collected in many local populations (brachi et al., 2013; frachon et al., 2017). in this framework, our upcoming resources coming soon will consist of 458 whole-genome sequenced accessions collected from 168 natural sites located in the southwest of france and characterized for a unique set of ecological factors, including climate, edaphic properties, bacterial communities (soil, root and leaf), plant communities and human activities including urbanization (bartoli et al., 2018; frachon, mayjonade, bartoli, hautekèete, & roux, 2019; roux, frachon, & bartoli, 2023). both whole-genome sequences and deep ecological characterization of their native habitats represent a strong added value to these resources. we wish the arabidopsis community to keep using the vasc resources. citing this article when publishing your results that use these resources will enable us to list the studies based on our collections, attest their usefulness, and therefore ensure the continuity of vasc funding. acknowledgements vasc benefits from the support of ijpb's plant observatory platform po-plants. it receives financial contributions from the inrae biology and plant breeding department. ijpb benefits from the support of saclay plant sciences-sps (anr-17-eur-0007). author contributions cc wrote the manuscript and ar, cg, ch and ol reviewed it conflict of interest statement the authors have declared that no conflicts of interest exist. notes https://www.ibisa.net/annuaire-crb/versailles-arabidopsis-stock-center-vasc-306.html references agorio, a., durand, s., fiume, e., brousse, c., gy, i., simon, m., anava, s., rechavi, o., loudet, o., camilleri, c. and bouché, n. 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(2021). heterochromatin is a quantitative trait associated with spontaneous epiallele formation. nature communications 12, 6958. https://doi.org/10.1038/s41467-021-27320-6 short communications genetic resources (2025), 6 (11), 14–23 doi: 10.46265/genresj.gbcl6863 https://www.genresj.org issn: 2708-3764 survey on threatened medicinal plants diversity of northwestern syria munzer aldarvish *, anas al kaddour a, akram bourgol, yasser ramazan, yousef hallak b, stephen cavers c and joan cottrelld a general organization for seed multiplication, syria b ministry of agriculture, syria c uk centre for ecology & hydrology, bush estate, penicuik, midlothian, eh26 0qb, uk d forest research, northern research station, roslin, midlothian, uk abstract: throughout history, many plant species have been used as natural medicines to prevent and treat human diseases. due to its geographical location, climate, and history, syria contains a remarkable diversity of medicinal plants. however, in recent years a prolonged period of conflict has resulted in widespread ecosystem destruction, human population displacement, and disruption of farming practices. although poorly documented this is believed to have resulted in a significant decline in medicinal plant populations. in this study, we used structured interviews with local agricultural experts to collect basic information on the current status and critical threats to medicinal plant species in northwest syria. our results show that many of these species have experienced genetic erosion and deterioration due to a combination of overuse (massive unmanaged gathering of medicinal plant material) and climatic changes, particularly those relating to more frequent droughts. to initiate ex situ conservation initiatives, the locations of medicinal plants exposed to deterioration were identified f rom the results of a questionnaire. seeds from seven species: chamomile (matricaria chamomilla l.), wild thyme (thymus capitatus l.), sage (salvia officinalis l.), hyssop (hyssopus officinalis l.), caper (capparis spinosa l.), basil (ocimum basilicum l.), and watercress (nasturtium officinale r. br.) were collected for the establishment of ex situ collections in the future. we discuss the potential for recovery initiatives to protect and conserve these species and to support the sustainable use of medicinal plant genetic resources in northern syria. such endeavours are vital for the continued well-being of the syrian population and humanity as a whole. keywords: plant genetic resources, medicinal plants, genetic erosion, in situ conservation, ex situ conservation citation: aldarvish, m., al kaddour, a., bourgol, a., ramazan, y., hallak, y., cavers, s., cottrell, j. (2025). survey on threatened medicinal plants diversity of northwestern syria. genetic resources 6 (11), 14–23. doi: 10.46265/genresj.gbcl6863. © copyright 2025 the authors. this is an open access article distributed under the terms of the creative commons attribution license (cc by 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. introduction medicinal and aromatic plants represent valuable global resources for the practice of traditional medicine as well as the development of novel pharmaceuticals (nalawade et al, 2003; hamilton, 2004; chacko et al, 2010; chen et al, 2010; alachkar et al, 2011; asiimwe et al, 2021; pakdemirli et al, 2021). several studies showed that there is a strong and continuing scientific and ∗corresponding author: munzer aldarvish (drgeneral2015@yahoo.com) commercial interest in documenting the traditional uses of medicinal plants collected from their natural habitats and in exploring new applications for them (daily, 1997; ecological society of america, 1997; nasrallah et al, 2020). the heightened demand for herbal medicines, natural health products and secondary compounds from medicinal plants is rapidly reshaping their use worldwide (saxena et al, 2007; khatib et al, 2021). in many regions of the world, the medicinal plant market is intimately connected with the livelihoods of people and their primary healthcare (saxena et al, 2007; valderrabano et al, 2018). collecting medicinal received: 31.08.2024 accepted: 24.01.2025 published online: 24.02.2025 https://www.genresj.org https://www.doi.org/10.46265/genresj.gbcl6863 https://www.genresj.org https://www.doi.org/10.46265/genresj.gbcl6863 mailto:drgeneral2015@yahoo.com genetic resources (2025), 6 (11), 14–23 threatened medicinal plants in syria 15 plants and plant materials from the wild contributes to the subsistence livelihoods of many indigenous people. even today, it is estimated that hundreds of millions of people, primarily in developing countries, derive a significant part of their sustenance and income from collecting plant and animal products. furthermore, a remarkable 70% of the global human population relies in some way on medicinal plants for their healthcare needs, underscoring the pivotal role these plants play in the economies of biodiversity-rich countries, through export and import (walter, 2001). in addition to the collection from wild populations, people cultivate various medicinal plants in their home gardens for everyday use, as their access to modern medicines and healthcare facilities is often limited (al-oudat and laham, 1994; agelet et al, 2000; kandari et al, 2012) the surge in demand for medicinal plants in urban areas and high unemployment rates in rural areas means a much wider range of people are now accessing wild medicinal plant resources beyond the restricted set of specialists who were previously dominant in this role (nahashon, 2013). as well as direct pressure from harvesting, many medicinal plants are also under pressure from bioprospecting – collection in pursuit of sources of new biochemicals. such unregulated commercialization may lead to these vital plant resources becoming inaccessible and unaffordable for populations that have relied on them for centuries, as well as denying them to people elsewhere in the world (roberson, 2008). furthermore, the profits from such commercial exploitation are rarely returned to the people who may have provided indigenous knowledge, neglecting the access and benefit-sharing aspects of the nagoya protocol (knight et al, 2023). finally, medicinal plant species also face general threats from habitat loss and climate change. as wild areas are destroyed or degraded, unique and valuable species are lost along with the habitat. this loss of biodiversity may also result in the loss of essential remedies for current and future diseases. due to all these pressures, medicinal plant populations are disappearing at an alarming rate, and conservation measures are urgently needed. there are well-established methods for the conservation and sustainable use of medicinal plant resources. rajasekharan and shabir (2020) assert that both conservation (in situ and ex situ conservation and cultivation practices) and resource management strategies (e.g. good agricultural practices and sustainable use solutions) should be developed in parallel and adhered to, in order to secure their long-term future. however, despite the availability of guidance, only a small fraction of medicinal plant species are being protected through traditional conservation methods in nature reserves or botanical gardens (chen et al, 2016). most collection practices for medicinal plants are unsustainable, with impacts including overharvesting, lack of replenishment in the wild, and poor cultivation practices. consequently, many species are now endangered or at risk of extinction and several have recently been included on the iucn red list (walter, 2001; baričevič et al, 2002; nahashon, 2013; rajasekharan and shabir, 2020) although extinction is the most serious impact on society, the loss of even a proportion of a species impacts diversity and translates into the loss of potential new medicinal discoveries (kumar, 2006). syria, situated within a region characterized by substantial topographical and climatic diversity in the area known as the fertile crescent, possesses a remarkable abundance of agro-biodiversity. of particular note, it has a rich diversity of plants used by local communities for medicinal purposes. the use of indigenous plants is widespread at the community and end-user levels (khatib et al, 2021). a study by kywan (2016) highlighted that among the 394 cultivated plant species grown in syria, 91 were classed as medicinal plants and were either collected from the wild or grown in home gardens. medicinal, aromatic wild plants represent a large part of syria’s flora. various plants, including matricaria sp., thymus sp., artemisia sp. are gathered directly from the wild by rural communities for use in traditional medicine (fao, 2001). traditional medicine is a significant healthcare system in syria, where people rely on various natural substances as sources of treatment (alamholo et al, 2023). these plants are integral to traditional medicine, especially in impoverished regions. at present, documentation of syria’s agro-biodiversity is inadequate, but what little information is available indicates the presence of numerous globally significant species. disturbingly, there have been documented declines in several species in syria, especially wheat, along with both cultivated and wild barley, as well as various types of legumes and vegetables – whether domesticated or wild – are at risk of degradation and extinction (fao & itpgrfa, 2022). also, handa et al (2006) noted that syria’s mild climate is ideal for growing a variety of plants. many of these plants are used in syrian culture for aromatherapy, perfumes and medicine. syria is home to approximately 3,459 plant species, spread across 865 genera and 131 families (fao, 2001). a significant number of these species are valued for their medicinal and aromatic properties. rural communities often gather these plants from the wild to prepare traditional medicines. however, syria’s medicinal and aromatic plants face threats from issues such as forest destruction due to fires, overgrazing, urban expansion, water scarcity, tree cutting for fuel and unsustainable harvesting (fao & itpgrfa, 2022). to safeguard these plants, there is a need for improved management, conservation, research into traditional knowledge and medicine, and the regulation of herbal medicine production and trade. within syria, a range of pressures are threatening the medicinal flora, including a changing climate (drought, rising temperature), wildfires, overgrazing, uncontrolled urban expansion, internal displacement, and extensive overharvesting. in particular, the expansion of agricultural cultivation into new areas, and the 16 aldarvish et al genetic resources (2025), 6 (11), 14–23 adoption of economically significant crops like cereals, forages and legumes, are driving substantial habitat loss (syrian government, undp, gef, 2009; aldarvish et al, 2022). although the deterioration of plant genetic resources in syria had begun before the ongoing political crisis started (fao, 1996), it has amplified existing threats and it will be imperative to quantify the scale of this effect (kywan, 2016; gaafar, 2021; geoglam crop monitor, 2021). alongside direct effects, such as armed conflicts in mountainous regions involving landmines and explosives, the civil war has also severely impaired the research and regulatory infrastructure, with institutions formerly dedicated to conserving and propagating genetic resources for future generations now closed. there are currently no conservation activities for medicinal plants taking place, not least because syrian botanists have been moved or diverted to other activities. the loss of competent authorities, along with any effective government oversight, has undermined a national process based on key biodiversity areas (kbas) and now management and conservation plans that address both ecological and social dimensions are urgently needed, to avoid extinction and promote restoration and sustainable use practices (valderrabano et al, 2018). as a first step, there is an urgent need to assess the current status of medicinal plant populations and identify cost-effective strategies for conserving those most at risk of genetic erosion and degradation (valderrabano et al, 2018). the research reported here was designed in accordance with the syrian national strategy for conservation and management of plant genetic resources for food and agriculture 2015–2035 (fao, 2015), and aimed to: • collect information about the current status and most pressing threats to medicinal plants in northern syria, using a questionnaire • characterize and map the distributions of medicinal plant species facing deterioration • collect seeds from multiple genotypes of each of the seven medicinal plant species for the future establishment of an in situ and ex situ collection. materials and methods the study was conducted from january to september 2023 across specific subdistricts in the idlib governorate (harim, mhambal, and darkosh) and the aleppo governorate (jebel saman and atareb) of northern syria (figure 1). these locations were identified after a series of focus group discussions (fgds) with local communities and stakeholders in northern syria, including agricultural experts, community leaders and senior farmers. representatives in each subdistrict nominated key informants (kis), allowing for the targeted selection of knowledgeable individuals with local expertise. the fgds revealed that these subdistricts were particularly exposed to ecosystem deterioration and disruption due to prolonged conflict, making them priority sites for assessing the status and threats facing medicinal plant species. data collection aimed to evaluate the current status and conservation needs of locally valuable medicinal plant species in these ecologically vulnerable areas. a questionnaire (supplemental data) was developed and administered to 50 kis between 1 april 2023 and 1 august 2023. the questionnaire was designed with diverse question types to gather detailed information. participants mainly chose answers from a predefined list, ensuring consistency across responses, but also had the option to add any additional answers not included in the predefined list. kis, nominated by subdistrict representatives, included senior farmers, agricultural engineers and researchers, chosen for their expertise and involvement with local agricultural practices. the participant composition consisted of 76% male and 24% female respondents aged 24–85, with 36% agricultural researchers, 34% agricultural engineers and 30% senior farmers, offering a comprehensive perspective on the perceived threats to the selected species and potential conservation interventions. survey and data analysis the survey aimed to capture respondents’ perceptions of critical threats to medicinal plant species – chamomile (matricaria chamomilla l.), wild thyme (thymus capitatus l.), sage (salvia officinalis l.), hyssop (hyssopus officinalis l.) and caper (capparis spinosa l.), which were considered by local experts to be the species most vulnerable to deterioration due to diverse factors such as overharvesting, unmanaged collection, and climatedriven droughts. collected data were analyzed in ms excel (microsoft office 2020) to assess the prevalence and distribution of responses for each question. the analysis included calculating percentages, frequency counts, and averages, providing a comprehensive view of response patterns, and highlighting key trends across participants’ answers. ex situ conservation methodology for ex situ conservation, seeds from five mature individual plant genotypes (1,000 seeds of each genotype) of each identified species were collected within each subdistrict, ensuring representation across ecological variations in northern syria. the seeds were harvested, dried and disinfected using a thiram fungicide. for proper tracking and future use, each seed sample was labelled with essential ‘passport’ information, including the species name, original collection location, and collection date, which includes the date the seeds were placed in storage. the seeds were then stored in paper bags, which were placed within airtight plastic containers along with dry silica gel to maintain optimal humidity levels at ambient room temperature (approximately 20–25◦c). seed samples have been temporarily stored in a designated room at the field staff’s home. . genetic resources (2025), 6 (11), 14–23 threatened medicinal plants in syria 17 results data collected from 50 key informants, including farmers, agricultural engineers and researchers in five t arget r egions w ithin t he a leppo a nd idlib governorates in northwestern syria are detailed in supplemental table. the results indicated that seven species mentioned in this study showed signs of deterioration across the locations surveyed (figure 1); the seven species showing this deterioration are given in table 1. figure 1. map of syria showing the study area (subdistricts within the aleppo and idlib governorates) in the north of the country. source: humanitarian data exchange across the study area, most (98%) of participants stated that wild thyme was susceptible to degradation, followed by chamomile (94%), hyssop (76%), sage (76 %), basil (42%), watercress (38%) and caper (14%) (figure 2). responses to the questionnaire (supplemental table) indicated that the species considered to be most in decline varied across the five s ubdistricts. m ost of the respondents in mhambal (86%) mentioned that caper was in decline, whereas those who mentioned the decline of chamomile (17–21%) and wild thyme (18–20%) were more evenly distributed across the five subdistricts (figure 3). although seven species were listed as being in decline in the study region, they were not in decline across all subdistricts. for example, none of the respondents from harim listed sage or hyssop as being in decline and none of the responders from jebel saman mentioned that caper and watercress were in decline. for all species except wild thyme, all respondents considered each species to be ’rarely available’. for wild thyme, 98% of respondents considered it to be ’rarely available’, and 2% considered it to be ’not available’. there was complete agreement among respondents regarding the natural habitat of the seven species. wild thyme, sage and hyssop were considered to grow in natural public spaces and forested areas. in contrast, caper and chamomile were considered to occur along agricultural roadsides, while watercress grew near freshwater streams. basil, on the other hand, is typically cultivated at home. respondents were asked to assess the importance of a list of reasons for the decline of medicinal plant species in northern syria. the results show that respondents considered the primary factor contributing to the deterioration of these species to be excessive use, characterized by unmanaged gathering of plant material, with 47% of respondents considering this to be a contributory factor. in addition, frequent droughts, climatic changes, desertification and the loss of forested areas to urban expansion were also identified as significant factors by a large number of respondents. to a lesser extent, respondents considered neglect by local authorities, lack of awareness regarding the importance and value of these species, and the repercussions of the syrian crisis, notably the lack of law enforcement, to play a role. the lack of interest and understanding in the use of medicinal plants among the younger generation and overgrazing were also noted by some as contributory factors but were only mentioned by 5% of the respondents. interestingly, deforestation and floods were not considered by any of the respondents to have a discernible impact on the deterioration of these species. when asked specifically about the effects of the syrian crisis in exacerbating the deterioration of these medicinal plant species, 60% of respondents indicated that the crisis had a substantial role. this was primarily attributed to the absence of both interest and legal measures aimed at safeguarding and preserving these botanical species. in contrast, 40% of participants expressed uncertainty regarding the impact of the crisis. it was notable that all participants stated that they continue to be interested in cultivating some of these species, with 66.7% stating that these species are for family use and 33.3% stating that these species are of economic use. according to respondents, the local population in northern syria uses these medicinal plant species to treat a range of complaints and diseases (table 2), with some complaints being treated by several species. for example, respiratory diseases were treated by chamomile, wild thyme, hyssop and basil. others were only treated by one species. for example, blood pressure is only treated with basil, and anorexia is only treated with sage. consequently, the loss of a certain species might mean that there is no other medicinal plant available to replace it as a treatment. according to respondents, the local population in northern syria uses these medicinal plant species to treat a range of complaints and diseases (table 2), with some complaints being treated by several species. for example, respiratory diseases were treated by chamomile, wild thyme, hyssop and basil. others were only treated by one species. for example, blood pressure is only treated with basil, and anorexia is only treated 18 aldarvish et al genetic resources (2025), 6 (11), 14–23 table 1. medicinal plant species identified by the key informants as most vulnerable to genetic erosion and deterioration. english name family scientific name life cycle basil lamiaceae ocimum basilicum l. annual caper capparaceae capparis spinosa l. perennial chamomile asteraceae annual hyssop lamiaceae perennial sage lamiaceae matricaria chamomilla l. hyssopus officinalis l. salvia officinalis l. perennial watercress brassicaceae nasturtium officinale r. br. annual wild thyme lamiaceae thymus capitatus l. perennial figure 2. the percentage of respondents who listed each medicinal plant species as being in decline in northern syria (n=50). figure 3. proportions of all respondents who listed a particular species as exposed to deterioration (100%) according to subdistrict (n=50). genetic resources (2025), 6 (11), 14–23 threatened medicinal plants in syria 19 table 2. list of diseases and complaints for which the seven medicinal species are used as treatment locally in northern syria, according to survey respondents. species use in traditional medicine chamomile infections of mouth and gums, respiratory diseases, colds, headaches; calming the nerves; protecting skin; regulating blood sugar; strengthening immune system wild thyme infections and inflammations of the digestive system, respiratory problems, coughs, worms; antispasmodic sage anorexia, flatulence, stomach pain or inflammation, diarrhoea, colic, stomach ulcers, indigestion hyssop respiratory diseases caper diabetes, skin diseases, anaemia, strengthening immunity, promoting weight loss basil enhance the functioning of the digestive system, cleanse the intestines, prevent constipation, relieve pain, enhance the health of the heart and arteries, treat colds and asthma, treat respiratory infections, and treat anaemia watercress or yellowcress high blood pressure, increases sexual energy in men, diuretics, anaemia, expectorant with sage. consequently, the loss of a certain species might mean that there is no other medicinal plant available to replace it as a treatment. to guarantee the availability of seeds from these varieties, ex situ conservation was employed in this study. seeds from five plant genotypes were collected, dried, disinfected, and placed in paper bags within airtight containers at ambient temperature. this approach allowed for accurate tracking of the samples, with vital passport information documented for future reference. the seeds are currently stored temporarily to preserve their viability until more sustainable long-term storage solutions can be adopted the data from the questionnaire revealed that every respondent displayed a strong commitment to safeguarding medicinal plant species. the results highlighted that the most popular method for protecting these seeds was seed collection and conservation of these species in the local gene bank. followed by raising awareness about the need to safeguard these species, as well as the implementation of practical protective measures in the areas where these species are found to counteract the factors leading to their decline. the idea of the establishment of local collections received significant support. in contrast, seed exchange was the least favoured method among respondents (figure 4). discussion syria, located in the fertile crescent, a region known for its significant topographical and climatic variety, boasts a rich diversity of plants used by local communities for medicinal purposes. these plants play a crucial role in plant diversity and contribute to global genetic resources, as highlighted by various studies (chacko et al, 2010; chen et al, 2010; alachkar et al, 2011; asiimwe et al, 2021; pakdemirli et al, 2021). however, the ongoing armed conflict has led to the overexploitation of local resources, including the excessive harvesting of medicinal plants. the absence of regulatory entities, protective laws and sustainable management practices leaves these resources unprotected, threatening their survival and regeneration. on top of this, forest fires and overgrazing have caused increased deterioration of plant populations in the region (handa et al, 2006; valderrabano et al, 2018; gaafar, 2021; khatib et al, 2021; al-darvish et al, 2022, 2023). this is a worldwide phenomenon, with loss of wild genotypes having been reported by several studies (walter, 2001; baričevič et al, 2002; kumar, 2006; roberson, 2008; nahashon, 2013; chen et al, 2016; rajasekharan and shabir, 2020; geoglam crop monitor, 2021) but the issue is particularly acute in syria. medicinal plant species, which have been used by humans for thousands of years, are a key component of traditional medicine practices worldwide. in syria, rural communities gather medicinal plants from the wild for use in traditional healing (fao, 2001; alamholo et al, 2023). these plants have gained increasing significance with the rise of folk healers who offer treatments with fewer side effects compared to synthetic drugs. medicinal plants also have economic value, as they can serve as a source of raw materials for the pharmaceutical industry. however, in northern syria, medicinal plant species are facing a decline. our questionnaire results have provided valuable insights into the current status of these plants and the perceived causes of their deterioration. according to data collected from a sample of 50 expert respondents, seven medicinal plant species in northern syria were identified as experiencing deterioration. these species are used to treat at least 24 different medical conditions, meaning their loss would significantly impact the local population, who often rely on these plants as their primary form of medical treatment. some conditions can be addressed by multiple species, while others depend on a single species for treatment. the loss of plants with unique medicinal properties would be especially detrimental to public health, particularly if no alternative plant species with similar properties are available. chamomile stands out among these species due to its broad medicinal applications for various complaints. it was one of the seven species identified as deteriorating across all five subdistricts. given its wide usage and the decline it is facing, chamomile warrants priority 20 aldarvish et al genetic resources (2025), 6 (11), 14–23 figure 4. the proportion of respondents supporting each proposed method for the protection of medicinal species (n=50). conservation efforts. in contrast, some of the other six species were reported as deteriorating in only one or two subdistricts, though the responses do not clarify the reasons for this. it could be due to more intense overharvesting in specific districts, but further investigation is needed to identify other potential causes. the data might also suggest that certain species are naturally absent from areas where they were not mentioned, indicating a restricted distribution that also calls for conservation attention. alternatively, uneven distribution could reflect increasingly unsuitable conditions in particular subdistricts for specific species. for instance, watercress, which requires freshwater streams, may be especially vulnerable to droughts in certain districts. a more comprehensive understanding of these patterns will help target conservation actions more effectively, ensuring that future plans are tailored to meet the specific needs of each subdistrict. the questionnaire also examined the conservation actions that respondents considered most appropriate. it was encouraging to find that nearly all respondents agreed on the importance of seed collection for the establishment of genebanks. this indicates broad support for the seed collection activities conducted as part of this study. however, there was less enthusiasm for practical in situ conservation methods, which may be due to perceptions that such actions are difficult or unfeasible in a conflict zone. for proper tracking and future use, each seed sample was labelled with essential ‘passport’ information, including the species name, original collection location, and collection date, which includes the date the seeds were placed in storage. the seeds were then stored in paper bags, which were placed within airtight plastic containers along with dry silica gel to maintain optimal humidity levels at ambient room temperature (approximately 20–25◦c). seed samples have been temporarily stored in a designated room at the field staff’s home the research team acknowledges that ambient temperature storage is not a suitable long-term conservation method, as it may lead to degradation over time. this decision was made due to limited access to cooling systems in northwestern syria, as well as immediate access requirements and preliminary storage needs. however, the team fully recognizes the limitations of this approach for long-term preservation. to address this, the team is exploring alternative conservation strategies. these may include transferring the samples to optimal cold storage conditions and collaborating with official authorities, such as agricultural research centres or the faculty of agriculture in idleb governorate, to ensure the long-term viability and accessibility of these samples for future research the findings of this study align with previous research on the deterioration of plant genetic resources and the conservation of medicinal plants. they reveal a consistent decline in biodiversity, particularly in regions like syria, where climate change, habitat destruction, and socio-political instability – especially the ongoing conflict – accelerate the loss of valuable plant genetic resources. these results echo earlier studies documenting similar patterns of biodiversity loss due to environmental and socio-political challenges. the study underscores the urgent need to preserve medicinal plants, which are vital for both medicinal and cultural purposes. environmental degradation, climate change, and conflict have significantly accelerated the decline of genetic resources (2025), 6 (11), 14–23 threatened medicinal plants in syria 21 plant biodiversity, emphasizing the necessity for targeted conservation efforts. in northern syria, the ongoing crisis, coupled with the lack of effective conservation measures, has placed medicinal plants at significant risk of extinction. this poses a threat not only to the region but also to the global community. the conservation of these plants is essential for healthcare, biodiversity, sustainable ecosystems, and cultural heritage. furthermore, the study highlights the critical need for immediate action to safeguard these plants, pointing to the absence of engagement from conservation organizations, which worsens their vulnerability. knowledge of the drivers behind the deterioration of these species is key to the development of appropriate conservation actions. the results of the questionnaire provide some useful pointers with the cause most frequently named by the respondents being ‘massive unmanaged gathering’ followed by ‘frequent droughts’. whilst recognizing the difficulty of implementing any management of the level of collecting whilst in a war situation, the results of the questionnaire nevertheless highlight the need to develop and implement a policy that will control and sustainably manage these key genetic resources. although the syrian crisis was only listed by 5% of the respondents as a cause of the deterioration, when participants were directly asked whether the syrian crisis contributed to the deterioration of medicinal plants, with options for ‘yes’ or ‘no’, 60% of the respondents considered it to have been a contributory factor which is closely associated with excessive use, characterized by unmanaged gathering of plant material. it also seems likely that the crisis impacts medicinal plant populations indirectly: for example, the supply of pharmaceutical drugs has been compromised by the crisis and has probably led to increased unmanaged gathering of medicinal plants by vulnerable people in search of costeffective treatments. while the crisis is a significant factor, the decline of plant genetic resources in syria began before the war that started in 2011 (fao, 1996; syrian government, undp, gef, 2009). respondents noted that, although overuse is the primary cause of this decline, other contributing factors include frequent droughts, climate change, desertification and the expansion of urban development into forested areas. moreover, neglect by local authorities, a lack of awareness about the importance and value of these species, and insufficient law enforcement further exacerbate the issue. additionally, there is a noticeable disinterest among the younger generation in using plants for medicinal purposes, potentially due to the unavailability of certain species in their natural habitats. the findings of this study align with previous research on the deterioration of plant genetic resources and the conservation of medicinal plants. they reveal a consistent decline in biodiversity, particularly in regions like syria, where climate change, habitat destruction, and socio-political instability – especially the ongoing conflict – accelerate the loss of valuable plant genetic resources. these results echo earlier studies documenting similar patterns of biodiversity loss due to environmental and socio-political challenges. the study underscores the urgent need to preserve medicinal plants, which are vital for both medicinal and cultural purposes. environmental degradation, climate change, and conflict have significantly accelerated the decline of plant biodiversity, emphasizing the necessity for targeted conservation efforts. in northern syria, the ongoing crisis, coupled with the lack of effective conservation measures, has placed medicinal plants at significant risk of extinction. this poses a threat not only to the region but also to the global community. the conservation of these plants is essential for healthcare, biodiversity, sustainable ecosystems, and cultural heritage. furthermore, the study highlights the critical need for immediate action to safeguard these plants, pointing to the absence of engagement from conservation organizations, which worsens their vulnerability. our findings point to a substantial gap in policy regarding syria’s indigenous genetic resources. while we have taken concrete steps to preserve what remains, there is an urgent need to develop a comprehensive national strategy to protect these species. such a strategy should include both in situ and ex situ seed conservation. also, awareness campaigns are essential to emphasize the importance of these plants, and the implementation of protective measures in the areas where they are found. these measures should be designed to specifically address the factors driving the decline of these species, with protocols tailored to reflect regional differences and local conditions. only by adopting a comprehensive approach can we ensure the long-term survival and genetic diversity of these invaluable medicinal plants. supplemental data questionnaire including responses received in the survey acknowledgements this research was supported by cara (the council for at-risk academics), united kingdom. author contributions munzer aldarvish coordinated the research and contributed to the research design and manuscript; anas al kaddour, akram bourgol and yasser ramazan contributed to the research design and manuscript and undertook the data analyses; yousef hallak contributed to the research design and carried out the field data collection; stephen caversand joan cottrellprovided academic guidance and support throughout the research process and contributed to the manuscript. https://www.genresj.org/index.php/grj/article/view/genresj.gbcl6863/suppdata217 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issn: 2708-3764 brazil’s implementation of access and benefit-sharing and the nagoya protocol: analyzing some trends and positions in the ongoing debate eduardo relly * friedrich-schiller-universität jena, sfb/trr 294 ‘strukturwandel des eigentums’, jentower, 23. og, leutragraben 1, 07743, jena, germany abstract: access and benefit-sharing (abs) arising from the utilization of biodiversity’s genetic resources and traditional knowledge is the third objective of the convention on biological diversity (cbd). since its inception, some of the parties to the cbd have enacted abs-national legislation and in 2014, the nagoya protocol came into force, providing a global standard among abs systems. given this, brazil has been working to implement abs since 2001, especially after the enactment of the national biodiversity law (law 13.123/2015), which is the domestic law for the nagoya protocol implementation. this paper examines how the implementation of abs and the nagoya protocol is viewed, discussed and debated by some stakeholders. based on qualitative semi-structured interviews, press releases, public declarations, legislation and grey literature, the paper reveals that although abs has faced strong criticism and delivered modest results, most stakeholders consider it strategic and important, especially in the face of the bioeconomy–biodiversity nexus. in general, positions on the implementation of abs policies and the nagoya protocol in brazil can be devised in the following categories: 1) acceptance and optimistic appreciation of abs, 2) acceptance of abs mechanisms but impending need for adjustments, 3) acceptance of abs mechanisms as a ‘bad with it, worse without it’ scenario, and 4) rejection of abs. our research also shows that when it comes to abs and providers of genetic resources, debates centred on the topic of biopiracy have declined, while debates characterized by compromise, institutionalization and the steering of abs via the implementation process are on the rise. keywords: abs, nagoya protocol, biodiversity, implementation, brazil, cbd, bioeconomy citation: relly, e. (2024). brazil’s implementation of access and benefit-sharing and the nagoya protocol: analyzing some trends and positions in the ongoing debate. genetic resources 5 (10), 65–80. doi: 10.46265/genresj.gkte3850. © copyright 2024 the authors. this is an open access article distributed under the terms of the creative commons attribution license (cc by 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. introduction: access and benefit-sharing and brazil brazil is a global environmental superpower, and how this megadiverse nation implements its policy on access and benefit-sharing (abs), along with the nagoya protocol (np), has far-reaching implications for the entire world. as both a major user and provider of genetic resources, brazil’s abs landscape is shaped by a dynamic industrial and agricultural sector, a vibrant academic community, and strong leadership from indigenous and local communities. few countries ∗corresponding author: eduardo relly (rellyeduardo@gmail.com) offer such a comprehensive microcosm of the challenges and opportunities surrounding the future of the nagoya protocol and domestic abs systems. access and benefit-sharing has been a pivotal concept that marks the transition from an age in which biological and genetic resources (gr) were regarded as a common heritage of mankind toward an international system based on the sovereignty of national states. emboldened by the convention on biological diversity (cbd) (unep, 1992), opened for signature during the earth summit in rio de janeiro in 1992 (entered into force in 1993), parties have started to formulate abs systems aiming to tackle different goals in the face of global inequalities. especially in the so-called global south, this included safeguarding genetic biodiversity by adopting cbd’s “mandate of justice (distributional, procedural, and received: 09.04.2024 accepted: 17.09.2024 published online: 10.10.2024 https://www.genresj.org https://www.doi.org/10.46265/genresj.gkte3850 https://www.genresj.org https://www.doi.org/10.46265/genresj.gkte3850 mailto:rellyeduardo@gmail.com 66 relly genetic resources (2024), 5 (10), 65–80 recognitional)” (suiseeya, 2014) in the face of unequal technological capacities between poor and rich countries as well as the establishment of abs-procedures for the compensation of indigenous peoples and traditional communities’ knowledge. last but not least, abs was to curb potential and actual biopiracy (rabitz, 2015). in fact, since the 1980s, researchers and activists have used the term ‘biopiracy’ to criticize the misappropriation and commodification of indigenous peoples and local communities’ (iplc) knowledge of seeds or plants by biotechnology companies, mainly from the global north (rafi, hsca, 1998; shiva, 2007; robinson, 2010). the principle of national sovereignty over gr was enshrined in article 3 and article 15 of the cbd and was also extended to gr (excluding human genetics) (unep, 1992). access and benefit-sharing is based on article 15 of the cbd. the latter was reinforced by the legally binding np which was agreed in 2010 (cbd, 2011) and came into force in 2014. the np provides the framework for national abs regulations to be respected by third parties, especially in the event that gr are utilized outside the provider country. broadly speaking, this is the most encompassing global abs mechanism that applies to gr of biodiversity. however, the abs concept was criticized from the outset. environmental activists, scientists and indigenous peoples feared that private forms of ownership would be imposed on iplc and that expropriation dynamics would be accelerated (shiva, 2004). contrary to the high expectations of megadiverse countries and of the np architects, on the one hand, and consistent with the concerns and hopes of activists and iplc, on the other, the np has not led to significant compensation payments (laird et al, 2020). the reasons for this are manifold, and both users and providers of gr have reasons to complain: a lack of global sanction mechanisms and the possibility of circumvention (rabitz, 2015; halewood et al, 2023); legal uncertainties on concepts such as access to and use of gr at the national level (vogel et al, 2018) which have resulted in limitations to international trade and research (braun, 2024); unresolved questions regarding transboundary traditional knowledge (tk) associated with gr as well as procedures to facilitate traceability (dutfield, 2015); structural conflicts and lack of trust between iplc and state institutions (hayden, 2003); the ambiguity of the concept of gr, both at the global and national levels (aubertin and filoche, 2011; müller, 2018). on the private sector side, non-harmonized implementation of the np tend to expose companies to high risks (michiels et al, 2022) and, last but not least, biotechnological innovations such as the new genomics tools, artificial intelligence and digital sequence information on genetic resources (dsi) pose further challenges to the successful implementation of the np as well as of the national abs policies (fdcl, gen, 2022). in brazil, the discussion on abs and np covers multifaceted aspects. in general, questions of justice and political constellations (dallagnol et al, 2016; feres et al, 2019), descriptive aspects of legislation (silva and oliveira, 2018), sectorial analysis (abihpec, 2017; costa, 2017; marinello, 2020), state transformations (eimer and donadelli, 2022), and comparative analyses of the legal situation (cni, 2017) have framed the debate. the private sector, consultancies, and third-sector organizations have been especially vocal in producing guidance for abs implementation. likewise, influential academics such as the brothers carlos and ismael nobre (nobre and nobre, 2019) and ricardo abramovay (abramovay, 2020) have urged for the implementation of abs in the amazon, seeing it as having the potential to represent a major breakthrough in promoting the brazilian bioeconomy. the topic of implementation in particular has very recently gained momentum as the expectation of the ratification of the np and its promulgation mounted and these then finally took place in 2021 and 2023, respectively. on the other hand, the implementation of national abs policies and the np were hampered by covid-19 pandemics and unfavourable political conditions during jair bolsonaro’s presidential (2019–2023) term (eimer and donadelli, 2022). worldwide, the literature on the implementation of abs systems has become more and more abundant as countries, blocs (e.g. the eu), scholars, scientists and activists have increasingly published their experiences and views (coolsaet, 2015; vanheusden and van den berghe, 2017; greiber, 2019; kamau, 2019; friso et al, 2020; kamau, 2022). in brazil, scientific interest in the implementation of abs policies mostly emerged in light of the ratification of the np (silva, 2019; ferreira, 2020; mozini, 2020; silva et al, 2021; eimer and donadelli, 2022). previous studies do exist, but they are either highly technical (rabitz, 2015; davis et al, 2016) or present purely national perspectives (segundo et al, 2018). furthermore, the research conducted on the brazilian case has also been dominated by legal analyses (ferreira and moraes, 2013; davis et al, 2016; ferreira, 2020; mozini, 2020). in this paper, i explore brazil’s effort to implement abs. brazil is particularly well-suited for this qualitative case study because it is one of the most passionate advocates of the np and a megadiverse nation, being both a provider and user of gr. moreover, the country has been regulating its genetic heritage since 2001 (brasil, 2001) and did not wait for the np (ratification of the np by brazil did not occur until 2021 (brasil, 2021b)) and promulgation only late 2023 (brasil, 2023c) to develop its own abs system. in addition to this, brazilian national legislation in the spirit of the np was enacted in 2015-2016 and the ministry of the environment has increasingly incorporated an institutional framework for abs. as one of the first examples of abs implementation, brazil has attracted considerable international attention. finally, brazil has become an important player in the genetic resources (2024), 5 (10), 65–80 abs and the nagoya protocol in brazil 67 emerging bioeconomy (backhouse et al, 2021) with direct influence on how abs and np are currently understood (queiroz-stein et al, 2024). the aim of this paper is to reconstruct the implementation process from 2015 to 2023 from the perspective of selected involved stakeholders. by analyzing their key positions in the debate, we will cluster and explore positions in depth that highlight current trends with potential to influence the implementation of brazil’s abs system and the np. given that brazil has a long experience with abs, its implementation hurdles, opportunities and shortcomings may reflect the challenges or even the limitations for the global implementation of abs policies. the paper is structured as follows: after a short introduction to our research methods and data, i summarize the abs mechanism, implementation process and governance in brazil. research on the national implementation of abs systems has largely overlooked the interaction between these systems, public policy and public debate. most studies have focused on implementation as a purely legal or procedural process, neglecting the complex sociological dynamics involved (matland, 1995). siebenhüner and suplie (2005) made an early attempt to bridge this gap by linking abs implementation, prior to the np, with the concept of “institutional learning”. they challenged the assumption of rationally bounded actors and extended the understanding of institutions beyond their formal structures. in the context of abs implementation, learning occurs within a network of actors historically shaped by “strong user interests”, “process facilitators”, and “provider interests” (siebenhüner and suplie, 2005). thus, implementation is a fluid process where trust, influence, expertise, and social capital play crucial roles in the realm of policy implementation (montgomery, 2000). according to this literature, implementation is largely processual and happens beyond purely managerial decisions; positions and trends on abs and the np thereby play a major role in the implementation and shall be taken into account especially due to collegiate governance of these issues in brazil. empirically, the topic of abs and np is managed in brazil by a relatively small community which spans activists, indigenous leaders, bureaucrats, (natural and social) scientists, attorneys, consultants and high-skilled employees of companies that engage with the use of biodiversity (more details in the next section). in the brazilian case, many of them have seats at the conselho de patrimônio genético nacional (cgen). cgen is the national committee in charge of the management of abs and the np; within cgen, experts represent their own organizations, sectors and communities. public servants who are also experts on abs and the np represent their ministries and secretaries at cgen. as said before, the community is small and lack of expertise within some groups may blur divisions and organized interests, as members of determined sectorial chambers may be instated to represent others for the sake of the quorum required for the cgen sessions. this diffused knowledge and multi-sited aspects of representation bring additional hurdles for the investigation, corroborating the fluidity of the implementation process. given this, interviews became the privileged tool for research. due to the specific expertise that the topic requires and given the reduced size of the abs community in brazil, we consider the expert interviews method the most adequate approach; according to meuser and nagel (2016), expert interviews reflect a tendency for institutionalization and a position in society that permits “free spaces for the construction of reality”. originally conceived upon the realities of an industrialized society (germany), and drawing on the topic of abs, whose interactions with tk and iplc are self-evident, one should ask whether such categorization is convenient in this case; we nevertheless deemed the expertise and experience of iplc of uttermost importance and drawing on kaiser (2012), iplc were considered fullright experts. to address these questions and following an intensive desk phase (analysis of grey literature such as publicministerial and sectorial reports, indigenous public declarations, internet homepages, legislation and current state of research we selected 12 expert interviews1 – carried out between august 2021 and march 2023 – with key representatives of different groups of the abs landscape in brazil (table 1). the interviews ranged from online sessions via zoom (zoom video communications, inc) to in-person meetings in different brazilian cities. some interviews were also carried out in montreal, canada, during the un biodiversity conference (cop15, december 2022). following meuser and nagel (2016), some experts hold a dominant role (in the form of social capital) in their fields, and the selection of interviewees for this paper was mostly based on this criterion. these dominant roles are established within specific contexts in the abs arena, and can be observed among both gr providers and stakeholders in the industrial and academic sectors (users of gr). consultants are part of this expert landscape, too. overall, a list of experts on abs was created by using the ‘snowball method’ (based on recommendations or approaching potential interviewees via existing ones and other contacts). recognition of abs expertise by the interviewees led us to a shortlist in which experts were categorized according to siebenhüner and suplie (2005) network of actors on abs (“strong user interests,” “process facilitators,” and “provider interests”). then we selected the interviewees for this paper according to the following criteria: they represent different interest groups at cgen and actively participate in the implementation process both of the np and the national abs system; in addition, 1 the interviews were held in brazilian portuguese and transcribed by a native speaker, ms. adriana mastrangelo ebecken. email: dri.mastrangelo@gmail.com. the author is also a brazilian portuguese native speaker and translated excerpts of the interviews into english. the text and the excerpts were subsequently proofread by an english native speaker. 68 relly genetic resources (2024), 5 (10), 65–80 they may also either observe or facilitate implementation. we developed the interview guideline based on a set of questions with which we sought to emphasize the interviewees’ positionality, expertise, awareness and sensitivity regarding abs. the questions included the following: “concerning abs, where do you stand professionally?”; “what are the biggest challenges and opportunities in the implementation of abs and the nagoya protocol in brazil and worldwide?”; and “what are the most important conflicts between users and providers of gr?”. the interviews, ranging in length from 30 to 90 minutes, were transcribed and then open-coded using the maxqda program (rädiker, 2023). in the second phase of the analysis, thematic clusters were formed by comparing the positions on abs articulated both in the interviews and the desk phase. the positions and clusters discussed here are not intended to exhaustively represent all perspectives; some also reflect historical arguments that have long been part of the controversies surrounding gr and abs. importantly, these clustered positions serve as tools to organize and deepen the analysis of certain qualitative trends within the broader debate. they can be understood as specific frameworks (concerning abs and the np) within the ongoing discussions on the biodiversity–bioeconomy nexus, as highlighted by lima (2021), lima and palme (2022) and queiroz-stein et al (2024), which consider the contentious landscape surrounding the use of biodiversity in brazil, as well as its distributive, political and ecological aspects. in this context, we emphasize the textual nature of the interviews and will explore their qualitative implications, with a particular focus on the logic of abs. context: abs implementation and governance in brazil the regulatory framework in which the abs mechanism is implemented in brazil is set down by the lei da biodiversidade (law 13.123/2015, lb) (brasil, 2015) and the subsequent national decree 8.772/2016 (brasil, 2016) which revoked the former provisional measure medida provisória 2.186-16/2001 (mp). the latter was the very first national-level attempt to regulate the issue of gr in brazil (bensusan, 2003). prior to the issue of the mp in 2001, the amazonian states of acre and amapá had created their own state laws on abs (santilli, 2004) (santilli, 2005). irrespective of regional efforts, the mp, the lb and the national decree 8.7772/2016 nationalized abs in the early 21st century. interestingly enough, the federal constitution of brazil (brasil, 1988) established in article 225 the protection of genetic heritage without regulating access to it. in addition, brazil’s constitution uses the concept of genetic heritage instead of genetic resources – as proposed by the cbd in 1992. the former is defined in article 225, caput, as a “common good for the use of the people” (brasil, 1988) and law 13.123/2015 says in article 2 (i) “information of genetic origin” (brasil, 2015). regulation by public law was only established after the ratification of the cbd (brasil, 1998), paving the way for the developments described above (segundo et al, 2018). the lb and the national decree inherited parts of the organizational structures of the former mp by reinstating and strengthening the role of the cgen, which falls under the ministry of the environment and climate change. this body manages the national system of genetic resource management and associated traditional knowledge (sisgen, https://sisgen.gov.br/) digital platform in which the whole process of access, prior informed consent (pic), mutually agreed terms (mat), research and development (r&d), notification of products, shipments of samples, auxiliary intellectual property procedures, and other elements of compliance shall be registered. the sisgen platform is primarily an instrument for declaring access (substituting the previous mandatory authorizations issued by the genetic heritage management council, cgen, under the mp) and management of brazilian gr dynamics. access to sisgen is only permitted to brazilian researchers and institutions; foreign entities or researchers are obliged to sign a partnership agreement with brazilian institutions (silva, 2019). the cgen is a collegiate board whose administration consists of four bodies: 1) a central plenary formed by twenty counsellors (11 are members of the federal administration and the remaining nine seats are allocated to civil society representatives) (brasil, 2016), 2) thematic councils are created by the plenary to assist in decision-making, 3) sectoral councils are established as a platform for the positions of organized groups such as scientists (câmara setorial da academia), iplc (câmara setorial das das guardiãs e guardiões da biodiversidade) and companies (câmara setorial das empresas), and 4) an executive board (led by the secretary of biodiversity, forests and animal rights) which is in charge of managing the activities of cgen, ensuring sisgen operates smoothly, setting the agenda for further discussions, etc. the financial body of the lb is the fundo nacional de repartição de benef́ıcios (brasil, 2016) (fnrb) and like cgen also falls under the ministry of the environment and climate change. funds originate from the annual budget of the ministry, donations, fines charged for illegal access and use of gr, benefit-sharing, etc. the fnrb funds are used exclusively for actions and activities that benefit holders of traditional knowledge and environmental conservation. analogous to cgen, the fnrb is also a collegiate instance with a similar structure to cgen. the internal regulations of the fnrb were only developed in 2022 with the manual de operação do fundo nacional para a repartição de benef́ıcios (brasil, 2022) outlining the procedures for benefit-sharing being completed in october 2023. at the time of writing (early 2024), this fund remains genetic resources (2024), 5 (10), 65–80 abs and the nagoya protocol in brazil 69 table 1. list of 12 expert interviews carried out from august 2021 to march 2023 with key representatives of different groups in the abs landscape in brazil interviewees occupation date place i1 employee at a cosmetics company 3 mar 2023 belém do pará, brazil i2 biologist and former member of cgen 1 dec 2021 braśılia (online), brazil i3 environmental analyst working in the branch of cosmetics 14 mar 2022 são paulo (online), brazil i4 representative of a national association of industries 10 mar 2022 são paulo (online), brazil i5 leading natural scientist 22 feb 2022 rio de janeiro, brazil i6 leading layer and consultant 14 mar 2022 curitiba, brazil i7 academic 14 dec 2021 juiz de fora (online), brazil i8 cgen member and representative of traditional community 18 dec 2022 montreal, canada i9 cgen member and representative of an indigenous group 13 dec 2022 montreal, canada i10 activist and member of an organization that defends indigenous rights 17 mar 2022 braśılia, brazil i11 indigenous lawyer and activist 28 jan 2023 porto alegre (online), brazil i12 legal scholar and activist 27 jan 2023 curitiba (online), brazil non-operational, that is, implementation has not yet occurred. after the ratification of the np by means of legislative decree n. 136/2020 and the ratification letter of 4 march 2021 (brasil, 2021b), brazil declared the lb as its domestic law for the implementation of the np. on 27 december 2023, the np was finally officially promulgated (brasil, 2023c). however, silva et al (2021) emphasize the urgent need for the np and lb to be harmonized, given that brazil is now obliged to comply with abs legislation from other np parties and the lb itself contradicts many of the dispositions of the np. questions arising from the retroactivity and temporal validity of the lb, cross-border gr, and the use of foreign gr opened a new chapter in the implementation of abs policies in the country, posing further challenges for policymakers and society (de souza dias, 2022). in terms of institutional strategies, there have been multiple sets of regulatory frameworks and national and regional strategies targeting abs. the national strategy for intellectual property (brasil, 2021a), the national strategy and action plan for biodiversity (brasil, 2017a), the national strategy on science, technology, and innovation (2016–2022) brasil (2017b), and regional initiatives such as the plano estadual de bioeconomia championed by the state of pará (governo do estado do pará, 2022) and the diretrizes para a construção conceitual da bioeconomia no amazonas are prime examples in this regard (governo do estado do amazonas, 2021). non-governmental organizations and state-sponsored institutions are also intertwined with the launch of abs especially in the amazon. institutions like the polo digital de manaus (https://polodigitaldemanaus.c om/), the hub de bioeconomia amazônica (https://fa s-amazonia.org/hub-de-bioeconomia-amazonica/), and the technological innovation clusters arranjo amoci (https://arranjoamoci.org/) and arranjo namor (ht tps://arranjonamor.org/) in manaus and belém do pará, respectively, both funded through the dispositions of the law of innovation (brasil, 2004) and linked to the federal ministry of science, technology, and innovation, are supposed to facilitate and speed up the implementation of abs and fostering the expansion of industrial intellectual property rights (iprs) in the amazon. the implementation of abs in brazil has gradually become part of the overarching discussion on the bioeconomy and the mainstreaming of biodiversity in national development strategies (whitehorn et al, 2019). the bioeconomy and abs generally form part of the “biotechnological vision” (bugge et al, 2016; lopes and chiavari, 2022) in the international debate on the bioeconomy. brazil’s iplc have however challenged such a definition by proposing the concept of “sociobioeconomy” (queiroz-stein et al, 2024) as outlined in the letter from the amazon (amazon socio-biodiversity meeting, 2021), written on 20 october 2021, as part of the position taken by the forest peoples alliance (h ttps://cnsbrasil.org/alianca-dos-povos-da-floresta/) and other organizations at cop26 in glasgow. there are ongoing disputes on the “bioeconomy–biodiversity nexus” (lima and palme, 2022) in brazil, however, the specific role of gr, the national abs system, and the np are clearly subordinate to the more generalist approaches on the bioeconomy, forest conservation issues, etc. particularly since lula da silva began his third presidential term, abs has also gained momentum in mainstream politics. with marina silva as minister of the environment, the new national secretariat for the bioeconomy issued by federal decree n. 11.349/2023 (brasil, 2023a) with a specific department for genetic heritage (brasil (2023a), chapter ii, art. 2., ii, d), 3) was established. referring to the prospects of the bioeconomy in brazil, silva stressed that “reindustrialization will come from the environment and ancestral knowledge” (relly, 2023b). in a similar https://fas-amazonia.org/hub-de-bioeconomia-amazonica/ https://fas-amazonia.org/hub-de-bioeconomia-amazonica/ https://polodigitaldemanaus.com https://polodigitaldemanaus.com/ https://arranjonamor.org/ https://arranjonamor.org/ 70 relly genetic resources (2024), 5 (10), 65–80 vein, the government has also fostered brazil’s research capabilities on biodiversity prospecting (bioprospecting) in the amazon region with the reorganization of the centro de biotecnologia da amazônia (melo, 2023), a research institution in charge of promoting biotechnology and thereby accelerating abs throughout the amazon basin. very recently, the federal government announced the plan nova indústria brasil (brasil, 2024) with the aim of increasing the technological and sustainable use of biodiversity by 1% per year until 2033. the country’s iplc seem also to have shifted their position on abs in recent years, following the possibilities of the bioeconomy and adding their own perspectives. their fears of biopiracy dominated stances on gr and tk in the early 2000s (relly, 2023a). open letters and declarations such as the carta de são lúıs do maranhão paved the way for a national abs regulation (the mp in 2001). this helped to politicize the issue, and iplc took a firm stand regarding their demands on the protection of tk coupled with the promotion of indigenous territorial rights. today, cgen has established its legitimacy among key stakeholders and has genuinely become the national forum for the issue (castro et al, 2022). critical voices do exist, especially targeting the weak dispositions for prior informed consent (pic) of the lb. sharp criticism or rejection of the ongoing implementation process is more commonly found on the side of iplc, activists and their supporters. in addition, overall criticism of the abs architecture has commonly been incorporated into the overarching topic of the bioeconomy, as indicated by the positions taken by the critical group carta de belém (carta de belém, 2022). nevertheless, risks to the outcomes of implementation also affect more powerful stakeholders. due to brazil’s position as an agricultural powerhouse, whose dependence on foreign (agro)genetic resources is huge (soybean, cattle genetics, fish, etc.) and not entirely covered by annex 1 of the fao’s international treaty on plant genetic resources for food and agriculture (fao, 2009), representatives of the agrobusiness sector tend to be more skeptical about the implementation or at least are more cautious about engaging with the whole process. the delay in the ratification of the np might also be seen as a symptom of such reluctance (eimer and donadelli, 2022). as brazil has had national abs legislation since 2001 and recently ratified and promulged the np, the global discussions on dsi and the corresponding dematerialization of gr have placed brazil’s abs system and experiences in the spotlight. the literature and our findings suggest that brazil’s legal concept of gr as genetic heritage (contrary to the “material” notion agreed in the cbd) has hampered discussions on this very issue since the topic has long been covered by lei da biodiversidade and previous regulation (silva and oliveira, 2018) aside from the definition of the lei da biodiversidade, actors continue to associate genetic heritage with material entities, especially plants. this understanding is dominated among iplc, in particular, since all five biocultural protocols drafted and published in brazil are based on plants (andrade, 2022). the results of the implementation are nevertheless disappointing. as stated above, the brazilian state has not yet paid any benefits to iplc via the fnrb. since the state has not yet begun to implement the fnrb, we cannot attest to the full-fledged implementation of abs in brazil. despite those shortcomings, we will see in the following pages that there is widespread acceptance of abs policies, and the np and many actors feel that it is time to seize the moment and drive the implementation processes. results and discussion: facts and positions on the implementation of abs in brazil exact facts and figures on the implementation of the np and abs in brazil are hard to obtain inasmuch as most abs contracts are confidential and only headers including related terms like “changes and regularization”, “terms of commitment”, and “non-monetary benefits” are published by cgen online (brasil, 2023b). a side event organized by cgen at the cop15 in montreal (december 2022) did, however, provide an opportunity to verify some figures. the data presented here are for the period 2001–2022 and do not differentiate the period after the lb. sisgen registration figures up to december 2022, a total of 68,764 access registrations were carried out by sisgen. of these, 56,909 (83%) referred to research on brazilian genetic heritage. a total of 3,203 registrations only involved acccessing tk and 8,652 entries stated that both gr and tk were accessed. thus, tk accounted for approximately 17% of the net registrations. interestingly enough, cgen individualized the registration of access to dsi – since dsi is covered by the brazilian abs legislation. of the 68,764 net registrations at sisgen, 1,411 entries declared in silico access, of which, 336 registrations stated commercial purposes, with 1,075 registrations declaring “access activities for commercial and non-commercial use of dsi on gr”. shipping figures were also published: gr were mostly shipped to the united states (35.90%), france (14.91%), the united kingdom (12.56%), and germany (7.07%). biodiversity samples or dsi were shipped from all brazilian ecological regions, but forested biomes predominated: the atlantic forest (39.73%) and the amazon (39.27%) led the statistics. benefit-sharing as of 2001, 3,116 users had declared products under the modality of non-monetary benefits, while 1,789 users selected monetary benefits. the overwhelming majority, 8,859 administrative declarations of products (the trigger for benefit-sharing under lb) were exempted genetic resources (2024), 5 (10), 65–80 abs and the nagoya protocol in brazil 71 due to the legal specifications of the lb – presumably related to agricultural research/product development which are exempted from benefit-sharing. whether monetary benefits were paid or not is hard to assess due to the confidentiality of abs agreements. however, when it comes to non-monetary benefit-sharing, cgen has published (brasil, 2023b) recent agreements. as of early 2024, in total, cgen registered 28 non-monetary agreements involving seven companies including the cosmetics giants natura & co (avon), l’oréaltm, and o boticáriotm. of the 28 non-monetary benefits agreements, 20 addressed traditional communities such as quilombolas, riverine communities, and other traditional communities, but no indigenous groups appear on the list. these abs agreements are worth around r$1,000,000 each (approx. us$200,000). at the fnrb, cgen amassed around us$1,250,000 to safeguard biodiversity via the payment of monetary benefits (e. relly, personal observation). as already mentioned, the brazilian state has not yet paid any benefits to iplc. notwithstanding this fact, benefits in brazil are only billed via the gr associated with tk modality, by which iplc and users of gr establish contractual obligations according to which the state receives a 0.5% share of the net commercialization revenues. this means that abs only occurs between private companies and providers of gr. companies such as natura & co have led the way in this regard and have therefore been seen as an efficient abs implementor. one outstanding example in this matter is the fundo médio juruá (https://institutojurua.org.b r/en) established in 2017 between natura & co and associations of communities living along the juruá river in the western amazon. an employee at natura & co (interviewee 1, i1) told us in early march 2023 that the modality of ‘gr associated with tk’ is the only possibility for effective benefit-sharing in brazil inasmuch as “we [the company] have to meet the condition that benefits will be directly transferred to the community”. moreover, most benefits that are currently being paid to iplc (monetary benefit-sharing) were agreed on under the previous mp and not under the current and valid lb. even under the old mp, benefits for iplc were very scarce. souza et al (2017) estimated that between 2004 and 2013, just one abs contract was concluded with indigenous peoples and 61 with traditional communities. overall, 86.4% of the total abs contracts in this period were a result of the state’s sovereignty over gr. nor do the figures provided by cgen in montreal during cop15 allow us to individualize abs under the new and valid regulation. however, as the figures collected by souza et al (2017) indicate, most benefits were paid to the state. stakeholder positions given the multifaceted debate on abs, our research has clustered some positions on the ongoing implementation of and future scenarios for abs. in so doing, i do not claim to have covered all positions of all stakeholders. consequently, the positions presented here acquire a normative character inasmuch as they are intended to provide a general insight into and characterize the process of implementation. they may reflect both local and global constellations, since views on nature, work, property, justice and distribution vary enormously. in general, the political economy of the cbd era is still quite relevant. siebenhüner and suplie (2005) seek to order the institutional learning regarding implementation and görg (2002) idea of a “field of conflicts” on gr still applies today. most positions reflect user, facilitator and provider interests, but topics such as biodiversity loss, iprs, national development, far-right anti-environmentalism, and the prospect of bioeconomic transitions have produced a more blurred landscape and “convivial conversations” (lima and palme, 2022) between former antagonists seem to be occurring continuously. thus, positions can be clustered in the following categories: 1) acceptance and optimistic appreciation of abs, 2) acceptance of abs mechanisms but impending need for adjustments, 3) acceptance of abs mechanisms as a ‘bad with it, worse without it’ scenario, and 4) rejection of abs (figure 1). positions here were extracted from the transcriptions of 12 interviews and clustered qualitatively according to scholarship on contemporary biodiversity debates in brazil provided by lima (2021), lima and palme (2022) and queirozstein et al (2023). according to these, terminologies like “territorial rights and social objectives” and “economic growth” frame biodiversity discussions in brazil and our research will move within this established scholarship. acceptance and optimistic appreciation of abs this cluster is mainly represented by natural scientists working both at universities and national research institutions (e.g. germplasm banks and crop genebanks) and sectorial organizations (e.g. industry, bioeconomy). this cluster can be seen as the group where users’ interests dominate. scientists working with genetic germplasm, for instance, may see themselves as bearers of a national mission to bring tk and gr from the “public domain as collecting sites” (hayden, 2003) and as legitimizing abs scenarios and practices vis-à-vis the idea of enfranchisement of iplc. a former cgen member and biologist working in this field (i2) stated that most iplc “neither know, nor even grasp how to value what they have”. others in industry tend to fully accept abs mechanisms, perceiving them as a tool for harmoniously reconciling nature conservation and economic development for iplc. interviewee i3, who works for a major cosmetics corporation in brazil, emphasized that: “abs conserves because he [an iplc individual] collects the fruit, the leaf, he’s not using it for timber. because he could be selling that wood, you know? but instead of selling the wood, what does he sell? he sells https://institutojurua.org.br/en https://institutojurua.org.br/en/ 72 relly genetic resources (2024), 5 (10), 65–80 figure 1. clustered groups, relationships and positions among stakeholders of the brazilian access and benefit-sharing landscape. terminologies were developed with the help of lima and palme (2022) and queiroz-stein et al (2023). the fruit, the leaf, the flower, which has a higher added value for the cosmetics industry and, in some way, he conserves it. so, for example, how much fruit do you take from the environment so that you don’t have a negative impact in the sense that you manage to keep a certain amount of seeds for other trees to grow. that’s management: “oh, you need to hire a professional to do this management”; with what money? so, do through abs!” positions emerging from this field tend also to be influenced by the prospects of bioinnovation and the biotechnological bioeconomy. in this realm, gr and tk are seen as a pool of resources for national development, a comparative advantage, and last but not least, a strategic asset (nogueira, 2022). cultural diversity seems somehow separated from biological diversity in this cluster. another interviewee (i4), a representative of a relevant brazilian association that fosters corporate use of national biodiversity, stressed his optimism: “(. . . ) first of all, i’m quite optimistic, okay? (. . . ) yeah, since brazil has the greatest biodiversity in the world, right? (. . . ) no country has the number of biomes that we have, right, with the reach that we have. (. . . ) we do have the greatest biodiversity and (. . . ) this amount of biodiversity, makes this diversity strategic for the country, right? so, the development of legislation that allows biodiversity to be exploited in a sustainable way, and that adds value to the country, is fundamental for economic development. it’s a wealth that the country possesses, right?” positions within this group tend to minimize the conflicting issues concerning pic and mat or even impacts on iplc livelihoods and cultural systems. there is a firm belief that abs is there for good and is thus, through its market-based logic, likely to achieve the goals of national and international abs mechanisms with regard to the utilization of biodiversity and ecological conservation. unlike natural scientists who typically work under the umbrella of state funding, actors from industry and representatives of powerful associations in the field of agriculture and industry deplore the poor performance of the state and blame it for the inefficiency and difficulties of the whole implementation process. this cluster does not argue that existing abs systems are flawless either, but the depiction of a win-win scenario prevails. last but not least, this position resembles lima (2021) “conservative ecological modernization” concept which he applied to the analysis of projects surrounding the bioeconomy in brazil. acceptance of abs mechanisms but impending need for adjustments according to our interview record, this group is the most heterogeneous and the largest. this cluster encompasses the largest number of experts on the issue. natural scientists, cgen members, lawyers, compliance firms, even members of industries, agroecologists and iplc fall into this group. people in this field can be seen mostly as facilitators who attempt to build bridges genetic resources (2024), 5 (10), 65–80 abs and the nagoya protocol in brazil 73 among diverging interests and possess relationships both with users and providers of genetic resources but strong user interests are also present in this group of positions. indigenous peoples and local communities in this cluster deviate from a more critical stance toward abs mechanisms and they are keen to identify advantages within the process of implementation. this cluster tends to reflect on the distinctions between the lb and the np and plead for further development of the national law, either by means of a new specific piece of legislation to address the integration of the np into the national abs system or by adapting implementation through guidelines created by cgen. another core issue for this cluster revolves around the view that abs systems are a better outcome than the previous principle of common heritage of mankind. in general, abs systems are deemed a game changer that represents a significant obstacle to biopiracy. that being said, scientists within this group complained about the hurdles created by the lb and the np, and articulated the more general view that science is a common benefit to all (bockmann et al, 2018). a common criticism of the lb is centred on the difficulties foreign and national scientists experience in conducting research on brazilian biodiversity. some within this cluster also postulated that conflicts between users and providers of gr have gradually diminished and commended the maturity and stability of the brazilian debate on the issue. this has led to institutionalization and the effective management of the conflicts, leaving behind the more politicized discussions on biopiracy, for instance. this last factor has significant positive consequences for the processes of implementation. looking back at brazil’s experience with abs so far, a leading scientist we interviewed (i5), who is an active participant in international networks, remarked: “if we consider 2001 until now (. . . ) there’s been a huge maturing process, and then the new legislation [lb] came along, which has a lot of problems, but is still much better than the other one (. . . ) so, i think all this has (. . . ) helped reduce conflicts. conflicts, most of the time, are not between the academy and industry, they are between the providers and industry, and even with us [scientists]. so, on this point, i’m even proud to say that in the mp we were often on the opposite side and now we’re together [with iplc] (. . . ) perhaps at the beginning there was some estrangement, i agree, but today we are very much together, defending the same things.” knowledge production and the role of facilitators are also pivotal within this position. at the cop15 in montreal, for example, natura & co, croda international plc and gss sustentabilidade e bioinovação (a leading compliance firm in brazil) launched the 2022 version of the project brogota, an outstanding piece of work, written in english, comparing the brazilian abs system with other national regulations around the world (gss sustentabilidade e bioinovação ltda, croda international plc, natura & co, 2022). despite the alleged progressiveness of the abs mechanisms (in terms of addressing justice issues), there is still a constant need for adjustments (whether that be harmonization between the np and the lb, or working out how to meet the expectations of providers), making them very much a work in progress. interviewee i6 who works in the compliance field also referred to these problems, affirming the many shortcomings of the implementation of abs worldwide, the cbd’s mandate of justice (suiseeya, 2014) in addressing global disparities, and last but not least, the necessity of impending changes with the aim of creating a multilateral system for benefit-sharing: “now, thinking about nagoya (. . . ), not just about brazil, about brazilian legislation, the big issues [to be solved], whether it’s brazilian legislation or nagoya, we have some structural problems, some conceptual problems. because ideologically, the legislation is wonderful, the intention of the nagoya protocol is wonderful, the fact that the cbd, back in 1992, brought in sovereignty for countries, is also fantastic, because at that time countries needed to have sovereignty over their genetic resources, not least because of the historical legacy of colonization. so, it was absolutely necessary. the big problem we face today is that soon there will be, in a way, a war over the availability of genetic resources. because some countries are stricter in the use of their genetic resources, others will see this as an opportunity to give away their genetic resources and facilitate access. and more than that, and this is one of the points that i’m fighting the most in relation to the nagoya protocol, is the overlapping of access. (. . . ) so this, for me, is one of the big bottlenecks of the nagoya protocol, the fact that we need a multilateral mechanism, a benefit-sharing fund that facilitates or makes it possible for this to happen in a more homogeneous way and for the countries to agree to it, right? indigenous peoples and local communities as well as agroecologists are in a minority in this cluster. however, their positions tend to point out the doubleedged nature of abs mechanisms which range from the social valorization of tk to the dangers of cultural exploitation (dutfield, 2015). changes must be carried out at the state level and should address providers’ interests. natural scientists deplore the bureaucratic hurdles and their positions express the need for a more clearly defined role of basic science or for the participation of foreign scientists who wish to use brazilian biodiversity. topics like pic and mat are more relevant and should form part of the discussion, inasmuch as they provide more legitimacy to the stabilization and management of conflicts between providers and users. in this position, we also observe “convivial conversations” with providers of gr, which do not preclude “market opportunities that may be reaped but to be open to the full spectrum 74 relly genetic resources (2024), 5 (10), 65–80 of possibilities, acknowledging and accepting multiple forms of social existence” (lima and palme, 2022). acceptance of abs mechanisms as representing a “bad with it, worse without it” scenario in the words of a distinguished scholar of the natural sciences (i7) based in the state of minas gerais: “in today’s context, it is bad with it [abs], but worse without it.” this “bad with it, worse without it” position has been adopted by a number of individuals predominantly from the fields of agroecology activism, environmental protection, science (especially anthropology, ethnobiology and the social sciences), law (including legal scholars), and members of governmental bodies and agencies who support or even promote the rights of iplc and public policies. this position clearly reflects the interests of the providers of gr and builds up the other interface for “convivial conversations” (lima and palme, 2022), a common aspect within this position. members of this cluster may express strong criticism of the current abs mechanisms and in the brazilian case, there is a common trend of addressing sharper critics of the national abs system due to its weaker stance on pic (vis-à-vis the nagoya protocol) and iplc’s lack of political representation both in the creation of the lb (2014–2016) and in ongoing implementation in general (guetta and bensusan, 2018). the proximities between specific sectors of academia and iplc’ demands in brazil was already noted by eimer and donadelli (2022). overall, the “bad with it, worse without it” position is a structural stance adopted by iplc and their supporters in relation to abs. it is indeed an utterly ambiguous position on the general concept of abs, and such ambiguity has accompanied the implementation process. in brazil at least, iplc, ngos and numerous grassroots organizations have nurtured a well-considered viewpoint – in the sense of the role of iprs among iplc as described by ido and valentini (2018) – on the possibilities of abs to address or instrumentalize iplc demands. although “bad with it, worse without it” may imply a lack of alternatives, limited room for manoeuvre, or even rejection, this position seems to nevertheless entail a predisposition to critical compromise and the creation of institutional spaces. this position was clearly taken by the signatories subscribers (many of them from iplc associations) of the carta aberta de recomendações da sociedade civil brasileira na 15ªconferência das partes da convenção da diversidade biológica e seus protocolos terra de direitos (2023), who stressed that: “despite all the criticism that the biodiversity law deserves, it also brings advances, such as the establishment of community biocultural protocols, the result of joint (. . . ) struggle by peasants, indigenous peoples, traditional and quilombola communities.” ambiguity marks the complex position of indigenous peoples in the face of the prospect of the commodification of tk and the rapprochement of market relations. in this sense, bonifácio josé baniwa’s argument, an indigenous scholar from the baniwa people from northern brazil, still resonates and it is quoted as an authorized and actual perspective on tk and its relationship to markets (alencar et al, 2003): “we work with crafts that have traditional values, traditional knowledge, and traditional meanings (. . . ) when we try to valorize these through sales, the market has its own requirements and rules. we will have to accept these rules if we want to improve the community’s income, but at the same time, we may end up breaking the law of traditional knowledge. it seems that one side is stronger than the other. we’re trying to work on the market understanding our way and not our knowledge having to fit the market. this is the main challenge for traditional knowledge.” in this cluster, the lb is seen as an unjust piece of legislation, a law that enabled access to instead of protection of tk. nevertheless, implementation of the lb by means of the national decree and the establishment of the new cgen provided iplc with some opportunities. our research was conducted at a rather difficult time, since jair bolsonaro’s presidential term and the covid19 pandemic prevented or hindered direct participation, leaving iplc with fewer tools to influence decisions at cgen level, inasmuch as health and security (conflicts on land) worries became a priority. irrespective of this, iplc participation at cgen is of utmost importance to tackle the unfavourable rules of the lb. interviewee i8, cgen member and representative of a traditional community in central-eastern brazil stated: “the implementation makes it possible for us to have an impact in some way. i think it’s an opportunity in the sense that we can influence the agenda (. . . ); we couldn’t do this for the last four years because of bolsonaro’s term, but now, with the new government [reference to the victory of lula da silva in the 2022 presidential election], we have this great opportunity to make implementation really go to the grassroots.” the fact that the lb allowed the development of biocultural protocols as valid documents for trade, access and benefit-sharing is seen as a reason why the law has not been rejected outright. interviewee i9, an indigenous woman from northern brazil who is also a cgen member, stated that these protocols are “our dream (. . . ) especially because they [the federal government of brazil] need to foster the biocultural protocols and respect the opinion of the peoples.” the “bad with it, worse without it” position predominates in our interviews with iplc. in this cluster, inequality, power relations, but also iplc’s leading role in the implementation and “biocultural jurisprudence” (bavikatte and robinson, 2011) are key. biopiracy is still a risk for some. however, we observed a structural shift toward institutionalization with a view to changing the abs mechanism from the inside, given that the complexity of the issue means it constantly takes on new forms, is reframed, and poses new challenges, such as dsi, transborder gr and overall harmonization genetic resources (2024), 5 (10), 65–80 abs and the nagoya protocol in brazil 75 with the np. there are, however, several issues that remain unresolved by legislation. these include weak pic dispositions, the question of unidentified tk, and the digital system enabling access through sisgen. last but not least, abs is seemingly expected to be a tool for territorial claims and iplc rights. this link between abs and territorial rights is illustrated by interviewee i10, an activist working at conselho indigenista missionário and an advocate of indigenous territorial rights, who told us that “from the moment you highlight people’s coexistence with biodiversity [referring to tk] (. . . ) you need to have a territory of your own.” rejection of abs in brazil, the outright rejection of abs systems only plays a minor role in the debate. the people who argue for the rejection of the concepts underlying the implementation of abs have long-standing critical expertise on the topic as well as excellent moral reputations. some are seen as intellectual authorities on tk and iplc rights. this cluster comprises indigenous peoples and legal scholars. their main argumentation against abs is rooted in the issue of individual iprs (against the background of indigenous collective forms of property) and the encroachment of capitalist logics on iplc’s collective social structures. in addition, questions regarding pic quickly come to the fore in this cluster. those positioning themselves against abs systems generally claim that cbd-based solutions are not suited to resolving the issues concerning tk and iplc rights. prior informed consent has been a structural argument in this regard and discussions on how the representation of indigenous peoples works tend to disregard abs mechanisms. in the brazilian debate, this position can be traced back to 2003 when the notorious brazilian anthropologist eduardo viveiros de castro questioned whether it was “the community that produces the consent or the consent that produces the community?” (alencar et al, 2003). rejecting any possibility of a stable pic concerning access to tk, viveiros de castro insisted on the structural nature of unsuccessful consent, given that indigenous communities do not operate according to the political models of liberal/western collective representativeness. casting doubt on the legitimacy of iprs emanating from gr associated with tk, this view clearly prevails among those who reject abs mechanisms. this is illustrated by interviewee i11, an indigenous lawyer and activist from southern brazil, who asked: “who is the juridical person who represents my people? this person does not exist”, making the case for the impossibility of a legitimate pic under brazilian biodiversity law. the issues concerning pic in this cluster are centred on the limitations of the liberal legal tradition and criticism of capitalism. interviewee i12, legal scholar, lawyer and long-standing activist for indigenous rights in brazil, noted that “the discussion on property (. . . ) is an essential discussion. if you introduce private property within a system in which private property does not exist, you will destroy this system.” drawing on his own remarks, i12 went on to state his position on abs mechanisms more clearly: “(. . . ) in any case, between not having any legislation and having legislation that regulates access, that is, that opens another door to usurpation, i would say the following: we have three forms of usurpation, right? free usurpation: everyone goes there and does what they want, a mess; legal usurpation; and illegal usurpation, which is against the law and may be punished. free usurpation ceases to exist when there is regulation, leaving just the other two. so, if this regulation opens up too much space for legal access, it’s better not to have it. i think the regulations we have in brazil open up a lot of space.” conclusion with brazil being both a major provider and user of gr, it is one of the few countries in the world that can illustrate the true complexity of the situation to help comprehend the challenges, opportunities and perhaps even limits of the abs mechanisms that were agreed upon at the cbd and in subsequent agreements (in our case, the np). in this regard, brazil can be seen as a microcosm of the (future) global abs landscape. notwithstanding this, abs has not unleashed its global potential and has frustrated some expectations, especially with regard to benefits, legal uncertainties and administrative failures (here reported in the case of the brazilian state). in this regard, brazil has also contributed to the alleged problems implementors and cop negotiators frequently express (silva et al, 2021). nevertheless, abs in brazil is robust. cgen has gained legitimacy among stakeholders and the majority adhere to its premises, notwithstanding the justified criticism of weak pic, high transaction costs, unequal power relations, and a lack of proper instruments of iprs that reflect indigenous sensitivities regarding the collective nature of their societies. we observe that the discussion on abs mechanisms has also changed considerably and has incorporated other transversal axes such as the bioeconomy and the mainstreaming of biodiversity in national strategies (within the bioeconomy–biodiversity nexus). politics have also shaped the spaces for debate and participation. having undergone massive political change in the last ten years, the role of biodiversity in brazil has been in the spotlight in a very different way. between the tug-of-war among powerful agrarian interests and the transition to a low-carbon economy, our research revealed that the abs and np topics, which predate the discussions on the bioeconomy, have also been increasingly appropriated by the latter. the topic of biopiracy, for instance, which characterized the debate (especially among iplc) during the early cbd years and the first regulation provided by the provisory measure n. 2186/2001 (2001-2015) (brasil, 2001), for instance, have been superseded by the negotiated participation of iplc in the national economy. in this regard, we observe increased entanglement of abs with 76 relly genetic resources (2024), 5 (10), 65–80 the prospects of a particular version of the bioeconomy, especially the biotechnological, bioecological and socalled bioeconomy of sociobiodiversity. these prospects provide opportunities but also pose risks to the actors involved. drawing on the literature on legal anthropology, and by crossing this with the interviews, we see a trend toward compromise via the multifaceted terms of implementation, enabled by the collegiate governance of cgen. the fact that the position that pleas for the outright rejection of abs systems is the least relevant in the debate suggests this trend. cgen’s authority and its position as a national forum indicate that too. although implementation has been disappointing, that has not prevented actors from attempting to influence the course of things. creating future scenarios and the possibility of shaping the course of implementation through cgen governance continue to offer actors an arena for adapting national legal provisions produced by the lb as well as guiding the contingencies of the np at the national level. in this particular regard and based on iplc’s positions on the issue of abs, we see a major shift toward the stabilization and institutionalization of the issue. the heated debates of the past are still relevant, but iplc are now acting pragmatically and programmatically, having even built alliances with some users and facilitators. the structural nature of the cbd arena, centred on users’, facilitators’ and providers’ interests, still holds true but the situation has become more blurred, inasmuch as actors are compromising more. on the side of iplc, compromise on abs tends to address another structural claim of the post-cbd era: territorial rights as the underlying basis of an effective abs system. the positions raised in our study represent only a fragment of a broader and more complex implementation process. due to the collegiate governance of abs in brazil, these positions tend to influence implementation. aligning to national development and reflecting more clearly users’ interests and the upper hand of natural sciences in the process, we clustered the position “acceptance and optimistic appreciation of abs”. the position “acceptance of abs mechanisms but impending need for adjustments” seems to encompass a wider range of stakeholders with diverse backgrounds and perspectives, from those advocating prioritizing users’ interests to those arguing for better protection of traditional knowledge. a more critical perspective, which leans toward providers’ interests while still seeking compromise with users and facilitators (such as the state and consulting companies), is captured in the position “acceptance of abs mechanisms as a ‘bad with it, worse without it’ scenario”. finally, the “rejection of abs” position, although less influential in the debate, is rooted in historical arguments about the inadequacy of tk to be accessed through abs. despite ongoing debates, trends toward implementation, institutionalization and governance of gr and tk in brazil, abs remain highly contested. conflict of interest the author declares that he has no competing interests or relationships that might have influenced the work reported in this paper. acknowledgements this research was funded by the collaborative research project sfb 294 ‘structural changes of property’ (friedrich schiller university jena and university of erfurt, germany) and conducted within its framework. i would like to extend my gratitude to all interviewees for their knowledge, insights and thoughtful contributions. special thanks are also due to my colleagues from the junior research group ‘jrt03 ownership of genetic resources: on the appropriation of traditional knowledge in the bio-economy’ under the sfb 294 project – pd dr anne tittor, prof. dr maria backhouse, leoni schlender, tila mendonça and zelda wenner – for their support and collaboration. furthermore, i am grateful to the members of the idsi research network for the enriching discussions we have had since cop15 in montreal. lastly, i would like to thank the cgen coordination for providing detailed information on this topic through our email exchange in april 2023. any errors in data interpretation remain my sole responsibility. references abihpec 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(2023). phenotypic diversity among finger millet (eleusine coracana (l.) gaertn.) landraces of nepal. genetic resources 4 (8), 1–14. doi: 10.46265/genresj.myza2446. © copyright 2023 the authors. this is an open access article distributed under the terms of the creative commons attribution license (cc by 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. introduction finger millet (eleusine coracana (l.) gaertn.) is an allotetraploid species (2n = 4x = 36; genome constitution aabb) of the grass family poaceae (dida et al, 2007). it evolved from a wild species (e. coracana ssp. africana (kenn.-o’bryne) (aabb)) which is the natural cross between wild species (e. indica (l.) gaertn. (aa)) and extinct unknown species (bb) (liu et al, 2014). east africa is considered its primary centre of diversity (phillips, 1972) where nine out of ten known ∗corresponding author: krishna hari ghimire (ghimirekh@gmail.com) species of the genus eleusine – coracana, africana, indica, floccifolia (spreng), intermedia (chiov.) (s.m.phillips), multiflora (hochst. ex a.rich), jaegeri (pilg.), kigeziensis (s.m.phillips) and semisterilis (s.m.phillips) – are found, except e. tristachya (lam.) (hilu and de-wet, 1976). ploidy and hybridization barriers suggest that tetraploid (2n = 4x = 36) species e. coracana (aabb) and e. africana (aabb) are in the primary gene pool, diploid (2n = 2x = 18) species e. indica (aa), e. tristachya (aa) and e. floccifolia (bb) formed the secondary gene pool and the rest of the species are in the tertiary gene pool (sood et al, 2019). it was domesticated about 5,000 years ago in eastern africa (ethiopian highlands) received: 12.04.2023 accepted: 20.06.2023 published online: 11.07.2023 https://www.genresj.org https://www.dx.doi.org/10.46265/genresj.myza2446 https://www.genresj.org https://www.dx.doi.org/10.46265/genresj.myza2446 mailto:ghimirekh@gmail.com 2 ghimire et al genetic resources (2023), 4 (8), 1–14 and introduced into the indian subcontinent 3,000 years ago (hilu et al, 1979; upadhyaya et al, 2006). globally, finger millet ranked fourth in importance among millet crops after sorghum, pearl millet and foxtail millet (upadhyaya et al, 2007). in many countries, precise data on cultivation area and production of finger millet are not available because the production statistics of this crop had often been combined with other millets (upadhyaya et al, 2010). it is cultivated on 3.8 million ha (12% of the total millet area) with coverage in more than 25 countries in africa (e.g. uganda, tanzania, kenya, ethiopia, rwanda, zaire, eritrea and somalia) and asia (e.g. india, nepal, sri lanka, myanmar, china and japan) (upadhyaya et al, 2010; bora, 2013; kumar et al, 2016; vetriventhan et al, 2016; hittalmani et al, 2017). it is grown in a wide range of environments from the tropical coastal regions of india (upadhyaya et al, 2006) to the high mountains (3,130 masl) of nepal (bastola et al, 2015; gaihre et al, 2021). having the c4 photosynthetic pathway (hittalmani et al, 2017; parvathi et al, 2019), it is a hardy crop grown in marginal land and stress environments with very low or minimum input (goron et al, 2015). finger millet (kodo in nepali) is the fourth most important cereal crop in nepal after rice, maize and wheat in terms of area and production, and occupies an average of 7.7% (265,401ha) of the total cultivated area covered by cereal crops and accounts for 2.9% (326,443t) of total cereal production with average yield of 1.23t/ha (moald, 2022). nutritionally, its importance is well recognized because of its high content of calcium (0.34%), dietary fiber (18%), protein (6–13%), minerals (2.5–3.5%), phytates (0.48%) and phenolic compounds (0.3–3%) (chandra et al, 2016). it is enriched with calcium, iron, zinc, proteins and calories (o’kennedy et al, 2006; upadhyaya et al, 2011). the crop is also valued for its health beneficial effects like anti-diabetic, anti-tumorigenic, antioxidant and antimicrobial properties (devi et al, 2011; kumar et al, 2016; nakarani et al, 2021). besides food and nutrition, it is an integral component of agrotourism in nepal due to local specialties made from it, such as dhindo (thick porridge) and high quality raksi (home-made wine) (ghimire et al, 2017; joshi et al, 2020; gaihre et al, 2021). vetriventhon et al (2020) reported a total of 36,873 finger millet accessions (including landraces, improved cultivars, wild and weedy relatives) conserved ex situ at global level and this number is ever increasing. most of these collections are yet to be characterized and utilized in breeding. the international crop research institute for semi-arid tropics (icrisat) has developed a finger millet core collection of 622 accessions, including 70 accessions from nepal, based on agromorphological diversity from their entire collection of 5,940 accessions (upadhyaya et al, 2006). two studies reported on the characterization of nepalese finger millet accessions (bhattarai et al, 2014) but the landraces used in those studies were not properly represented in the entire genebank collection. characterization of collected landraces is the most important avenue to open the door for their utilization. however, less utilization of local genetic resources for crop improvement programmes is evident in nepal due to lack of information about the desirable accessions in the genebank resulting from poor characterization and evaluation data. three out of six finger millet varieties notified in the country were improved from native landraces, which include okhle-1, kabre kodo1 and rato kodo. the present study describes the characterization of finger millet accessions conserved at the national agriculture genetic resources centre (nagrc, genebank) of nepal, grouping of accessions with similar characters using a range of multivariate statistical tools and identifying potential landraces to be utilized in finger millet improvement programmes. materials and methods plant materials and experimental sites this study used 300 finger millet accessions (supplemental table 1) received from nagrc which include 295 landraces collected from 54 districts of 6 provinces, and 5 released varieties (okhle-1, dalle kodo-1, kabre kodo-1, kabre kodo-2 and shailung kodo-1) of nepal. a total of 295 landraces were selected from nearly 1,000 accessions of 54 districts based on proportions and representation, so that there was minimum repetition in local name and at least one accession from each district. experiments were conducted at three mountain locations of nepal, namely agriculture research station (ars) vijayanagar, jumla (2,350 masl); nagrc khumaltar, lalitpur (1,360 masl) and hill crops research programme (hcrp), kabre, dolakha (1,740 masl). geocoordinates of experimental locations and collection sites of landraces were mapped (figure 1). all three sites had coarse textured sandy loam soil. general methodology the experiments were laid out in alpha lattice design with 300 entries and two replications having 15 blocks within replications and 20 plots in each block. each plot was constituted by 20 plants in a single row of 2m length with 25cm spacing between rows. during 2017 and 2018 respectively, seeding was done on 24 and 19 april at jumla, 3 june and 26 may at dolakha and 17 june and 7 june at khumaltar. direct seeding was done with the application of chemical fertilizers at the rate of 20:10:10 kg/ha n:p2o5:k2o as basal doses. thinning was applied within 25–30 days after seeding to maintain a plant-to-plant spacing of 10cm within rows. manual weeding was done as per requirement but no irrigation and pesticides were applied. data recording morphological data of nine qualitative and 17 quantitative traits were recorded as per the standard descriptors of finger millet (ibpgr, 1985). the qualitative traits were recorded from a single replication of 2017 at khugenetic resources (2023), 4 (8), 1–14 nepalese finger millet diversity 3 figure 1. map of nepal showing collection sites of characterized finger millet accessions in six provinces, coded as indicated in the legend. the three experimental sites where field trials were conducted are indicated by stars. maltar only, based on observations as per the descriptor states. observations on days to 50% flowering, days to 80% maturity, grain yield (kg/ha) and straw yield (t/ha) were based on whole plot data whereas measurements on other quantitative traits such as plant height (cm), tillers per hill (n), flag leaf length (cm), flag leaf width (cm), flag leaf sheath length (cm), ear exsertion (cm), ear head length (cm), ear head width (cm), fingers per head (n), length of the longest finger (cm), width of the longest finger (cm), weight per head (g) and weight of 1,000 grains (g) were made from five randomly selected plants. observations for days to maturity of those accessions which did not set grains due to extremely low temperature after flowering at jumla were considered as missing values whereas grain yield of those accessions were estimated as zero. data analysis the frequency of each descriptor state for all qualitative traits were tabulated with their proportions whereas observations for all quantitative traits of each year and locations were subjected to unbalanced analysis of variance (anova) using regression model with the software genstat version 15 (vsn international, 2015). the combined mean data were subjected to descriptive statistical analysis such as minimum, maximum, mean, standard error and coefficient of variation using minitab17 (minitab, 2010). hierarchical clustering of observations and construction of dendrogram were done based on unweighted pair group method with arithmetic mean (upgma) by using factominer package (sebastien et al, 2008) and factor map visualization was made by using factoextra package (kassambara and mundt, 2020) of r statistical software (r core team, 2020). correlation analysis (pearson’s coefficient with probability) and principal component analysis (pca) were done using minitab-17 (minitab, 2010). standardized shannonweaver diversity indices (h’) (shannon and weaver, 1949) were calculated for each trait with microsoft excel (ghimire et al, 2018a,b) . results diversity index and frequency distribution of qualitative traits based on shannon-weaver diversity index (h’), we observed very low diversity for finger branching and grain shattering (0.122) but it was highest for plant pigmentation (0.908) followed by grain covering (0.793), ear shape (0.790) and seed colour (0.722) (table 1). the data showed that the predominant ear shapes in nepalese finger millet are open (42.7%) and semi-compact (41.7%) types. more than two-thirds (68%) of accessions had intermediate ear size followed by large ear size (29.7%). less than 2% of the accessions had branching in fingers and grain shattering. similarly, one-third of the accessions had pigmented plants while very few accessions were highly susceptible to lodging (3.3%). seed colour varied from white to purplebrown (figure 2). the predominant seed colour in the collection was light-brown (53%) followed by purplebrown (37%). descriptive statistics and diversity indices of quantitative traits range, mean, standard error of mean (se), coefficient of variation (cv) and shannon-weaver diversity index (h’) of each quantitative trait are presented in table 2. a wide range of variation in agronomic performance 4 ghimire et al genetic resources (2023), 4 (8), 1–14 table 1. shannon-weaver diversity indices (h’), descriptor states and frequency of nine qualitative traits. qualitative traits h’ descriptor states frequency (n) proportion (%) ear shape 0.790 droopy 38 12.7 open 128 42.7 semi-compact 125 41.7 compact 9 3.0 ear size 0.647 small 7 2.3 intermediate 204 68.0 large 89 29.7 finger branching 0.122 absent 295 98.3 present 5 1.7 grain covering 0.793 exposed 67 22.3 intermediate 196 65.3 enclosed 37 12.3 lodging susceptibility 0.654 low 209 69.7 intermediate 81 27.0 high 10 3.3 plant pigmentation 0.908 not pigmented 203 67.7 pigmented 97 32.3 seed colour 0.722 white 11 3.7 light-brown 159 53.0 copper-brown 19 6.3 purple-brown 111 37.0 spikelet density 0.676 sparse 89 29.7 intermediate 201 67.0 dense 10 3.3 spikelet shattering 0.122 absent 295 98.3 present 5 1.7 figure 2. seed colour variation (white to purple-brown) on different finger millet accessions (in case of mixed seeds, colour of the majority of the seed is considered, e.g. colour of ngrc04793 is recorded as white). genetic resources (2023), 4 (8), 1–14 nepalese finger millet diversity 5 was observed among the evaluated accessions. the early maturing accessions started flowering at 75 days after seeding and the late maturing accessions flowered at 140 days whereas the average plant height ranged from 61 to 119cm. the average adjusted grain yield and straw yield ranged from 230 to 3,494kg/ha and 2.0 to 20.2t/ha, respectively. the cv varied from 6.2% for flag leaf length to 33.3% for grain yield. h’ ranged from 0.864 to 0.907 suggesting high diversity in finger millet accessions for all quantitative traits. clustering observations a upgma hierarchical clustering divided the entire 300 accessions into four clusters (figure 3). the number of accessions in each cluster and cluster characteristics for each quantitative trait are presented in table 3. cluster 4 was the largest cluster with 111 (37%) accessions having the highest cluster means for grain yield (1,858 kg/ha), straw yield (10.6t/ha), plant height (100cm), 1,000grain weight (2.4g), weight per head (6.5g) and ear length (6.6cm). cluster 3 was the second largest cluster with 107 (35.6%) accessions having the lowest mean grain yield (1,147kg/ha) but the longest mean flowering days (122) and maturity days (161 days). cluster 1 was the smallest cluster with 16 (5.3%) accessions characterized by the lowest cluster mean for straw yield (4.9t/ha), weight per head (4.5g), finger length (5.0cm), ear length (5.5cm), fingers per head (5.6), plant height (78cm), flowering (85 days) and maturity (132 days). non-significant difference was observed between cluster mean and overall mean for flag leaf width. principal component analysis the contribution of various traits in total phenotypic variation among 300 finger millet accessions was evaluated by principal component analysis (pca). the first five principal components with eigenvalue ~1 or more, explained 61.8% of the total variation (table 4). the first principal component (pc-1) explained 22.6% of the total variation which was positively attributed to days to maturity (0.421), days to flowering (0.401), straw yield (0.338), fingers per head (0.303), finger length (0.295), ear length (0.290), plant height (0.280), leaf length (0.255), weight per head (0.252) and ear width (0.229). the second component (pc-2) explained an additional 14.9% of the total variation.the maximum variation in this pc was primarily due to the lower grain yield (-0,496), sheath length (-0.389), plant height (0.313), 1,000-grain weight (-0.278), ear exsertion (0.256), and tillers per plant (-0.255) but higher value of days to flowering (0.314) and maturity (0.267). the third component (pc-3), which explained 9.1% of the total variation, differentiated the accessions by higher finger length and ear length but lower leaf length. pca using cluster means showed that the first three components explained 100% of the total variability with 60.5 and 28.2% contribution by pc-1 and pc2, respectively (table 4). most of the traits occupied the right side of the bi-plot and thus contributed with positive loadings to the variation explained by pc-1 (figure 4b). a clear-cut elbow on the fourth component in the scree plot (figure 4a) as well as the two-dimension scatterplot of pc1 and pc2 (figure 5) revealed strong support for the clustering result since we can see the visible groupings of accessions as per the clusters (figure 3). correlation between traits the pearson’s correlation coefficients between traits are presented in table 5. a total of 136 trait associations were estimated among the 17 quantitative traits. out of these, associations between days to 50% flowering and days to 80% maturity (0.93) as well as between ear length and length of the longest finger (0.63) had high estimates. this indicates that only one trait from each of these pairs could be recorded and assessed during future characterization work. grain yield was positively correlated with plant height, productive tillers per plant, flag leaf length, leaf sheath length, ear exsertion, ear length, finger length, ear weight and 1,000-grain weight but negatively correlated with days to flowering and maturity. when selecting for grain yield, we should consider these traits strongly associated with grain yield while when selecting for straw yield, our focus should be on taller plant height and late maturity since straw yield had strong positive association with days to flowering, maturity and plant height. promising landraces promising trait-specific donors were identified (table 6) based on combined mean data of six environments (supplemental table 2). some landraces were good for multiple traits (highlighted in table 6) and some others for particular traits. for instance, ngrc06490 was high yielding with higher number of tillers; ngrc04849 was high yielding with higher 1,000-grain weight; ngrc04871 was high yielding with taller plant, longer ears and higher 1,000-grains weight; kabre kodo-2 was high yielding with higher 1,000-grain weight and higher number of tillers; and ngrc04818 was high yielding with taller plant, higher 1,000-grain weight and higher weight per head. landraces ngrc04849 and ngrc06490 produced 121.6% and 120.1% higher grain yield respectively, than the overall mean (1,577kg/ha) and 10.4% and 9.7% higher yield respectively, compared to kabre kodo-2, a newest and best among the five released varieties. mean days to flowering was 112 but 11 accessions flowered before 90 days after seeding. early flowering genotypes may be selected for droughtprone lowlands as well as higher altitudes since they can escape both drought and cold stress during reproductive stage. these landraces showed potential to be efficiently utilized in breeding programmes for the improvement of finger millet. 6 ghimire et al genetic resources (2023), 4 (8), 1–14 table 2. variability statistics and shannon-weaver diversity indices (h’) of 17 quantitative traits. se, standard error; cv, coefficient of variation. trait minimum maximum mean se cv (%) h’ days to 50% flowering (n) 75 140 112.2 0.70 10.8 0.870 days to 80% maturity (n) 122 174 154.4 0.59 6.6 0.871 plant height (cm) 61 119 95.4 0.44 8.0 0.864 tillers per hill (n) 2.5 5.9 4.4 0.03 11.1 0.898 flag leaf length (cm) 22 32 27.2 0.10 6.2 0.880 flag leaf width (cm) 0.5 1.2 0.91 0.01 9.7 0.906 flag leaf sheath length (cm) 11 22 15.1 0.08 8.7 0.872 ear exsertion (cm) 7.2 14 10.8 0.07 10.7 0.899 ear head length (cm) 4.5 8.8 6.2 0.04 11.0 0.880 ear head width (cm) 3.0 6.5 4.2 0.03 13.3 0.867 fingers per head (n) 4.1 8.9 7.0 0.04 9.4 0.878 length of the longest finger (cm) 4.0 9.8 5.9 0.06 16.9 0.875 width of the longest finger (cm) 0.48 0.93 0.68 0.005 11.9 0.902 weight per head (g) 3.1 10 5.9 0.06 17.6 0.897 weight of 1,000 grains (g) 1.8 3.2 2.3 0.01 9.5 0.882 grain yield (kg/ha) 230 3,494 1,577 30.3 33.3 0.884 straw yield (t/ha) 2.0 20.2 9.3 0.17 30.7 0.907 table 3. number of accessions and characteristics of each cluster in comparison with population mean. sd, standard deviation; *, significant (p = 0.01–0.05) and **, highly significant (p ≤ 0.001) difference with overall mean. trait cluster 1 cluster 2 cluster 3 cluster 4 overall mean sd mean sd mean sd mean sd mean sd number of accessions 16 66 107 111 300 days to 50% flowering (n) 85** 6.56 101** 6.13 122** 7.14 116** 6.39 112 12.1 days to 80% maturity (n) 132** 5.86 145** 6.01 161** 5.53 158** 5.71 154 10.2 plant height (cm) 78** 8.92 – – 93** 5.90 100** 5.69 95.4 7.6 tillers per hill (n) – – – – 4.2** 0.44 4.5** 0.45 4.4 0.49 flag leaf length (cm) 24.4** 1.07 26.8* 1.46 – – 27.9** 1.44 27.2 1.68 flag leaf width (cm) – – – – – – – – 0.91 0.09 flag leaf sheath length(cm) 13.8** 0.91 16.0** 1.18 14.4** 1.17 – – 15.1 1.31 ear exsertion (cm) 10.1* 1.18 – – 10.3** 1.08 11.2** 1.10 10.8 1.15 ear head length (cm) 5.5** 0.45 5.8** 0.53 – – 6.6** 0.63 6.2 0.68 ear head width (cm) 3.8** 0.56 4.0** 0.48 – – 4.4** 0.50 4.2 0.56 fingers per head (n) 5.6** 0.88 6.8** 0.46 7.3** 0.52 – – 7.0 0.66 length of longest finger (cm) 5.0** 0.70 5.4** 0.80 – – 6.6** 0.96 5.9 1.01 width of longest finger (cm) – – – – 0.67* 0.09 – – 0.68 0.08 weight per head (g) 4.5** 0.72 5.6** 0.80 5.6** 0.84 6.5** 0.96 5.9 1.03 weight of 1,000 grains (g) – – – – 2.2** 0.19 2.4** 0.22 2.3 0.22 grain yield (kg/ha) 1,181** 379.8 1,756** 359.5 1,147** 385.1 1,858** 473.9 1,577 524.4 straw yield (t/ha) 4.9** 2.22 7.3** 1.98 10.1** 2.35 10.6** 2.42 9.3 2.85 discussion genetically diverse accessions conserved in ex situ genebanks are tremendous genetic resources for breeding high-yielding and stable crop varieties to ensure global food security. enormous morphological and genetic diversity exists among finger millet accessions but their utilization in breeding programmes is very weak in most countries including nepal because this crop has received very little attention for characterization, evaluation and pre-breeding activities. only about 10% of genetic resources including finger millet stored in genebanks have been utilized in crop improvement programmes, which is mainly due to a lack of information about the desirable accessions resulting from the poor characterization and evaluation data (hodgkin et al, 2003; nguyen and norton, 2020). although nagrc holds nearly 1,000 finger millet accessions, we characterized only 300 accessions which might not represent the total genetic diversity of the genetic resources (2023), 4 (8), 1–14 nepalese finger millet diversity 7 table 4. eigenvalues and eigenvectors under five principal components (pc) for entry means and cluster means. eigen analysis entry means cluster means pc-1 pc-2 pc-3 pc-4 pc-5 pc-1 pc-2 pc-3 pc-4 pc-5 eigenvalue 3.763 2.691 1.461 1.237 0.998 10.28 4.79 1.93 0 0 proportion 0.226 0.149 0.091 0.083 0.069 0.605 0.282 0.113 0 0 cumulative (%) 22.6 37.5 46.6 54.9 61.8 60.5 88.7 100 100 100 eigenvectors days to 50% flowering 0.401 0.314 -0.044 -0.057 0.088 0.296 -0.114 0.141 0.083 -0.081 days to 80% maturity 0.421 0.267 -0.046 -0.086 0.044 0.300 -0.099 0.116 0.057 -0.454 plant height 0.280 -0.313 -0.214 0.065 0.166 0.279 0.189 0.124 0.042 0.308 tillers per hill 0.032 -0.255 0.103 -0.383 0.091 -0.123 0.419 -0.032 0.148 -0.007 flag leaf length 0.255 -0.102 -0.318 0.206 -0.343 0.304 0.079 0.097 -0.005 -0.256 flag leaf width -0.014 -0.022 -0.142 -0.442 -0.780 -0.130 0.007 0.654 -0.054 -0.001 flag leaf sheath length -0.021 -0.389 -0.283 0.042 0.147 0.147 0.392 0.150 0.149 0.305 ear exsertion 0.124 -0.256 -0.124 0.396 -0.136 0.260 0.252 0.034 0.177 -0.456 ear head length 0.290 -0.145 0.456 -0.017 -0.005 0.274 0.022 -0.343 -0.068 -0.168 ear head width 0.229 -0.006 0.258 0.206 -0.343 0.266 -0.230 -0.101 -0.102 0.298 fingers per head 0.303 0.118 -0.340 -0.132 0.101 0.294 0.015 0.240 -0.090 -0.071 length of longest finger 0.295 -0.094 0.468 -0.100 0.070 0.275 -0.065 -0.322 0.025 0.039 width of longest finger -0.010 -0.087 0.218 0.519 -0.200 -0.098 0.340 -0.425 0.197 -0.023 weight per head 0.252 -0.218 -0.088 -0.159 0.095 0.297 0.141 0.013 -0.030 0.327 weight of 1,000 grains 0.041 -0.278 0.220 -0.255 -0.074 -0.124 0.412 0.122 0.287 -0.088 grain yield 0.073 -0.496 -0.061 -0.048 0.036 0.131 0.414 -0.009 -0.776 0.016 straw yield 0.338 0.131 -0.073 0.097 -0.010 0.305 -0.089 0.066 0.403 0.295 table 5. correlation coefficients among grain yield and other associated quantitative traits in finger millet based on combined mean data. fd, days to 50% flowering; md, days to 80% maturity; ph, plant height; t/p, number of tillers per plant; ll, flag leaf length; lw, flag leaf width; sl, flag leaf sheath length; ee, ear exsertion; el, ear length; ew, ear width; f/h, number of fingers per head; fl, length of longest finger; fw, width of longest finger; w/h, weight per head; tw, 1000-grain weight; gy, grain yield; sy, straw yield; **, significant at 1% level; *, significant at 5% level. trait fd md ph t/p ll lw sl ee el ew f/h fl fw w/h tw gy md 0.93** ph 0.21** 0.26** t/p –0.06 –0.06 0.11 ll 0.27** 0.32** 0.33** 0.01 lw –0.03 0.04 0.02 0.02 0.07 sl –0.26** –0.25** 0.33** 0.05 0.16** –0.01 ee –0.04 –0.06 0.33** 0.04 0.20** –0.05 0.21** el 0.24** 0.29** 0.30** 0.10 0.13* 0.00 –0.03 0.10 ew 0.25** 0.26** 0.10 0.05 0.22** 0.00 –0.14* 0.09 0.37** f/h 0.53** 0.50** 0.31** 0.00 0.28** 0.02 0.03 0.02 0.14* 0.17** fl 0.34** 0.38** 0.29** 0.10 0.11 0.01 –0.04 0.06 0.63** 0.27** 0.11 fw –0.08 –0.09 0.01 –0.01 0.05 –0.10 0.01 0.06 0.07 0.06 –0.10 0.00 w/h 0.20** 0.23** 0.39** 0.17** 0.22** 0.05 0.07 0.12* 0.32** 0.12* 0.27** 0.24** 0.02 tw –0.08 –0.06 0.10 0.20 0.04 0.08 0.12* 0.02 0.12* 0.04 –0.18** 0.15* 0.04 0.16* gy –0.26** –0.13* 0.46** 0.33** 0.23** 0.09 0.35** 0.23** 0.19** 0.02 –0.03 0.16* 0.10 0.30** 0.36** sy 0.59** 0.58** 0.25** –0.03 0.30** –0.03 –0.13* 0.13* 0.21** 0.15* 0.26** 0.28** 0.00 0.21** 0.04 –0.01 8 ghimire et al genetic resources (2023), 4 (8), 1–14 figure 3. upgma hierarchical clustering divided 300 finger millet accessions into four clusters. landraces are coded with their accession number whereas released varieties are with their name. country. shannon-weaver diversity index (h’) considers both richness and evenness of the phenotypic classes of the qualitative traits but emphasizes the normality in observation of quantitative traits (ghimire et al, 2018b; upadhyaya et al, 2010). we estimated as low, medium and high diversity if the values of h’ were ≤ 0.400, 0.401–0.600 and ≥ 0.601, respectively (eticha et al, 2005). very low h’ for grain shattering and finger branching was observed since > 98% of the accessions didn’t have shattering type of grains and branching type of fingers. according to the findings of dasanayaka and kaluthanthri (2017), a very small proportion of sri lankan finger millet accessions exhibited shattering and finger branching traits. the rest of the qualitative traits (h’ = 0.645–0.908) as well as all 17 quantitative traits (h’ = 0.864–0.907) showed very high polymorphism. similar diversity indices were reported in the entire global collections of 5,940 accessions (3,567 from 14 african countries, 2,163 from five south-asian countries, seven from the usa, 22 from three european countries and 181 from unknown origin), a core collection of 622 accessions and a mini-core collection of 80 accessions (upadhyaya et al, 2006, 2010) as well as in an east african collection of 1,993 accessions (reddy et al, 2009). phenotypic proportions of qualitative traits were calculated as the frequencies of each descriptor state for the traits. observations on ear shape with droopy, open, compact and semi-compact suggested the presence of all cultivated races (elongata, plana, compacta and vulgaris) of e. coracana ssp. coracana (upadhyaya et al, 2006; bharathi, 2011; sood et al, 2019; backiyalakshmi et al, 2021). our observations for other qualitative traits were similar as in core and minigenetic resources (2023), 4 (8), 1–14 nepalese finger millet diversity 9 figure 4. scree plot (a) and loading plot (b) of pc-1 and pc-2 for 300 finger millet accessions based on cluster means, showing association between the traits. ew, ear head width; fd, days to flowering; md, days to maturity; sy, straw yield; fl, length of the longest finger; f/h, fingers per head; el, ear head length; ll, flag leaf length; w/h, weight per head; pht, plant height; ee, ear exsertion; sl, flag leaf sheath length; gy, grain yield; fw, width of the longest finger; t/p, tillers per plant; tw, weight of 1,000 grains; lw, flag leaf width. figure 5. scatter plot of the first two dimensions of principal component analysis using entry means for 300 finger millet accessions. landraces are coded with their accession number whereas released varieties are with their name. cluster centroid in respective cluster is indicated with larger symbol. the representative accessions of each cluster are indicated with accession numbers and are colour-coded as in figure 3. 10 ghimire et al genetic resources (2023), 4 (8), 1–14 table 6. promising finger millet trait donors selected based on combined mean data of six environments. accessions with multiple promising traits are highlighted in bold. trait promising accessions days to flowering (< 90 d) ngrc03581, ngrc03540, ngrc03502, ngrc01516, ngrc01489, ngrc03635, ngrc03636, ngrc03539, ngrc06503, ngrc06485, ngrc03650 days to maturity (> 130 d) ngrc03502, ngrc03540, ngrc01489, ngrc03581, ngrc03636, ngrc03539, ngrc06503 plant height (< 70cm) ngrc06503, ngrc03502, ngrc03639, ngrc04814 plant height (> 110cm) dalle-1, ngrc04871, ngrc03511, ngrc04818, ngrc05764 number of tillers/plant (> 5.4) ngrc06490, kabre kodo-2, ngrc04852, ngrc01609, ngrc03579, ngrc05739 flag leaf length (> 30cm) ngrc05109, ngrc01490, ngrc03605, ngrc04852, ngrc06493, kabre kodo-1, ngrc03678, ngrc03690, ngrc04746, ngrc01401 flag leaf sheath length (> 17.5cm) ngrc01655, ngrc04871, ngrc04724, ngrc06498, ngrc04818, ngrc03528, ngrc06504, ngrc04863 ear exsertion (> 14cm) ngrc06493, ngrc01527, ngrc01610, ngrc03693 ear length (> 7.5cm) ngrc04871, ngrc01406, ngrc04821, ngrc01447, ngrc04817, ngrc01458, ngrc01609, ngrc01451 weight per head (> 8g) ngrc04818, dalle-1, ngrc04804, ngrc01446, ngrc04806, ngrc01401, ngrc01639, ngrc01487 1000-grain weight (> 2.8g) kabre kodo-2, ngrc04816, ngrc04849, ngrc01418, ngrc04873, ngrc04850, ngrc04824, ngrc04871, ngrc04818 grain yield (> 2,769kg/ha) ngrc04849, ngrc06490, ngrc04871, kabre kodo-2, ngrc06487, ngrc04727, ngrc04836, ngrc04806, ngrc04818 straw yield (> 13t/ha) kabre kodo-1, ngrc01456, ngrc01452, okhle-1, ngrc01455, ngrc01539, ngrc05758 core collections of global accessions (upadhyaya et al, 2010), in sri lankan accessions (dasanayaka and kaluthanthri, 2017; kumari et al, 2018), in north-west indian accessions (kumar et al, 2019) and in global collections from icrisat (malambane and jaisil, 2015). descriptive statistics such as range, mean and cv of various quantitative variables suggested wide variability within 300 accessions of the nepalese collection. mean flowering data ranged from 75 to 140 days which was narrower than in the global collections which included wild species (41–164 days) (bharathi, 2011) but wider than in the core collection (51–96 days), the minicore subsets (51–93 days) (upadhyaya et al, 2010) and in a global collection of 314 accessions (51–97 days) (backiyalakshmi et al, 2021). since our research was conducted at higher altitudes, we observed mean flowering time of 112 days (pooled mean of three sites) which is higher than in studies conducted at lower elevations, which reported 79 days (bhattarai et al, 2014), 89 days (bastola et al, 2015) and 74 days (backiyalakshmi et al, 2021). our range for average plant height (61–119cm) was narrower compared to the range of 23–155cm observed in a much larger sample (537) of nepalese accessions (bhattarai et al, 2014), 55–240cm in east african accessions (reddy et al, 2009), 84–143cm in a global collection (backiyalakshmi et al, 2021) and 45–180cm in a global composite collection of 1,000 accessions (bharathi, 2011), but wider than the range of 73–113cm in the mini-core collection (upadhyaya et al, 2010). principal component analysis partitions the total variation into components and measures how each component contributes to the total phenotypic variation. the important traits in the evaluation of our landraces are days to maturity and flowering, straw yield, fingers per head, finger length, plant height, grain yield and 1,000grain weight. the scatter plot (figure 5) suggested that pc-2 has efficiently divided the accessions based on grain yield. high-yielding landraces and all released varieties (shailung kodo-1, dalle-1, kabre kodo-1, kabre kodo-1 and okhle-1) were in the upper half of the scatter plot. clustering of observations gives the average of a variable in the cluster, the average of the variable for the whole data set, the associated standard deviations and the p-value to test the hypothesis: the cluster mean is equal to the overall mean. rejection of this hypothesis (p < 0.05) means there is significant difference between the clusters for multiple quantitative traits. the representative genotypes of each cluster illustrate the overall characters of that cluster. pearson’s correlation coefficient suggested that grain yield is positively correlated with plant height, tillers, leaf length, ear exsertion, ear and finger length, ear weight and 1,000-grain weight. this suggests that our focus when selecting landraces for grain yield should be on accessions with early flowering, taller height, high tillering, big ears and bold grains. previous findings suggested a positive correlation between grain yield vs. days to flowering and maturity (bharathi, 2011; lule et al, 2012; bastola et al, 2015; patel et al, 2017), but we observed contrasting results i.e. negative correlation of grain yield with flowering and maturity days. this can be explained because the early flowering genotypes escaped cold stress in higher altitudes like jumla where many late maturing accessions yielded genetic resources (2023), 4 (8), 1–14 nepalese finger millet diversity 11 zero due to susceptibility to low temperatures. early maturity is, therefore, a very important trait for mountain farmers cultivating millets in dry and rainfed conditions to escape drought as well as cold stress during the grain-filling stage. straw yield had a strong positive association with days to flowering, maturity, plant height, leaf length, ear length, finger length, ear weight and number of fingers which is in support of the findings of bharathi (2011); bastola et al (2015); patel et al (2017); backiyalakshmi et al (2021). this means the accessions with taller plant height and late maturity produced higher straw yield which is a very important trait for farmers who are growing livestock in their farm and use millet straw for feed. although finger millet is the fourth most important cereal in nepal, its cultivation area has been shrinking over the last decade (moald, 2022). one of the reasons behind this is the limited options of highyielding varieties accessible to farmers. average grain yield in our research ranged from 230–3,494kg/ha but 15 accessions produced an average grain yield of more than 2,500kg/ha, which is comparable to commercial varieties. research conducted by anuradha et al (2022) on indian genotypes and by wolie and belete (2012) on ethiopian genotypes also reported some promising genotypes with more than 3,000kg/ha grain yield. since nepal is a member of the international treaty on plant genetic resources for food and agriculture (itpgrfa), exotic high-yielding genetic resources through the multilateral system could be introduced and used in crossings between asian and african genotypes to develop high-yielding lines exploiting the wide genetic variability to improve finger millet productivity of the country. conclusion we observed wide phenotypic variation within nepalese finger millet landraces conserved at nagrc for various qualitative and quantitative traits. we identified landraces with high potential for functional agronomic traits such as higher grain and straw yield, early maturity, bold grains, which could be utilized as trait-specific donors in paving the breeding pathway of finger millet. these selected landraces are currently being tested under national coordinated varietal trials by hcrp throughout the country. furthermore, these landraces could be deployed to similar hilly environments without further delay to enrich the varietal options for finger millet-growing farmers of nepal. acknowledgements this research was funded by unep/gef and technically coordinated by the alliance of bioversity international and ciat. we acknowledge the logistic support received from nagrc lalitpur, hcrp dolakha and ars jumla for the field experiments. authors’ contribution khg conceived the idea, conducted the field experiments, analyzed the data and drafted the manuscript. all co-authors guided in the field experiment and contributed to writing and revising the manuscript. conflict of interest we declare that there is no conflict o f i nterest among authors regarding this work. supplemental data supplemental table 1. list of the 300 finger millet accessions used in the study with their local names and collection sites. the accessions with asterisk (*) are released varieties whereas others are landraces. supplemental table 2. entry means of 300 finger millet accessions for 17 quantitative traits (combined over 6 environments). the accessions with asterisk (*) are released varieties whereas others are 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(2012). genetic divergence and variability studies in some ethiopian finger millet germplasm collections. scholarly j. agric. sci 3(4), 110–116. url: https://www.researchgate.net/ publication/315892606. https://doi.org/10.1007/s13237-020-00322-3 https://www.genstat.co.uk/ https://www.researchgate.net/publication/315892606 https://www.researchgate.net/publication/315892606 introduction materials and methods plant materials and experimental sites general methodology data recording data analysis results diversity index and frequency distribution of qualitative traits descriptive statistics and diversity indices of quantitative traits clustering observations principal component analysis correlation between traits promising landraces discussion conclusion acknowledgements authors' contribution conflict of interest supplemental data genebank report genetic resources (2025), (s2), 49–57 doi: 10.46265/genresj.ebbb2856 https://www.genresj.org issn: 2708-3764 the svalbard global seed vault – conserving plant genetic resources for european and global food security åsmund asdal * nordgen, alnarp, sweden abstract: the svalbard global seed vault is the largest safety backup of the world´s crop diversity. it offers safe storage for duplicates of seed samples conserved in genebanks worldwide. since its opening in 2008, 123 institutes located in 87 different countries have deposited 1,331,458 seed samples of 6,297 crop and crop wild relative species in the seed vault. european institutes have very actively taken advantage of the opportunity to back up their genetic seed material. as of today, 38 institutes located in 30 different european countries have deposited 178,999 seed samples in the seed vault. details about seed samples, depositors and species are publicly available through the seed portal website. the seed samples that are deposited in the seed vault remain the property of the depositing institute. the germplasm is at their disposal if they should need it, e.g. if the material in their home collections is damaged or inaccessible. the seed vault has already proved its value and importance for securing plant genetic resources when the international center for agricultural research in the dry areas (icarda), formerly having its headquarters and genebank in aleppo, syria had to relocate their genebank activities, due to the syrian civil war. deposited seeds withdrawn from the seed vault formed the basis for establishing new genebank functions at icarda units in morocco and lebanon. keywords: svalbard global seed vault, plant genetic resources, genebank, ex situ conservation citation: asdal, å. (2025). the svalbard global seed vault – conserving plant genetic resources for european and global food security. genetic resources (s2), 49–57. doi: 10.46265/genresj.ebbb2856. © copyright 2025 the authors. this is an open access article distributed under the terms of the creative commons attribution license (cc by 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. rationale and history plant genetic resources (pgr) are the raw material needed for the development of improved crop plant varieties, and therefore a precondition for securing future food supplies. plant genetic material is conserved and made available for research and plant breeding by national, regional or international genebanks and other institutions and organizations holding plant and seed collections (sanchez et al, 2023). the first genebanks for seed conservation were established as early as in the 1920s, but most countries established their genebanks or genetic resource centres from the 1960s onwards. the nordic genebank (ngb) for plants was established in 1979 as a regional genebank for the five nordic countries (denmark, ∗corresponding author: åsmund asdal (asmund.asdal@nordgen.org) finland, iceland, norway and sweden). ngb was merged with nordic programmes for domestic animal genetic resources (angr) and forest genetic resources (fgr) in 2008, into the nordic genetic resource centre (nordgen) located at the swedish agricultural university in alnarp campus outside malmö in sweden. many genebanks are vulnerable to different types of natural and man-made disasters, war and conflicts or simply to lack of resources, and over the years valuable genetic material has been lost. insurance policies are needed to ensure their protection (asdal, 2021). the simplest and best way of securing this kind of material is to ensure the conservation of duplicates in storage facilities located in other geographical locations. the further away, the lower the risk for the same accident, natural disaster, political unrest etc. to occur both at the genebank and at the deposit location. considerations along these lines were the basis for the nordic genebank decision in 1984 to begin backing received: 31.10.2024 accepted: 20.01.2025 published online: 12.02.2025 https://www.genresj.org https://www.doi.org/10.46265/genresj.ebbb2856 https://www.genresj.org https://www.doi.org/10.46265/genresj.ebbb2856 mailto:asmund.asdal@nordgen.org 50 asdal genetic resources (2025), (s2), 49–57 up the nordic seed collection in an abandoned part of a coal mine in the svalbard islands (figure 1), an arctic archipelago administered by norway, halfway between the mainland and the north pole (asdal (2021), figure 2). the coal mine offered stable permafrost (i.e. permanently frozen ground) between -3 and 4◦c that secured the freezing of seeds without any cooling equipment, energy supplies or even regular surveillance (asdal, 2021). over the years, around 13,000 seed samples of important nordic agricultural and horticultural crops were placed in the coal mine and stayed there until the seed vault opened in 2008. this low-tech and rather cheap solution gained extensive attention among genebanks and institutions committed to the conservation and use of pgr worldwide, fao among these. the idea of storing backup copies of seed collections in a similar type of setting also from other national and international genebanks was launched quite soon and discussed and elaborated further through the 1990s (qvenild, 2005). when the international treaty on plant genetic resources for food and agriculture (itpgrfa) came into force in 2004, the international framework for this kind of international cooperation became established and discussions about a global seed storage facility in svalbard were revived. a report was commissioned by the norwegian ministry of foreign affairs to investigate the feasibility of establishing an international seed facility in svalbard based on political, legal, technical and practical considerations (fowler et al, 2004). based on the recommendation of this report, norway presented the offer to build and manage a global seed vault in svalbard at the fao commission on genetic resources for food and agriculture (cgrfa) meeting in rome, italy in 2004. the offer comprised secure black-box storage of backup seed samples of genebank collections conserved in optimal storage conditions at 18◦c. black-box conservation in this case implies that the deposited seed samples remain the property of the depositing genebank, the seed material is conserved in sealed boxes and the material can only be returned to the owner if needed. after comprehensive international support, orally expressed already in the cgrfa meeting, the seed vault was funded and built by the norwegian government in 2007 (asdal, 2021) (figure 3). the broad support also materialized in the fact that many genebanks quite quickly began to prepare seeds for the opening event. already at the seed vault opening on the 26 february 2008, 19 genebanks deposited 237,106 seed samples, which was a significant manifestation of the international commitment to increase the security of pgr and an acknowledgement of the role of the svalbard global seed vault. the main objective of the svalbard global seed vault is to conserve security duplicates of unique seed samples that are conserved in regular genebanks. over the years, the seed vault has also become a strong and iconic symbol of the importance of conserving pgr, and it is now actively used in public and media outreach to increase public awareness about the importance of pgr conservation and use. this is illustrated by the fact that the seed vault today is, even when the interior is closed to visitors, one of the top tourist destinations in svalbard. building the svalbard global seed vault fits in a longterm norwegian commitment to support international efforts and projects on conserving biological and genetic diversity. svalbard is and will, despite severe climate change in the arctic, also in the future be a cold place suitable for frozen storage for seeds. together with good infrastructure and public services in svalbard and international confidence in norwegian management, svalbard appears to be an ideal location for a facility like this. ownership and cooperation the svalbard global seed vault is owned by norway and the norwegian government guarantees its continuous long-term management and conservation of the seeds. seed operations, i.e. anything related to the dispatching and depositing of the seeds, are managed in partnership between the norwegian ministry of agriculture and food, the global crop diversity trust and the nordic genetic resource centre (nordgen). communication with current and potential depositor genebanks, seed deposit routines and logistics are taken care of by nordgen while the crop trust supports seed multiplication and shipment from genebanks in developing countries. the three partners cover the management costs with larger parts of the budget coming from the ministry and crop trust. the three partners work together on increasing awareness of the svalbard global seed vault and encouraging more genebanks to back up their genetic resources in svalbard. information is disseminated on different platforms, through websites and social media, by conferences and online lectures, through genebank visits and media interviews. despite access inside the seed vault has been closed for all visitors due to security reasons since 2018, a broad diversity of visitors goes to svalbard on the occasions of seed vault openings when seeds are brought into the seed vault, including genebank representatives, scientists, politicians, international organization officials, journalists and tv teams. the seed vault operates in close connection with fao and its cgrfa and itpgrfa. information about the seed vault and its offer to secure genetic resources from genebanks worldwide is regularly conveyed in meetings and side events in fao bodies and itpgrfa governing body meetings. the seed vault mission is reported and presented in documents and reports from these organizations. the itpgrfa benefit-sharing fund has supported several projects aiming at securing seed collections in the vault. genetic resources (2025), (s2), 49–57 the svalbard global seed vault 51 figure 1. the location of the global seed vault and the location of the svalbard archipelago (map developed by claudio ballerini, using qgis). figure 2. the history of conserving seeds in permafrost started in 1984, when backup samples of the nordic gene bank seed collection were deposited in a steel container placed in an abandoned shaft of cole mine #3. the container is still in the mine, hosting some experimental seed samples while the backup collection has been moved to the seed vault. (photo: nordgen) 52 asdal genetic resources (2025), (s2), 49–57 figure 3. the svalbard global seed vault was constructed in 2007 and opened in february 2008. it is located on the hillside of the plateau mountain above longyearbyen airport, around 4km outside longyearbyen town centre. (photo: nordgen) the facility the seed vault consists of an outer so-called portal building, situated 130m above sea level, leading into an 80m-long tunnel ending in a large transverse hall with doors leading into three seed chambers. the total depth of the construction is about 130m, which includes the portal building, the tunnel, the transverse hall and the seed chambers, each 12m wide and 27m long. the seed chambers have between 40 and 60m of solid rock between the roof and the hillside surface (figure 4). natural permafrost in the ground provides between 3◦c and -5◦c. the seed chambers are artificially cooled down to -18◦c, which is the recommended temperature for long-term conservation of seeds in genebanks (fao, 2014). during the first years of operation, melting water in the hillside in the summer seasons and rainfalls caused water intrusion in the entrance tunnel. pumps were installed in the lower part of the tunnel to pump out the water. heavy rainfall in october 2016 caused more water to enter the tunnel than ever before. although the chambers containing the seeds were not affected by the water, the incident convinced the partners and the norwegian government of the need for a new watertight entrance tunnel. knowledge about expected increased temperatures in svalbard also contributed to the decision to construct an improved entrance tunnel. a new watertight tunnel was funded by the norwegian government and completed in 2019. in the same project, a new building for housing technical equipment such as ventilation and new cooling machines was constructed, and a range of security measures and surveillance systems were installed. the renewed seed vault is now well-prepared for all kinds of threats including climate change and possible hostile actions. warmer temperatures in the arctic will not threaten the security of the seeds in the seed vault but will require some more electric power to cool down the interior of the seed vault. each of the three seed chambers in the seed vault has the capacity to store 1,5 million seed samples, which is approximately twice the number of unique samples that are currently maintained globally in one or more genebanks (fao, 2010) (figure 5). however, genebanks are expected to expand their seed collections through new collecting missions aimed at securing crop wild relatives from natural habitats and farmer material in regions that so far have not yet been fully covered. in addition, research and plant breeding programmes will create new genetic material, and older varieties that are replaced will need to be conserved. nevertheless, the svalbard global seed vault will have the capacity to conserve duplicate samples of unique genebank accessions for several decades to come. over the years, the seed vault has probably become norway’s internationally most well-known building and, by that, contributed significantly to increased public awareness about the importance of pgr conservation and use. as one of the preconditions for storing seeds in the seed vault is that the safety-duplicated genetic material should be available for plant breeding and research from the depositing genebank, the seed vault genetic resources (2025), (s2), 49–57 the svalbard global seed vault 53 figure 4. this model drawing shows the different parts of the seed vault, from the concrete portal building, visible from the outside, via the tunnel, transverse hall and three seed chambers. (drawing: norwegian ministry of agriculture and food.) figure 5. deposited seed samples are conserved in ordinary warehouse shelves in three seed chambers. the first chamber was filled up in 2020, and the second chamber was taken into use. the third chamber has not been taken into use yet. (photo: riccardo gangale/the norwegian ministry of agriculture and food) mission may also indirectly contribute to keeping genetic resources in the public domain. seed deposit management the svalbard global seed vault offers a free-of-charge service to store duplicate samples of seeds that are conserved in genebanks worldwide. the seed vault is normally unstaffed, but nordgen organizes three socalled seed vault opening weeks each year to receive seed shipments for deposit in the vault. current and potential depositor genebanks are informed about the dates and encouraged to ship backup seed samples in advance of these occasions. on average, during the last few years around 12–15 genebanks have shipped seeds to the seed vault on each of these occasions. the seed samples deposited in the vault remain the property of the genebank. only the owner genebank has access to the seeds and can require seed boxes to be returned if needed. all genebanks making their genetic resources available for research, breeding and education are eligible to conserve backup copies of their seed collections in the seed vault. in addition, conditions for depositing seeds in the seed vault are that the genetic resource should be under long-term storage in a genebank and backed up in a second suitable genebank at another location, making the seed vault the second security backup (nordgen, 2024). guidelines for depositing seeds in the vault recommend that a safety duplicate should contain at least 500 viable seeds for outbreeders and heterogeneous accessions with high diversity and a minimum of 300 seeds for genetically uniform accessions. seeds for long-term storage must be well-dried and packed in watertight aluminium pouches according to the fao genebank standards (fao, 2014). seed pouches are packed in sealed 54 asdal genetic resources (2025), (s2), 49–57 boxes or sent in packages that are put in standard plastic boxes upon arrival. different box materials are accepted as long as they are robust enough to remain solid through the shipment process. genebanks should compile and submit an accession list of the seed material to be deposited 4–6 weeks ahead of the opening date and then ship the seeds, ensuring that the seed boxes or packages are received in longyearbyen, svalbard before the announced seed deposit event takes place. before deploying seed boxes in the seed vault, all sealed seed boxes are x-rayed for any unwanted and potentially harmful items inside the box. seed boxes are sealed and not opened when conserved in the seed vault. the depositing genebank is responsible for monitoring the quality of the seeds over time, normally by performing germination tests on seed samples of the same yield that are conserved in their home genebanks. depositors are also allowed to deposit boxes with test samples that can be returned if a genebank wants to test seeds that actually have been conserved in the seed vault for a shorter or longer time. when germination falls under the genebanks’ own threshold, new seeds must be produced and sent to svalbard. some genebanks have started to send newer samples of previously deposited accessions, coordinated with their plans for the multiplication of accessions for their own needs. when all accessions in a previously deposited box have been re-deposited, the whole box in question will be removed from the seed vault. seed vault holdings between the opening in 2008 and november 2024, 123 institutes located in 87 different countries have deposited 1,331,458 seed samples of 6,297 crop and crop wild relative species in the seed vault (figures 6 and 7). depositor genebank and institutes are international agricultural centres (iarcs), two regional genebanks (nordgen and spgrc in southern africa), many national genebanks and universities, a few ngos and so far, one private company. information about all depositing institutes is publicly available on the seed portal search page https://seedvault.nordgen.org/sear ch. about two-thirds of deposited samples are owned by iarcs and one-third by national genebanks including university collections (figure 8). as most genebanks conserve seeds originating from many countries, there are seeds originating from 229 different countries and territories in the seed vault. as genebanks tend to keep the original country name as the country of origin in their databases – even when countries split, merge or change names – former countries are also included in this large number. european depositing institutes are mainly national genebanks of different sizes and with different responsibilities regarding crop conservation. some countries have one centralized seed genebank while others have a more decentralized genebank system. as of november 2024, 193,660 seed samples of 1,914 different species originating from 53 countries and territories in europe were deposited in the seed vault. thirty-eight (38) institutes/genebanks located in 30 different european countries have deposited 178,999 seed samples in the seed vault. the seed portal database as mentioned, information about seed samples conserved in the seed vault is saved in a specially developed and designed database called the seed portal. genebanks provide accession lists with some basic data about all seed samples they want to deposit before each seed vault opening occasion. a template has been developed for this purpose, requiring information about accession number, scientific species name, country of origin, year of regeneration and number of seeds for each sample. accession lists are uploaded to the database, and a validation process detects typos, errors and invalid information. basic data is publicly available at the seed portal website https://seedvault.nordgen.org/. the first and only withdrawal of seeds the seed vault has already proven its value and importance for securing pgr when the international center for agricultural research in the dry areas (icarda), formerly having its headquarters and genebank in aleppo, syria had to relocate its genebank activities, due to the syrian civil war. seeds deposited from aleppo between 2008 and 2014 were withdrawn from the seed vault in 2015, 2017 and 2019 and formed the basis for establishing new genebank functions at icarda units in morocco and lebanon (westengen et al, 2020). concluding remarks the svalbard global seed vault is welcoming all genebanks to deposit backup samples of the collection in this very safe place in the arctic, and the ultimate goal is that all unique plant genetic material of orthodoxseeded species conserved in genebanks is secured and copied in the seed vault. in a troubled world, genebanks should implement safety measures for securing their genetic resources and conserving samples in more than one place is an easy solution. the icarda case illustrated that genebanks in relatively stable countries, which was the common assumption for syria up to the arabic spring, should ensure that backup samples are saved at one or more other locations. in addition, natural disasters and lack of resources can cause the loss of seeds and genetic resources in genebanks. the icarda case also showed that the concept of the seed vault set up for conservation and withdrawals of duplicate seed samples works well and meets the need for extra security for pgr. however, like any other insurance policy, the hope is that no genetic resources (2025), (s2), 49–57 the svalbard global seed vault 55 figure 6. graph showing the increase of seed vault holdings year by year from the opening in 2008 until the end of 2024. icarda withdrawals took place in 2015, 2017 and 2019, explaining a reduction or low increase in conserved numbers these years. figure 7. by the end of 2024, seed samples belonging to 6,297 species were conserved in the svalbard global seed vault. the graph shows the 25 genera having the highest numbers of samples conserved in the seed vault. three genera are represented with more than 100,000 samples; triticum (wheat): 268,353 samples, oryza (rice): 190,758 samples and hordeum (barley): 117,551 samples. 56 asdal genetic resources (2025), (s2), 49–57 figure 8. diagram showing proportions of seed samples deposited by five different categories of genebanks and institutes. further seed withdrawals from the seed vault will be needed. luckily, the 17-year story of the seed vault shows that many genebanks, along with international organizations and partners, acknowledge both the need for security measures for their genetic resources and appreciate the option provided by the seed vault. it is evident that the seed vault enjoys a high level of confidence among genebanks, ministries, scientists and other stakeholders. from the seed vault partners’ point of view, it is highly appreciated that the number of genebanks that deposit their valuable genetic material in the seed vault is increasing year by year. it is also very satisfying to observe that the global support of the seed vault mission is quite unanimous, also in the media. positive media coverage has undoubtedly contributed to increased public awareness about the importance of genetic resources and encouraged new genebanks to make plans for depositing seeds in the seed vault. it is also inspiring to see that, in a world marked by conflicts, securing pgr in the seed vault is still an international effort where countries in different parts of the world cooperate despite disagreements in other fields. the author of this article hopes that the seed vault mission can inspire and pave the way for peaceful international cooperation also in other sectors. acknowledgements a big thank you to the editor of this special issue on ex situ conservation of plant genetic resources in europe in the genetic resources journal filippo guzzon, for his patience and for contributing to significant improvements of the manuscript. thanks also for the comments from constructive reviewers. the author is also grateful to claudio ballerini for developing the map in figure 1. conflict of interest statement the author declares that there are no conflicts of interest references asdal, å. 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(2004). study to assess the feasibility of establishing a svalbard arctic seed depository for the international community prepared for the ministry of foreign affairs, center for international environment and development studies (noragric) at the agricultural university of norway and nordic gene bank. nordgen (2024). information to depositors. nordgen seed vault website. url: https: //www.nordgen.org/our-work/svalbard-globalhttps://www.fao.org/4/i1500e/i1500e.pdf https://www.fao.org/4/i1500e/i1500e.pdf https://www.fao.org/4/i3704e/i3704e.pdf https://www.fao.org/4/i3704e/i3704e.pdf https://www.nordgen.org/our-work/svalbard-global-seed-vault/information-for-depositors/ https://www.nordgen.org/our-work/svalbard-global-seed-vault/information-for-depositors/ https://www.nordgen.org/our-work/svalbard-global-seed-vault/information-for-depositors/ genetic resources (2025), (s2), 49–57 the svalbard global seed vault 57 seed-vault/information-for-depositors/. accessed date: 2024-12-17 qvenild, m. (2005). sowing seeds in permafrost: an idea whose time has come. master of science thesis. department of international environment and development studies, norwegian university of life sciences. url: http://www.umb.no/statisk/noragric/ publications/master/2006 marte qvenild.pdf. sanchez, d., sadoun, s. b., mary-huard, t., allier, a., moreau, l., and charcosset, a. (2023). improving the use of plant genetic resources to sustain breeding programs’ efficiency. pnas 120(14), 2205780119. doi: https://doi.org/10.1073/pnas.2205780119 westengen, o. t., lusty, c., and yazbek, m. (2020). safeguarding a global seed heritage from syria to svalbard. nat. plants 6, 1311–1317. doi: https://doi. org/10.1038/s41477-020-00802 https://www.nordgen.org/our-work/svalbard-global-seed-vault/information-for-depositors/ http://www.umb.no/statisk/noragric/publications/master/2006_marte_qvenild.pdf http://www.umb.no/statisk/noragric/publications/master/2006_marte_qvenild.pdf https://doi.org/10.1073/pnas.2205780119 https://doi.org/10.1038/s41477-020-00802 https://doi.org/10.1038/s41477-020-00802 rationale and history ownership and cooperation the facility seed deposit management seed vault holdings the seed portal database the first and only withdrawal of seeds concluding remarks acknowledgements conflict of interest statement original article genetic resources (2025), 6 (12), 211–220 doi: 10.46265/genresj.pegr2598 https://www.genresj.org issn: 2708-3764 received: 12.03.2025 | accepted: 03.09.2025 | published online: 12.12.2025 what farmers value matters for the management of local breeds: a case study of the pyrenean goat breed abstract: local breeds are often kept in farming systems where their locally adapted traits are an asset. the agroecological transition movement has brought renewed interest in these breeds with their locally adapted traits. better understanding the factors underpinning trait preferences of local-breed goat farmers can inform efforts to manage genetic diversity and animal selection. this article examines what goat farmers value in their animals to gain a better understanding of their decisions and expectations. based on evidence from 20 interviews with farmers using the local pyrenean goat breed, we show that although breeders attach importance to various animal performance factors, hardiness consistently stands out as a key value. we also show that they attach importance to other factors, such as animal diversity, temperament and appearance (breed standard and aesthetics). breeders relate to their animals in a way that mobilizes senses, feeling, experiential or emotional dimensions more than just a set of hard traits, and when breeders voice what they value and how that value manifests in practice, their narratives translate the importance of balance and trade-offs. keywords: local breeds, goats, france, hardiness, values citation: lauvie, a., nozières-petit, m.-o., thuault, f. and couix, n. (2025) “what farmers value matters for the management of local breeds: a case study of the pyrenean goat breed”, genetic resources, 6(12), pp. 211–220. doi: 10.46265/genresj. pegr2598. © copyright 2025 the authors. this is an open access article distributed under the terms of the creative commons attribution license (cc by 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. anne lauviea,*, marie-odile nozières-petita, fanny thuaultb,c, nathalie couixd,e a selmet, univ montpellier, cirad, inrae, institut agro, montpellier, france b association la chèvre de race pyrénéenne, foix, france c present affiliation: aredear occitanie, foix, france d agir, université de toulouse, inrae, castanet-tolosan, france e present affiliation: lessem, inrae, grenoble, france * corresponding author: anne lauvie (anne.lauvie@inrae.fr) introduction livestock selection must address new challenges around animal genetics to move the agroecological transition forward. the genetics community highlights the need to design breed selection programmes with specific objectives, such as resilience, relevant for livestock in transition toward agroecology. additionally, they should consider the reduced control over environmental conditions in agroecological farming systems (phocas et al, 2016). there is consequently growing interest in efforts to improve breed hardiness and integrate diverse objectives that combine a wider set of traits (for example, production-related traits with traits tied to reproduction, survival, health and welfare). animal genetics is experiencing a fast-paced change in methodologies, with the development of genomics soon to be followed by high-throughput genotyping and phenotyping workflows (boichard et al, 2015). local breeds, and especially those with smaller populations, have been relatively sidelined from selection programmes that have focused on dominant breeds. however, localbreed animals often thrived in farming systems such as agropastoralism, where their locally adapted traits and hardiness proved an asset (moulin and perucho, 2023). the agroecological transition movement has brought renewed interest in these breeds with their locally adapted traits (dumont et al, 2013). for any goat farm, deciding which breed or breeds to select as a starting point and deciding which individual animals to use to produce the next generation are pivotal decisions that shape and scaffold a coherent production system (vissac, 2002). https://doi.org/10.46265/genresj.pegr2598 https://www.genresj.org https://doi.org/10.46265/genresj.pegr2598 https://doi.org/10.46265/genresj.pegr2598 genetic resources (2025), 6(12), 211–220212 lauvie et al better understanding the factors underpinning these traitpreference decisions of local-breed farmers can valuably inform the work of geneticists who deliver the tools and strategies driving genetic selection programmes. what the farmers want and expect to see in their herds – in other words, what they value in each animal – is a key factor driving these decisions. however, beyond the market value of their products that fundamentally drives revenues, local breeds – more than other breeds – are attributed a variety of other values that reflect each breeder’s relationship with individual animals from that breed and with the breed itself (couix et al, 2023). we posit that examining these values can bring key insights to inform the design of genetic resources management programmes geared to address the challenges facing livestock farming today. this inquiry is especially relevant in the case of local and rare breeds investigated here, as they have so far remained sidelined from the big selection programmes but are now attracting renewed interest to help agroecological transition move forward. here we addressed these challenges by studying farmers’ objects of value around the local and rare pyrenean goat breed from south-western france. the pyrenean goat is a local breed that farmers have progressively abandoned. a pyrenean goat conservation organization was created in 2004, and pyrenean goat numbers today amount to just 5,000 animals kept by around 200 farmers, mostly in farms across the french pyrenees (pyrenean goat breed association, 2024a). in 2023, most of the farmers (207) were situated in the two regions concerned by the pyrenean mountain range, occitanie and nouvelleaquitaine regions. twenty-two flocks were located in other french regions (pyrenean goat breed association, 2024b). about a third of these farmers raise their flock for milk and cheese. the remaining two-thirds produce kid meat, often alongside other farm products or additional professional activities (‘pluriactivity farmers’). the pyrenean goat exhibits strong phenotypic variability, especially in terms of colour. a breed standard describing the defining traits was adopted in 2008 (pyrenean goat breed association, 2024a). adopting the same kind of pragmatist stance as couix et al (2023), we considered value formation as a series of what john dewey (1939) theorized as valuation processes, where the formation of values spans both the immediate valuing appraisal and the higher-level evaluation (bidet et al, 2011) and considers the dynamic dimension of values as they change over the course of time and in the course of action. this led us to examine both the way farmers appraise and qualify the animals they tend, and the related livestock farming practices. consequently, the aim of this article was to study what goat farmers value in their breed and in their animals. first, we present the approach adopted, which is based on analyzing evidence captured through interviews. we then show that the farmers voiced a diversity of objects of value that accommodate diverse biological and even non-biological traits beyond pure animal performance factors – and thus reveal a diversity of values. we then discuss how the diverse values attached to local-breed animals, driven by the farming system in which they are raised, can help inform strategies for addressing the challenges in managing farm-animal genetic resources. materials and methods theoretical background we based our work on 20 semi-structured interviews led in 2021 with pyrenean goat farmers. the objective of this analysis was to sift through their discourses to identify how the values they attach to their individual animals and the animal breed are formed (lauvie et al, 2022). here we mobilized john dewey’s theory of valuation (dewey, 1939), which posits that values are shaped by projected consequences of actions (the means) led – in this case, by the goat farmers – to bring about desirable outcomes (the ends). we therefore focused our attention on what farmers said they wanted and expected to see in their flock animals, in other words, what they valued in each animal. however, we went beyond simply surfacing the diversity of sources of value that emerged from farmers’ discourses, to focus on what they told us about how this diversity of values manifests into practice, especially when they connected their expectations of the animals to practices they employ to get a flock that suits them. we also attended to what they said about the responses they saw in the animals. john dewey (1939) argued that values are not immutable but always potentially open to revision as they can be made to change in the course of interactions between individuals and their environment. consequently, we also prepared to focus on the way farmers revised their objects of value in response to interactions with their animals. data collection in order to capture ‘what farmers value’ in their work with a local breed, we collected data using a process designed to capture the diversity of farming situations found in our case study. we selected the sample of farmers to cover various criteria. these included the diversity of geographical configurations, by choosing farmers in different locations, and both dairy systems and suckling systems. the sample included mainly pyrenean goat breed association members, as well as non-members and former members. additionally, it included breed-association board or committee members, and farmers with varying lengths of experience with pyrenean goats. the interview guide helped to collect focused data on the history of the goat farm, the reasons that prompted the farmer to choose the pyrenean goat and flock management system – particularly reproduction and culling/replacement – and the farmer’s relationship with the pyrenean goat breed association. a student (senior internship), under the supervision of the authors, performed 20 interviews: 11 with suckling-system farmers and 9 with dairy-system farmers. the vast majority of the farmers surveyed (i.e. 16 out of 20) were members of the pyrenean goat breed association. six of the interviews were attended by two people from the farm (goatherd, partner or associate) who conferred and spoke together. one of the interviews led into further exchange with a second (former) farmer (who worked on another farm) who joined the first interviewee. we counted these two exchanges as two separate interviews. all the interviews were recorded and transcribed, except one which was the object of a very detailed report. an interdisciplinary and transdisciplinary team supervised the student and later conducted the analysis presented in this paper (two researchers in animal sciences with a systemic genetic resources (2025), 6(12), 211–220 considering local breed farmers’ values 213 approach, one researcher in organization sciences and the person in charge of the facilitation and extension for the pyreanean goat breed association). data analysis we explored the qualitative interviews by coding the data using nvivo v11 software to develop themes. coding produced the six themes that feature in the node map in figure 1, and each of these six themes contained subthemes. here, we focused our analysis more specifically on the core theme tagged ‘objects of value in the animal’ and its nodemapped subthemes (see figure 1). we did not set out to work on speech as an object, but like macé, we considered spoken discourse as a space that operates connections and attachments and therefore warrants attentive analysis (macé, 2021). it is for this reason that here, unlike in most livestock science papers, we organized the presentation of our results around verbatim content. the original versions of those verbatim (in french) are available as supplemental material 1. results diverse considerations attached to animal performance factors farmers have varied expectations regarding milk yield the farmers who saw milk yield as an object of value in the animals referred to both milk quantity and milk quality. certain farmers mentioned milk protein yields or figure 1. node map featuring the six core themes identified and the subthemes identified specifically for theme no. 3. milk fat yields, whereas others referred to a more global, end-in-view criterion, i.e. cheese yield, which in some cases they connected to specific milk parameters and, therefore, to frames of evaluation involving other, non-yield-related criteria. one farmer, for example, talked about the acidity of their flock’s milk, while another – likely inspired by a previous study in the same breed – cited casein polymorphism in their flock’s milk, and several farmers spoke about the taste of the milk as a dimension of milk quality. still connected to milk production but going beyond quantitative and qualitative traits of milk output, some farmers stated that they attached importance to having goats that were easy to milk, which they sometimes connected to udder conformation, but sometimes not. looking at the process of evaluation for these objects of value, certain farmers talked about their own criteria, especially on milk quality, while others also mentioned the standardized national milk test records system called ‘contrôle laitier’ that some saw as a valuable tool for community-wide pyrenean goat breed conservation and management efforts. on a farm, they highlighted that the milk records tool provided benefits for the wider breed community as well as for their own personal management. lastly, certain interviewees explained the link between the performance of their animals and their flock management decisions: “this year, well, increasingly, but especially this year, maybe because we give alfalfa now, as we have an alfalfa plot, but it’s now 30–40 crottins [type of cheese] a day. with no weight differential. that’s a huge yield right there.” genetic resources (2025), 6(12), 211–220214 lauvie et al meat production: it’s not just average daily gain that counts farmers also emphasized the importance of factors related to kid meat production. these factors include size, weight, growth and conformation, which one farmer summed up as the need to have kids “that grow out well”. these size and conformation factors that work towards zootechnic performances were sometimes more or less explicitly verbalized as connected to the breed standard, which we discuss in further detail below. some farmers attached importance to more specific criteria, such as birth weight, minimum expectations on liveweight at a given age milestone, average daily gain (adg), or carcass weight. as seen above for milk yield, certain interviewees explained the link between the performance of their animals and their flock management decisions: “because if you care for your does, then the kid will automatically birth big, so you weigh it. it might weigh 5 kg at birth or 3 depending on how you’ve cared for the doe”. coherency between conformation and dairying: a challenge for a breed claimed as dual-purpose? the pyrenean goat farmers studied here have the option of running a dairy system or a suckling system, as the breed is considered dual-purpose. however, certain farmers mentioned that, in the past, the breed had to navigate periods of tension between the two production uses that proved challenging to manage as a community: “in fact, the buck committee1 was originally set up about 10 or 15 years ago when everyone in the breed association realized that divisions were emerging between the dairy farmers who only wanted to select for milk, and the suckling systems farmers who only wanted to select for type”. several farmers advanced a linkage here between kid health and/or growth and milk quality of the nursing does. for instance, one farmer, who farms their flock for milk and cheese, said: “kid growth […] is important [to consider] because if the mother is giving milk packed with protein, then i reckon the kid is also going to grow out a lot faster. so that’s another way [suckling system farmers] can look at it, even if they’re not dairying”. another farmer formulated the same idea in a different way: “but a dairy breed and a meat breed are just the same thing. because goats that produce the most meat also give the most milk. that’s just common sense.” 1 group of farmers within the breed conservation association that sets and selects breeding bucks prolificacy: a flock trait framed by the system the last category of animal-factor performances that farmers discussed is prolificacy of the flock, which is a trait one farmer actively worked on: “for does farmed 100% free-range and browsing a ‘nutrient-poor’ environment […] prolificacy isn’t even two. so, for me, if there was something to improve my production, it would be about that, some improvement in prolificacy”. however, high prolificacy is not always a preference, and farmers are conscious that it comes with limits: “we have a crazy-high prolificacy rate, which has downsides too, because we end up with triplets. we’ve had triplets making up 30% of the herd, and that’s not great. […] it screws up the lactation cycles, the animals are exhausted, and then you have triplet kids weighing in at 4kg.” hardiness is valued as a constant a notion that accommodates a diversity of traits hardiness was a term widely used by the farmers interviewed, and for many, it was one of the features that led them to choose the breed, and sometimes even the first factor they mentioned. one farmer put it in these terms: “ah! hardiness! well, that’s what we like about the pyrenean goat, its hardiness. it can eat woody brush, and never really get sick. hardiness means goats that range and graze what they need, never getting sick, kept outside all the time – you just arrange to bring them in for kidding, and that’s pretty much it, that’s what we want.” when expanding on what they mean by hardiness, the farmers talked about a wide range of abilities: ability to range and use all vegetation resources available on the farm, ability to stay outside even in harsh weather, ability to mother their kids, ability to take care of their own needs, ability to tolerate parasitism problems and health problems in general, and so on. certain abilities, such as ranging widely and using all vegetation resources available on the farm, were more widely recognized among breeders. depending on farmers, various abilities were emphasized in different ways. a notion that pulls together animal–environment–herd management factors several farmers made a connection between their vision of hardiness and their animal management practices or even their livestock system performance goals, with one farmer asserting that: “it’s that opportunity to exploit that hardiness, with prospects for saving on costs, that ultimately pushed me to look for the friction point that would mean it ends up making economic sense”. genetic resources (2025), 6(12), 211–220 considering local breed farmers’ values 215 some farmers talked about the benefit of finding a compromise between animal-factor performances and hardiness, as illustrated here: “obviously, you need your animals [...] to give you enough milk to get by, to make a livelihood, put food on the table. that’s your bottom line – the rest is just about finding the right compromise, the right balance. but for us it’s also super important to have well-adapted animals that will happily stay outside without any kind of problems”. certain interviewees also underlined that hardiness drives animal-factor performances. for example, one farmer made the connection between the ability to use specific resources and production performances: “it would be a hardy goat, that has learnt very young to range for all the food around in its environment, like woods, brush and wildland, and yet still manages to give you a goodish adg.” an important feature of this notion of hardiness is that it is tightly connected to how the animals are managed, such as free-ranging for example, or in resource-squeezed systems, and some farmers also make the connection to local range habitat, such as this farmer who explained: “the breed is adapted to our work conditions and the local topography, even if today […] it’s been raining nonstop for a month now, and there’s no other breed that could withstand the wet like she did, that’s for sure. plus, it’s a goat that uses little if any bought feed, so it’s pretty cheap to keep.” hardiness as all-round self-sufficient animals finally, in addition to underpinning a range of abilities and traits linked to farm-system conditions, the notion of hardiness was also articulated in more global approaches that refer to what might be described as self-sufficiency, i.e. the fact that the animals demand less human care and attention, and fend for themselves: “hardiness also means needing as little care from us as possible.” one of the farmers surveyed expanded on this idea by bringing the domestication factor into play: “that’s what it is: livestock that can... listen, i’d be exaggerating if i thought my goats could happily live without me, i don’t think they could, because there’s been some domestication. they are only there to collaborate with me. but my goats can stay outside without getting infested with parasites”. note that this self-sufficiency can also be identified as a specific trait in a given animal, in which case it makes that individual animal particularly valued. in one interview, when the interviewer and farmer were standing facing the flock and the farmer was asked which of the goats had the most merit, the farmer answered: “i think it’s the white one with the black neck. her name is [name of the goat]. in milk, hair, type, hardiness, easy, stood in the middle of the herd, never needing special care. she just blends in, so independent that... well, for me, that’s a good balance, a good compromise.” this self-sufficiency also overlaps into a degree of stubbornness, resistance or refusal to yield to human command – a point touched upon by a farmer: “it’s that part of hardiness that has its downsides. just try and separate the kids at birth, and see how far you get! nowhere! there’s just no way!”. on the same farm, the following testimony explained why they aim for and prize self-sufficiency: “like us, an animal will adapt to indoor comfort, but we just cannot keep them penned up indoors around the clock. it’s just impossible. they would turn violent. they’re too accustomed to being outside… […] we favour that. because they need to know how to go out and forage, find their own food. look, here’s your proof: we give them a yoghurt pot of barley, it’s there on offer, and they don’t even look at it. zero cereal feed”. note that in this case, the fact that the goats resist the farmers’ command is considered a valued trait. other objects of value beyond zootechnical traits diversity valued in and of itself while one farmer spoke of how milk yield in his flock was widely heterogeneous and how they wanted to bring about more homogeneity by removing the less milk-driven stock, most of the farmers who talked about intra-breed diversity approached the issue from the angle of working to conserve this genetic diversity: “otherwise, you are also impoverishing the gene pool. because if we keep pushing the population through a funnel, one day we’ll end up with clones” or again: “because i really liked those animals, plus i’m naturally for conservation and against losing stuff, like agricultural diversity and even biological diversity in general. [...] but there’s also the fact, when you’re working on a local breed, you have to be really careful not to bottleneck the population. which means you just can’t let any family die off. it’s our job to improve every goat – even poor ones. that’s part of what the conservation effort is about – taking a poor goat and improving it. sometimes it drags on, and sometimes nothing works. there are bad families, hopelessly bad families.  [….] that said, for me, there’s that overriding criterion of vital genetic variability, as you really can’t let any family disappear, because a breed that counts just 4,000 animals is a desperately vulnerable breed.” genetic resources (2025), 6(12), 211–220216 lauvie et al the farmer in that last example, like others interviewed, voiced the fact that they are ready to push less hard on animal performance criteria in order to conserve the breed’s genetic diversity. one farmer said they used to inbreed heavily, but got to a point where they needed to “look around elsewhere”. importance attached to the animal’s temperament the animal’s behaviour and temperament proved to be equally important objects of value, and the farmers often frame these traits in terms of the type of human–animal relationship they potentiate, whether positive or negative. this is seen in the words of one farmer, who said: “[at the] close of the season, we set away three goats, one of which, [name of a goat], is at practically 2l. we just can’t get along at milking. she’s a right bitch! […]. outside of milking, she’s great, but at milking, […] she just straight refuses.” another puts it in these terms: “as a rule, the one i like best is the one that communicates the most, the one you share most with, the one you call by first name.” these temperament factors can also be gain importance depending on the types of activities carried out on the farm. one farmer, for instance, highlighted how the goats play a doubly important role because they host visitors on his farm. the appearance factor: between breed standard and aesthetic preferences the farmers’ accounts featured a substantial number of references to the animal’s appearance, either in terms of the breed standard or ‘type’, i.e. the defining physical traits of the breed, or in terms of the aesthetics factor. note that several farmers employed the adjective ‘beautiful’ for both typerelated and aesthetics-related traits: “this year we’ve had several newborns that really are beautiful. when i say beautiful, i don’t just mean beautiful as in pretty to look at. i mean, they also show good conformation, real potential... in terms of breed standard and such.” farmers who explicitly mentioned the breed standard sometimes voiced the fact that it was co-constructed by collective agreement, and one interviewee distinguished a component based on personal criteria, on top of the other foundational criteria that had been set by the breed community: “that’s personal criteria, not pyrenean goat breed criteria.” note too that several farmers outlined how these breedstandard criteria or breed-type criteria are in flux and workable, and that they aim to improve that dimension in their own flocks. several farmers prized the aesthetics factor of the breed, as shown in this exchange: “and i just fell in love with the breed. instantly. the beautiful coat, the lovely long hair, the body form. plus the fact that it’s threatened with extinction – that was added motivation.” traits that farmers attach aesthetic importance to or say are a part of what makes the animals beautiful include not just the striking horn set and coat colouring (sometimes with a preference for a palette of colours) but also, for example, the ears, the hair, and the shape of the head or the legs. some statements also referenced an aesthetics of the goat roaming free outside. one farmer said: “they are prettier when they are ranging free, that’s where they are beautiful.” and another said: “outside they ’re just magnificent”. it is noteworthy that the role of the senses, feelings, the experiential and/or emotional dimensions emerge strongly in these accounts. what breeders value: how valuation processes involve more than just hard traits the experiential and emotional dimensions in livestock farming practice the previous paragraph, highlighting the aesthetics factor in goat farming, clearly reveals a prominent role of senses or feelings to the goat farmers’ decisions. the responses to questions addressing what farmers want and expect to see in their animals also expressed more holistic considerations tied to all-around satisfaction with goat farming. in other words, the farmers’ objects of value accommodate more than just a hard set of diverse biological traits, as illustrated in this exchange: “but what i expect from the goats is that everyone is happy – myself and the goats – and that we all get a decent living because, well, because they give good milk. because i try to make sure that they get the best life possible, and give us milk, and then we sell the cheese and we are happy with that.”  both the aesthetics factor and the considerations around overall satisfaction, as expressed in the interviews, carry an emotional dimension, and this emotional dimension will translate into how an animal population is managed. it is worth noting that the emotions expressed formed a subtheme of the ‘human-animal relationship’ theme. although this is not the focus of this paper, it highlights how emotions are connected to attachment to animals. in an illustrative example, one farmer said: “it’s the city; i don’t like it there and i miss my goats, so i don’t like being away from them for too long.” and one added: genetic resources (2025), 6(12), 211–220 considering local breed farmers’ values 217 “in time, you start to love these animals. there’s really something special about goat farming.” farmer narratives on balance and trade-offs finally, listening to the farmer's accounts of the broad diversity of features they value, a challenge emerged: how to integrate these diverse features, or in other words, how to find balance or manage trade-offs. the farmers’ narratives are effectively punctuated with stories that talk about balance and trade-offs. we identified several ways in which this is expressed that employ a number of terms, such as “package”, “rounded”, or “balance”: “you ask what i’m looking for in particular? it’s a combination, a package” or “when i bought her, she was an instant favourite. she was a goat that had an angular, bone-led frame with a big head, which matched exactly what i was looking for in the pyrenean. very very long hair. she only had one udder, but she had been bucks dam for several years in a row. she had so much, she gave so much, gave a lot of milk too. so for me, she counted among the goats that i’d call ‘rounded’. she was a real favourite. gentle, easy temperament – and she even lived to 16 years old.” “you learn pretty quick that a goat that’s giving 3–4l of milk puts everything into milk and forgets to invest in immunity. so you have to find the right balance.” how efforts to find balance and manage trade-offs manifest into practice this effort to find balance is explained through farmers’ statements on their selection decisions within the pyrenean goat breed population, or for renewal of their own herd. note that any trade-offs made will primarily depend on the perception of the focal traits and on whether selection can serve as a lever to achieving satisfactory goals. for example, one interviewee said they lend more importance to certain selection criteria on the grounds that other criteria, although important, are considered innate (i.e. inherently part of the breed’s make-up, and therefore not requiring active selection): “quite honestly, me, personally, i put type first: good legs, a big muzzle, good ears, hair. for me, type comes first. i know, it doesn’t sound logical, but... it’s because i think any pyrenean goat has good yields already, it’s innate”. in terms of ways to construct a compromise between traits to select for, the process can translate, for example, as (1) expressing a set of several traits to be co-selected together as a set, or (2) prioritizing criteria by first selecting for one and then selecting for others. the following farmer’s narrative illustrates, for instance, approach (1): “always looking for ways to continue selecting for both type and milk, without putting one ahead of the other. i really think you can manage to get both. you have to work on it, but you can get both.” this other farmer’s narrative is more illustrative of approach (2): “i’m in a performance recording programme, so obviously i look at growth. growth in my herd first. then i’ll adjust depending on maternal prolificacy, maternal hardiness.” discussion a diversity of farmers’ objects of value in animals the variety of farmers’ objects of value lead to a reconsideration of how animal diversity is characterized. multiple objects of value were often observed grouped together, which means that farmers’ concerns revolve around accommodating balances and trade-offs. this diversity also shows that while typical zootechnical categories, such as performances and abilities, may work for a number of elements that farmers wish to see, these categories fail to encompass the full range of elements valued. this insight prompted us to further explore the notion of ‘breed attribute’, as proposed in our previous research, to account for zootechnical characteristics of local-breed animals alongside characteristics connected to other dimensions, such as their importance for sustaining local cultures and communities (nozières-petit & lauvie, 2018; lauvie et al, 2023). although these attributes effectively connect to diverse functionalities provisioned by local-breed animals, here we found that this purely functionality-driven vision is too narrow, as our results showed that experiential or emotional dimensions may also come into play. the objects of value expressed by farmers are more than just ‘zootechnical’, reflecting a diverse set of potential reasons for choosing one animal or breed over another. farming livestock as a livelihood is also associated with other dimensions that carry meaning to farmers. from this perspective, the features of discourse that translate the relational dimension and the emotions involved warrant attentive investigation. there is a compelling rationale for developing research at the intersection of zootechnics and sociology or zootechnics and philosophy that looks at relational bonds between humans and farm animals (see porcher, 2001; despret and meuret, 2016). in a report on the results of her thesis research, porcher (2001) argues that affectivity is an integral part of working with livestock and that this affectivity component has been underconsidered in research on animal welfare (which was her original starting point). the object of related research has thus shifted towards the study of work, i.e. the study of how animals and humans work together. to refocus research on the management of farm-animal populations, a promising approach is to better account for human–animal relationships in the analysis of livestock farming practices, starting by looking at how breeders talk about and describe these relationships and the way these relationships interact with specific practices and contribute to the satisfaction breeders get out of their work. it is worth noting the example of a farmer who considered the occasional stubbornness of animals in his herd – when genetic resources (2025), 6(12), 211–220218 lauvie et al they refused to obey his command – as a positive trait. although this only emerged from one interview, it offers an insightful starting point for defining the perimeters (in terms of objects addressed), strategies and paradigms engaged by disciplines dealing with the management of genetic resources. this is because this kind of discourse challenges the traditional strategy. rather than projecting methodical control, it embraces the idea of ‘working with’ (composer avec in french, borrowing from the title of despret and meuret (2016) cited above), of ‘following/guiding nature’ rather than trying to control it (morin, 1980). more broadly, attending to what it means for humans to work with living beings would help address a question that is pivotal to the principles of agroecology (hubert, 2020). from a methodological perspective, one way to further pursue this research would be to produce the most accurate description possible of farmers’ practices, particularly in terms of decisions around which animals to retain in the herd, but also, more broadly, the relationships that form between farmers and animals at key points in livestock management. this methodological approach could usefully combine interviews tailored to capture these points with direct observation of practices. equally important would be a diachronic approach enabling specific attention to the way farmers effectuate changes in their practices (integrating their own analysis of the outcomes of their practices), in order to introduce a dynamic perspective where values are not seen as immutable. it is at this juncture that we address the formation of values as theorized by john dewey, and as stated by bidet et al (2011), who conceptualize valuations purely as behaviours that are situationally observable; consequently, a valuation cannot be reduced to a representation. pulling together everything that farmers value: selection as a lever in multidimensional and relational approaches as stated in the introduction, one of the key motivations for focusing on what farmers value in their herds was to inform fresh thought on managing genetic resources in farmanimal populations. however, our findings resonate strongly with the questions being raised in selection programmes for farm animals. one challenge identified for genetics is diversifying the objectives of selection programmes. this involves defining breeding objectives that balance production traits with functional abilities. such diversification is crucial for advancing the agroecological transition (phocas et al, 2016). this trend finds confirmation in the diversity of traits that the farmers surveyed considered important. at the same time, they emphasize the effort to find balance and trade-offs, which aligns with models that consider herd management and genetic selection together while examining trade-offs among multiple performances and abilities within a herd (see douhart, 2013). to complement these modelling approaches, characterizing how farmers translate this effort to find the right balance and trade-offs into practice could provide insight into the different ways these choices come together. furthermore, the strong links between animal traits and flock management, and between animal traits and flock environments, that emerged here in the farmers’ narratives are also in line with recent work by geneticists to better characterize livestock breeding environments and account for genotype–environment interactions (phocas et al, 2016; hazard et al, 2017). this observation argues for bridging disciplines to bring scientific communities together – the genetics community and the livestock farming system community, and even reaching out to ecologists and social scientists. mobilizing farmers’ field knowledge of their animals and their local farm habitats – and possibly even the ways they describe how these two factors, i.e. animals and habitats, intersect and interplay – also appears to be a promising direction for developing this research front. finally, diversity has also been identified as a pivotal issue and a key resource for making genetic selection better adapted to addressing agroecology challenges (phocas et al, 2016). however, the local-breed farmers studied here considered and valued such a diversity of dimensions that they cannot viably be reduced simply to a gene pool; therefore, it is important to consider the genetic dimension in conjunction with other dimensions of value embodied by local-breed animals. ducos et al (2021) stress that a contribution of animal genetics to the diversity principle of agroecology requires going beyond approaches based on the search for an optimal animal with calibrated performances for standardized and controlled breeding environments, and adopting a more systemic view. the convergence and complementarity between our findings and some of the central challenges for animal genetics highlight that the plurality of dimensions and relationships emerging from this work demonstrate how overly reductionist approaches fail to capture the full complexity of questions concerning the suitability of livestock animals to farming practices, environments and systems. we arrived at the idea that progress could be made by developing a relational approach that repositions genetics as one of several levers intersecting with livestock animals, farming practices and environments in the broadest sense, i.e. in their ecological dimensions, technical dimensions, social dimensions, and beyond. this would require even greater interdisciplinarity and transdisciplinarity, so that we move beyond simply devising selection programmes toward producing knowledge about a nexus of suitability between animals, habitats/environments, and the objectives breeders attach to their livestock practice. in this new approach, the lever of genetic selection would be reframed to articulate with other actionable levers by crossing boundaries to mobilize disciplines that develop systemic approaches, but also social sciences and the field knowledge of farmers and the wider stakeholder community involved in managing farm-animal populations. the type of approach envisioned here would aim to produce knowledge that is both more global (i.e. holistically including a diversity of knowledge sets and dimensions), situated (to address agroecology challenges) and operable (i.e. borrowing from practices that operate at animal, herd, and breed population scales). a further avenue for extending this work would be to attach greater importance to the observable valuation processes at work at the breed-society scale. indeed, the methodology developed in this article rests upon interviews and mainly focuses on individual values and valuation processes. the composition of our work team, built through a transdisciplinary process, included the facilitator of the association, who had good knowledge of the collective dynamics and discussions. this allowed us to check a certain consistency of the analysis. however, to better genetic resources (2025), 6(12), 211–220 considering local breed farmers’ values 219 tackle the collective dimension of valuation, complementary methodologies could be used, such as focus groups, to investigate how farmers talk about what they value in their animals when they discuss this theme together. participant observation of moments when collective decisions are made at the scale of the breed association could also be a relevant methodology. it would allow a better understanding of how those values are discussed (through which objects and themes, which kind of actions), and help identify around which values consensus is built (which play a role in holding the collective together). we take the stance that closer attention to situations involving collective management of animal populations that have been sidelined from major selection programmes is a potentially fertile approach to help identify configurations, relationships, questions and practices already at work, which could usefully inform processes of rethinking better ways to mobilize genetic resources – a challenge that is now high on the agenda in the field of genetic selection for both animal and plant species. conclusion this study aimed to describe the full diversity of what farmers value in their local-breed animals. this entry point enabled us to characterize a wide-ranging diversity of their objects of value spanning zootechnical performances, adaptive capacities, behavioural factors, self-sufficiency (requiring little human intervention), appearance and aesthetics, genetic diversity, and more. we also demonstrated the overarching importance of trade-offs and efforts to find balance among different traits. managing genetics to meet these expectations involves more dimensions than simply focusing on improving individual traits; it requires an integrated approach to livestock farming. this approach is situated, or, in other words, it takes into account the specificities of each situation, and it connects people with tools, practices and animals. supplemental data supplemental material 1. verbatim accounts from the interviews in french, and their translation into english, as reported in the article. author contributions conceptualization/design of the research questions: al, nc, monp, ft; conceptualization/theoretical framework: nc, al; methodology: al, nc, monp, ft; formal analysis: al, nc, monp, ft; supervision of the investigation (interviews conducted by a student): al, monp, ft, nc; writing – first version of the original draft: al; writing – completed original draft: al, nc, monp, ft; writing – review and editing: al, nc, monp, ft. acknowledgements the authors thank senior-year intern blaise dupuis for conducting the interviews that provided the material for this research. we also thank all the farmers who graciously set aside their time for these interviews. metaform langues translated the french draft of this paper into english. we thank the reviewers for their useful comments. this work was supported by the joint technology unit ‘umt pasto’ [‘resources and transformations for pastoral livestock farming in mediterranean regions’]. conflict of interest statement the authors declare no conflict of interest. ethics statement farmers gave permission to conduct the interviews for the purposes of this research, and were fully informed about its purposes. references bidet, a., quéré, l., truc, g. 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(2002). les vaches de la république: saisons et raisons d'un chercheur citoyen, inra editions. https://doi.org/10.46265/genresj.mrbt4299 https://doi.org/10.46265/genresj.mrbt4299 https://aoc.media/opinion/2021/01/28/parole-et-pollution/ https://aoc.media/opinion/2021/01/28/parole-et-pollution/ https://doi.org/10.1017/s1751731116000926 https://doi.org/10.1017/s1751731116000926 https://journals.openedition.org/ruralia/278 https://journals.openedition.org/ruralia/278 https://www.chevredespyrenees.org/ genebank report genetic resources (2025), (s2), 58–69 doi: 10.46265/genresj.evef5522 https://www.genresj.org issn: 2708-3764 vir: from a small bureau in the russian empire to the present-day national center for plant genetic resources igor g loskutov *, yulia v ukhatova and elena k khlestkina n.i. vavilov all-russian institute of plant genetic resources (vir), st. petersburg, russia abstract: in 2024, the all-russian institute of plant genetic resources (vir), the world’s oldest scientific institution dedicated to plant genetic resources (pgr), celebrates its 130th anniversary. founded as the bureau of applied botany in the late 19th century, vir has evolved into a globally recognized institute, currently also known as the national center for plant genetic resources. it preserves a collection of more than 320,000 unique accessions from 64 botanical families, 376 genera and 2,169 species. the institute’s mission includes the systematic collection, comprehensive study, long-term conservation and sustainable utilization of pgr. the foundation for most of vir’s research endeavours was laid by nikolay vavilov, a prominent botanist, geneticist, plant breeder and scientist with an encyclopedic scope of knowledge. a key strength of the institute has always been its extensive cooperation with global, regional and international organizations, as well as research institutes, universities and breeding centres in many countries around the world. today, as the national center for pgr, vir continues to build on this legacy. its efforts include the development of a legislative platform for genetic resources in russia and fostering international scientific cooperation on pgr, continuing the vision of nikolay vavilov. the purpose of this review is to show the milestones that marked the development of pgr studies and utilization in russia as well as to highlight some of the institute’s achievements since several conceptual approaches to these issues remain highly relevant. keywords: bureau of applied botany, vir, vavilov, collection, history, study, conservation, genetic resources, plants citation: loskutov, i. g., ukhatova, y. v., khlestkina, e. k. (2025). vir: from a small bureau in the russian empire to the present-day national center for plant genetic resources. genetic resources (s2), 58–69. doi: 10.46265/genresj.evef5522. © copyright 2025 the authors. this is an open access article distributed under the terms of the creative commons attribution license (cc by 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. introduction and historical events the bureau of applied botany was founded in 1894 in st. petersburg under the auspices of the scientific committee of the ministry of agriculture and state property, intended as an institution for collecting and studying the diversity of cultivated plants in the russian empire. all the measures towards its establishment were initiated and accomplished by prof. a.f. batalin and prof. i.p. borodin, who would both become the bureau’s first heads. the bureau made significant progress starting in 1900 with the arrival of r.e. regel, an expert in botany and agriculture. the most significant changes ∗corresponding author: igor g loskutov (i.loskutov@vir.nw.ru) occurred in 1905, when regel was elected as the new head of the bureau (loskutov, 1999). the main concrete outcome of the bureau’s activities under regel’s leadership was the collection, identification and description of the varietal diversity of cultivated plants grown in the russian empire. those efforts helped to restore the lost diversity of cereal crop varieties and populations – particularly malting barley – following the devastating droughts in the volga region. the racial, varietal and specific composition of local cultivars and populations was documented for wheat, barley, oats, rye, some legumes, vegetable crops and others (regel and proceedings on applied botany and breeding, 1915). the bureau’s plant studies resulted in identifying hereditary morphological and agronomic traits, performing successful crosses, and clarifying the genetic nature of some of those, following gregor received: 29.10.2024 accepted: 31.12.2024 published online: 14.02.2025 https://www.genresj.org https://www.doi.org/10.46265/genresj.evef5522 https://www.genresj.org https://www.doi.org/10.46265/genresj.evef5522 mailto:i.loskutov@vir.nw.ru genetic resources (2025), (s2), 58–69 vir: from a small bureau to national pgr center 59 mendel’s principles of genetic analysis. comprehensive research on plant collections data enabled the development of the bureau’s own original botanical classification systems for a number of priority crops, based on the studied morphological, anatomical, cytological, biochemical, immunological and agronomic plant characters (regel and proceedings on applied botany and breeding, 1915). in 1906, the bureau of applied botany received the highest award (diploma d’onore) at the world exhibition in milan (esposizione internazionale di milano) for the presentation of the barley collection and the results of its study, later summarized by regel in his publication les orges cultivées de l’empire russe in french (loskutov, 1999). regel laid the foundations for collecting missions, both within the empire and beyond, to supply the collections with new accessions. in 1908, he launched the publication of proceedings on applied botany, the first scientific journal dedicated to the use of botany in agricultural practice (loskutov, 2009). in the following years, the exchange of publications gained significant momentum: by 1928, the proceedings on applied botany were sent to 175 research institutions worldwide, while in exchange the local scientific library received publications from 136 foreign research centres. in 2023, the journal founded by regel (currently named proceedings on applied botany, genetics and breeding) celebrated its 115th anniversary. today, the journal is indexed in the web of science, the russian science citation index (rsci), scopus, and doaj (supplements, 2023). when regel unexpectedly passed away from typhus in 1920, a talented young researcher, nikolay ivanovich vavilov, was elected in his place. his works on the immunity of cereal crops were highly valued by leading russian scientists at that time. from those days onward, the bureau’s activities expanded to a completely different scale: as early as 1924, it was raised to the status of an institute (the all-union institute of applied botany and new crops). vavilov established a network of experiment stations across the ussr, where all the collected crop materials were studied under various environmental conditions (vir’s current experimental network consists of 15 branches in 12 regions of russia, from the southernmost site in dagestan to the arctic). he organized collecting missions abroad, to all the world’s continents, as well as to the remotest nooks over the vast territory of the ussr. vavilov enhanced the international exchange of plant germplasm among leading plant breeders and botanists on a global scale. while collecting plant resources and studying them, he continued to work on his scientific theories and fundamental principles. based on these research endeavours, he promoted plans for himself and his associates to conduct further plant explorations across the soviet union and in foreign countries. he developed a scientific framework to support the expansion of the cultivation area for individual crops and the introduction of new cultivars and plant species into the ussr territory. unfortunately, those ambitious plans failed to come to life to their fullest (loskutov, 2020). in 1930, the institute was renamed the all-union research institute of plant industry, and during this time it adopted its world-famous acronym, ‘vir’. however, the new name did not fully encompass the institute’s principal activities and objectives. looking ahead, it would be only at the end of 2014, when vir gained the status of one of the first federal research centres in russia, that the concept of ‘plant genetic resources’ (pgr) entered the institute’s official name (see a photo of vir’s headquarters in figure 1). the great patriotic war (world war ii) of 1941–1945 was a dramatic period for the survival of the institute’s staff members and the safeguarding of its unique collections. with the outbreak of the war, even before the city was beleaguered by the nazis, the government decided to evacuate a number of factories and institutes from leningrad, including vir. however, it was not until the winter of 1942 that the institute started a partial evacuation of its employees and holdings, although the arrangements to prepare the move had been underway for a long time. eventually, the largest and most important part of the collections was left in the besieged city. the remaining staff members had to toil under the harshest conditions of the siege, in unheated premises. the severe winter of 1941–1942 brought drastic reductions in the daily bread allowance, and the city faced starvation. hundreds of thousands of people died from hunger, including vir’s employees, who kept seeds and tubers in the collections safe and intact. only their heroic efforts saved the institute’s collection from destruction and loss of viability. such heroism cost the lives of more than 20 scientists and specialists, but as irreparable as that cost might be, vir managed to survive the most dangerous period in its existence. suffering extreme physical exhaustion in frozen rooms, without water or electricity, under continuous shelling, the institute’s staff members secured the collection of cultivated plants and their wild relatives, the herbarium and the scientific library for future generations, often paying with their own lives (loskutov, 2021). the current situation vavilov paid special attention to the conservation of the genetic diversity of cultivated plants and their wild relatives, collected from all over the world, for future generations. the need for safe preservation was triggered by the concern that, over time, the valuable global collection could lose its qualities or genetic homogeneity due to frequent regenerations of its accessions, which were necessary to restore high germination rates. to minimize the frequency of regenerations while ensuring the seed viability of accessions, it became essential to maintain them under controlled conditions in specialized low-temperature storage facilities. 60 loskutov et al genetic resources (2025), (s2), 58–69 figure 1. vir’s headquarters, st. petersburg. photo: vir archives the geographic approach to plant germplasm conservation at the institute’s experimental stations has remained predominant since vavilov’s times. all accessions, mostly those from the larger cereal collections, have been distributed among the duplicate collections maintained at vir’s experimental stations for regeneration and conservation, following vavilov’s agroecological classification of crops (vavilov, 1957). by combining long-term storage in the national genebank with short-term storage in working and duplicate collections at experimental stations and active collections at the institute (figure 2), vir has ensured integrated and highly secure germplasm conservation. in addition, the institute’s collection management is based on a thoroughly developed (to the level of varieties) botanical classification of each genus and species. while managing the preserved biodiversity, much attention is paid to the passport data of each incoming accession, especially to the true geographic origin and its correct (original) name, which are important for the timely identification of duplicate accessions in the collection (loskutov, 2009). storage facilities since 1946, vir has been experimenting with the longterm storage of various crop accessions from its global collection under controlled conditions. the results of such experiments made it possible to find the optimal storage conditions for germplasm accessions, preserved as dried seed samples in hermetically sealed containers. long-term and medium-term storage of seed samples at low temperatures is a safe and relatively inexpensive method for pgr conservation (khoroshailov, 1978; filipenko, 2007). pgr storage at low temperatures began at vir in the mid-1950s with systems under various temperature conditions. in 1969, the storage facility was reconstructed and updated. special premises were allocated in the institute’s headquarters for this purpose, where a temperature of +5/7◦c was maintained, and where a significant part of the vir collection was preserved until the late 1980s (khoroshailov, 1978). in 1976, the national seed store was built at the kuban experiment station of the institute (krasnodar territory) to house the vir core collection under controlled conditions. the design of the national seed store, occupying the underground section of the building, allowed for the storage of seed samples in hermetically sealed glass containers at +4◦c in 24 rooms without relative humidity control, with a total estimated capacity of 400,000 accessions. for a long time, the vir collection was successfully stored in the national seed store. by the mid-1990s, about 70% of the accessions held within the base seed collection had been placed for storage in the kuban experiment station facility. in 1994–1997, the international community (international plant genetic resources institute (ipgri) and united states department of agriculture (usda)) helped to renovate, including the sealing of underground floors, the building and equipment of the national seed store, so that constant and stable storage could be maintained there. this programme included the delivery of modern refrigeration equipment and computer hardware, the latter serving as the platform whereupon the electronic databases of the vir collections were developed (loskutov, 2009). in 2008, the kuban seed genebank (as the national seed store had been renamed), a branch of vir, was supplied with new technological equipment and larger refrigeration chambers for pgr conservation, designed to perform effective operations at a higher scientific genetic resources (2025), (s2), 58–69 vir: from a small bureau to national pgr center 61 figure 2. working collection at vir, st. petersburg. photo: vir archives. and methodological level. today, the repositories of the kuban seed genebank hold 336,396 storage seed samples of various crop seed samples, including about 17,000 accessions from other scientific institutions, at low positive (+4.5◦c) and negative temperatures (–5.0◦c and –20.0◦c) in a ratio of 10 to 1. modern low-temperature storage facilities were installed in the vir headquarters (st. petersburg) in 2000, with support from the international community (ipgri, usda). the temperature regime of +4◦c is maintained in two seed storage rooms (437m3 in volume), and –10◦c in three others (434m3). the research conducted at the vir genebank resulted in partial development and introduction of more advanced technologies (lamination, sealing) for long-term pgr conservation, which led to a significant decrease in labour costs and funding, required to maintain the collections in a viable state, reducing the need for periodic seed regeneration. of late, significant efforts have been made to transfer seed accessions under controlled storage conditions. the seed material is now hermetically packed in laminated foil bags or glass containers in all types of storage. as of early 2024, 438,951 seed accessions from the vir pgr collection were stored in the institute’s low-temperature seed storage facilities in st. petersburg. information about these accessions is available in the database of stored materials: 308,481 accessions are placed under medium-term storage (–10◦c), and 130,470 under long-term storage (–20◦c) conditions (table 1). at present, a total of over 750,000 storage units (including safety duplicates) with genetic resources accessions from the unique global collection of vir are placed for safe conservation in one or the other type of temperature-controlled storage facilities within the institute’s genebank network (loskutov, 2025). in 2023, as part of the instrumentation upgrade programme under the national project ‘science and universities’, vir installed new updated seed storage facilities, with a temperature regime of –18◦c and a capacity to store over 300,000 units. plant exploration the revival of vavilov’s name and ideas in the second half of the 20th century catalyzed a thorough exploration of plant resources all over the globe. not only soviet and russian researchers were credited with this endeavour, but also scientists and experts from many foreign countries (zeven and zhukovsky, 1975; sazonova et al, 1994; frankel et al, 1995; harlan, 1995; pistorius, 1997; pistorius and van wijk, 1999; loskutova and ozerskaya, 2018, 2019, 2020, 2021; solberg et al, 2023). 62 loskutov et al genetic resources (2025), (s2), 58–69 table 1. structure of preservation of the world collection of vir, 2024. *, seed samples. № type of storage place of storage number of accessions 1 ex situ vir, st. petersburg, working collection 290,000 2 network of vir stations, field genebank 30,000 3 vir, st. petersburg, medium-term storage 308,481* 4 vir, st. petersburg, long-term storage 130,470* 5 kuban seed genebank, medium-term storage 290,692* 6 kuban seed genebank, long-term storage 45,704 7 in vitro st. petersburg 1,134 8 cryo st. petersburg 3,677 besides, vavilov’s life and work, the activities of the institute he led, and the importance of the vir collections for the global community have been a subject of interest for russian and foreign researchers (rokityansky et al, 1999; pringle, 2008; nabhan, 2011; cohen and loskutov, 2016; goncharov, 2017; reznik, 2017, 2021; loskutov et al, 2023). the new knowledge obtained from contemporary plant explorations inspired the institute’s scientists to develop and expand vavilov’s concept of the centres of crop origin. the data accumulated through comprehensive research on the enormous diversity of crop species and their wild relatives helped to develop new botanical classifications or adjust the existing taxonomies for the most important crops, published in new volumes of the cultivated flora, a series of publications started by vavilov. an indepth study of intraspecies diversity made it possible to identify or artificially develop the plant forms predicted by vavilov’s law of homologous series in hereditary variation (loskutov, 2025). a crucial part of vir’s international activities in the 1960s–1980s was organizing and implementing the work on collecting, studying and conserving pgr within the framework of the council for mutual economic assistance (comecon), which bound together the ussr, the socialist countries of eastern europe, and mongolia. their joint collecting missions and seed exchange efforts enlarged the genebank collections of such countries as czechoslovakia (1.5 times), hungary (by twofold), bulgaria and east germany (threefold), and poland (sevenfold). in addition, the collections of mongolia and romania were also expanded (alexanian, 2002). after the dissolution of the ussr in 1991, independent pgr genebanks emerged in the former soviet republics. in some cases, national genebanks were established based on agricultural, plant breeding or botanical research institutes that had been closely cooperating with vir for decades in various areas, including plant germplasm exchange, such as those in estonia, latvia, lithuania, moldova, georgia, armenia, tajikistan and kyrgyzstan. in other cases, national genebanks developed from collections previously maintained at experimental stations or base sites within the vir network before 1991. these genebanks were established in ukraine, belarus, azerbaijan, uzbekistan, kazakhstan and turkmenistan (loskutov, 2025). only in the past ten years, 110 collecting missions have explored the russian federation and foreign territories to monitor pgr in situ and enrich vir’s ex situ collections (state research centers, 2023). composition of the collections and research on pgr diversity thanks to the large-scale activities undertaken by vavilov and his associates, vir is now the holder of two unique biological collections: • the vir collection, a global collection of genetic resources of cultivated plants and their wild relatives. it is one of the world’s largest in terms of the botanical, genetic, geographic and ecological diversity, and includes samples from 64 botanical families, 376 genera and 2,169 species. in terms of crop groups, the collection is composed of cereals (137,500 samples), legumes (46,500), forage crops (32,000), vegetable and melon crops (52,000), industrial crops (28,500), tuber crops (8,300), fruit crops (23,000), totalling 327,800 samples. as of 1 november 2024, the volume of vir’s world collection amounted to more than 320,000 unique samples (loskutov, 2025); • the vir herbarium (acronym ‘wir’), a collection known as the herbarium of cultivated plants of the world, their wild relatives and weeds of the n.i. vavilov all-russian institute of plant genetic resources. it is one of the world’s richest herbaria, specializing in cultivated plants, with a status corresponding to a specialized herbarium of global significance. as of 1 november 2024, it includes 141,293 herbarium specimens and 379,292 herbarium sheets (khlestkina et al, 2022). currently, vir employs over 1,200 people, of which about 500 conduct research in st. petersburg and over 700 work at experimental stations – the vir branches. the institute’s structure includes several components. in st. petersburg, the departments of genetic resources focus on collecting, studying in the field, propagating, preserving and distributing samples of the collection. additionally, methodological departments and laboratories conduct in-depth research genetic resources (2025), (s2), 58–69 vir: from a small bureau to national pgr center 63 on the collection samples from the point of view of genetics, physiology, biochemistry and molecular biology. the structure of vir also includes a genebank, represented by the laboratory for long-term storage of pgr, collective-use centres and a network consisting of 15 vir experimental stations located in different parts of the russian federation. every year, vir distributes more than 5,000 collection samples free of charge to more than 200 requests from state research, breeding institutes and universities of the russian federation. vavilov’s plans for genetic research into plant species diversity, which he was unable to fully realize during his lifetime, became one of the main priorities of the institute. this mission formed the basis of a programme aimed at selecting and developing sources and donors for important agronomic traits. the resulting data underpinned the establishment of trait-specific and genetic collections with identified genes) (merezhko, 1994; mitrofanova, 1994; vir, 2005; porokhovinova et al, 2013). in the 2000s, modern methods of molecular biology started to be widely introduced for the in-depth study of the genetic diversity preserved in the vir global collection (anisimova et al, 2011; antonova et al, 2011, 2016; artemyeva et al, 2012, 2017; zlotina et al, 2013; burlyaeva, 2014; gavrilenko et al, 2014; teplyakova et al, 2017; novakazi et al, 2019; sallam et al, 2021). today, the institute is engaged in extensive genomic and postgenomic research activities, including those within the framework of projects under the auspices of the federal scientific and technical program for the development of genetic technologies for 2019–2030, and the national project ‘science and universities’, targeted at different crop groups: cereals (antonova et al, 2022; porotnikov et al, 2022; khlestkina et al, 2022; chikida et al, 2023; gnutikov et al, 2023; loskutov et al, 2023; rozanova et al, 2023; semilet et al, 2023; lukina et al, 2024; shvachko et al, 2024), grain legumes (krylova et al, 2023, 2024a,b), oilseeds and industrial crops (mikhailova et al, 2024, 2022; anisimova et al, 2023), vegetables (strygina and khlestkina, 2022; berensen et al, 2023; fateev et al, 2023), forage crops (malysheva et al, 2023), fruit crops (kamnev et al, 2023; razgonova et al, 2023a,b), potato (antonova et al, 2020; fomina et al, 2020; rybakov et al, 2020; gavrilenko et al, 2023; gurina et al, 2022), and many others. following the implementation of those programmes and projects, new knowledge was obtained in the field of plant genetics, physiology, biochemistry and taxonomy, including the identification, mapping and/or labelling of more than 100 target genes and loci of quantitative traits. only from 2018 to 2022, vir’s scientists identified 8,566 sources of important agronomic traits and identified 32 donors of valuable genes. twelve edited lines of various crops have been released and are being studied using genetic technologies under vir’s projects jointly with the institute’s partners. sustainable use of pgr from 2007 to 2023, based on the assessment of more than 100,000 accessions of cereals, grain legumes, oilseeds, fruit and berry, and vegetable crops for the most important economically valuable and biochemical quality traits, more than 6,700 sources for various breeding areas were identified. more than 26,000 accessions of various crops were studied for resistance to diseases and pests against harsh infectious backgrounds. more than 970 sources of high resistance were identified. the effectiveness of resistance sources of various crops to populations of harmful organisms was also studied – causative agents of leaf rust, dark brown leaf spot and fusarium head blight in wheat, barley and oats; net spot, loose and stone smut in barley; coccomycosis in cherry; common cereal aphid in sorghum. more than 5,250 accessions of the gene pool of cultivated plants were screened for resistance to abiotic environmental factors, and more than 500 sources of valuable physiological traits were identified (weak photoperiod response and early maturity, tolerance to excess mobile aluminium, cold and drought resistance). over the past 5 years (2019–2023), vir identified 6,820 sources of economically valuable traits and created 19 donors of valuable genes, which were sent to the leading breeding centres of the russian federation. since 2013, vir has registered almost 130 patents (varieties and technologies for implementation in production in the agro-industrial complex). all selected and created genetic sources and donors were sent to leading breeding institutions of the russian federation. during the same period, over 300 new varieties of major agricultural crops were created and approved for use in agricultural production of the russian federation based on vir sources and donors in breeding centres and research institutes. vir employees created 98 varieties of economically significant crops included in the state register of breeding achievements of the russian federation, and received 47 patents and 102 author’s certificates for varieties. implementation of modern technologies in 2004, the genebank of vir was equipped with cryogenic systems (figure 3), and the institute was able to start cryopreservation activities. presently, pollen samples and cuttings of fruit and berry crops are preserved in liquid nitrogen vapours, and cuttings are also placed under liquid nitrogen (filipenko, 2007). currently, biotech and digital approaches are widely applied at vir to develop methodologies for maintaining ex situ pgr collections, including their conservation and twoor three-fold safety duplications under controlled conditions (short-, mediumand longterm storage of seed accessions in specialized lowtemperature facilities, cryopreservation, and in vitro conservation of vegetatively propagated crop samples), safe maintenance of perennial crop accessions in the field, regeneration of accessions to ensure seed 64 loskutov et al genetic resources (2025), (s2), 58–69 figure 3. storage facilities, cryopreservation, st. petersburg. photo: vir archives. germination and obtain fresh material (figure 4), etc. (khlestkina et al, 2022). by now, vir has used biotechnologies to maintain 1,134 accessions in vitro, including 1,081 accessions of vegetatively propagated temperate-climate crops (656 potato, 21 onions, 348 berry and fruit plants, and 53 ornamentals), as well as 2,045 pollen samples, 1,184 cuttings of various fruit plants and grapes, and 448 samples of apical meristems (415 of potato, and 33 of raspberry) for a total of 3,677 accessions under cryopreservation (table 1). the methods developed at the institute to preserve and evaluate pgr are published in the format of guidelines (filipenko, 2007; loskutov et al, 2012). international events vir has organized more than ten major international events in recent years, including the international wheat conference in 2010, the international oat conference in 2016, regular international vavilov conferences (the last one in 2022; khlestkina et al (2022) ) held every five years, and major international conferences dedicated to the institute’s birthday, the last of which was successfully held in november 2024. besides, vir was the initiator and co-organizer of two major fora ‘genetic resources of russia’, bringing together the holders of biological collections of all specializations (tikhonovich et al, 2022, 2023). partly owing to this initiative and following broad multidisciplinary discussions at those forums, the national law on bioresource centers and biological collections was drafted in russia (tikhonovich et al, 2024). sustainable future and an intensified research programme over the past five years, the institute has significantly intensified comprehensive research on the vir collection through the implementation of large-scale research programmes, including collaboration with partner institutions participating in programmes coordinated by vir, as well as through vir’s active involvement in consortia. examples of such partnerships are the ‘national network collection of plant genetic resources for effective scientific and technological development of the russian federation in the field of genetic technologies’, and ‘breads of russia’, supported in the framework of the federal scientific and technical program for the development of genetic technologies for 2019–2030. an example of participation in a consortium is the programme of the world-class scientific center, ‘agrotechnologies for the future’. in 2022, two decrees were issued by the president of the russian federation. the first founded the national center for plant genetic resources based on vir, while the second established the interdepartmental commission on the formation, preservation and use of plant genetic resources collections (khlestkina et al, 2022). in 2023, the government of the russian federation approved the program for the development of the national center for plant genetic resources for 2023–2030. this programme provides conducive conditions for the development of scientific, research and development activities in the field of pgr to ensure scientific and technological development of the russian federation, finding integrated solutions to the questions genetic resources (2025), (s2), 58–69 vir: from a small bureau to national pgr center 65 figure 4. regeneration field, harvest time, pushkin, st. petersburg. photo: vir archives. associated with the accelerated progress of genetic technologies. the law on bioresource centers and biological (bioresource) collections was adopted by the state duma of the russian federation on 26 november 2024, and on 30 november 2024, russian president vladimir putin signed the federal law. within the framework of the program for the development of the national center for plant genetic resources, annual collecting missions are planned to be conducted abroad from 2024 to 2030. the first mission was carried out in 2024, jointly with the national research institute of plant genetic resources of the republic of uzbekistan. in 2023, vir signed an agreement on scientific cooperation with the national council of humanities, sciences, and technologies (conahcyt) of the united mexican states; among other objectives of the agreement, a joint collecting mission is planned to be organized in mexico. vir is open to discussions on undertaking important international collecting missions together with other countries within the framework of planning for the period up to 2030. the vir world collection, which began as the russian collection of cultivated plants, has grown into one of the most systematically collected, comprehensively studied and representative collections of pgr in the world. at present, the activities of vir as the national center for plant genetic resources, and the development of the legislative system on genetic resources in russia will be conducive to fostering effective international scientific cooperation in the field of pgr, thus keeping vavilov’s legacy alive. the national center will provide effective solutions to new challenges in the systematic collection, comprehensive study, reliable preservation and sustainable use of pgr in the russian federation at a new level. authors contributions conceptualization, i.g.l., yu.v.u. and e.k. kh.; writing—original draft preparation, i.g.l., yu.v.u. and e.k. kh; writing—review and editing, i.g.l., yu.v.u. and e.k. kh. all authors have read and agreed to the published version of the manuscript. conflict of interest statement the authors declare no conflict of interest references alexanian, s. m. 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(2013). use of allele-specific markers of the ppd and vrn genes for predicting growing season duration in barley cultivars. vavilovskii zhurnal genetiki i selektsii=vavilov journal of genetics and breeding 17(1), 50–62. https://doi.org/10.3390/plants12010143 https://doi.org/10.3390/plants12010143 https://doi.org/10.3390/biom12050689 https://doi.org/10.30901/2227-8834-2023-4-251-261 https://doi.org/10.30901/2227-8834-2023-4-251-261 https://doi.org/10.1186/s12870-017-1121-4 https://doi.org/10.1186/s12870-017-1121-4 https://doi.org/10.30901/2658-6266-2023-2-o1 https://doi.org/10.30901/2658-6266-2023-2-o1 https://doi.org/10.30901/2658-6266-2022-2-o4 https://doi.org/10.30901/2658-6266-2022-2-o4 https://doi.org/10.30901/2658-6266-2024-2-o4 https://doi.org/10.30901/2658-6266-2024-2-o4 introduction and historical events the current situation storage facilities plant exploration composition of the collections and research on pgr diversity sustainable use of pgr implementation of modern technologies international events sustainable future and an intensified research programme authors contributions conflict of interest statement preserving, improving and rediscovering: the role of the research centre of viticulture and enology in safeguarding grapevine genetic resources in italy preserving, improving and rediscovering: the role of the research centre of viticulture and enology in safeguarding grapevine genetic resources in italy maria antonietta palombi ​✉ 1 , vittorio alba 2 , marco ammoniaci 3 , noemi bevilacqua 1 , a raffaele caputo 2 , roberto carraro 4 , stefano favale 1 , simone garavelloni 3 , massimo gardiman 4 , massimo morassut 1 , marina niero 4 , roberto nuti 1 , giuseppina pipitone 4 , sergio puccioni 3 , alessandra zombardo 3 1 crea-ve, council for agricultural research and economics, research centre for viticulture and enology, velletri, 00049, italy 2 crea-ve, council for agricultural research and economics, research centre for viticulture and enology, turi, 70010, italy 3 crea-ve, council for agricultural research and economics, research centre for viticulture and enology, arezzo, 52100, italy 4 crea-ve, council for agricultural research and economics, research centre for viticulture and enology, conegliano, 31015, italy abstract grapevine is one the most cultivated species worldwide, with 8,000 estimated varieties. protecting this biodiversity is of utmost importance, especially in countries historically devoted to viticulture, such as italy. one of the richest italian ampelographic collections, spread in different regions from the north to the south of the peninsula, is owned and managed by the research centre of viticulture and enology (crea-ve). vitis vinifera l., but also other species of the vitis genus. since 2004, the italian ministry of agriculture, food sovereignty and forestry has financed a specific programme named ‘risorse genetiche vegetali – trattato fao (rgv-fao)’ [plant genetic resources – fao treaty] to collect, conserve, characterize and document plant genetic resources for food and agriculture. this paper presents the processes undertaken to enhance the collection, characterize its accessions, preserve and foster the genetic diversity and adaptability in grapevines, with particular emphasis on how this material is managed, evaluated and valorized in terms of different perspectives and practical uses. keywords plant genetic resources, vitis spp, biodiversity, genebank, ex situ conservation introduction grapevine is one of the most important crop species in the mediterranean area and its value is not only economic but also historic. archaeobotanical findings document the presence of wild grapes in the old continent since the neolithic age (rottoli, 1993; savo, kumbaric, & caneva, 2016). moreover, there is evidence of grape domestication during the viii-vii century bce, confirmed by the discovery of seeds of cultivated varieties in central italy (motta, 2002). in this area, a well-established wine tradition probably began during the etruscan period and roman empire (delle-donne, 2017), which has continued until the present. if historical aspects are important, the environmental value of plant biodiversity is especially relevant in marginal areas (biasi & brunori, 2015), where autochthonous grapevine varieties are traditionally grown and represent an ecosystem service with agroecological benefits (giffard et al., 2022; oiv, 2018). it is estimated that there are more than 70 vitis species, with approximately 8,000 different cultivated varieties: 6,000 vitis vinifera l. and 2,000 interspecific hybrids (lacombe, 2023). the most recent report from the un food and agriculture organization (fao) on the status of global plant genetic resources for food and agriculture indicates that approximately 60,000 accessions of the vitis genus are currently maintained in genebanks worldwide (fao, 2010). this number will be soon updated since a new report is under revision (document n. cc5227/en). the objective of germplasm conservation is to safeguard diversity through the implementation of effective techniques that reduce the risk of losses. the sustainable utilization and conservation of plant genetic resources relies on the efficient management of germplasm collections, which is essential to ensure the survival of the resources and their accessibility to relevant stakeholders, including researchers, breeders and farmers (fao, 2014). currently, wine production is highly concentrated in a few grape varieties that dominate the market. it is estimated that in 2016, the top 17 varieties covered half of the world's grapevine planted area (anderson & nelgen, 2021), and within these, few clones are in use leading to a strong erosion of grapevine biodiversity. one of the primary challenges in the conservation of genetic resources is the necessity for long-term commitment and the integration of such activities into continuously funded, non-periodic programmes. the main objective is to guarantee the continued preservation of local viticultural genetic resources, which, regardless of potential commercial interests, represent a heritage of humanity and necessitate the involvement of specialized institutions capable of upholding internationally agreed standards. in harmony with the convention on biological diversity (cbd, 2005), the international treaty on plant genetic resources for food and agriculture (itpgrfa) was adopted by fao and came into force in june 2004. in italy, the itpgrfa was ratified in 2004 with a specific law (l. 06, april 2004, n. 101) and, consequently, all the collections maintained in the country under the supervision of the italian ministry of agriculture, food sovereignty and forestry (masaf) were rationalized and included in a specific programme for collection, conservation, characterization and documentation of pgrfa, known as rgv-fao (vaccino et al., 2024). the main objectives are the conservation and sustainable use of agricultural plant genetic resources maintained in italian repositories, essential for food security and safety. in this specific framework, the main concern for public research in viticulture is the need to safeguard grapevine biodiversity. origin of crea-ve repository one of the main vitis collections in italy is maintained by crea (council for agriculture research and economics) at the research centre for viticulture and enology. the primary, historic core of the ampelographic collection was established in the 1900s (gardiman & bavaresco, 2015) in conegliano (veneto). other subcollections in arezzo (tuscany), velletri (latium) and turi (apulia) were included over time as the research centre evolved. in conegliano, the repository was founded in 1923 with the establishment of the experimental station of viticulture and enology, to provide the material necessary for the future work of the station. by the end of 1924, this collection included 350 european varieties, 246 direct-producer hybrids and 65 american rootstocks. the other collections are more recent; in velletri (latium, central italy), a collaboration between arsial (agenzia regionale per lo sviluppo e l'innovazione dell'agricoltura del lazio) and the crea-ve research centre started in 1994, with the aim of recovering autochthonous grapevine material present in different area of latium region. the exploration of the principal grape growing area in latium contributed to collecting autochthonous varieties and, in 1998, after a minimum characterization and phytosanitary screening of all the plant material collected, an ex situ collection was established. in arezzo (tuscany, central italy), the grapevine collection was set up starting in 1992. the vineyard covers an area of about 6ha and contains accessions mainly belonging to the autochthonous germplasm of central italy, collected and propagated from mother plants found mainly in tuscany and umbria. over the decades, the management of the grapevine collection has been carried out thanks to funds from different regional and national projects. in apulia, the first collection was established in 1970 using regional funding and renewed in 2004; currently, it covers an area of about 10ha and includes both wine and table grape autochthonous varieties. during the last decade, the collection has therefore been implemented with additional accessions recovered within the fao programme for the protection and valorization of genetic resources. over the years, the collection has been constantly updated and enriched with new accessions resulting from research in various cultivation areas and exchanges with other national and international institutes. as the collection has been expanded and enriched over the years, it has also been characterized and rationalized through the identification of duplicates, synonyms, homonyms, and unique genotypic and phenotypic characteristics. this paper focuses on the various activities carried out to maintain, characterize and utilize the grapevine genetic resources conserved in the crea-ve ampelographic collection. plant genetic resources conservation at council for agricultural research and economics, research centre for viticulture and enology (creave) currently, the crea-ve collection maintains over 3,000 accessions, including distinct species of the genus vitis l., both cultivated and relatives, as reported in table 1. an exhaustive list of grape accessions maintained at crea-ve is available in the european database eurisco (http://eurisco.ecpgr.org/). table 1: vitis accessions maintained at crea-ve species number of accessions v. vinifera l. 2,906 hybrids of vitis spp. 393 v. aestivalis michx. 1 v. andersonii rehder 1 v. arizonica engelm. 1 v. baileyana munson 1 v. berlandieri planch. 2 v. betulifolia diels & gilg 1 v. champinii planch. 1 v. cinerea (engelm.) millardet 1 v. coignetiae pulliat 1 v. doaniana munson 1 v. longii prince 3 v. monticola engelm. 1 v. novae angliae fernald 1 v. riparia michx. 25 v. rubra michx. 1 v. rupestris scheele 12 v. slavinii rehder 1 v. treleasei munson 1 due to the introduction of new accessions and/or a more in-depth identification of the material collected, the number of accessions reported should be considered very dynamic. of the 19 species of the genus vitis conserved in the crea-ve collection, the most represented is vitis vinifera l.; the other species are exploited primarily to produce rootstocks or as genetic material for breeding. an example of morphological diversity of different species of vitis spp. is shown in figure 1. figure 1: some specific characteristics of different species of vitis spp.: a, vitis doaniana muns.; b, vitis bayleiana munson; c, vitis berlandieri planch.; d, vitis vinifera cv. ‘garnacha tinta’. considering the main use of the accessions maintained at the crea-ve repository, it is possible to distinguish wine grapes (61.6%), table grapes (17.7%), accessions used both as wine and table grapes (0.6%), rootstocks (5.7%) and grape material not defined (14.4%). with respect to the biological status of the accessions, we have traditional varieties (70%), advanced varieties (1.5%), and breeding or research material (15%), with 13.5% of the accessions for which the biological status remains not defined. all accessions are maintained ex situ in dedicated vineyards with a minimum of five vines. part of the germplasm is maintained in containers in screenhouse facilities to comply with phytosanitary legislation. different accessions of certain varieties are preserved to maintain some intra-varietal diversity. grape accessions at the crea-ve repository have different origins, mainly from italy (more than 60%). many of these accessions represent rare or neglected grapevine varieties found throughout various wine-producing regions of italy (bergamini et al., 2017; gasparro et al., 2020; giust & caputo, 2014; palombi et al., 2023; zombardo et al., 2022; zombardo et al., 2024). materials from other european countries, including georgia and armenia, represent a consistent percentage of the conserved germplasm (24%), and americas (usa, argentina and brazil), asia (china and japan) and africa (algeria and south africa) are represented in the collection (figure 2). figure 2: country of origin of the vitis accessions maintained at the crea-ve repository. ordinary agronomic interventions are carried out during the cultivation cycle (winter pruning, soil management, pest control, fertilization, emergency irrigation, spring suckering, summer tying, and topping, crown management) to maintain the vines in a good vegetative-productive and phytosanitary state. management of vitis genetic resources the management of large germplasm collections is a complex task that requires a great deal of technical, agronomic and scientific expertise and it must be carried out in accordance with international standards (maghradze, maletic, maul, faltus, & failla, 2015; oiv, 2007). primarily, the objective is to preserve the grapevine heritage, as well as to collect data on physiological and phenotypic characteristics of the germplasm (boursiquot, 2000; lacombe, 2023; maul et al., 2012). the first step in managing a collection is to correctly identify the collected plant material by carefully recording information about each accession, such as genotypic fingerprints and morphological characteristics. the next step is to collect information to record the 'passport data' according to the fao multi-crop passport descriptor list for vitis species (alercia, diulgheroff, & mackay, 2015; oiv, 2007). these data include basic information such as a unique code, pedigree, origin, donor and others, in addition to specific descriptors that are relevant for the grapevine varieties and species. according to itpgrfa, every biological accession must also be linked to a digital object identifier name (doi), an international standard adapted to identify plant germplasm worldwide (alercia, lópez, hamilton, & marsella, 2018), to facilitate the exchange of biological material and access to the information on crops and research around the world. the acquisition of dois for all conserved grapevine accessions at crea is planned, and it is currently in progress. the identification and characterization of the grapevine accessions represent the fundamental actions to be carried out in a rational germplasm conservation plan. these are achieved through a range of analytical techniques, including ampelographic description based on the analysis of traits that are highly heritable, dna analyses, and agronomic and resistance trait evaluations. the traditional approach for identifying and classifying grapevine varieties is ampelography (galet, 1976; this, lacombe, & thomas, 2006), which relies primarily on the visual examination of morphological features. these observations are conducted by experts in the field, based on international standardized descriptors (ipgri, 1997; oiv, 2007; upov, 2008). the international organisation of vine and wine (oiv) experts have also introduced a ‘primary priority descriptors list’ encompassing only 14 descriptors (maul & this, 2008; oiv, 2007), with a highly discriminating power, to reduce time in the characterization process. using the descriptors and methods defined by oiv and upov, the ampelographic characteristics of many accessions, maintained at crea, have been recorded over the years (alba et al., 2014; alba et al., 2015; labagnara, bergamini, caputo, & cirigliano, 2018; palombi et al., 2023; zombardo et al., 2021) and their information was useful for conducting distinctness, uniformity and stability (dus) tests, studies on somatic variants (crespan, carraro, giust, & migliaro, 2016) and characterization of italian variety families distributed in the peninsula (costacurta et al., 2003; costacurta et al., 2004). the phyllometric method (also known as leaf ampelometry) is based on the measurement of specific leaf characteristics, such as the length of the veins and the angles formed between them (bodor-pesti, taranyi, deák, sárdy, & varga, 2023). this technique, firstly proposed bygoethe (1876) and then set up by ravaz (1902), is performed using specific ampelometric software (soldavini, stefanini, dallaserra, policarpo, & schneider, 2006), although, in recent years, it has been improved with the adoption of leaf morphometric methods (chitwood, 2021) and image analyses by means of artificial intelligence (liu et al., 2021; nart et al., 2024). ampelography was long the only method for identifying varieties, but dna fingerprinting, especially if performed by microsatellites has proved suitable for both the rapid and reliable identification of varieties and the comparison of data between different laboratories, using reference data codification (sefc et al., 2001; this et al., 2004). the crea grapevine collection was genetically characterized during the last decade using at least 11 simple sequence repeats (ssr or microsatellites) markers (migliaro, morreale, gardiman, landolfo, & crespan, 2013). this work unveils duplicates, cases of mislabelling, homonyms and synonyms (cipriani et al., 2010; lorenzis et al., 2019; pipitone, migliaro, morassut, & palombi, 2024; storchi et al., 2016). a more detailed genetic characterization using 18k single nucleotide polimorphic (snp) markers was recently carried out on a subset of the conegliano collection, comprising more than 600 accessions (d’onofrio et al., 2021). genotyping is also useful for defining the pedigree of varieties and the area of origin, and accessions from crea's ampelographic collection have been successfully used in phylogenetic studies of many varieties (bergamini et al., 2012; bergamini et al., 2016; crespan, giannetto, coletta, & antonacci, 2009; d’onofrio et al., 2021). the recording of characteristics, the expression of which is often influenced by environmental conditions (agronomic traits and quality), is another action that will be undertaken. these data are crucial for the potential use of the material in breeding programmes. moreover, during the vegetative season, visual inspections are conducted to evaluate the health status of the vines and to find out fungal disease (mildew and esca, in particular), virus infections and grapevine yellows symptoms. in figure 3, a scheme highlighting the primary processes of grape collection and conservation activities is shown. figure 3: scheme of genebank activities. the data collected are partly included in various databases and can be accessed via the websites of the european vitis database (http://www.eu-vitis.de), the vitis international variety catalogue (https://www.vivc.de), the eurisco web catalogue (https://eurisco.ecpgr.org). the maintained accessions show very high phenotypic variability for many characters, including leaf (figure 4) and cluster shape and size (figure 5), berry colour, shape and size, seed presence, berry skin thickness, sugar accumulation, phenological periods (alba, roccotelli, gasparro, & caputo, 2023) and susceptibility to various pathogens. another indicative example is the average weight of the bunch: in some accessions, it is less than 50g, while in others it can exceed 600g. examples include fruit colour (table 2) and range of phenological stages (figure 6). figure 4: variability in leaves can be observed in blade size and shape, number and depth of lobes, shape and size of teeth, petiolar sinus, hairs, etc. a, ‘ramsey’; b, vitis cinerea engelmann; c, ‘malbo gentile’; d, ‘pinot meunier’; e, 'badacsonyi somszoeloe'; f, ‘chasselas cioutat’. figure 5: morphological variety differences in grapes for colour, shape, size and other traits. table 2: distribution in colour classes of the accessions maintained in the different subcollections (%). black white rose red grey conegliano 48 47 3 1 1 arezzo 59 37 4 velletri 28 65 2 5 turi 37 59 4 figure 6: variability in the date of occurrence of the main phenological periods. the final stages of the development of a common and shared information system among the repositories concerned by the rgv-fao programme are currently underway. at present, the various facilities of crea-ve collate their data in shared spreadsheet files, where passport data, morpho-phenological data and genetic data, when available, are reported. over the past two decades, grape accessions have been exchanged for various purposes at the national and international levels. at the national level, autochthonous cultivars have been requested with the objective of reintroducing them into cultivation (growers) or incorporating them into national breeding programmes (researchers); at the international level, the majority of germplasm of foreign origin was received from several research centres, using specific agreements. the number of exchanges is estimated at 100 accessions. valorization of the collections within the itpgrfa framework, recovery, characterization and conservation are to be considered priority components in the management of the collection (fao, 2010). moreover, another cornerstone is the sustainable use of agrobiodiversity. italian biodiversity of autochthonous vines represents the heritage of a territory, due to its long history from the first domestication to modern cultivation. the discovery and rescue of autochthonous grape varieties promote the valorisation of the wine-growing territory and thus preserve its traditional and cultural legacy. public and private research centres have undertaken this challenge with the primary objective of safeguarding the italian ampelographic assortment. measures for the sustainable use of genetic resources include expanding the genetic base of cultivated varieties and increasing the diversity available to farmers. to be commercially employed, a grape variety must first be listed in the national register. for wine grapes, this is additionally contingent upon classification at the regional administrative level, while varieties intended for fresh consumption (e.g. table grapes) require only registration to obtain the certification of vegetative propagation material. the process of registration and classification is accomplished through the morphological, physiological and agronomic characterization of the variety, which must be conducted in accordance with precise legislative national guidelines. in italy, the interest of winegrowers in local varieties linked to the history of their territory is growing steadily and significantly: varietal wines are appreciated niche products and marketed profitably. as a result of the various national and regional projects for the conservation and valorization of vine genetic resources carried out by crea, many local and historical italian varieties have been included in the national register, and it is now possible for winegrowers to use them commercially. in central italy (tuscany) significant examples are ‘orpicchio’, ‘morellone’, ‘nocchianello bianco’, ‘nocchianello nero’, and ‘gralima’, while other varieties are currently being evaluated for their interesting characteristics, for example, ‘tané’, a variety from massa carrara (tuscany) with a bright rose berry colour, that at first evaluation seems to be suitable for producing easy-to-drink rosé wines. also, in central italy (latium) example are nine different clones of ‘cesanese di affile’. in southern italy, we can mention ‘santa sofia’, a white grape variety that was registered in 2019 and can now be grown in basilicata and campania, ‘sabato’, a black grape variety, and ‘agostina’, with white grapes, to cite a few examples. as far as apulia is concerned, ‘negro dolce’ is an interesting variety in the process of being registered. in northern italy, the most recent cases concern varieties that are characteristic of the veneto region, including ‘rabosa bianca’, ‘recaldina’, ‘pecolo scuro’, ‘pattaresca’, ‘mattarella’ and ‘benedina’. the vines present in the vineyard collection also served as plant material used for the propagation and subsequent planting of the varieties in other germplasm collections, hopefully also with custodian winegrowers, or to bring back some varieties to the territories of origin with a past viticultural vocation (i.e. ‘biancone’, transferred back to the place of origin, elba island in tuscan archipelago; zombardo, personal communication). in addition to its primary function as a genebank, the grapevine germplasm collection is indispensable for the implementation of numerous national and international research projects and the activities of the vitis working group of the european cooperative programme for plant genetic resources (ecpgr), as it ensures the availability of essential samples and data. the multi-year phenological data set is of paramount importance for the creation and validation of phenological models, as well as for the investigation of vine adaptation to climate change (fila et al., 2012; parker, cortázar-atauri, leeuwen, & chuine, 2011; tomasi, jones, giust, lovat, & gaiotti, 2011; valori et al., 2023). finally, the grapevine germplasm collection allowed us to raise awareness of the existence of almost forgotten vines that deserve attention, at a scientific but also general public level (pagano, zombardo, valentini, & storchi, 2014; storchi, zombardo, valentini, puccioni, & perria, 2022; zombardo, valentini, puccioni, pagano, & storchi, 2017). conclusions and perspectives over the years, a great deal of effort has gone into characterizing the preserved varieties, using the most advanced phenology, morphology, biochemistry and molecular tools for various groups of varieties. however, many phenotypic and ampelographic aspects need to be explored further, to understand also better how some of these could influence interesting traits (i.e. berry quality, resistance/tolerance to biotic and abiotic stresses) to help researchers to better understand the genetic basis of traits and, consequently, for useful traits introgression for varietal constitution. however, several aspects remain to be fulfilled. these include creating a core collection, duplicating unique accessions to enhance security, filling the gaps in the genetic and geographical distribution of conserved biodiversity, and improving the sharing and dissemination of information. as reported, the crea-ve repository maintained the collection as a field genebank. however, this conservation strategy is financially demanding due to the intensive management requirements, and there is an inherent risk of material loss due to pests and diseases. to address these challenges, novel approaches combining in vitro storage (slow growth) and cryopreservation may offer a solution for the long-term maintenance of grape genetic resources. these techniques represent the optimal strategy for the long-term storage of plant genetic resources, offering the greatest safety and cost-effectiveness. however, in the case of vegetatively propagated species, such as grapevine, they present the disadvantage of being genotype dependent. should these issues be resolved in the future, cryopreservation could be effectively applied at the crea-ve repository. finally, researchers involved worldwide in different topics of grape could work for a possible ‘global grape diversity platform’ to secure the long-term conservation and use of these genetic resources. in this perspective, crea-ve (and other research centres of crea) is involved in the european project pro-grace (https://www.grace-ri.eu/). the project addresses different topics, such as developing and testing unified strategies, procedures and standards for evaluating phenotypic traits of plant genetic resources both in situ and ex situ, and providing the information to end-users, including breeders and farmers. the aim is to create a new concept and governance model for sharing information on plant genetic resources at the european level, enabling the construction of an integrated european plant genetic resources information system. this approach seeks to ensure the safeguarding, use, valorization and cost reduction of grape genetic resources. acknowledgements this work was supported by the italian ministry of agriculture, food sovereignty and forestry (masaf) within the rgv-fao programme (grant d.m. n.50045/2023). author contributions m. antonietta palombi: conceptualization and ideation; vittorio alba, a. raffaele caputo, roberto carraro, massimo gardiman, m. antonietta palombi and alessandra zombardo: writing, original draft preparation and data analysis; massimo gardiman, tables and figure preparation; roberto carraro: pictures; vittorio alba, roberto carraro, massimo gardiman, m. antonietta palombi and alessandra zombardo: writing, review and editing; marco ammoniaci, noemi bevilacqua, roberto carraro, stefano favale, simone garavelloni, massimo morassut, marina niero, roberto nuti, giuseppina pipitone, sergio puccioni: collect data; m. antonietta palombi: supervision. all authors have read and agreed to the published version of the manuscript. conflict of interest statement the authors declare that they have no competing interests. references alba, v., bergamini, c., cardone, m. f., gasparro, m., perniola, r., genghi, r. and antonacci, d. 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(2017). diversité du matériel génétique en italie un exemple de valorisation au sein de la collection du crea-vic en toscane. revue des oenologues 162, 16–17. retrieved from https://www.researchgate.net/publication/317236409 short communication genetic resources (2025), 6 (12), 205–210 doi: 10.46265/genresj.srql5271 https://www.genresj.org issn: 2708-3764 received: 03.09.2025 | accepted: 27.10.2025 | published online: 12.12.2025 a small-scale assessment of the availability of eurisco accessions abstract: a critical assessment of plant genetic resource (pgr) availability in europe reveals a significant gap between documented accessions and those that are practically obtainable for researchers and breeders. while the eurisco database lists over two million accessions, a study of 100 random accessions found nearly 60% to be unavailable, challenging the assumption that a large number of documented accessions equals usability. material from 52% of the approached genebanks could not be obtained within five months. the primary barrier was the inability to contact genebank staff, which points to a systemic issue in which pgr access is not always prioritized. insufficient material for distribution and geopolitical issues were further causes of low availability. this indicates a threat to the effective utilization of european pgr and highlights an urgent need for genebanks to improve communication and operational capacity to ensure these vital resources become accessible for crop development and food security. keywords: availability, eurisco, pgr, plant genetic resources, smta citation: wijnker, e. and van hintum, t. (2025) “a small-scale assessment of the availability of eurisco accessions”, genetic resources, 6(12), pp. 205–210. doi: 10.46265/genresj.srql5271 © copyright 2025 the authors. this is an open access article distributed under the terms of the creative commons attribution license (cc by 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. erik wijnker*, theo van hintum centre for genetic resources, the netherlands, wageningen university and research, p.o. box 16, 6700aa wageningen, the netherlands * corresponding author: erik wijnker (erik.wijnker@wur.nl) introduction the international treaty on plant genetic resources for food and agriculture (itpgrfa) (fao, 2009) defines plant genetic resources (pgr) for food and agriculture as: “any genetic material of plant origin of actual or potential value for food and agriculture.” pgr underpin global food security by enabling crop improvement to face the challenges of future food production. in europe alone, hundreds of genebanks have been established to conserve pgr ex situ. their holdings are listed in aggregated databases such as genesys pgr (genesys, 2025) and fao’s world information and early warning system on plant genetic resources for food and agriculture (wiews) (fao, 2025a). according to policy documents addressing the ex situ conservation of plant genetic resources (pgr), such as the influential third report on the state of the world's plant genetic resources for food and agriculture, over four million accessions are documented in genesys with over two million accessions conserved in europe (fao, 2025b). global crop conservation strategies also use the aggregated databases as a source of information to determine the conservation status of crops and set priorities for future activities (dulloo and khoury, 2023). at first glance, such numbers suggest that a wealth of pgr is available and conserved. however, this assumption may be misleading as conserved material is not always accessible for use. because pgr are tangible assets, it is of crucial importance that stakeholders can access and use them. the availability of material in genebanks, however, is an issue. personal communications with the user community, but also rare https://doi.org/10.46265/genresj.srql5271 https://www.genresj.org https://doi.org/10.46265/genresj.srql5271 mailto:erik.wijnker%40wur.nl?subject= genetic resources (2025), 6(12), 205–210206 wijnker and van hintum studies like bjørnstad et al (2013), make it apparent that pgr access is limited. bjørnstad et al (2013) requested pgr from genebanks globally that are contracting parties to the itpgrfa and observed that ‘facilitated access’ is not that straightforward. they received material from only 44 countries out of the 121 approached. if pgr are to be of “value for food and agriculture” as noted in the definition of pgr by fao, they must be available. defining pgr availability is not easy, as it has many dimensions. one prerequisite is that genebanks physically have material for distribution. ex situ collections require a targeted infrastructure to ensure long-term safekeeping of material, which depends on stable genebank funding. accessions are to be held under good conditions and be subjected to good genebank practice: proper documentation, regular viability monitoring, proper regeneration practices and the creation of safety backups. the fao genebank standards (fao, 2014; 2022) provide excellent guidance on such practices. equally important, however, is the ability of genebanks to deliver material to users. this requires efficient distribution systems, compliance with phytosanitary and legal requirements, and ensuring clear conditions of use. for example, material that is distributed for research purposes only – and therefore not eligible for commercialization – may be considered ’available’ but has little, if any, value to commercial breeding and food production. in practice, availability means that users have good access to accession information, are able to order and obtain accessions quickly (i.e. within weeks) under permissive and uniform conditions. the standard material transfer agreement (smta) is a well-established, standardized contract used for plant genetic resources shared under the provisions of the itpgrfa. the intention of this research was to provide an impression of current pgr availability in europe for policymakers, researchers and the genebank community. to better understand the actual availability of pgr material in europe, a small-scale study was conducted in which plant genetic resources were requested to assess their availability. pgr were selected from eurisco, a large and complete european pgr database that is often used by stakeholders (kotni et al, 2023). this study was not designed to evaluate the functioning of individual genebanks. the names of individual genebanks and requested accessions have therefore purposely been omitted from this report. this study should also not be interpreted as a critique of eurisco. eurisco itself notes that the presence of data does not guarantee that the material will be supplied: “the presence of data listed in eurisco does not provide any warranty that the respective collection holders will be able to provide any plant material to interested parties” (ecpgr, 2025). materials and methods selection of the material to be requested the general approach involved directly requesting a random set of pgr accessions and systematically observing the requesting process, the receipt of material and the conditions governing its use. material was selected from eurisco, the european search catalogue for plant genetic resources that is maintained by the european cooperative programme for plant genetic resources (ecpgr) (kotni et al, 2023), and serves as an important data source for both genesys and wiews. because of its completeness and frequent use by stakeholders, the eurisco database provided a perfect starting point for this research. availability was defined pragmatically as the ability to order material and receive it within five months. material that was not obtainable within this timeframe was considered unavailable. given the high costs associated with maintaining and regenerating genebank materials, the number of requested accessions was deliberately kept low. a total of 100 accessions was randomly selected, representing 0.005% of records in eurisco. anticipating that no more than half of the requested accessions would be successfully obtained, the study was expected to use material from approximately 50 accessions or fewer. while the limited sample size constrains the statistical reliability of the availability estimate, it was deemed sufficient to provide a general impression of the availability of pgr accessions currently conserved by european genebanks. on 14 january 2025, a complete eurisco dataset in csv format was downloaded from the eurisco website (ecpgr, 2025), comprising 2,101,833 accession records. to refine the dataset and focus on plant genetic resources (pgr) that are conserved ex situ, two filtering steps were applied. first, the 682,541 records from the nottingham arabidopsis stock centre (eurisco descriptor instcode = “gbr140”) were excluded. additionally, 5,697 records identified as in situ conserved material (eurisco descriptor storage = 60) were removed. following these steps, 1,413,596 accessions remained. to ensure proportional representation of genebanks while selecting 100 accessions at random, the 418 contributing institutes were sorted according to the number of accessions they recorded in eurisco. one accession was randomly selected from the smallest institutes contributing to the cumulative first 1% of the total records, another from the next 1%, and so forth, up to the point where an individual institute contributed 1% or more of the total accessions. for those larger institutes, the number of selected accessions was determined proportionally to their contribution. for instance, the largest institute contributed 14.2% of the accessions in eurisco; therefore, 14 accessions were randomly selected from its records. this selection methodology ultimately resulted in 100 accessions being drawn from 52 institutes (of which 38 contributed only one accession). with 52 institutes (spread over 23 countries), this selection includes 12.4% of all genebanks that contribute to eurisco. it comprises 52 different species of plants, of which barley (12 accessions), bread wheat (7), common bean (6) and maize (6) were most prevalent. requesting the material guidelines for requesting seeds were established in advance, including the use of a standardized text for all requests through email. this standard text did not disclose the purpose of our requests, but when a statement of purpose was explicitly required (occasionally during online requests or later communication, the following text was provided: “in the framework of a methodological research project we are exploring the content of european genebanks in eurisco and genetic resources (2025), 6(12), 205–210 availability of pgr 207 access to these valuable resources.” the depletion of valuable seed stocks was minimized by requesting small quantities (< 25 seeds) whenever the requesting process allowed specification of seed amounts. requests for vegetatively propagated material were withdrawn once the holding genebank indicated that the requested accession could be provided. the requesting procedure consisted of two rounds of requests: an initial request in which all genebanks were approached, and a follow-up in which contact was sought with genebanks in which the initial request did not result in a response. the initial request was made on 28 february 2025. an online search was conducted for the 52 selected genebanks to identify contact details or online ordering facilities. material was preferentially requested via online ordering. when no clear online order form or selection system was available, requests were submitted via email or online contact/feedback forms using standardized texts. requests were directed primarily to the email address indicated for ordering; if unavailable, contact was sought with the genebank manager, the genebank, or the associated institute, in that order. in the absence of a clear email address, the wiews listing corresponding to fao of the institute (fao, 2025a) was used. in five cases, emails were returned as “undeliverable,” prompting a search for alternative addresses. for one accession, a renewed search identified an online ordering facility, and in two cases, the initial email request led to referral to the online order site. the request cycle concluded once all institutions had been contacted via online order, email or online forms. approximately four weeks later (between 21 and 25 march 2025), follow-up emails were sent to the 23 genebanks that had not responded, or to inquire further about the status of our request. these reminders were directed to the previously used email addresses or submitted via online forms. where possible, the institutes’ email addresses listed in the fao wiews database were included in cc if no contact had been established during the initial request. when the initial request had been submitted through an online form, a separate email was sent to the wiews-listed address. the received seed material was stored in the drying chamber of the centre for genetic resources, the netherlands (cgn), and seed amounts were determined. two vegetatively propagated accessions were received, of which the number of viable cuttings was recorded. results requesting the material for two institutions (with one selected accession each), online searches for the genebanks yielded no leads, and wiews contact details were incomplete (no website or email). in a third case (one accession), the genebank indicated on its website that it was unable to accommodate new requests due to resource constraints. consequently, material could be requested from 49 genebanks, covering 97 accessions. when possible, material was requested through online ordering. of the 52 selected genebanks (conserving 100 selected accessions), 17 (maintaining 55 accessions) allowed online searches of their holdings, including 4 genebanks (conserving a total of 9 accessions) using the grin-global interface. not all institutes permitting online searches offered online ordering. seven initial requests were placed online, with an additional 5 following further inquiries, resulting in 12 institutions (23%) providing 31 accessions via online ordering. online ordering was not always straightforward, often requiring prior registration, navigating counterintuitive interfaces, or addressing technical errors that necessitated email contact. some failures to order had other causes: one accession could not be located via the online interface and was later confirmed as no longer held, while two accessions listed as ‘unavailable’ on a grin-global interface were requested via a contact form but received no response. overall, experiences with online ordering varied from straightforward to frustrating. of the 49 institutions (97 accessions) that could be approached, 7 (14%) were initially contacted through online ordering. the remaining 42 were contacted through email (39 genebanks) or online forms (3 genebanks), making email the principal mode of communication for requesting genebank material. email communication presented its very specific challenges: replies to requests were often sent from alternative addresses, causing changes in subject lines that complicated tracking of individual requests. telephone requests were not attempted. the initial 42 messages resulted in a reply in 18 cases (43%) but were not answered in most cases (57%): either no response was received (19 cases; 45%) or emails were returned as ’undeliverable’ (5 cases; 12%). repeated efforts, including renewed searches, alternative addresses and reminder emails, allowed us to establish contact with 33 institutions (including the one indicating unavailability of all material in the collection on its website), while 19 genebanks (37%) remained unreachable, representing 40 accessions. effectiveness of our requests highly depended on live and actively monitored addresses. during the request process, wiews-listed addresses associated with the fao code for the genebank used in eurisco were often necessary, as no other contacts were available online. of the 52 selected institutions, 6 had no email addresses listed in wiews, 6 addresses resulted in undeliverable emails, and 9 did not respond. thus, at least 21 of 52 institutes (40%, representing 29 accessions) could not be reached via wiews-listed addresses. some were nevertheless contactable through other online addresses. no systematic assessment of wiews address functionality was conducted; therefore, it cannot be assumed that the remaining 60% are reliably reachable. four requests (four accessions) were withdrawn by requestors during the requesting process. in two cases, requests were terminated after verification that material was available. this concerned a vegetatively propagated rhododendron accession and an accession for which the holding institute indicated that the requested seeds were in short supply, but that a small amount could nevertheless be distributed. two other requests (two accessions) were terminated without clarity on the availability status. these both concerned accessions that required special permissions to be granted (either by government or by breeders) prior to distribution: one cites-protected species and one commercial grape variety. both these accessions would – if permission were sought – be subject to mtas rather than standard smtas and be available for research only. these accessions were classified as ‘possibly available.’ genetic resources (2025), 6(12), 205–210208 wijnker and van hintum availability of accessions on 31 july 2025, five months after the initial request, the request period for this research was stopped. starting from an initial list of 52 genebanks (100 accessions), it was possible to assess the availability of selected accessions at 33 genebanks (63%; 60 accessions). contact was made with 32 institutes, and for one genebank (one accession), information for availability was derived from the website that indicated distribution was not possible. the remainder of 19 genebanks (37%; 40 accessions) could not be contacted through the channels used. data on accession availability are presented in table 1, in which material is listed as ‘available’ if it could be obtained within a 5-month period, given the used communication/ order methods. of the 100 selected accessions, 38 were classified as ‘available’. of these, 33 accessions were physically received from 19 genebanks (37%). five accessions were not physically received, but availability was inferred from communication with genebanks: two accession requests were withdrawn (because of low stocks or vegetative material) and three accessions were lost in transit (sent but not received). a further three accessions were considered ‘possibly available’: one delivered as the wrong accession and two requiring additional, special permissions. the remaining 59 accessions were considered ‘unavailable’: 1 accession still in process, 18 accessions explicitly unavailable according to genebank feedback, and 40 accessions held by institutes that could not be contacted (table 1). table 1. availability status of requested material. accessions have been grouped according to availability status, in which availability was defined as the ability to receive material within a 5-month period. *, four genebanks could not provide all accessions requested and appear in both cells marked with an asterisk; **, the genebank total indicates the number of genebanks included in this study. availability subgroup no. of genebanks no. of accessions notes available   18 + 4* 38 material was either received (33 accessions), the request was withdrawn by requestors to save seed stock of prevent vegetative propagation (2 accessions) or material was lost in the mail (3 accessions) possibly available request withdrawn 2 2 material may be obtainable if special permission is requested and granted (availability for research only; mta)   wrong accession was received 1 1   not available genebank confirmed non-availability 7 + 4* 18 material not available at genebank   long delivery time 1 1 ordering time exceeds the research limit of 5 months   genebank did not respond 19 40 material could not be requested due to no response total   52** 100   for 60 accessions (33 genebanks), we obtained information on their availability (other genebanks could not be contacted or did not respond). of those, 18 accessions (30%) were unavailable. reasons for their unavailability were provided for 14 accessions, with a ‘lack of material’ being the most prevalent reason (8 accessions, 3 genebanks). legal requirements prevented the distribution of two accessions, international armed conflicts prevented distribution of two accessions (two genebanks), one genebank indicated the inability to distribute any material on their website, and one accession was no longer maintained in the collection (table 2). availability via online ordering did not appear to correlate with seed availability. of 31 accessions requested online (12 genebanks), 8 accessions (26%) were unavailable, similar to the overall unavailability rate of 30% for genebanks that provided feedback on holdings. table 2. reasons for non-availability of requested material as provided by genebanks. reason for not sending no. of genebanks no. of accessions genebank cannot distribute 1 1 international agreements prevent distribution 1 1 lack of material/too few seeds 3 8 material is not disease tested 1 1 no longer in collection 1 1 war prevents sending 2 2 no reason provided 2 4 total 11 18 . distribution details, payments and conditions for use by recording the moment of parcel reception, the approximate delivery time for the requested pgr could be inferred (figure 1). the majority of seed shipments (14 out of 17 packages) were received between two and eight weeks after initiating the request. the average time from first contact to accession receipt was six weeks. all received material arrived undamaged. seed quantities per accession varied widely, ranging from 11 seeds (three accessions) to 381 seeds (one accession), with a median of 50 seeds. clonal material (cuttings) was received from two institutes (two accessions) and was alive and viable upon arrival. genetic resources (2025), 6(12), 205–210 availability of pgr 209 in three cases, payments were required as part of the request process. two genebanks (two stations of the same institute) charged handling fees (€10 per request plus €2 per accession), and one genebank charged handling fees (€17) plus phytosanitary testing costs (€177) for two accessions. the majority of received accessions (obtained from 19 genebanks) required a material transfer agreement (mta). for 16 genebanks (84%; 30 accessions), a standard mta (smta) was used, either signed manually with electronic exchange of documents (eight cases) or via click-wrap/easysmta (eight cases). two genebanks (providing one accession each) provided accessions without any user restrictions, and one genebank distributed under a different mta. all 30 accessions received under smta were obtained from genebanks located in countries that ratified the itpgrfa (fao, 2009). the one accession obtained under a different mta was received from a genebank located in a country that did not ratify the itpgrfa. discussion access to pgr is a fundamental prerequisite for their effective utilization, and constitutes a central theme in international policy discourse, particularly in relation to access and benefit-sharing (abs) mechanisms under the itpgrfa. despite the prominence of this issue, the practical conditions governing physical access to pgr remain insufficiently documented. empirical investigations of this domain are scarce, with bjørnstad et al (2013) being among the few to demonstrate that presumed availability of pgr is not always substantiated in practice. in light of this, a systematic assessment of pgr availability within europe is both timely and imperative. although locating accessions via the eurisco catalogue is relatively straightforward, the process of requesting material figure 1. time required for receiving ordered ex situ accessions from 17 genebanks. the histogram shows the time (in weeks) between the moment a genebank was contacted and the receipt of the parcel. from the respective holding institutions proved considerably more challenging. for 22 genebanks (42%), we did not find a website dedicated to genebank activities (like describing collections and featuring genebank contact details). when websites did exist, the procedures for ordering material were frequently ambiguous. as a result, email emerged as the primary mode of communication. the request protocol was standardized and limited to two contact attempts per genebank, which was deemed a reasonable threshold for effort. while alternative strategies, such as translating emails into the local language, telephone outreach or leveraging personal networks, might have improved response rates, they were not employed in this study. of the 52 approached genebanks, 22 genebanks (42%) sent (at least part of) the requested material, which might have increased to 25 genebanks (48%) with additional effort (see table 1). likewise, it is estimated that the current availability of pgr accessions ranges between 38% and 41%, with the upper bound including accessions that may be obtainable through additional effort (see table 1). because of the small sample size, we suggest that this number be interpreted with caution. the average time required to receive seed material was approximately 6–7 weeks, a duration considered acceptable for most research and breeding purposes. nevertheless, the study reveals that material from 27 genebanks (52%) could not be obtained, leaving 59% of accessions presently inaccessible. this highlights a significant and alarming proportion of inaccessible material in european genebanks. the principal barrier to accessing pgr appears to be the difficulty in establishing communication with genebank personnel. the initial round of requests elicited responses from only half of the 52 genebanks contacted, which increased to two-thirds (33 genebanks) following a second round. notably, one-third of the genebanks (19 out of 52; 37%) remained entirely unresponsive. the difficulty of contacting genetic resources (2025), 6(12), 205–210210 wijnker and van hintum genebanks was also due to the unclarity and unreliability of contact information on websites or in databases, like wiews. while the reasons for non-responsiveness of genebanks are speculative, the data suggest that not all genebanks prioritize the facilitation of pgr access. these findings align with those of bjørnstad et al (2013), who reported a similarly high rate of non-responsiveness among contracting parties in a global study on facilitated access. although eurisco serves as a valuable repository for documenting pgr holdings, our findings indicate that actual access to these resources remains a significant challenge. extrapolating the estimated availability rate of 38–41% to the broader eurisco database (excluding arabidopsis accessions) suggests that only approximately 481,000 to 594,000 accessions may be readily obtainable. this figure stands in stark contrast to the more than two million accessions currently documented, highlighting a substantial gap between nominal documentation and actual accessibility. the sample size for our research was deliberately kept small: 0.005% of eurisco accessions and 12.4% of contributing genebanks (including all those that contribute > 1% of all eurisco accessions). our estimate for availability is therefore to be interpreted with caution. likewise, the small dataset limits the ability to statistically discern possible causes underlying seed availability. for example, requests to ten genebanks (19%) were directed to genebanks in four countries that did not ratify the itpgrfa. of the 25 requested accessions, 92% were classified as non-available. but assessing any effect of ratification of the itpgrfa on availability would be confounded by the fact that nine of these requests were made to institutions in countries involved in armed conflicts during our research period (israel, russia and ukraine). in this case, feedback provides more insight. of the four genebanks from these countries that replied to our emails, itpgrfa ratification was not mentioned as prohibiting seed distribution. two of the genebanks did explicitly mention that the ongoing war prevented distribution (table 2), indicating that pgr availability can be affected by geopolitical conflicts. europe hosts over 400 genebanks and collection holders in 43 countries that are listed in eurisco, yet it remains unclear whether all institutions are actively conserving and distributing their holdings. this raises critical questions regarding institutional capacity and commitment to resource sharing. the observed challenges in distribution point to a disconnect between the documented inventory of accessions and their actual availability to users. many factors affect the availability of accessions from genebanks, ranging from expired email addresses to war. even though this research could not address the underlying causes, elucidating these will be of the greatest interest to both genebanks and their stakeholders. for many genebanks, having material available for users touches directly on their raison d’être and may affect societal support for their work. for other stakeholders, a clear grasp of the underlying causes may help take measures to improve and promote pgr availability. in conclusion, the findings of this study underscore the urgent need to address systemic barriers to pgr access in europe. failure to do so risks eroding valuable genetic resources, decreasing societal support for genebanks and impeding the capacity of researchers and breeders to develop resilient crop varieties essential for food security. author contributions both authors contributed equally to the manuscript acknowledgements we would like to thank the many enthusiastic genebank staff we interacted with during the course of our research. this work was carried out in the framework of the programme genetic resources (wot-03 and kb-34) funded by the dutch ministry of agriculture, fisheries, food security and nature. conflict of interest statement the authors have no relevant financial or non-financial interests to disclose. data availability no data accompany this manuscript, but details on sampling procedures and collected data may be requested by contacting the authors. references bjørnstad a., tekle s., goransson m. (2013). ‘‘facilitated access’’ to plant genetic resources: does it work? genet. resour. and crop evol. 60, 1959-1965. doi: https://doi. org/10.1007/s10722-013-0029-6 dulloo e., khoury c. k. (2023). towards mainstreaming global crop conservation strategies (germany, bonn: global crop diversity trust). doi: https://doi.org/10.5281/ zenodo.7610356 ecpgr (2025). european search catalogue for plant genetic resources (eurisco). http://eurisco.ecpgr.org fao (2009). international treaty on plant genetic resources for food and agriculture (itpgrfa) (italy, rome; food and agriculture organization of the united nations). https:// www.fao.org/plant-treaty/overview/text-treaty/en fao (2014). genebank standards for plant genetic resources for food and agriculture. rev. ed. (italy, rome: food and agriculture organization of the united nations). https:// www.fao.org/3/i3704e/i3704e.pdf fao (2022). practical guide for the application of the genebank standards for plant genetic resources for food and agriculture: conservation of orthodox seeds in seed genebanks (italy, rome: fao commission on genetic resources for food and agriculture). doi: https://doi. org/10.4060/cc0021en fao (2025a). world information and early warning system on plant genetic resources for food and agriculture (wiews). https://www.fao.org/wiews/data/ex-situ-sdg-251/search/en fao (2025b). the third report on the state of the world's plant genetic resources for food and agriculture (italy, rome: fao commission on genetic resources for food and agriculture), 374 p. doi: https://doi.org/10.4060/cd4711en genesys (2025). genesys plant genetic resources portal (genesys pgr). https://www.genesys-pgr.org/ kotni p., van hintum t. j. l., maggioni l., oppermann m., weise s. (2023). eurisco update 2023: the european search catalogue for plant genetic resources, a pillar for documentation of genebank material. nucleic acid res. gkac852. doi: https://doi.org/10.1093/nar/gkac852 https://doi.org/10.1007/s10722-013-0029-6 https://doi.org/10.1007/s10722-013-0029-6 https://doi.org/10.5281/zenodo.7610356 https://doi.org/10.5281/zenodo.7610356 http://eurisco.ecpgr.org https://www.fao.org/plant-treaty/overview/text-treaty/en https://www.fao.org/plant-treaty/overview/text-treaty/en https://www.fao.org/3/i3704e/i3704e.pdf https://www.fao.org/3/i3704e/i3704e.pdf https://doi.org/10.4060/cc0021en https://doi.org/10.4060/cc0021en https://www.fao.org/wiews/data/ex-situ-sdg-251/search/en https://doi.org/10.4060/cd4711en https://www.genesys-pgr.org/ https://doi.org/10.1093/nar/gkac852 _hlk206773289 _hlk206150314 _hlk211340048 _hlk207785654 original article genetic resources (2025), 6 (12), 1–13 doi: 10.46265/genresj.jcdc4631 https://www.genresj.org issn: 2708-3764 cultivar loss and conservation of genetic resources of the phureja potato (solanum phureja l., phureja group) in peru juan f seminario *,a, leónidas s concha-tupayachi b, alejandro seminario-cunya c, tulio medina-hinostrosad and wilsser silva *,e a facultad de ciencias agrarias, programa de ráıces y tubérculos andinos, universidad nacional de cajamarca, peru b universidad tecnológica de los andes, cusco, peru c universidad nacional autónoma de chota, peru d dirección de recursos genéticos y bioseguridad del ministerio del ambiente, lima, peru e programa de doctorado en ingenieŕıa y ciencias ambientales, universidad nacional agraria la molina, av. la molina s/n, la molina-lima, peru abstract: solanum tuberosum l. phureja group, known in peru as ‘phureja potato’ or ‘chaucha potato’ and as ‘criolla’ in colombia, is characterized by its earliness and the absence of dormancy in the tubers. it stands out for its nutritional value and its contribution to food security. however, it faces a high risk of disappearance in peru. this study assessed its current status by collecting historical data, ex situ and in situ conservation analyses, and genetic erosion studies in local communities. historical information suggests that phureja was relevant and abundant in the past. currently, ex situ collections include 69 accessions, of which the international potato center conserves a significant portion. as for in situ conservation, 116 accessions have been identified. however, since 1992, genetic erosion has been documented in six departments of peru. the main causes include: lack of time for continuous cultivation, prioritization of dairy farming, low seed quality, preference for more commercial modern or traditional cultivars, and the expansion of mining projects. the critical situation of the phureja potato requires urgent measures to collect new information and evaluate the remaining genetic variability. this assessment is essential to develop conservation and sustainability strategies to ensure its survival and its contribution to peru’s food and cultural well-being. keywords: genetic erosion; potato diploid, solanum phureja, yellow potato, chaucha citation: seminario, j. f., concha-tupayachi, l. s., seminario-cunya, a., medina-hinostrosa, t., silva, w. (2025). cultivar loss and conservation of genetic resources of the phureja potato (solanum phureja l., phureja group) in peru. genetic resources 6 (12), 1–13. doi: 10.46265/genresj.jcdc4631. © copyright 2025 the authors. this is an open access article distributed under the terms of the creative commons attribution license (cc by 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. introduction the understanding of global biodiversity remains limited, reaching only 20% of species. especially in the centres of origin of cultivated plants, there are gaps in knowledge about how farmers preserve local cultivars. this, in turn, hinders the implementation ∗corresponding authors: juan f seminario (jfseminario@yahoo.es), wilsser silva (20240313@lamolina.edu.pe) of methodologies that could favour conservation, such as establishing baselines, conducting monitoring and collecting evidence on cultivar dynamics (losses and additions) and the likely genetic erosion involved (de carvalho et al, 2016; dawson et al, 2023). genetic erosion – the loss of crop genetic diversity in specific contexts of time and space – is a persistent concern in the agri-food field. its dynamics, triggers, measurement methods and magnitude of losses are still not fully understood (van de wouw et al, 2009; khoury et al, 2022). this problem is especially critical received: 22.02.2025 accepted: 19.05.2025 published online: 07.07.2025 https://www.genresj.org https://www.doi.org/10.46265/genresj.jcdc4631 https://www.genresj.org https://www.doi.org/10.46265/genresj.jcdc4631 mailto:jfseminario@yahoo.es mailto:20240313@lamolina.edu.pe 2 seminario et al genetic resources (2025), 6 (12), 1–13 in key crops such as potato, which is key to food security, nutrition and sustainability of global agrifood systems. the reduction of the crop’s genetic diversity limits its ability to adapt to environmental changes and phytosanitary challenges, threatening both agricultural productivity and the livelihoods of dependent communities (burgos et al, 2020; devaux et al, 2020). the phureja potato is classified under the international code of botanical nomenclature (icbn) as the species solanum phureja (juz. & buk.). its taxonomic key indicators are: corolla lobes wider than half the petal length, short acuminata, leaves somewhat glossy, less densely pubescent (ochoa, 2001). on the other hand, according to the international code of nomenclature for cultivated plants (icncp), this potato is located within the phureja group, as solanum tuberosum l. phureja group, and its taxonomic key indicates that tubers sprouted at harvest (huamán and spooner, 2002). its most notable features are its rapid maturation, from three to four months, and the lack of a resting period for its tubers (ochoa, 2001; huamán and spooner, 2002). the phureja potato stands out for its high nutritional and culinary value, and its richness in secondary metabolites with antioxidant properties. it is an important source of essential minerals such as potassium, iron and zinc, which reinforces its nutritional value (cerónlasso et al, 2018; beltrán-penagos et al, 2020; peña et al, 2021). its tolerance to adverse conditions, such as poor soils and high altitudes, makes it a resilient option in the face of climate change. it also has great potential in the produce industry. its role in genetic improvement is crucial, contributing valuable genes to develop more resistant and sustainable varieties (gabriel and franco, 2013; ñústez and rodŕıguez, 2020). it has been considered as one of the world’s 50 foods of the future (ñústez, 2021). despite its importance, the phureja potato has received little conservation attention in peru. this situation is evident in the south and centre of the country, where several studies conducted in the last three decades (zimmerer, 1991; de haan and thiele, 2004; cip and fedech, 2006; plasencia et al, 2018) have documented significant losses. similarly, a report focusing on northern peru (seminario and zarpán, 2011) suggest that this potato could be at risk of disappearing. based on this background, the objective of this research was to gather evidence to evaluate its relative importance in the past and to analyze the loss of cultivars that occurred in the last decades in peru. materials and methods records of the presence, importance and relative abundance of the phureja potato in peru historical sources on ancient peru were used, including the works of varcárcel (1985), the report by j.b. martinet at the end of the 19th century (martinet, 1977), and the works of herrera (1921) and vargas (1936, 1946, 1955), among others. historical information was also gathered on one of the pioneers in potato conservation and genetic improvement in the paucartambo region (cusco). this is mr. yabar, recognized for his valuable work between the 1930s and 1945, during which time he promoted early practices of conservation and selection of local potato cultivars (yabarvillagarćıa, 2004). another key source was the database of ochoa (2003), which contains information on passport data from native potato collections carried out in peru between 1947 and 1997. information about ex situ conservation of the phureja potato in peru in 2020, a contact was established with personnel responsible for the potato collections of the experimental stations at the national institute for agrarian innovation (inia), the institution in charge of preserving the genetic resources of cultivated plants in peru, located in the localities of puno, cusco, juńın, ayacucho and cajamarca. during this period, detailed information was collected on the number of accessions (units of genetic material) of phureja potatoes conserved in each station. this work allowed us to understand the practices and criteria applied for the conservation of this germplasm in different regions of the country. in addition, in 2023, the database of the international potato center (cip) was accessed to obtain the number of phureja potato entries and those corresponding to peru. in cusco, access was granted to the germplasm database managed by a group of communities there known as the ‘parque de la papa’, managed by the ngo andes del cusco association. this database is an exhaustive register of native potato cultivars, including phureja varieties, conserved ex situ. among these materials are samples repatriated from cip, preserved in collaboration with local communities associated with the park. in addition, information was collected from the regional center for andean biodiversity research (criba) of the universidad nacional de san antonio abad del cusco (unsaac), specifically on the number of native potato and phureja varieties stored in its germplasm bank. in the city of paucartambo, the salcedo rojas family coordinates the activities of the paucartambo conservationists association. after an interview with two family members, we were able to access (for systematization and analysis) the database on native potatoes maintained by 30 conservationists from the districts of paucartambo, challabamba, colquepata, and huancarani. in addition, an interview was conducted with julio hancco, an outstanding conservationist from the community of pampacorral, located in the province of lares, who is widely recognized for his work in the conservation of agricultural biodiversity. in the cajamarca region, interviews were conducted in 2011 and 2021 with 20 potato conservation farmers genetic resources (2025), 6 (12), 1–13 conservation status of phureja potato in peru 3 in the provinces of cajamarca, celend́ın, hualgayoc and san marcos, previously identified in a study by seminario and zarpán (2011). the objective of these interviews was to determine the number of germplasm entries they maintain and to analyze the socioeconomic factors that affect their conservation work. information about in situ conservation of the phureja potato in peru the catalogues of native potatoes produced in peru during the last two decades were compiled and analyzed. those catalogues that specify the species to which the accessions belong or that, at least, record the duration of tuber dormancy were included. although these documents do not provide direct information on cultivar erosion, the presence or absence of phureja potatoes in these collections is considered a relevant indicator of the level of conservation of this group on farmers’ farms. in cajamarca region, information was collected on phureja potato collections carried out since 2005 in several provinces of this region. these data provided a comprehensive overview of the conservation efforts of potato cultivars and varieties by the communities. in addition, these activities made it possible to explore the challenges, strategies and traditional knowledge associated with in situ conservation, especially highlighting the crucial role of communities in the preservation of the genetic diversity of the phureja potato. also in 2021, in the provinces of chota and cutervo, interviews were conducted with 45 potato farmers and information was collected on the phureja cultivars planted in the previous season and the varieties lost in the last two to three decades (figure 1). in the locality of waqanqa, located in the district of paucartambo, cusco, direct observation was carried out between 16 and 19 february 2020 with the objective of identifying potato cultivars. in addition, 16 farmers from the districts of paucartambo and challabamba, who participated in the xix agricultural fair, held on 11-12 september 2023, were interviewed (figure 1). empirical evidence on genetic erosion of the phureja potato in peru an exhaustive literature search for research related to the genetic erosion of the phureja potato was carried out, covering articles and other scientific publications produced between 1980 and 2023 that document the loss of native potato germplasm in peru. for this purpose, physical libraries and bibliographic databases of wide coverage, such as google scholar, scielo, web of science and scopus, were consulted in order to gather and analyze information on this topic. results presence, importance and relative abundance of the phureja potato in peru according to information gathered from existing literature, catalogues, germplasm banks and farmers’ knowledge, the phureja potato is grown or present in 11 of peru’s 25 regions. these regions are mainly located in the andean zone, at altitudes ranging from 2,000 to 3,500 meters above sea level (figure 1). since ancient times, the phureja potato has been an essential source of food and a key component in the food security of andean communities (forbes et al, 2020). in ancient peru, five types of potato were identified, classified in quechua language according to their morphological characteristics and agroecological adaptations: hatun papa (large potato), chaucha [= phureja] papa (early maturing potato), maguay papa (early planting potato), capo papa (possibly qjapo papa, associated with elevated areas and dark soils) and chiri papa (potato cultivated in cold regions) (varcárcel, 1985). herrera (1921) described the diversity and quality of potatoes in paucartambo (cusco), classifying non-bitter potatoes into four groups. within the group of elongated potatoes, he included the chaucha or phureja potato, characterized by its rapid growth, watery tubers and reddish buds, although there were also varieties with round tubers and white buds. these potatoes have a cultivation cycle of approximately three months. vargas (1936) also highlighted the importance of phureja potatoes in paucartambo. these, known locally as papa nueva, mosoc papa or misca mahuay, are sown early and are used both for immediate consumption and starch extraction. in addition, vargas (1946) gathered a collection of 774 potato samples mainly from puno, apuŕımac and cusco, of which 79% corresponded to solanum andigenum and 10% to solanum stenotomum. later, in las papas sudperuanas, parte ii, vargas identified four clones of chaucha potatoes (1206, 1207, 1208 and 1209) belonging to s. stenotomum, and one clone (549) classified within s. andigenum (vargas, 1946). luis á. yabar ordóñez (1886-1965), a horticulturist originally from paucartambo, stood out as a pioneer in the conservation of native potatoes in peru during the period from 1930 to 1945. yabar compiled and maintained a valuable collection of approximately 250 native potato cultivars, including the churumpis, chauchas or phurejas, miskillas, thumillas, tokolos and phasñachas groups (yabar-villagarćıa, 2004). his work was widely recognized by specialists in the study of this tuber. in 1944, j. g. hawkes acknowledged his contribution by naming a species in his honour: s. yabari, which included two varieties, s. yabari var. pepino and s. yabari var. cuzcoense. these species were later reclassified as part of s. stenotomum (ochoa, 1999; watanabe et al, 2008). 4 seminario et al genetic resources (2025), 6 (12), 1–13 figure 1. map of the 25 regions of peru showing the location of in situ studies, as well as the presence and erosion of the phureja potato. a report from the mid-1940s noted that commercial potato varieties in peru included chata blanca, chata rosada, chata negra, chata negra, amarilla, shitu, mauna and chaucha (derteano, 1944). in the 1960s, c. ochoa documented the presence of specimens of chaucha blanca, chaucha colorada and chaucha negra in the province of sandia, puno (ochoa, 1964). subsequently, soukup (1970) described the chaucha potato or phureja potato as an early maturing variety characterized by its rapid cooking, even at the first boiling. in cajamarca, iberico (1981) noted that the chaucha potato is distinguished by its characteristic colour, similar to that of egg yolk, as well as its extremely smooth texture and pleasant flavour. this tuber is commonly consumed parboiled and with skin. leiva et al (1990) documented its use in traditional medicine, specifically to relieve headaches. in this context, it is applied sliced with salt as a poultice on the temples, or ground in combination with olluco (ullucus tuberosus), white corn (zea mays), salt and vinegar for therapeutic purposes. c. ochoa, noted for his extensive potato collections between 1947 and 1997, collected a total of 322 phureja potato samples from 15 departments of peru. these samples were organized according to their relative abundance as follows: amazonas (140), puno (52), cajamarca (30), ayacucho (28), piura (16), huánuco (12), juńın (11), lima (11), la libertad (6), apuŕımac (4), huancavelica (4), ancash (2), cusco (2), pasco (1) and lambayeque (1). these data reflect the distribution and prevalence of the phureja potato during this period. however, ochoa (2003) noted that these samples were preserved only as herbarium specimens in various museums in peru and other parts of the world. ex situ conservation of the phureja potato in peru in peru, 6,295 native potato accessions have been identified in eight databases of working collections, genetic resources (2025), 6 (12), 1–13 conservation status of phureja potato in peru 5 including a university, a civil association and four experimental stations of inia located in the sierra (table 1). of these, only 0.3% correspond to the phureja group, which shows its minimal representation, even in the three main collections of the cusco region, where its presence is insignificant. at the international level, cip, which houses the world collection of potato germplasm, conserves only nine accessions of solanum tuberosum l. phureja group from peru, out of a total of 181 accessions of this species, most of which originate from colombia and ecuador (cip, 2023). the database of the paucartambo native potato growers’ association initially registered 2,500 cultivars. each farmer maintains between 38 and 140 cultivars on average. within this group, only two cultivars belong to the phureja group: puka q’achirma and k’ello q’achirma. both varieties are kept by a single farmer in the locality of challabamba. julio hancco, a well-known conservationist from pampacorral, cusco, reported that he had a collection of 350 potato cultivars, although none belong to the phureja group. he also reported having limited knowledge of this category of potato. consistently, the analysis of the catalogue of a lot composed of 115 documented cultivars from his collection did not show the presence of cultivars of the phureja group either (hancco et al, 2008). in 2010, 20 conservationists of the cajamarca potato producers association dedicated to the conservation of native potatoes, including cultivars of the phureja or chaucha group, were identified in cajamarca. of these, 17 maintained between 1 and 12 cultivars of the phureja group (table 2). by 2021, the number of active conservationists decreased to 18, of which only 11 preserved between 1 and 10 cultivars of this group (table 2). the total reduction in the number of native potato accessions in general and of the phureja group in particular was 50% and 52%, respectively. the decrease in cultivars observed in 2021, together with the reduction in the number of active conservationists, is partly attributed to the advanced age of the latter, with an average age of 62 years, which limited their ability to continue conservation work. in addition, there were significant changes in the main economic activities of the conservationists: five focused primarily on livestock, four replaced agriculture with other occupations (such as carpentry, salaried work or temporary migration), three moved to urban areas while maintaining only partial agricultural activities, and one completely abandoned his activities due to chronic illness. together, the remaining small collections total 35 accessions conserved ex situ in cajamarca (table 2). in situ conservation of the phureja potato in peru between 2006 and 2023, 27 regional catalogues of native potato were prepared, of which 19 met the established inclusion criteria. a total of 2,102 native potato accessions were identified in these catalogues, of which 4.3% correspond to the phureja group (table 3). among the most outstanding catalogues are: cuyo cuyo (puno), in the south, with 13 cultivars of the phureja group (wildlife conservation society , 2022); huánuco, in the centre of the country, with 17 cultivars of phureja potato (egúsquiza, 2015); and cajamarca, in the north, which records 43 cultivars of phureja. the cajamarca collection was morphologically characterized, highlighting the first 15 accessions collected in three districts of hualgayoc, which achieved a successful harvest (figure 2) (seminario and zarpán, 2011; seminario et al, 2019). in addition, in 2021, in the cajamarca provinces of chota and cutervo, 24 cultivars of phureja planted in local farms during the last agricultural seasons were identified, and identified by their traditional names. the exploration conducted in the community of waqanqa (paucartambo, cusco) indicated that potato cultivation has been reduced in area and cultivars. no phureja potato cultivars were found. on the contrary, corn, apple and pasture crops stood out. also, 16 farmers participating in the xix agricultural fair of paucartambo (11-12 september 2023), who were interviewed, indicated that they maintained between 2 to 250 varieties of native potato, and nine of them planted one to two chaucha cultivars (puka chaucha or k’ello chaucha). they mentioned that these potatoes had been lost due to lack of time to attend to the crop, because of their rapid sprouting and the scarcity of seed. empirical evidence of genetic erosion of the phureja potato in peru genetic erosion in potatoes of the phureja group had its first documented evidence in waqanqa, the largest community in the mapacho river valley, in paucartambo. this group of potatoes, traditionally cultivated in the region, showed a progressive disappearance since the 1960s, being reduced to only four or five varieties in 1987. factors such as labour shortages, migration to urban areas and low local appreciation of these cultivars led to their replacement by improved varieties (zimmerer, 1991, 1992). canahua et al (2002) conducted a study in regions with extensive areas of potato and quinoa cultivation in six provinces of puno. the results indicated that the cultivation of potatoes of the phureja group is restricted to small areas in the localities of moho, yunguyo and viquechico. in addition, it was concluded that these cultivars are in danger of disappearing due to their low yields, despite being valued for their short maturity cycle and remarkable regeneration capacity. de haan and thiele (2004) documented a decline in the frequency of cultivation of cultivars of the phureja group in the district of yauyos, lima. this phenomenon was attributed to factors such as limited seed availability, low capacity for germplasm exchange, labour shortages, and farmers’ increasing preference for more commercial cultivars such as ‘huayro’ and ‘peruanita’. 6 seminario et al genetic resources (2025), 6 (12), 1–13 table 1. accessions of native potato and phureja potato, maintained ex situ in regional genebanks in peru, 2020. a, 778 collected within the potato park, 410 repatriated from cip and 150 obtained through seed exchange; b, collected in puno. n/a: information not available. location native potato phureja potato source institutions cusco 2,500 12 l. lizárraga, interview, 18 feb 2020 rcabr -unsaac pisac, cusco 1,345a n/a a. argumedo, interview, 15 feb 2020 andes association, potato park cusco 1,300 5b l. palomino, interview, 20 apr 2020 inia, andenes ayacucho 400 n/a m. morote, interview, 2 may 2020 inia, canán juńın 300 3 n. zúñiga, interview, 2 mar 2020 inia, santa ana puno 450 1 r. cahuana, interview, 15 may 2020 inia, illpa cajamarca n/a n/a h. roncal, interview, 25 may 2020 inia, baños del inca total 6,295 21 table 2. native potato accessions (total and phurejas) maintained by conservationist farmers in cajamarca in 2010 and 2021. source: interviews conducted in 2010 (seminario and zarpán, 2011) and 2021, respectively. n/a: not available. conservationist name province/district age in 2021 2010 2021 total acc. phureja acc. total acc. phureja acc. juan huaccha san marcos/ pedro gálvez 62 200 2 90 2 santos abanto san marcos/gregorio pita 61 90 3 30 1 pedro i. abanto san marcos/pedro gálvez 49 45 3 25 0 orestes dávila san marcos/ josé sabogal 65 40 1 20 1 termópilo arévalo celend́ın/sorochuco 59 90 4 100 10 sergio rodŕıguez celend́ın/sorochuco 65 82 n/a 30 3 armando vergara celend́ın/huazmı́n 54 100 10 n/a n/a segundo d. gil celend́ın/huazmı́n 66 83 12 50 5 idelso garay celend́ın/huazmı́n 71 65 2 80 0 alindor d́ıaz cajamarca/ la encañada 74 45 3 15 3 miguel riquelme cajamarca/la encañada 73 45 4 50 2 gumercindo zelada cajamarca/encañada 55 45 4 10 2 josé i. ayay valdez cajamarca/ cajamarca 70 75 0 70 1 germán sangay cajamarca/ encañada 58 35 3 60 0 emilio huamán cajamarca/namora 48 295 4 0 0 abel maŕın ŕıos cajamarca/namora 61 180 1 15 0 juan e. mendoza hualgayoc/bambamarca 50 15 6 20 5 josé telmo cabrera san marcos/gregorio pita 65 80 3 70 0 luis cabrera ocas san marcos/gregorio pita 70 180 2 160 0 wilson pastor maŕın san marcos/gregorio pita 67 50 0 20 0 total 1,840 67 915 35 cip and fedech (2006), together with de haan et al (2010), conducted a study in huancavelica on two potato groups, identifying the presence of 144 and 557 cultivars from four provinces and eight communities, but found no cultivars belonging to the phureja group. this absence was attributed to factors such as seed loss, temporary migration of inhabitants, substitution by modern cultivars and the lack of dormancy characteristic of this species. however, brush et al (1981) documented the existence of a single cultivar of solanum phureja called ‘pujuya’ in the community of aymará, tayacaja district, huancavelica, known for its frost resistance and also cultivated by farmers in nearby regions such as chongos alto, in huancayo, juńın. the ministry of agriculture conducted research in the southeastern juńın department on native potatoes, identifying that it was currently difficult to find cultivars of the phureja group (minagri, grupo yanapai, inia and cip, 2017). similarly, plasencia et al (2018), in a study on the diversity of native potatoes in challabamba (paucartambo) and quillcas (juńın), reported the presence of all the species studied, except s. tuberosum l. phureja group, although the causes of its absence were not determined. seminario and zarpán (2011) and seminario et al (2019) estimated a 17% reduction in cultivars of the phureja group in five provinces of cajamarca over the previous two decades. the main causes of this decline include the lack of time to attend to the crop, due to its short sowing and harvesting cycles; the preference of local communities for livestock activities rather than agriculture; the low quality of seed, which in many cases genetic resources (2025), 6 (12), 1–13 conservation status of phureja potato in peru 7 table 3. number of potato phureja cultivars in 19 peruvian native potato catalogues in 2023. regions/communities total cultivars phureja potato source huancavelica/ huayta corral, tupac amaru, villa hermosa, pucara, dos de mayo 144 0 cip and fedech (2006) cusco/ huama, huarqui, poques, patacancha, willoc, tauca 260 0 cosio (2006) cusco/ palccoyo, acco acco phalla y quisini (district of sicuani) 141 0 gutiérrez and valencia (2010) cajamarca/ chota and lajas 23 5 incap jorge basadre (nd) cajamarca/ three communities of shitamalca 24 1 programa bioandes (nd) cajamarca 28 5 cabrera and pando (2011) cajamarca/22 communities 43 43 seminario et al (2019) puno 86 0 muñoz and estaña (2012) cusco/ quescay, kcallacancha, sipascancha alta, miscahuara 30 4 revilla (2014) apuŕımac y huancavelica. 24 0 fonseca et al (2014) la libertad/ san juan, la soledad, canucubamba, macullida, las colpas, arcopampa y chugay 129 1 cip, asociación pataz., and inia (2015) huánuco/ 35 communities 296 17 egúsquiza (2015) huánuco, juńın, huancavelica, ayacucho, apurimac 12 0 riveros and peralta (2015) juńın/ seven communities and 14 families in the southeastern part of the department 146 1 minagri, grupo yanapai, inia and cip (2017) apuŕımac/ huayana y pomacochas 119 0 proderin (2018) apurimac, cusco y puno/113 communities of apurimac, 58 de cusco and 8 of puno 200 0 roldan et al (2019) huancavelica/ castillapata, paltamachay, huachhua, paccho molinos, santa rosa de pacchacclla y pumaranra 184 0 cip, grupo yanapai, gobierno regional de huancavelica and aguapan (2021) puno/ cuyocuyo 91 13 wildlife conservation society (2022) la libertad/ la victoria 122 0 asociación-pataz, cip, inia and aguapan (2023) total 2,102 90 was depleted and with poor yields; the expansion of mining projects in the region; the trend towards the cultivation of modern varieties and more commercial native cultivars; labour shortages, caused by emigration and employment in non-agricultural activities; and the limited availability of seed for crop regeneration. in 2021, a field study in the provinces of chota and cutervo (cajamarca) revealed the loss of eight phureja potato cultivars in the last two decades, reducing the total recorded from 32 to 24 (table 2). the missing cultivars included huevo de perdiz, baya, cemelina, rosada, morada, negra, chilopa and amapola. the main causes identified are the rapid growth cycle of this group, which makes its management difficult; the preference for modern cultivars that are more competitive in the market; the prioritization of livestock; and the low quality of the available seeds, known as ‘tired seeds’. currently, the remaining germplasm of phureja potato in cajamarca comprises 67 cultivars, of which 43 have been morphologically characterized in five provinces, while 24 are only nominally registered in chota and cutervo. it is essential to extend studies to other provinces to evaluate diversity and promote its conservation. discussion historical evidence (vargas, 1936, 1946, 1955; herrera, 1921; varcárcel, 1985) and ochoa (2003) collections in 15 of the 19 departments where potatoes are grown in peru highlight the historical relevance of the phureja potato. its distinctive characteristics, such as the absence of dormancy, precocity and adaptation to early harvests, underline its agricultural importance. however, it is necessary to thoroughly review the historical data (van de wouw et al, 2009) and to explore again the sites visited by ochoa to confirm the persistence of phureja in these regions. this will allow updating knowledge about its persistence, as well as its agricultural and cultural value in the current context. ex situ collections are essential to preserve genetic diversity and prevent its loss, acting as a vital complement to in situ conservation. both strategies 8 seminario et al genetic resources (2025), 6 (12), 1–13 figure 2. phureja or chaucha potato cultivars harvested in the province of hualgayoc, cajamarca, northern peru, in 2005: 1.chaucha (ch). chachapoyana, 2. ch. blanca, 3. ch. huagalina, 4. ch. porpora, 5. ch. conda, 6. ch. amarilla, 7. ch. negra, 8. ch. colombiana, 9. ch. colombina negra, 10. ch. montañera, 11. ch. shoga, 12. ch. pabla, 13. ch. pimpinela, 14. ch. blanca peruanita, 15. ch. clavelina. in northern peru, the names of phureja potato cultivars are usually composed of two elements: a primary common name, such as chaucha, and a secondary name which, in many cases, may coincide with the names of cultivars belonging to other potato groups. for example, ch. huagalina, whose secondary names may be related to specific physical characteristics. are integrated to ensure the availability of these resources for future generations (priyanka et al, 2021). a key advantage of ex situ collections is their accessibility to researchers and users, allowing their use in training, genetic improvement, research and repatriation to source communities (fu, 2017; ellis et al, 2020; nagel et al, 2022). however, in peru, ex situ germplasm of phureja potato is limited, with only 65 accessions, distributed among state institutions (21), conservationists (35) and cip (9). the reasons for this scarcity of ex situ samples need to be investigated. this could be due to the scarcity of these materials on farms or to a lack of interest among researchers and conservationists, due to the difficulty of preserving them because of their lack of dormancy. this germplasm does not represent a significant complement for in situ conservation, nor does it constitute a solid base for the repatriation of cultivars (joshi et al, 2020; lüttringhaus et al, 2021). the most prominent collections of phureja potato in the andes are in colombia: that of nariño and northern ecuador, together with the colombian central collection, which together conserve 348 accessions (rodŕıguez, 2010; lasso et al, 2018). surprisingly, the united states potato germplasm bank (uspg) houses 144 phureja potato accessions, all unique and without duplicates (ŕıo and bamberg, 2021). this highlights the importance of integrating international efforts for the conservation and study of this valuable genetic diversity. phureja potato cultivars maintained by conservationists or potato guardians in cusco are scarce, while in cajamarca they have experienced a drastic reduction of 48% between 2010 and 2021. furthermore, the socioeconomic conditions of the 20 conservationists studied in cajamarca are not favourable to guarantee efficient conservation. this evidences the need to fill a critical gap through studies on in situ conservation of phureja potatoes in other unexplored regions and to delve deeper into conservation dynamics in cusco and cajamarca. addressing these areas will allow a more comprehensive understanding of the strategies needed to preserve this valuable genetic diversity in the context of the peruvian andes. the use of tools such as the fouror five-cell methodology offers an efficient way to obtain this information in a short period (rana et al, 2006; padulosi and dulloo, 2012). for in situ conservation, information from catalogues on native potatoes and specific reports in cajamarca and cusco were used. however, these catalogues present high variability in their content, influenced by the approach, purposes and descriptors used in their elaboration. despite these differences, they proved to be genetic resources (2025), 6 (12), 1–13 conservation status of phureja potato in peru 9 a valuable resource for the objectives of this research, as they provided an approximate view of the materials found in situ. although they reflect information from a specific time, these documents also record cultural aspects and traditional knowledge, highlighting the work of potato conservationists. in addition, they can serve as an essential baseline for ongoing monitoring and evaluation of genetic diversity (minagri, grupo yanapai, inia and cip, 2017). the evidence gathered in this research suggests that, in peru, ex situ and in situ conservation strategies for phureja potato are not operating in a complementary and efficient manner (nagel et al, 2022). however, the data obtained may constitute a valuable reference for monitoring cultivar conservation, a priority aspect that has received little attention in cultivars in general (padulosi and dulloo, 2012). the information available on the genetic erosion of the phureja potato in peru is limited, with research conducted in only a few communities in six potatoproducing departments. however, these studies provide an indication of the situation that may be occurring at the national level. broader regional research that addresses remaining genetic variability and its relationship to the environment is essential. the loss of cultivars is associated with factors such as the lack of time to plant and harvest crops in short periods, reflecting migration, and the prioritization of more profitable activities, such as dairy cattle ranching. in cajamarca, areas previously dedicated to annual crops and potatoes are now used as pasture for dairy cattle. this trend, observed since the early 2000s (winters et al, 2006), has been encouraged by the presence of three large milk collection companies and 1,052 artisanal dairy plants (indecopi, 2023), which guarantee investment security, attractive prices and immediate income for producers. cultivar loss is also attributed, in part, to poor seed quality (farmers say, “it no longer yields, it’s tired”), reflecting seed degeneration due to pathogen accumulation after prolonged vegetative propagation (forbes et al, 2020; sierra et al, 2020). the preference for modern cultivars and some native cultivars of greater commercial acceptance, observed in waqanqa, yauyos, huancavelica and cajamarca, is also a contributing factor, although studies are required to assess their impact. in addition, mining projects, especially in cajamarca, where 53.6% of the territory of the sierra provinces is concessioned to 33 mining companies (gpc, 2014), affect cultivars. this includes direct effects, such as employment (40% of workers in yanacocha are local) (yanacocha, 2018), and indirect effects, such as land sales and migration, which weaken agricultural sustainability. in peru, the possible occurrence of allelic or gene erosion, defined as the loss of alleles and their combinations, and genomic erosion, which implies the complete loss of the genome, has been observed in the phureja potato (thormann and engels, 2015). allelic erosion occurs mainly due to the replacement of these cultivars with modern or traditional varieties of higher commercial value. genomic erosion, on the other hand, is manifested through genetic displacement due to the elimination of the phureja potato from cropping systems. if this process continues, the implications would be serious for the country, as it would face the definitive loss of this species (s. tuberosum l. phureja group), which would have a significant impact on agricultural biodiversity and food security. conclusion the phureja potato, is present in 11 of peru’s 25 regions. despite its historical relevance and former abundance, this valuable crop is now in danger of disappearing in the country. the germplasm of the phureja potato in peru includes 90 accessions conserved in situ, 21 ex situ accessions maintained by state institutions, and 35 accessions safeguarded by farmers in the cajamarca region. this research represents the first systematic effort to document the genetic diversity and erosion of this resource. it is crucial to complement this initial analysis with new regional data to validate its consistency and to develop a comprehensive national inventory to identify and preserve the remaining genetic variability. the loss of genetic resources and the erosion of the phureja potato are influenced by several interrelated factors. among them, farmers’ lack of time to attend to this crop, which requires continuous planting and harvesting, is aggravated by labour shortages due to the emigration of the most skilled members of rural families. in addition, the shift to more profitable activities, such as dairy farming, and the priority given to improved cultivars or commercial varieties displace the phureja potato. other factors include the scarcity and poor quality of seed, and the presence of mining projects that affect cultivation areas. acknowledgements the authors extend special thanks to: the native potato conservationists of cajamarca and paucartambo (cusco); the professionals in charge of potato collections at inia experimental stations; the farmers of cajamarca and paucartambo who participated in the interviews; to alejandro argumedo, director of the asociación andes del cusco, for facilitating access to the database of native potato from the potato park; to luis lizárraga, director of the biodiversity research center of unsaac; and to the saucedo rojas family of paucartambo, for allowing us to examine the database of the collections that the native potato conservationists of this province maintain. author contributions jfsc: conceptualization, methodology, writing original draft, revision. lsct: data collection, formal analysis. asc: writing original draft, review. tmh: data collection 10 seminario et al genetic resources (2025), 6 (12), 1–13 and curation, ws: methodology, visualization, review and editing. ethics statement the authors declare that this research did not require the approval of an ethics committee, as no clinical or experimental procedures requiring such approval were performed. however, the ethical principles applicable to research involving human subjects were strictly adhered to. before each interview, participants were clearly informed about the objectives of the study, the voluntary nature of their participation, and their right to withdraw at any time without penalty. informed consent was obtained orally and in language accessible to each interviewee. each person was also given the option of authorizing the use of their name in the research. their dignity, rights, and autonomy were respected at all times. conflict of interest statement the authors declare no known conflicts of interest, financial or personal relationships that could influence the work or materials presented in this article. references asociación-pataz, cip, inia and aguapan (2023). catálogo de variedades de papa nativa de tayabamba, la libertad, perú . beltrán-penagos, m. a., sánchez-camargo, a. p., and narváez-cuenca, c. e. 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(1992). the loss and maintenance of native crops in mountain agriculture. geojournal 27(1), 61–72. doi: https://doi.org/10.1007/ bf00150635 https://doi.org/10.1007/bf00150635 https://doi.org/10.1007/bf00150635 introduction materials and methods records of the presence, importance and relative abundance of the phureja potato in peru information about ex situ conservation of the phureja potato in peru information about in situ conservation of the phureja potato in peru empirical evidence on genetic erosion of the phureja potato in peru results presence, importance and relative abundance of the phureja potato in peru ex situ conservation of the phureja potato in peru in situ conservation of the phureja potato in peru empirical evidence of genetic erosion of the phureja potato in peru discussion conclusion acknowledgements author contributions ethics statement conflict of interest statement original article genetic resources (2025), 6 (11), 41–56 doi: 10.46265/genresj.zfee2261 https://www.genresj.org issn: 2708-3764 morphological and biochemical characterization of ethiopian mustard (brassica carinata a. braun) germplasm grown in central ethiopia ermias estifanos *,a, tileye feyissa b, kassahun tesfaye c, alemu lencho a and christina eynckd a department of plant science, ambo university, guder, ethiopia b institute of biotechnology, addis ababa university, addis ababa, ethiopia c bio and emerging technology institute, addis ababa, ethiopia d saskatoon research and development centre, agriculture and agri-food canada, saskatoon, sk, canada abstract: ethiopian mustard (brassica carinata a. braun) shows potential for diverse applications, including as leafy greens, green manure and oilseed feedstock for biofuels. this study evaluated the seed and oil production potential and phenotypic diversity of 49 b. carinata accessions through trials conducted in 2018 at the holeta and asela research centers in ethiopia, using a lattice design. data were collected on phenological, morphological, agronomic and seed quality traits. the analysis revealed significant variability across most traits, except for silique width and oil and protein content at asela, and main raceme length and total glucosinolate content at holeta. combined analysis showed significant genotype-by-location interactions for flowering date, seeds per silique and seed yield per hectare, indicating a strong environmental influence on these traits. phenotypic and genotypic correlation analyses identified strong positive correlations between leaf traits and phenology, seed yield and seed quality, while oil content was negatively associated with protein and glucosinolate content. principal component analysis identified five components at asela and six components at holeta with eigenvalues greater than one, explaining over 77% of the total variation at both locations. key traits such as plant height, seed yield and oil content contributed significantly to these principal components. cluster analysis grouped the accessions into three clusters based on distinct trait combinations. accessions 17545, 21373, 24203 and 24495 consistently performed well across multiple traits across sites, making them strong candidates for breeding programmes focused on improving seed yield and quality in b. carinata. keywords: brassica carinata, germplasm characterization, oil content, seed yield citation: estifanos, e., feyissa, t., tesfaye, k., lencho, a., eynck, c. (2025). morphological and biochemical characterization of ethiopian mustard (brassica carinata a. braun) germplasm grown in central ethiopia. genetic resources 6 (11), 41–56. doi: 10.46265/genresj.zfee2261. © copyright 2025 the authors. this is an open access article distributed under the terms of the creative commons attribution license (cc by 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. introduction brassica carinata a. braun (ethiopian mustard, carinata) (bbcc) known as ‘gomenzer’ in amharic, a natural amphidiploid hybrid between b. nigra (bb) and b. oleracea (cc), is believed to have originated in the ethiopian highlands (warwick and black, 1991). it is ∗corresponding author: ermias estifanos (ermiasestifo@yahoo.com) well adapted to these highlands (prakash and hinata, 1980; rakow, 2004) and to a wider range of climatic conditions (montemurro et al, 2016). the crop is grown globally for diverse applications. in the horn of africa, it is primarily utilized as leafy greens and as oilseed for culinary use (basili and rossi, 2018; hagos et al, 2020), while in europe it is employed for green manure and biofuel production (cosentino et al, 2008; montemurro et al, 2016). in north america, b. carinata is grown for biofuel and oleochemical applications (blackshaw et al, received: 06.11.2024 accepted: 28.03.2025 published online: 16.05.2025 https://www.genresj.org https://www.doi.org/10.46265/genresj.zfee2261 https://www.genresj.org https://www.doi.org/10.46265/genresj.zfee2261 mailto:ermiasestifo@yahoo.com 42 estifanos et al genetic resources (2025), 6 (11), 41–56 2011; marillia et al, 2014; gesch et al, 2015; lawton, 2019; seepaul et al, 2021; nóia-júnior et al, 2022), whereas in asia it is primarily cultivated for oilseed production (katiyar et al, 1986; lawton, 2019; thakur et al, 2019). b. carinata seeds are rich in oil, with contents ranging from 25% to 47% by weight (taylor et al, 2010; redda et al, 2022; ambaw et al, 2024a). the oil is used as an edible oil in ethiopia after being blended with nuge (guizotia abyssinica) oil and/or other vegetable oils (belete et al, 2012; bosekeng, 2019; thakur et al, 2019; alemaw and gurmu, 2023). characterized by a high erucic acid content, the oil possesses desirable physicochemical properties, such as low free fatty acid content and abundant unsaponifiable matter, enhancing its stability and quality (cardone et al, 2003; mohdaly and ramadan, 2022). these attributes, combined with the low moisture and acidity levels (< 1% and 0.98% as oleic acid, respectively), make b. carinata oil a versatile feedstock. its industrial applications encompass biodiesel and bio-jet fuel production, as well as the manufacturing of lubricants, plastics, soaps and detergents (mohdaly and ramadan, 2022; redda et al, 2022). the biomass, particularly its high-protein content seed meal, serves as a nutritious supplement for livestock, poultry and swine (paula et al, 2019; george et al, 2021). it also helps manage soil-borne pests through biofumigation, releasing biocidal compounds, particularly isothiocyanates, which are effective against various soil pathogens (dos santos et al, 2021; iboyi et al, 2022). studies on b. carinata revealed a complex interplay between genetic diversity, phenotypic variation and agronomic potential. population structure analyses using snp (tesfaye et al, 2023) and ssr markers (thakur et al, 2020; zhou et al, 2022) indicated moderate genetic diversity, low heterozygosity and limited gene flow. tesfaye et al (2023) reported heterozygosity of 0.30 and nucleotide diversity of 1.31 × 10-5, while thakur et al (2020) observed an average of 3.03 alleles per marker. phenotypic studies highlighted significant morphological diversity, including oil content (mohdaly and ramadan, 2022; ambaw et al, 2024b). b. carinata also demonstrated superior productivity, economic returns and environmental performance compared to other mustard species (rathore et al, 2022). further research has explored trait correlations and identified markers for seed quality (zhang et al, 2017), and qtl for traits like flowering time and oil content (mohdaly and ramadan, 2022). characterizing germplasm is essential for understanding phenotypic and genetic diversity, and is crucial for selecting accessions for breeding programmes and designing conservation strategies (kumari et al, 2023; salgotra and chauhan, 2023). morphological traits, such as seed size and plant height, are primary criteria for selecting accessions in breeding programmes due to their ease of measurement (el-esawi, 2018; swarup et al, 2021). traits such as seed oil content and quality, as well as biomass production, help to select genotypes meeting standards for consumption or industrial use (zhang et al, 2017; tesfaye et al, 2019). some studies have assessed several morphological traits of b. carinata, such as plant height, leaf shape, seed size and flowering time. zada et al (2013) evaluated 132 accessions and documented significant phenotypic variability. hagos et al (2020) also reported significant differences in leaf size attributes (p < 0.001) and phenological traits of 313 accessions. despite extensive research on the potential of ethiopian mustard for biofuel applications, a comprehensive understanding of its morphological, agronomic, seed and oil content traits remains crucial for developing effective breeding strategies for a range of purposes, as these traits have a significant impact on seed and oil production. this study aims to evaluate b. carinata accessions to determine their seed and oil production potential and diversity to be utilized in breeding programmes. materials and methods plant material a total of 49 ethiopian mustard accessions were evaluated in this study. the planting materials comprised 47 landraces, randomly selected from eight geographically clustered regions within the collections of the ethiopian biodiversity institute, addis ababa ethiopian biodiversity institute (ebi, https://ebi.gov.et). additionally, two released varieties, ‘holetta-1’ and ‘s-67’, developed by the holeta agricultural research center, were included. the passport data of the accessions used and the pedigree of the released varieties are provided in supplemental table 1. the ethiopian biodiversity institute (ebi), the source of the landrace accessions, provided limited quantities of seed for each accession. specifically, each accession was provided with only 4g of seed, which was insufficient for establishing trials at more than two locations. this limitation restricts the assessment of genotype-byenvironment (g×e) interactions across a wider range of environmental conditions. experimental site field experiments were conducted at two agricultural research centres in ethiopia during the 2018 cropping season. the first location, holeta, is situated 40km southwest of addis ababa at an altitude of 2,400m above sea level (m.a.s.l.), with coordinates of 9◦3′n latitude and 38◦30′e longitude. the soils in this area are predominantly nitosols and vertisols with the region receiving an average annual rainfall of 1,008.14mm (tura et al, 2021) and a mean maximum and minimum temperature of 22.3◦c and 6.15◦c, respectively (geleta et al, 2024). the second location, asela, is located 170km south of addis ababa at an altitude of 2,200 m.a.s.l., with coordinates of 8◦1′n latitude and 39◦9′e longitude. asela’s soils are primarily genetic resources (2025), 6 (11), 41–56 ethiopian mustard germplasm characterization 43 lateritic and black cotton, with an average annual rainfall of approximately 1,000mm (etwrdec (2017); fekadu (2021)) and a mean minimum and maximum temperature of 5.0◦c and 28.0◦c, respectively (megersa et al, 2024). experimental procedure experiments were conducted at two sites utilizing a 7×7 lattice design with two replications of 49 accessions. the design was chosen due to the limited seed availability from ebi. the plot size was 1.8m2, consisting of four rows 30cm apart and measuring 1.5m in length. seeds were sown by hand drilling in rows at a rate of 5kg/ha. at both sites, fertilizers at a rate of 46kg/ha nitrogen and 69kg/ha p2o5 were applied. following earo (2004) guidelines, conventional agricultural practices, encompassing field preparation, nutrient application and weed control measures were implemented. data collection plant-based data: measurements were conducted from five randomly selected plants per plot. leaf petiole length, leaf length and leaf width were measured from five middle leaves of each selected plant. number of leaves per plant, plant height, number of primary branches per plant and seed yield per plant were recorded from the same five plants. main raceme length and number of siliques per main raceme were recorded from five main racemes of the same selected plants. silique length, silique width, and number of seeds per silique were measured for five siliques selected from the main racemes of each of the same five plants. mean values for the measured traits were calculated from these measurements. measurements of selected traits were conducted according to the international board for plant genetic resources descriptors for brassica and raphanus (ibpgr, 1990). plot-based data: for each plot, the following data were collected from the two central rows: days to flowering, days to maturity, thousand seed weight and seed yield per hectare. seed quality analysis was conducted at the agriculture and agri-food canada – saskatoon research and development centre in saskatchewan, canada. the oil and protein content of the seeds was determined using near-infrared reflectance spectroscopy (foss model 6500). oil and protein concentrations were expressed as percentages on a moisture-free basis (hossain et al, 2018). total glucosinolates were extracted and purified using ion-exchange chromatography and on-column enzymatic desulfation (thies, 1980). trimethylsilyl derivatives were prepared using the acetone and 1methylimidazole-based method of landerouin et al (1987). in this study, data were collected at various growth stages according to the biologische bundesanstalt, bundessortenamt und chemische industrie (bbch) scale. early leaf development traits were recorded at bbch 19, branching and flowering traits at bbch 50, and silique, seed, and maturity traits at bbch 90 (meier, 2018). data analysis data analysis was performed using r software (r core team, 2023) packages. analysis of variance (anova) was conducted using the ’pbib.test’ function from the agricolae package (mendoza, 2023) for individual location analyses, while the ’aov’ function from the same package was used for the combined analysis across locations. scatter plot mean seed yield per hectare comparison was performed by r software ggplot2 package (wickham, 2016). mean separation was carried out using tukey’s honestly significant difference (hsd) test, also implemented in the agricolae package (mendoza, 2023). before doing the combined analysis of variance, a test for homogeneity of variance was performed (ftest) (mead et al, 2003). the phenotypic and genotypic correlation coefficients were calculated using the r software variability package (popat et al, 2020), which implements the methods described by singh and chaudhary (1977) as described below: phenotypic correlation coefficient: rp = covp(xy)√ vp(x)vp(y) where covp(xy) is the phenotypic covariance between trait x and trait y, vp(x) and vp(y) are the phenotypic variances of x and y, respectively. genotypic correlation coefficient: rg = covg(xy)√ vg(x)vg(y) where covg(xy) is the genotypic covariance between trait x and trait y, vg(x) and vg(y) are the genotypic variances of x and y, respectively. heatmaps of the correlation results were created by the r software pheatmap package (kolde, 2019). principal component analysis (pca) and ward’s d2 linkage and euclidean distance hierarchical method cluster analysis were performed using the ‘factoextra’ (kassambara and mundt, 2020) and ‘factominer’ (le et al, 2008) packages. the optimal number of clusters was determined using ‘nbclust’ package (charrad et al, 2014). results and discussion overview of trait variation across locations accessions showed coefficients of variation (cv) ranging from 4.8% to 32.8% (table 1), consistent with the high coefficients of variation (> 20) observed for traits such as leaf petiole length, leaf length, leaf width, primary branches per plant, number of siliques per main raceme, and seed yield per hectare at both locations in this study. ambaw et al (2024b) also reported similarly high cvs, suggesting that these traits exhibit substantial phenotypic plasticity and/or are strongly influenced by genetic factors segregating within our accessions (table 1). the fact that high cvs were observed at both locations implies a consistent influence, hinting at significant g×e interactions. the average seed yield per hectare was slightly higher at asela, at 44 estifanos et al genetic resources (2025), 6 (11), 41–56 table 1. summary statistics of agronomic, morphological, and quality traits for 49 brassica carinata accessions evaluated at asela and holeta. lpl, leaf petiole length (cm); ll, leaf length (cm); lw, leaf width (cm); lpp, leaves per plant; df, days to flowering; dm, days to maturity; ph, plant height (cm); pb, primary branches per plant; mrl, main raceme length (cm); spmr, number of siliques per main raceme; sl, silique length (mm); sw, silique width (mm); sps, number of seeds per silique; tsw, thousand seed weight (g); syppl, seed yield per plant (g); sypha, seed yield per hectare (kg); oil, oil content (%); pro, protein content (%); tg, total glucosinolate content (µmol/g); min, minimum; max, maximum; sd, standard deviation; cv, coefficients of variation. trait asela holeta mean ± sd min max cv mean ± sd min max cv lpl 13.5 ± 3.7 4.9 21.7 27.6 11.4 ± 3.7 2.7 19.5 32.8 ll 28.2 ± 6.5 9 40.7 23.1 26.3 ± 6.5 7 38.2 24.5 lw 10.9 ± 2.2 5.8 15.8 19.9 12.4 ± 2.5 5.4 18.5 20.1 lpp 99 ± 14.2 67 123.1 14.3 92.9 ± 14.4 59.5 118.1 15.5 fd 86.7 ± 10.4 61 109 12 92 ± 9.7 66 114.5 10.5 md 153.1 ± 14.5 127 180.5 9.5 174.4 ± 8.4 148.5 188.5 4.8 ph 197.7 ± 13.9 162 220.5 7 188.7 ± 19.2 114 219.5 10.2 pb 14.3 ± 3 8.2 20.3 21.2 14.5 ± 3.1 8 20.3 21.2 mrl 5.5 ± 0.6 4.5 6.6 10.8 5.2 ± 0.4 4.4 6.1 8 spmr 18.4 ± 4.5 11.2 28.6 24.6 18.6 ± 5.1 10 32.8 27.3 sl 28.5 ± 4.4 19.8 38.6 15.5 30.3 ± 4.9 20.3 40.2 16 sw 2.2 ± 0.2 1.7 2.8 10.6 2.4 ± 0.2 2.1 3.1 8.4 sps 12.8 ± 1.4 9.8 15.6 11.1 12.7 ± 1.5 9.5 15.6 12.2 tsw 3 ± 0.5 1.9 4 16 3.1 ± 0.5 2.2 4.1 14.8 syppl 3.6 ± 0.8 2.2 5.2 22.6 3.1 ± 0.8 1.6 4.7 26.2 sypha 2,245.4 ± 500.3 1,262.1 3,074.2 22.3 2,161.6 ± 500 1,173.9 2,950.5 23.1 oil 37.2 ± 2.5 32.6 43.1 6.7 44 ± 2.2 37.4 49.1 5 pro 29.9 ± 2.1 25.8 34.6 7.1 26 ± 1.8 22.7 31.5 6.8 tg 99.9 ± 9.7 82.6 123.3 9.7 83.3 ± 8.5 61.6 99.2 10.2 2,245.4± 500.3kg (ranging from 1,262.1 to 3,074.2kg), compared to 2,161.6 ± 500kg at holeta (ranging from 1,173.9 to 2,950.5kg) (table 1). this slight increase in mean yield at asela, along with consistent standard deviations across locations, indicates relative similarity in environmental conditions. however, the average oil content was higher at holeta, at 44 ± 2.2% (ranging from 37.4 to 49.1%), compared to 37.2± 2.5% (ranging from 32.6 to 43.1%) at asela. the difference in trend between seed yield and oil content may be attributed to environmental differences, while the broad range in yields and location-based variation in oil content highlights the genotypic variability of b. carinata, offering valuable potential for selection in future crop improvement efforts. comparable yields for b. carinata have been reported in other studies. seepaul et al (2021), in a literature review of multiple field trials, summarized yields ranging from 1,929kg/ha to 2,732kg/ha. furthermore, tesfaye et al (2023) found a seed yield average of 3,185kg/ha on selected b. carinata genotypes in ethiopia. similarly, iboyi et al (2023) reported a range from a minimum seeding rate of 732kg/ha at a 1.12kg/ha to a maximum seeding rate of 1,087kg/ha at 5.6kg/hain southeastern united states. for oil content, previous studies on b. carinata in ethiopia have also reported comparable findings. ambaw et al (2024b) reported oil content values ranging from 37.88% to 46.98%, while redda et al (2022) observed oil content between 35% and 45% in ethiopian germplasm. these results are consistent with the findings of this study, highlighting the rich phenotypic diversity and potential for improving seed yield and oil quality in b. carinata. analysis of variance individual location analysis results showed significant variability among tested b. carinata accessions for most of the traits except silique width, oil and protein content at asela and main raceme length and total glucosinolate content at holeta (table 2). combined location analysis of variance revealed significant variation among accessions for all traits, while the interaction between accession and location showed significant variation for the traits flowering date, number of seeds per silique and seed yield per hectare (table 3). the significant variations observed in most studied traits among b. carinata accessions indicate substantial genetic diversity within the species. this aligns with tesfaye et al (2023), who reported that 93% of the genetic variation was attributed to molecular variance, and thakur et al (2020), who found an average genetic diversity of 0.37. this diversity provides opportunities for breeding programmes to develop cultivars with desirable traits. the significant interaction between genotype and location for certain traits suggests that the performance of b. carinata accessions is influenced by environmental factors. this implies that genetic resources (2025), 6 (11), 41–56 ethiopian mustard germplasm characterization 45 table 2. analysis of variance mean squares for replication, genotype, block, and error across agronomic, morphological, and quality traits for 49 b. carinata accessions evaluated at asela and holeta. lpl, leaf petiole length (cm); ll, leaf length (cm); lw, leaf width (cm); lpp, leaves per plant; df, days to flowering; dm, days to maturity; ph, plant height (cm); pb, primary branches per plant; mrl, main raceme length (cm); spmr, number of siliques per main raceme; sl, silique length (mm); sw, silique width (mm); sps, number of seeds per silique; tsw, thousand seed weight (g); syppl, seed yield per plant (g); sypha, seed yield per hectare (kg); oil, oil content (%); pro, protein content (%); tg, total glucosinolate content (µmol/g); cv, coefficients of variation. **, and *, denote significance at p ≤ 0.01, and p ≤ 0.05 respectively. trait asela holeta replication genotype block error cv (%) replication genotype block error cv (%) lpl 0.98 27.7** 0.5 0.5 5.2 56.94** 27.7** 0.6 0.5 6.5 ll 169.28** 84.8** 20.7 20.4 16.0 379.7** 83.4** 19.2 21.1 17.4 lw 44.72** 9.5** 2.7 2.1 13.4 31.09** 12.4** 3.2 2.5 12.7 lpp 263.19** 403.4** 10.8 13.5 3.7 26.13 416.7** 12.9 14.8 4.1 fd 32 217** 6.7 7.9 3.2 54.38** 186.1** 11.1* 4.7 2.4 md 2.3 420.4** 51.1 43.9 4.3 3.31 142.3** 35 23.3 2.8 ph 1,389.4** 387.3** 84.6 141.6 6.0 11,100.5** 733.3** 120.8 153.3 6.6 pb 0.09 18.5** 2.2 2.9 11.8 266.48** 18.9** 3.3 3 11.9 mrl 0.5 0.7** 0.4 0.2 8.8 71.54** 0.4 0.2 0.3 10.1 spmr 5.93 40.8** 2.8 3.1 9.6 6.9 51.5** 2.3 5.4 12.5 sl 4.57 39** 18.6 17.2 14.5 188.3** 47.2** 23.6 18.2 14.1 sw 0.06 0.1 0.1 0.1 14.4 0.01 0.1** 0.1 0.04 8.0 sps 0.03 4** 1.6 1.4 9.4 0.74 4.7** 0.8 1.5 9.7 tsw 0.11 0.4** 0.1 0.1 11.9 0.29 0.4** 0.1 0.1 11.6 syppl 0 1.3** 0.1 0.1 7.3 0.17 1.3** 0.04 0.1 7.9 sypha 5,437.06 500,587.4** 30,678.6 28,324.7 7.5 134,332.25* 499,973.6** 35,619.7 18,572.4 6.3 oil 19.2 12.5 7.4 9.7 8.4 0.52 9.5** 3.9 3.2 4.1 pro 29.85 9.1 3.4 6.1 8.3 15.35* 6.3* 5.2 3.7 7.4 tg 710.02** 186.6** 56.4 84.2 9.2 1,142.64** 145.1 137.2 110.5 12.6 table 3. analysis of variance mean squares for location, genotype and their interaction across agronomic, morphological and quality traits in the combined evaluation of 49 brassica carinata accessions. lpl, leaf petiole length (cm); ll, leaf length (cm); lw, leaf width (cm); lpp, leaves per plant; df, days to flowering; dm, days to maturity; ph, plant height (cm); pb, primary branches per plant; mrl, main raceme length (cm); spmr, number of siliques per main raceme; sl, silique length (mm); sw, silique width (mm); sps, number of seeds per silique; tsw, thousand seed weight (g); syppl, seed yield per plant (g); sypha, seed yield per hectare (kg); oil, oil content (%); pro, protein content (%); tg, total glucosinolate content (µmol/g); cv, coefficients of variation. **, and *, denote significance at p ≤ 0.01, and p ≤ 0.05 respectively. trait location genotype (g×l) error cv (%) lpl 223.93** 55.4** 0.02 1.1 8.5 ll 180.13** 168.1** 0.04 25.7 18.6 lw 107.86** 21.6** 0.3 3.2 15.3 lpp 1,836.73** 818.5** 1.6 16.2 4.2 fd 1,363.72** 391.6** 11.5* 7.7 3.1 pb 2.17 37.2** 0.2 5.5 16.3 spmr 2.87 90.3** 2 3.9 10.6 sl 167.15** 83.9** 2.3 20.1 15.3 sw 3.12** 0.13** 0.06 0.07 11.3 sps 0.75 6.4** 2.4* 1.4 9.3 tsw 0.62* 0.8** 0 0.1 11.2 syppl 13.31** 2.5** 0.1 0.1 7.7 sypha 344,348.37** 948,487.3** 52,073.7** 26,771.9 7.4 oil 14,693,939.17** 99,738** 44,010.9 38,121.2 11.7 pro 765.71** 9.4** 6.1 5.1 8.1 tg 13,474.13** 206.2** 125.5 114.1 11.7 46 estifanos et al genetic resources (2025), 6 (11), 41–56 selecting genotypes for specific growing conditions is crucial for optimal yield and quality. the lack of significant variation in silique width and oil and protein content at asela (table 2) might indicate a relatively narrow genetic variation or a strong genetic control over these traits; however, in the case of silique width, although the genotypic mean square was similar between asela and holeta, significance was detected only at holeta due to its lower experimental error, which increased the f-ratio and statistical power to reveal genotypic differences. meanwhile, the significant variation in main raceme length and total glucosinolate content recorded at asela but not at holeta (table 2) suggests that there is a high environmental influence on the expression of these traits. the significant interactions between genotype and location for flowering date, seeds per silique and seed yield per hectare highlight the importance of considering both genetic factors and environmental conditions when selecting genotypes for specific regions. similar significant variability among b. carinata genotypes has been reported by belete (2011); walle et al (2014); dhaliwal et al (2019); amsalu (2020b,c); mahendra-salam et al (2021) and khan et al (2022). the observation that the majority of genotype-bylocation (g×l) interactions in our combined analysis of variance were non-significant (table 3) aligns with the findings of adeniji and aloyce (2012), but contrasts with the reports of significant g×l interactions by kumar et al (2020) and tadesse et al (2021). this discrepancy in results could stem from several factors related to the experimental design, the germplasm studied and the environmental conditions. correlation analyses phenotypic correlation analysis at the two locations revealed significant positive and negative associations among various agronomic and seed traits (figures 1 and 2). leaf and phenological traits exhibited the strongest positive phenotypic correlations at both locations, including leaf petiole length with leaf length (0.80, 0.80), leaf width (0.80, 0.78), flowering date (0.78, 0.72), and maturity date (0.73, 0.58); leaf length with leaf width (0.90, 0.89), flowering date (0.72, 0.70), and maturity date (0.65, 0.58); leaf width with flowering date (0.70, 0.66) and maturity date (0.64, 0.61); and flowering date with maturity date (0.81, 0.79). additionally, strong positive phenotypic correlations were observed for seed yield per plant with seed yield per hectare (0.87, 0.92) and protein content with total glucosinolate content (0.81, 0.80) at asela and holeta, respectively. similarly, consistently significant negative phenotypic correlations were recorded for oil content with protein content (0.83, -0.78) and with total glucosinolate content (-0.80, -0.52) at asela and holeta, respectively. to fully understand the genetic relationships underlying the observed phenotypic variation, analyzing genotypic correlations among the studied traits is essential. this analysis offers valuable insights into the genetic linkages between traits, aiding in the identification of potential breeding targets and providing a deeper understanding of the genetic limitations within b. carinata. the genotypic correlation analyses of this study revealed several significant associations among the considered traits for both locations (figures 1 and 2). strong positive correlations were again observed among leaf and phenological traits, including leaf petiole length with leaf length (0.99, 0.99), leaf width (0.98, 0.92), flowering date (0.80, 0.77) and maturity date (0.81, 0.70); leaf length with leaf width (0.98, 0.96), flowering date (0.89, 0.98) and maturity date (0.91, 0.99); and leaf width with flowering date (0.82, 0.89) and maturity date (0.88, 0.98). flowering date also showed high correlations with maturity date (0.92, 0.97). additionally, strong positive genotypic correlations were recorded for seed yield per plant with seed yield per hectare (0.99, 0.99) and plant height (0.61, 0.58); and for thousand seed weight with leaf length (0.67, 0.72), leaf width (0.77, 0.78), maturity date (0.57, 0.61), and plant height (0.72, 0.66) at asela and holeta, respectively. seed yield per hectare showed significant positive associations with the number of siliques per main raceme (0.52, 0.52) and thousand seed weight (0.53, 0.49). negative significant correlations were recorded for oil content with protein content (-0.50, 0.66) and total glucosinolate content (-0.84, -0.59) at asela and holeta, respectively. the strong positive correlations observed in this study between leaf traits (leaf length, leaf width, and leaf petiole length) are consistent with previous research by yimer et al (2021). this suggests a consistent genetic and environmental influence on these traits in b. carinata. as expected, the positive correlation between plant height and phenological traits (flowering and maturity dates) observed in this study aligns with walle et al (2014). this relationship is likely due to the natural elongation of the flowering spike contributing to plant height. however, confirming this correlation in b. carinata under the studied conditions helps strengthen its role in breeding for synchronized flowering and uniform plant architecture. the positive correlations between seed yield per hectare and seed yield per plant are further supported by amsalu (2020a) and belete (2011). these findings highlight the importance of seed yield per plant and the number of siliques per main raceme as key determinants of overall yield. marjanović-jeromela et al (2007) provide additional evidence for the significance of these traits in brassica species. the positive correlation of thousand seed weight with various traits, including leaf length and width, maturity date and plant height, aligns with the findings of tadessel and alemu (2019). this underscores the importance of seed weight in yield improvement. the inverse relationship between oil content and protein and glucosinolate contents observed in this study is consistent with previous research by tadessel genetic resources (2025), 6 (11), 41–56 ethiopian mustard germplasm characterization 47 figure 1. heatmap displaying phenotypic (above diagonal) and genotypic (below diagonal) correlation coefficients for 19 traits measured in brassica carinata accessions grown at asela. lpl, leaf petiole length (cm); ll, leaf length (cm); lw, leaf width (cm); lpp, leaves per plant; df, days to flowering; dm, days to maturity; ph, plant height (cm); pb, primary branches per plant; mrl, main raceme length (cm); spmr, number of siliques per main raceme; sl, silique length (mm); sw, silique width (mm); sps, number of seeds per silique; tsw, thousand seed weight (g); syppl, seed yield per plant (g); sypha, seed yield per hectare (kg); oil, oil content (%); pro, protein content (%); tg, total glucosinolate content (µmol/g). **, and *, indicates significant at p ≤ 0.01, and p ≤ 0.05 , respectively. and alemu (2019), confirming the common trade-off between these seed quality traits in oilseed crops. however, the positive genotypic correlations between thousand seed weight and phenological traits in this study differ from the negative associations reported by khan et al (2022) and kumar-singh et al (2018). different b. carinata accessions possess distinct genetic architectures (tesfaye et al, 2023). discrepancies in correlation results may be partly attributed to the simple lattice design employed. residual environmental heterogeneity within blocks, location-by-block interactions and incomplete blocking effects could have influenced trait correlations. future studies should consider spatial analysis techniques to account for these factors. the observed inconsistencies in the correlations between main raceme length and protein and oil content, as well as silique width and total glucosinolate content, across different locations, suggest that environmental factors play a significant role in influencing the expression of these traits. this result highlights the need for future research that specifically investigates the g×e interactions governing these complex trait relationships in b. carinata. the strong associations observed between leaf, phenological and seed traits in this study highlight their potential as target traits for breeding programmes aimed at improving b. carinata yield and quality. however, the influence of environmental factors on trait expression underscores the importance of conducting multi-location trials and considering genotype–environment interactions when selecting genotypes for specific regions. principal component analysis a principal component analysis (pca) was employed to examine the combined data from asela and holeta. this analysis identified six principal components (pcs), each with eigenvalues greater than 1 according to kaiser’s criterion (jolliffe, 2002). together, these six pcs accounted for 82.0% of the total variability (supplemental table 2). the first two components (pc1 and pc2) represented 52.7% of the overall variation and were visualized in a biplot (supplemental table 2, figure 3). the distribution of accessions on the biplot reveals considerable genetic diversity among the accessions. pc1, accounting for 31% of the variance, is characterized by strong positive loadings for leaf petiole length (0.90), leaf length (0.94), leaf width (0.92), days to flowering (0.89), and days to maturity (0.88), indicating that accessions with greater values for traits that influence plant structure and phenology grouped together in pc1 and suggests that selecting for plant leaf area could lead to later maturity. pc2, which explained a further 21.7% of the variation, was characterized by substantial contributions from plant height (0.69), seed yield per plant (0.73), seed yield per hectare (0.75) and the number of siliques per main raceme (0.61), implying a connection between taller plants with a 48 estifanos et al genetic resources (2025), 6 (11), 41–56 figure 2. heatmap displaying phenotypic (above diagonal) and genotypic (below diagonal) correlation coefficients for 19 traits measured in brassica carinata accessions grown at holeta. lpl, leaf petiole length (cm); ll, leaf length (cm); lw, leaf width (cm); lpp, leaves per plant; df, days to flowering; dm, days to maturity; ph, plant height (cm); pb, primary branches per plant; mrl, main raceme length (cm); spmr, number of siliques per main raceme; sl, silique length (mm); sw, silique width (mm); sps, number of seeds per silique; tsw, thousand seed weight (g); syppl, seed yield per plant (g); sypha, seed yield per hectare (kg); oil, oil content (%); pro, protein content (%); tg, total glucosinolate content (µmol/g). **, and *, indicates significant at p ≤ 0.01, and p ≤ 0.05 , respectively. prolific density of siliques that led to high seed yield. this association points to a potential breeding target: increasing plant height and silique density to enhance overall seed yield. however, it is crucial to consider the potential for lodging associated with taller plants and high silique density. breeding efforts should also focus on improving stem strength and lodging resistance to ensure that increased yield is not compromised by plant instability. the distribution of accessions on the biplot reveals considerable genetic diversity, suggesting a rich resource for future breeding efforts and highlighting the potential for selecting lines with desirable trait combinations. the pca biplot demonstrated that accessions 24494, 24495, 21373, 24203 and 17545 were closely aligned with traits such as plant height, seed yield per plant, seed yield per hectare and thousand seed weight, all of which exhibited strong positive loadings on pc2. this close relationship suggests that these accessions are strongly associated with increased plant height and seed yield potential. similarly, accessions 21383, 208404, 212665, 19959 and 208412 were positioned near the oil content (figure 3), demonstrating the potential for enhanced seed oil production. in contrast, the released varieties ‘s-67’ and ‘h1’ were not best suited for seed-related and oil traits. abraha et al (2024) used pca for the classification of 313 b. carinata accessions for 18 traits and reported the first and second pcs accounted for 34.3% of the observed variability. pc1 explained 22.2% of the variability between the morphological attributes and most strongly accounted for the differences between the accessions, which is a lower proportion than this study pc1 variation (31.03%). the difference may be attributed to differences in the genetic diversity and population structure of the germplasms analyzed. belete (2011) analyzed 36 b. carinata accessions for nine agro-morphological traits and reported that 91.4% of the total variation was contributed by the first five principal components, which is a comparable result to the 82.03% cumulative variance explained by the first six pcs in our study. kumar et al (2020) also applied principal component analysis using seven traits of 11 b. carinata accessions and found that only the first four principal components showed eigenvalues greater than one and they cumulatively explained a similar proportion (82.46%) of the total variability in this study. this suggests that all three datasets capture a large proportion of the total variability within a relatively small number of underlying pcs. the closeness in variability proportion suggests that, despite the differences in sample size and specific traits considered, the patterns of diversity within b. genetic resources (2025), 6 (11), 41–56 ethiopian mustard germplasm characterization 49 figure 3. ca biplot of the first two principal components of b. carinata accessions, highlighting trait loadings (red arrows) and accession distribution (blue dots) across combined locations. lpl, leaf petiole length (cm); ll, leaf length (cm); lw, leaf width (cm); lpp, leaves per plant; df, days to flowering; dm, days to maturity; ph, plant height (cm); pb, primary branches per plant; mrl, main raceme length (cm); spmr, number of siliques per main raceme; sl, silique length (mm); sw, silique width (mm); sps, number of seeds per silique; tsw, thousand seed weight (g); syppl, seed yield per plant (g); sypha, seed yield per hectare (kg); oil, oil content (%); pro, protein content (%); tg, total glucosinolate content (µmol/g). direction of red arrow indicates the association of the corresponding trait with the principal components, while the length of the arrow reflects the magnitude of the trait’s contribution (longer arrows = stronger influence); light blue accessions with higher cos2 values (well-represented by pc1 and pc2); deep blue, accessions with lower cos2 values (poorly represented by pc1 and pc2). carinata germplasms are consistently captured by a relatively small number of pcs. these pca results hold significant agricultural implications for informing b. carinata breeding programmes aimed at enhancing seed yield, oil content and overall agronomic performance. cluster analysis cluster analysis can be applied to various samples and descriptors to examine the relationships and distances among them. in germplasm collections, it is useful for assessing genetic similarities and differences. understanding genetic distance, the measure of dissimilarity between accessions, is key to predicting the success of crop improvement efforts (peeters and martinelli, 1989). cluster analysis conducted on the combined data from asela and holeta grouped the tested accessions into three distinct clusters of 21, 21, and 7 members, respectively (figure 4). the two released varieties were both placed in cluster i. cluster i is characterized by the highest mean scores for seed yield per hectare, seed yield per plant, plant height and main raceme length (supplemental table 3). cluster ii is distinguished by the highest mean values for leaf petiole length, leaf length, leaf width, number of leaves per plant, days to flowering, days to maturity, number of primary branches per plant, silique length, protein content and total glucosinolate content. cluster iii has the highest mean values for number of siliques per main raceme, number of seeds per silique and seed oil content. cluster iii exhibited the highest intra-cluster distance (5.6), indicating a high level of genetic diversity among the accessions (supplemental table 3). the greatest inter-cluster distance was recorded between cluster ii and cluster iii (7.4), which suggests a high level of genetic divergence, making accessions from these clusters promising for hybridization. this was followed by the distance between cluster i and cluster iii (7.3), and between cluster i and cluster ii (6.0). the clustering results from the dendrogram corresponded to the findings of both principal component analysis (pca) and correlation studies. for instance, 50 estifanos et al genetic resources (2025), 6 (11), 41–56 accessions 17545, 17562, 24203, 21373, 24494, 24495, 212665, 20913 and 24491 fall in the same quadrant of the pca biplot and are closely associated with plant height, seed yield per plant, seed yield per hectare, thousand seed weight, number of siliques per main raceme, primary branches per plant, leaf length and leaf width; these traits are significantly positively correlated with seed yield per hectare. the primary distinctions between clusters are influenced by the same characteristics that are significant contributors to the first and second principal components. accessions grouped within the same cluster exhibited greater similarity to each other than those in separate clusters, further confirming the relationships identified through these analytical methods. the diversity in cluster numbers identified across b. carinata research, can be attributed to variations in genetic diversity, accession selection and study design. for example, the lower cluster counts reported by muthoni (2010) (two clusters in 47 genotypes) and adeniji and aloyce (2012) (three clusters in 14 genotypes)are likely due to smaller sample sizes and limited genetic representation. conversely, studies with larger numbers of more diverse accessions, such as those by zada et al (2013), abraha et al (2024) (eight clusters in 313 genotypes) and ambaw et al (2024a) (seven clusters in 386 genotypes), captured greater genetic diversity by incorporating genotypes from multiple geographic regions, agroecological zones and possibly wild relatives. our study, which identified three distinct clusters among 47 b. carinata landraces, aligns with the trend observed in these previous studies. the number of clusters we found is consistent with studies that utilized a moderate sample size and landrace accessions, which are known to exhibit substantial genetic diversity. this suggests that while our sample size was comparable to muthoni (2010), the inclusion of diverse landraces contributed to the identification of a greater number of distinct groups. these disparities also highlight the impact of methodological choices: more recent studies employing advanced genotyping tools (e.g. snps or ssrs) and sensitive clustering algorithms (e.g. structure) have enabled a more precise analysis of population structure. in our study, we used phenotypic data for clustering, which, while informative, may not have captured the same level of genetic resolution as molecular markers. however, the congruence between our cluster analysis, pca, and correlation studies suggests that the phenotypic data effectively revealed meaningful genetic relationships among the accessions. the variability in cluster numbers underlines the need for standardized experimental approaches that integrate extensive, diverse germplasm collections with high-resolution markers to enhance genetic diversity, refine breeding strategies and preserve adaptive alleles in b. carinata. mean performance comparison mean seed yield per hectare values for the evaluated accessions showed a clear positive relationship between asela and holeta (supplemental figure 1). this finding is consistent with studies like abu et al (2022), who used the additive main effects and multiplicative interaction (ammi) analysis to identify six b. carinata accessions exhibiting relatively stable performance across environments. as mean yield increased at asela, it also tended to increase at holeta, indicating that accessions with high seed yield per hectare in one location generally performed well in the other. tukey’s mean difference test was applied to traits that showed significant results in the analysis of variance. based on mean performance accessions 17545, 21373, 21378, 24203, 24493, 24494, 208609, 212665 and 216845 exhibited increased leaf length, increased leaf width, a greater number of primary branches, longer siliques, more seeds per silique, higher thousand seed weight, higher seed yield per hectare, higher oil content, higher protein content and a high total glucosinolate content at both locations (supplemental tables 4 and 5, respectively). however, there were two exceptions: accession 21378 did not rank first in terms of the number of primary branches per plant at either location, and accession 24493 did not rank first for total glucosinolate content at asela with respect to the traits under consideration. despite these exceptions, the identified accessions showed strong potential for improving key traits like seed yield, oil content and protein content, all of which are economically valuable. the variability in rankings for 21378 and 24493 accessions with regard to specific traits suggests that these accessions may exhibit traitspecific responses depending on the environment. in this study, genotypic variation was assessed based on phenotypic differences in the studied traits. significant variations among the b. carinata accessions were observed, which is consistent with the findings of tesfaye et al (2023), who reported moderate genetic diversity in b. carinata populations (average expected heterozygosity = 0.31, polymorphism information content = 0.26), indicating variability among accessions. while our study assessed phenotypic diversity based on mean trait values, thakur et al (2020) reported a gene diversity of 0.37, which reflects the average genetic variation within populations. khedikar et al (2020) reported low molecular genetic diversity in b. carinata accessions (heterozygosity = 0.30, nucleotide diversity = 1.31×10−5), suggesting limited variability due to a narrow genetic base and potential inbreeding effects. phenotypic diversity reflects the combined effects of genetic and environmental factors, while molecular diversity captures variation at the dna level. differences in sampling strategies, molecular markers and potential inbreeding effects can influence molecular genetic diversity estimates and contribute to discrepancies between studies. it is important to acknowledge that direct comparisons between phenotypic and genetic resources (2025), 6 (11), 41–56 ethiopian mustard germplasm characterization 51 figure 4. dendrogram of 49 brassica carinata accessions assessed for 19 studied traits at asela and holeta using ward’s d2 linkage and euclidean distance. molecular diversity estimates should be interpreted with caution, as they reflect different aspects of genetic variation. these differences in genetic diversity estimates could be attributed to variations in the b. carinata populations studied, the types of molecular markers used and the sampling strategies employed. further research is needed to elucidate the underlying factors contributing to these discrepancies. positive correlations between seed yield per plant and siliques per main raceme suggest opportunities for enhancing overall yield (dwivedi et al, 2023; saini et al, 2023). pca effectively identified key traits: plant height, seed yield per plant, seed yield per hectare, thousand seed weight and oil content driving variation within the b. carinata accessions. this helps in pinpointing promising genotypes for crop improvement, as the initial principal components capture a significant percentage of the total variance, focusing on genetic differences that influence important agronomic traits like seed and oil yield. moreover, clustering accessions based on trait similarities supports targeted breeding strategies. however, the variability in genotype rankings across different environments highlights the importance of considering g×e interactions in breeding programmes (kumar et al, 2020; tesfaye et al, 2024). thus, these findings underscore the need for robust multi-location and multiseason trials to effectively evaluate genotype adaptability and optimize crop improvement efforts for b. carinata. while our study revealed variability in accession rankings across the two locations, suggesting potential g×e interactions, the limited number of experimental sites (haleta and asela) restricts our ability to draw strong conclusions. as detailed in the materials and methods, seed limitations from ebi constrained our study to these two locations. consequently, the observed variation may reflect random environmental 52 estifanos et al genetic resources (2025), 6 (11), 41–56 noise rather than robust g×e patterns. future studies should prioritize multi-location trials to capture a wider range of environmental influences and provide more reliable insights into genotype adaptability. conclusion the study emphasizes the importance of genetic diversity within b. carinata and the need for multi-location trials to ensure the identification of accessions with stable performance and adaptability across different environments. however, due to limited seed availability from ebi, this study was restricted to two experimental locations, limiting the scope of g×e interaction analysis. based on our findings, crop improvement efforts should prioritize accessions 24494, 24495, 21373, 24203 and 17545 for yield improvement (based on plant height, seed yield per plant, seed yield per hectare, and thousand seed weight) and accessions 21383, 208404, 212665, 19959 and 208412 for enhanced oil content (based on plant height, seed yield per plant, seed yield per hectare, and oil content). to maximize trait expression, especially for environmentally influenced traits like flowering date and seed yield, location-specific selection is crucial. additionally, conducting multi-location trials is essential for understanding genotype adaptability and ensuring consistent performance across diverse environments, ultimately supporting more effective crop improvement strategies for b. carinata. our cluster analysis aligns with the trend of moderate cluster numbers. however, the use of phenotypic data for clustering, rather than high-resolution molecular markers, may have limited the precision of our genetic diversity assessment. future studies should integrate advanced genotyping tools and diverse germplasm collections to enhance the understanding of genetic diversity and refine breeding strategies in b. carinata. supplemental data supplemental table 1. passport data of the accessions used and pedigree information of the released varieties supplemental table 2. loadings, eigenvalues, and variances of principal components with eigenvalues greater than one from pca of 49 brassica carinata accessions at asela and holeta supplemental table 3. cluster mean values and intraand inter-cluster distances for 49 brassica carinata accessions at asela and holeta supplemental table 4. mean performance evaluation results for 19 traits of 49 brassica carinata accessions at asela supplemental table 5. mean performance evaluation results for 19 traits of 49 brassica carinata accessions at holeta supplemental figure 1. mean seed yield per hectare (sypha, in kg) comparison of 49 brassica carinata accessions in two locations: asela vs. holeta author contributions ermias estifanos was responsible for material preparation, data collection, analysis, and drafting the first version of the manuscript. kassahun tesfaye contributed to the study conception and design. tileye feyissa supervised the study and provided feedback on previous versions of the manuscript. alemu lencho facilitated the research process. christina eynck conducted the seed analysis, provided resources and provided comments on earlier drafts of the manuscript. all authors read and approved the final manuscript. acknowledgements we are grateful to the ethiopian biodiversity institute, addis ababa, ethiopia, for providing brassica carinata accessions. we also extend our thanks to holeta and kulumsa agricultural research centers for facilitating the field trials, with special thanks to holeta arc for supplying seeds of released varieties. our gratitude goes to ambo university for partially covering the field trial costs. lastly, we appreciate the collaboration of saskatoon research and development centre, saskatchewan, canada in determining seed quality traits. funding the fieldwork was partially supported by ambo university’s student research support fund, and the seed analysis was supported by saskatoon research and development centre, agriculture and agri-food canada, saskatoon, sk, canada. conflict of interest statement the 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(2022). genetic diversity analysis reveals potential of the green peach aphid (myzus persicae) resistance in ethiopian mustard. international journal of molecular sciences 23(22), 1– 21. doi: https://doi.org/10.3390/ijms232213736 https://doi.org/10.1007/s10722-020-00988-3 https://doi.org/10.1007/s10722-020-00988-3 https://doi.org/10.2139/ssrn.3901848 https://doi.org/10.11648/j.jps.20140202.12 https://doi.org/10.11648/j.jps.20140202.12 https://doi.org/10.1007/bf00231281 https://doi.org/10.1007/bf00231281 https://doi.org/10.5897/ajps2021.2156 https://doi.org/10.3389/fpls.2017.00615 https://doi.org/10.3390/ijms232213736 introduction materials and methods plant material experimental site experimental procedure data collection data analysis results and discussion overview of trait variation across locations analysis of variance correlation analyses principal component analysis cluster analysis mean performance comparison conclusion supplemental data author contributions acknowledgements funding conflict of interest statement data availability short communication genetic resources (2025), 6 (12), 111–119 doi: 10.46265/genresj.skpz5680 https://www.genresj.org issn: 2708-3764 received: 14.04.2025 | accepted: 23.08.2025 | published online: 08.10.2025 a public mid-density genotyping platform for north american atlantic salmon (salmo salar l.) abstract: genomics-enabled selective animal breeding has become common in recent years, prompting a growing need for diverse genotyping tools that facilitate collaboration among research groups while meeting specific programme needs and objectives. here, we report the development of a medium-density amplicon panel (dartag) of 2,950 loci for north american atlantic salmon. it includes loci distributed across the genome and loci useful for distinguishing the continent-of-origin, parentage, and sex determination. this mid-density panel offers more cost-effective and rapid genotyping capabilities for atlantic salmon researchers and breeders. the open access provided by this platform facilitates comparisons and enhances data reusability across projects, institutions and countries that use different genomic tools for genotyping. this genotyping panel can make routine genotyping a viable tool for breeding and research programmes. keywords: salmon, aquaculture, amplicon-sequencing, selective breeding, dartag genotyping citation: zhao, d., chinchilla-vargas, j., sandercock, a. m., taniguti, c. h., long, r., palti, y., gao, g., pietrak, m., may, s. a., heller-uszynska, k., beil, c. t. and sheehan, m. j. (2025) “a public mid-density genotyping platform for north american atlantic salmon (salmo salar l.)”, genetic resources, 6(12), pp. 111–119. doi: 10.46265/genresj.skpz5680. © copyright 2025 the authors. this is an open access article distributed under the terms of the creative commons attribution license (cc by 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. dongyan zhaoa,†, josue chinchilla-vargasa,†, alexander m. sandercocka, cristiane h. tanigutia, roseanna longb, yniv paltib, guangtu gaob, michael pietrakc, samuel a. mayc‡, kasia helleruszynskad, craig t. beila, moira j. sheehana,* abreeding insight, cornell university, ny, 14853, ithaca, usa bnational center for cool and cold water aquaculture research, usda-ars, wv, leetown, 25430, usa cnational cold water marine aquaculture center, usda-ars, me, 04634, franklin, usa ddiversity arrays technology, act 2617, bruce, australia ‡current address: the center for aquaculture technologies, 8445 camino santa fe suite 104, san diego, ca, usa * corresponding author: moira sheehan (moirasheehan@cornell.edu) †these authors have contributed equally to this work introduction commercial aquaculture of atlantic salmon (salmo salar l.) accounts for approximately 70% of the global total salmon production (pandey et al, 2023). atlantic salmon has long been the focus of selective breeding programmes aimed at improving production traits related to growth, disease resistance and fillet quality (vallejo et al, 2024; kristjánsson et al, 2020). the past two decades have witnessed a rapid adoption of molecular methods to enhance breeding programmes, particularly the application of genomic selection methods (meuwissen et al, 2001). these molecular advancements require developing genotyping tools for various applications, most often in the form of panels of preidentified single nucleotide polymorphisms (snps). in plant breeding, these targeted genotyping technologies can be characterized into low density (i.e. hundreds of loci), medium density (i.e. hundreds to thousands of loci) and high density (i.e. tens of thousands to millions of loci). this classification differs notably from livestock standards, where medium-density arrays typically contain around 50k markers and highdensity arrays can exceed 700k markers. for aquaculture study and breeding, until recently, the majority of these resources were designed and owned by private companies (gao et al, 2023; kijas et al, 2017), slowing the efficiency of atlantic salmon genomic research. therefore, publicly accessible genomic resources are needed to facilitate open, reproducible research for atlantic salmon, with applications in aquaculture, conservation and fisheries management. https://doi.org/10.46265/genresj.skpz5680 https://www.genresj.org https://doi.org/10.46265/genresj.skpz5680 mailto:moirasheehan%40cornell.edu?subject= genetic resources (2025), 6(12), 111–119112 zhao et al north american atlantic (naa) salmon are genetically distinct from european and baltic atlantic strains. the common ancestor of today’s salmonids underwent a lineagespecific whole genome duplication event at ~96 million years ago (allendorf and thorgaard, 1984; danzmann et al, 2008; berthelot et al, 2014). since that time, the re-diploidization of salmonids has proceeded independently in the old world and the new world. today, the genetic divergence of european and baltic atlantic salmon (2n = 2x = 58) from naa salmon (2n = 2x = 54) is characterized by large structural changes and unique karyotypes that designate them as subspecies (de boer et al, 2007; brenna-hansen et al, 2012). despite high syntenic conservation, naa salmon exhibited significant genomic differences compared to its european counterpart (brenna-hansen et al, 2012; gao et al, 2020). atlantic salmon farming in the eastern united states and canada is limited to north american (na) genetic stocks due to ecological and conservation concerns. therefore, genotyping panels developed for european atlantic salmon are less effective when applied to na populations, underscoring the need for origin-specific genomic tools (yáñez et al, 2016). in the united states, the only remaining wild populations of atlantic salmon are found in the gulf of maine and are listed as endangered under the endangered species act (https://www.fisheries.noaa.gov/species/atlantic-salmon/ protected). the usda-ars national cold water marine aquaculture center (ncwmac) has operated a selective breeding programme for the st. john river (sjr) strain of naa salmon since 2003 for traits such as growth, fillet quality and resistance to sea lice (a major pathogen in marine aquaculture) (peterson et al, 2020; vallejo et al, 2024). the sjr strain, chosen for its rapid growth and suitability for captive aquaculture, undergoes a 4-year lifecycle involving specialized systems for egg incubation, fry growth, and maturation, culminating in spawning mature broodstock weighing approximately 3–8kg. up to 150 families are cultured annually, with fish evaluated in biosecure tanks and commercial net pens for performance. in 2022, ncwmac adopted a genomic selection index weighted 70% for growth and 30% for sea lice resistance, supported by a 50k snp chip developed for the na salmon genome (gao et al, 2023; vallejo et al, 2024). the cost of using the 50k snp chip remains a barrier to widespread usage. in contrast, lowor medium-density genotyping panels may meet many of the same goals at a reduced price with reduced lab equipment requirements and lower overhead. here, we developed a multi-purpose mediumdensity dartag panel with 3k markers by subsetting markers already included in the 50k naa salmon snp array (gao et al, 2023). dartag is a targeted amplicon sequencing platform developed by diversity arrays technology, llc, which provides low-cost and reproducible genotyping results across sequencing projects (blyton et al, 2023; “dartag,” n.d.). we validated the dartag panel by genotyping 3,710 naa salmon from the united states department of agriculture (usda) aquaculture stock and show that (1) the 3k dartag panel can be used to obtain high-quality snps across genotyped individuals, (2) the panel accurately identifies relationships between individuals, and (3) the 3k panel can be effectively used for linkage analysis comparable to the high-density array. this open-source 3k dartag panel can increase the accessibility of genotyping for programmes without access to in-house genotyping technology or specialized labour. it may also reduce the cost of genotyping by lowering the marker density without sacrificing much information, thereby increasing breeder access to genotyping services to allow for more intensive, routine and effective usage of genomic resources in naa salmon breeding. materials and methods selection of 3k marker loci for building the dartag genotyping panel we previously published the results of the 50k snp affymetrix array (gao et al, 2023) developed based on the alignment of whole-genome re-sequencing of 80 naa salmon fish from three distinct aquaculture stocks to the naa salmon reference genome (genbank accession gca_021399835.1). from the 50k array, 10,353 snps were selected for their even genome distribution and functional annotations, including 8,803 snps from the naa-based snp dataset (gao et al, 2023), 1,462 highly informative snps from a european-based snp array (houston et al, 2014), 64 snps for distinguishing the continent of origin (coo), 20 mitochondrial snps, and four sex determination snps. the evenly distributed 10k snp set was submitted to diversity arrays technology (dart) for proprietary in silico quality control. dart recommended the loci that passed quality control to produce the final 3k snp panel. upon initial testing of the 3k panel, it was observed that the 20 mitochondrial markers consumed ~40% of the reads per sample, indicating the abundance of mitochondrial dna and undesirable preferential amplification. therefore, these 20 markers were removed, leaving a final set of 2,980 genomic loci (figure 1). notably, the 50k snp dataset was initially developed based on the naa salmon’s contig-level assembly, and gao et al, 2023 mapped these snps to the final chromosome-level assembly. among the 2,980 dartag markers, 2,911 were assigned chromosome coordinates by gao et al, 2023. for the remaining 69 markers, we used blast to align the 180bp flanking sequences to the reference genome. we confirmed the positions of 49 of the 69 markers, bringing the number of markers with known pseudomolecule physical locations in the chromosomes up to 2,950 (supplemental table 1). the unmapped snps are likely due to several factors, such as their contigs not being included in the final assembly, contig splits due to hi-c or bionano scaffolding, and/or error corrections at the scaffold level that altered the reference sequences. to compare the 50k snp array with the 3k dartag panel, we aligned the two datasets into a consistent genomic framework, including matching target snp positions and reference and alternative base calls. the axiom and dartag arrays can include probes on the plus or minus strand. thus, correct inference of reference and alternative alleles depends upon the oligo orientation, especially for a/t and c/g (i.e. snps ambiguous to dna strand) snps. we established contig orientation through blast alignment of the 180bp flanking sequences of the 3k snps against the reference genome. reference and alternative bases were designated based on the contig orientations and the axiom and dartag probe orientations. figure 1. a) filters and criteria applied to produce the 3k dartag marker panel from the 50k north american atlantic (naa) salmon snp array. abbreviations: k is thousands. b) the distribution of 2,950 snps on the 3k dartag panel across the 27 chromosomes of the naa salmon genome. https://www.fisheries.noaa.gov/species/atlantic-salmon/protected https://www.fisheries.noaa.gov/species/atlantic-salmon/protected genetic resources (2025), 6(12), 111–119 robust genotyping for na atlantic salmon 113 in-house genotyping technology or specialized labour. it may also reduce the cost of genotyping by lowering the marker density without sacrificing much information, thereby increasing breeder access to genotyping services to allow for more intensive, routine and effective usage of genomic resources in naa salmon breeding. materials and methods selection of 3k marker loci for building the dartag genotyping panel we previously published the results of the 50k snp affymetrix array (gao et al, 2023) developed based on the alignment of whole-genome re-sequencing of 80 naa salmon fish from three distinct aquaculture stocks to the naa salmon reference genome (genbank accession gca_021399835.1). from the 50k array, 10,353 snps were selected for their even genome distribution and functional annotations, including 8,803 snps from the naa-based snp dataset (gao et al, 2023), 1,462 highly informative snps from a european-based snp array (houston et al, 2014), 64 snps for distinguishing the continent of origin (coo), 20 mitochondrial snps, and four sex determination snps. the evenly distributed 10k snp set was submitted to diversity arrays technology (dart) for proprietary in silico quality control. dart recommended the loci that passed quality control to produce the final 3k snp panel. upon initial testing of the 3k panel, it was observed that the 20 mitochondrial markers consumed ~40% of the reads per sample, indicating the abundance of mitochondrial dna and undesirable preferential amplification. therefore, these 20 markers were removed, leaving a final set of 2,980 genomic loci (figure 1). notably, the 50k snp dataset was initially developed based on the naa salmon’s contig-level assembly, and gao et al, 2023 mapped these snps to the final chromosome-level assembly. among the 2,980 dartag markers, 2,911 were assigned chromosome coordinates by gao et al, 2023. for the remaining 69 markers, we used blast to align the 180bp flanking sequences to the reference genome. we confirmed the positions of 49 of the 69 markers, bringing the number of markers with known pseudomolecule physical locations in the chromosomes up to 2,950 (supplemental table 1). the unmapped snps are likely due to several factors, such as their contigs not being included in the final assembly, contig splits due to hi-c or bionano scaffolding, and/or error corrections at the scaffold level that altered the reference sequences. to compare the 50k snp array with the 3k dartag panel, we aligned the two datasets into a consistent genomic framework, including matching target snp positions and reference and alternative base calls. the axiom and dartag arrays can include probes on the plus or minus strand. thus, correct inference of reference and alternative alleles depends upon the oligo orientation, especially for a/t and c/g (i.e. snps ambiguous to dna strand) snps. we established contig orientation through blast alignment of the 180bp flanking sequences of the 3k snps against the reference genome. reference and alternative bases were designated based on the contig orientations and the axiom and dartag probe orientations. figure 1. a) filters and criteria applied to produce the 3k dartag marker panel from the 50k north american atlantic (naa) salmon snp array. abbreviations: k is thousands. b) the distribution of 2,950 snps on the 3k dartag panel across the 27 chromosomes of the naa salmon genome. methodology and procedure of the dartag genotyping platform the dartag genotyping assay consists of four steps based on principles described in krishnakumar et al, 2008 and implemented as described (zhao et al, 2023; sandercock et al, 2025a). briefly, the pool of 3k naa salmon oligos, each targeting one genetic variant plus adjacent flanking sequence, are hybridized to denatured gdna in step 1, followed by snp/indel copying into dartag molecules by dna polymerase in step 2. also in step 2, amplicons are ligated to create circularized molecules. in step 3, the reaction content is treated with nucleases to remove any un-circularized molecules. dartag products are subsequently amplified in step 4 with the simultaneous addition of sample-unique barcodes used downstream for demultiplexing. the products of the dartag assay, after purification and quantification, are sequenced on ngs platforms (e.g. novaseq 6000, illumina) with a depth of around 200x. sequences are demultiplexed, quality controlled, and the genetic variants are detected using dart’s proprietary analytical pipelines. genetic resources (2025), 6(12), 111–119114 zhao et al selection of samples for validating the dartag panel and genotyping results the salmon 3k marker panel was tested using a set of 3,710 fish from the sjr strain reared at ncwmac (supplemental table 2). this set included 118 fish from year classes (yc) fertilized in yc2009 and yc2010, 1,099 fish from yc2014, 2,487 fish from yc2018, and two individuals lacking year class information. the dataset represented three generations of fish from the sjr strain of naa salmon. genotyping was processed in two batches: (1) 1,105 fish from 2014 and (2) the remaining 2,605 fish. dart provided genotypes in vcf format, read counts for all markers, dosage calls, and missing allele discovery counts (madc) for the second batch, which contained the read count for all 54bp microhaplotype alleles discovered in the samples. read count data for reference (ref) and alternative (alt) alleles from both batches underwent a 2-step quality control and filtering process. first, we removed samples with high missing data rates (≥ 95%), where a marker was considered missing if it had fewer than 10 reads. subsequently, we filtered out marker loci that were present in < 10 samples. to enable accurate comparative analysis between the two batches, we concatenated read count data into a single file and conducted dosage calls using the updog r package (gerard et al, 2018). the original dartag marker ids were converted to the chromosome-level marker ids in the 50k array and lookup table of the dartag vs. axiom ids is provided (supplemental table 1). pedigree verification and parentage testing before verifying parentage, duplicated ids, individuals appearing both as male and female, and any circular dependencies in the pedigree were removed using the clean_pedigree() function from bigr (rrid: scr_026677; v0.3.4) (sandercock et al, 2025a; sandercock et al, 2025b). parentage testing was performed with the seekparentsf90 module in blupf90 (misztal et al, 2014) with an allowed maximum threshold of 1% of markers showing mendelian errors between parent-offspring pairs proposed by pedigree. percentages over the threshold were flagged as a pedigree error. to identify potential parents in the genotyped set of individuals, the –seektype 2 flag was used in the analysis. additionally, within-family clustering of individuals was performed with a principal component analysis (pca) via the breeding insight genomics app (rrid v0.6.2) (beygelzimer et al, 2019; sandercock et al, 2025a). supervised clustering and k nearest neighbor (knn) an initial parentage verification analysis of the dartag datasets found discordance between pedigree records and genotypes, suggesting that the individuals in batch 2 were not labelled with the correct sample ids. a 2-step approach was implemented to estimate the correct ids: (1) match the sample ids between the axiom 50k and 3k dartag datasets based on genetic similarity, and (2) validate the estimated sample ids through a second parentage verification analysis. before matching, missing genotypic data in batch 2 samples were imputed using beagle v5.4 with the default parameters (browning, zhou, and browning 2018). the 50k dataset and the 3k dataset of fish born in 2018 were filtered to retain only shared loci in both datasets. to estimate potential matches (step 1), we used the k-nearest neighbors (knn) algorithm as implemented in the fast nearest neighbor search algorithms and applications (fnn) r package (v1.1.4.1) (beygelzimer et al, 2019). the individual samples on the 50k panel were paired with the sample exhibiting the smallest genetic distance (euclidean distance) in the 2018 dartag dataset. this was accomplished using the knn.dist() function of the fnn package (parameter k = 1), effectively assigning the single, most likely sample id from the 50k dataset to its counterpart in the 2018 3k dataset. to assess the accuracy of this first step, we performed the same steps above with the 50k and 3k genotype data for the 2014 salmon samples (batch 1). we found 99.8% agreement between the putative sample id and the matched sample id using only this initial knn match. despite the assessed accuracy of step 1, several individuals in the 50k dataset did not pair with a unique sample in the 2018 3k dataset. in these cases, only the match with the lowest genetic distance was retained for step 2. finally, a second parentage analysis (step 2) was performed using the sample ids estimated by knn for mislabelled individuals in the 2018 dartag dataset. the sample ids that passed this verification confirmed that the revised ids were consistent with mendelian expectations, enabling the accurate identification of the parents of the mislabelled samples. only validated samples that passed both steps were included for genetic map construction. genetic map construction to evaluate the utility of the dartag marker panel for closed-population marker-assisted selection (mas), a linkage map was generated using lep-map3 (v0.5.0; rastas, 2017) from the validated salmon samples. samples were retained if they belonged to a family with at least ten individuals, resulting in 1,035 samples, 55 families, and 2,806 informative snp loci. first, the parentcall2 function was used to call missing parental genotypes, with halfsibs = 1 to include half-sib information. then, filtering2 checked snps for non-informative markers or non-mendelian markers (i.e. segregation distortion), although no additional snps were removed due to the previous, more stringent filtering in plink 1.9 (purcell et al, 2007). markers were categorized into 33 linkage groups (lgs) using separatechromosome2, with lodlimit = 27 set as the expected number of haploid chromosomes in naa salmon (1n = 1x = 27). of the 33 lgs, 27 lgs contained markers aligned with their expected physical chromosomes. the six remaining lgs contained four or fewer markers, so the markers from these six lgs were categorized as ‘single’ markers for the next step. the ‘single’ markers were added to one of the 27 lgs with a more relaxed lodlimit = 10 and loddifference = 3. lastly, the genomic positions of the markers within each lg were ordered with ordermarkers2. additional filtering was performed to remove markers with (1) a physical position that deviated significantly from the other markers in the lg and (2) a lower pairwise lod score with closely positioned markers (supplemental figure 1). genetic resources (2025), 6(12), 111–119 robust genotyping for na atlantic salmon 115 results creation of the 3k naa salmon dartag panel the 3k dartag panel (salmon dartag3k bi cornell university (1.0)) is comprised of 514 snps from a europeanbased snp array (houston et al, 2014), 64 snps for identifying coo, 4 snps from the sex determination (sd) locus, and 2,418 genic snps from the naa salmon snp dataset. to enhance comparability, the 3k snps were mapped to the naa salmon chromosomes, and 2,950 snps were assigned unambiguously to physical positions (supplemental table 1). the 514 european-based markers were mapped across the 27 chromosomes, with an additional small fraction (0.6%) remaining in unplaced sequences based on the naa salmon reference genome (supplemental table 3). the majority of chromosomes (18/27; 66.7%) maintained moderate to high marker coverage, containing between 15 and 30 markers each, indicating robust coverage across most of the genome. validation of the 3k salmon dartag panel and genotyping results to assess the 3k panel, a validation set of 3,710 samples was genotyped in two batches using the 3k dartag panel to: (1) construct a genetic linkage map and (2) evaluate the usefulness of the dartag panel for downstream genetic analyses. we established a minimum threshold of ten read counts for a marker locus to be considered valid. under this criterion, 1,077 (97%) of 1,105 samples from batch 1 and 2,470 (95%) of the 2,605 samples from batch 2 retained data for 75% of the total markers. batch 2 showed particularly robust performance, with 2,181 (84%) samples containing data for ≥ 90% of the total markers (supplemental table 4). of the 2,950 markers, 2,495 (85%) and 2,827 (96%) were present in ≥ 50% of the samples from batches 1 and 2, respectively, suggesting they are highly conserved sequences within the naa salmon population. batch 2 demonstrated superior marker performance, with 2,410 (82%) markers present in ≥ 90% of the samples compared to 1,857 (63%) in batch 1 (supplemental table 4). this disparity in missing data rates between batches was likely due to lower dna quality in the batch 1 samples. overall, the panel demonstrated the robustness and applicability of the panel for high-throughput genotyping in naa salmon populations. for comprehensive analyses, we merge read count data from both batches. of the 2,950 snps assigned to physical positions, 2,278 markers were successfully genotyped in ≥ 85% of samples. additionally, 2,493 samples retained figure 2. principle component analysis (pca) plots in the first two dimensions of the validation families. a) pca plot of the nine full-sibling families with the largest members of 2018-born fish before parentage testing and knn analysis. b) pca plot of the same nine full-sibling families of 2018-born fish after pedigree correction with knn. genetic resources (2025), 6(12), 111–119116 zhao et al genotype data for ≥ 85% of the markers. the merged dataset showed lower data rates compared to batch 2 independently, reflecting the impact of batch 1’s lower performance. after filtering for missing data and concatenating the allele dosage results from both batches, 2,806 markers were retained for downstream analyses. pedigree verification, parentage testing, and correction via knn analysis an initial parentage testing run to verify pedigree accuracy showed that 99.6% of offspring-parent pairs in the dataset were erroneous. to better visualize the within-family clustering, the principal components of genotypes of 276 fish belonging to the nine families with the most siblings were plotted. no identifiable clustering by family was found (figure 2a). this led us to identify widespread mislabeling of samples from fish born in 2018. to identify the best-matching id in the dartag genotyping results, 3k genotype calls were compared to the same markers in the 50k array using knn analysis. genetic distances between matched samples ranged from 27.2 to 53.63, with a maximum value of 45 selected as a filtering threshold that limited the number of samples with multiple matches, retaining 1,493 fish. parentage testing of the knninformed parent-offspring pairs found 992 samples (66.5%) fulfilled mendelian expectations with their proposed parents. this two-step approach produced a set of 1,013 individuals, composed of 55 full-sib families of at least ten individuals and their respective parents, which were then used to generate the linkage map. figure 2b shows the clustering of the nine families with the most individuals, as assigned by knn. creation of a linkage map the final salmon dartag linkage map (figure 3) consisted of 27 lgs with 2,642 markers and a total length of 1,983.81cm for the female map, and 927.8cm for the male map (with an average density of 1.33 markers/cm and 2.85 markers/cm, respectively). lg length from the female map ranged from 52.44cm to 101.02cm, with an average of 73.5cm. the male map linkage group length ranged from 2.42cm to 75.3cm, with an average length of 34.4cm. consistent with findings from the same fish tested on the 50k marker panel (gao et al, 2023), paternal and maternal recombination patterns differed (figure 3b). in paternal chromosomes, recombination was elevated at the telomeres with strong interference near the centromere. in contrast, maternal chromosomes exhibited distinct patterns based on chromosome type: in acrocentric chromosomes, recombination was elevated around the centromere and decreased toward the telomeres, whereas in metacentric chromosomes, interference was pronounced at the centromere with comparable recombination patterns extending toward both telomeric ends. markers were generally well distributed across the 27 lgs, with ~50% of the markers located within the first 10 lgs (table 1). additional mapping details are summarized in supplemental table 5. figure 3. genetic map of naa salmon constructed from 1,035 individuals from 55 families. a) distribution of 2,642 snps across 27 linkage groups of the north american atlantic salmon linkage map. b) relationship plots of physical map distance (mb; x-axes) to genetic map distance (cm; y-axes) for each of the 27 chromosomes in the male and female genetic maps. table 1. linkage map from 1,035 fish spanning 55 families and 2,642 uniquely mapped snps by chromosome on the male and female maps (in cm). chromosome marker count male (cm) female (cm) chr01 148 56.8 78.7 chr02 55 10.1 92.6 chr03 107 50.4 99.1 chr04 90 26.7 93.7 chr05 75 52.0 96.8 chr06 80 20.8 99.4 chr07 54 21.4 85.7 chr08 75 61.8 87.2 chr09 163 17.1 83.2 chr10 151 43.6 75.7 chr11 103 47.7 67.4 chr12 114 29.6 67.3 chr13 129 36.4 70.0 chr14 131 22.8 60.8 chr15 146 42.7 62.2 chr16 93 5.6 56.6 chr17 51 2.4 58.0 chr18 98 36.3 68.9 chr19 103 44.7 57.2 chr20 113 17.1 58.5 chr21 86 47.5 53.5 chr22 79 55.3 53.6 chr23 131 75.3 101.0 chr24 64 34.8 56.2 chr25 54 6.4 52.6 chr26 90 49.2 95.6 chr27 59 13.5 52.4 min 51 2.4 52.4 max 163 75.3 101.0 average 97.9 34.4 73.5 total 2,642 927.8 1,983.8 discussion and conclusion the naa salmon 3k dartag panel serves as a robust and versatile tool for genetic applications, providing reliable data for pedigree verification, parentage assignment and linkage map construction. its mid-density design fills a gap in community resources between the high-density 50k array and the low-density option of 384 snps (center for aquaculture technologies, personal communication). the 3k panel achieves comparable genome coverage to the 50k genetic resources (2025), 6(12), 111–119 robust genotyping for na atlantic salmon 117 genotype data for ≥ 85% of the markers. the merged dataset showed lower data rates compared to batch 2 independently, reflecting the impact of batch 1’s lower performance. after filtering for missing data and concatenating the allele dosage results from both batches, 2,806 markers were retained for downstream analyses. pedigree verification, parentage testing, and correction via knn analysis an initial parentage testing run to verify pedigree accuracy showed that 99.6% of offspring-parent pairs in the dataset were erroneous. to better visualize the within-family clustering, the principal components of genotypes of 276 fish belonging to the nine families with the most siblings were plotted. no identifiable clustering by family was found (figure 2a). this led us to identify widespread mislabeling of samples from fish born in 2018. to identify the best-matching id in the dartag genotyping results, 3k genotype calls were compared to the same markers in the 50k array using knn analysis. genetic distances between matched samples ranged from 27.2 to 53.63, with a maximum value of 45 selected as a filtering threshold that limited the number of samples with multiple matches, retaining 1,493 fish. parentage testing of the knninformed parent-offspring pairs found 992 samples (66.5%) fulfilled mendelian expectations with their proposed parents. this two-step approach produced a set of 1,013 individuals, composed of 55 full-sib families of at least ten individuals and their respective parents, which were then used to generate the linkage map. figure 2b shows the clustering of the nine families with the most individuals, as assigned by knn. creation of a linkage map the final salmon dartag linkage map (figure 3) consisted of 27 lgs with 2,642 markers and a total length of 1,983.81cm for the female map, and 927.8cm for the male map (with an average density of 1.33 markers/cm and 2.85 markers/cm, respectively). lg length from the female map ranged from 52.44cm to 101.02cm, with an average of 73.5cm. the male map linkage group length ranged from 2.42cm to 75.3cm, with an average length of 34.4cm. consistent with findings from the same fish tested on the 50k marker panel (gao et al, 2023), paternal and maternal recombination patterns differed (figure 3b). in paternal chromosomes, recombination was elevated at the telomeres with strong interference near the centromere. in contrast, maternal chromosomes exhibited distinct patterns based on chromosome type: in acrocentric chromosomes, recombination was elevated around the centromere and decreased toward the telomeres, whereas in metacentric chromosomes, interference was pronounced at the centromere with comparable recombination patterns extending toward both telomeric ends. markers were generally well distributed across the 27 lgs, with ~50% of the markers located within the first 10 lgs (table 1). additional mapping details are summarized in supplemental table 5. figure 3. genetic map of naa salmon constructed from 1,035 individuals from 55 families. a) distribution of 2,642 snps across 27 linkage groups of the north american atlantic salmon linkage map. b) relationship plots of physical map distance (mb; x-axes) to genetic map distance (cm; y-axes) for each of the 27 chromosomes in the male and female genetic maps. panel described by gao et al (2023). because it is a subset of loci on the 50k, it could be used for sparse testing plus imputation on higher numbers of progeny when parents are genotyped on the 50k in parent-progeny studies. the inclusion of four sex-linked markers and 68 coo markers further enhances its applicability across diverse research and breeding scenarios. we acknowledge that the fewer markers in the mid-density panel relative to the 50k array may result in a substantial loss of resolution for fine-scale mapping applications such as genome-wide association studies (gwas), as qtl detection and mapping accuracy are highly dependent on marker density. however, the reduction in marker density is expected to have only a modest impact on genomic selection applications, where prediction accuracy may show only a slight decrease. while the 3k panel was developed and validated for naa salmon, its transferability to european salmon populations would need empirical validation. importantly, the inclusion of 514 markers from a european salmon-based affymetrix snp array could potentially be useful for european salmon populations. the potential utility of this panel might be particularly relevant for comparative genomic studies, population structure analyses, or preliminary screening purposes for both north american and european atlantic salmon. however, users should consider possible limitations when applying it to european populations, including: (1) potentially reduced marker polymorphism in european populations, (2) different linkage disequilibrium patterns genetic resources (2025), 6(12), 111–119118 zhao et al that might affect marker informativeness, and (3) possible ascertainment bias due to the north american-focused marker selection. the naa salmon dartag panel is publicly available and open for any researcher or breeder to order through dart (https://www.diversityarrays.com), with a cost midway between the 50k high-density array and the 384-snp lowdensity options. the high detection rate and repeatability make this panel suitable for genetic map construction, markerassisted selection, whole-genome association mapping, reconstruction of recombination patterns, allele dosage estimation, and parental confirmation in naa salmon from the northeast us. the panel’s efficacy on breeding materials or populations outside the northeast us has not been tested. one benefit dartag has over fixed array platforms is the ability to update and improve the marker panel as needed. the panel is a pool of 2,950 oligos, one per locus, which are used to generate sequencing libraries from assayed material. because the pool is created from individual oligo stocks, removing suboptimal loci or adding new loci can be quickly done by creating a new pool. independently, as new significant trait markers and/or markers specific to other germplasm are detected, they can be included in the original pool in the panel’s next version(s). due to our budgetary restrictions, we created a panel of 3,000 loci; however, smaller, complementary panels can be made at lower up-front and downstream usage costs. subpanels of a few hundred loci may also be developed using other amplicon techniques, such as genotyping by thousands (gtseq), for lower genotyping costs (campbell et al, 2015). the practical upper limit for the number of probes on a dartag panel is 7,000 loci. however, the optimal maximum may differ by species and genome complexity, and read depth required to sufficiently call genotypes (andrzej kilian dart, personal communication). supplemental data supplemental file 1. genotypic data in vcf format for the 1,013 individuals used to produce the linkage map supplemental figure 1. relationship plots of physical map distance (mb; x-axes) to genetic map distance (cm; y-axes). supplemental table 1. physical position and identification of the 2950 snps included in the dartag panel. supplemental table 2. accessions used in the testing of the salmon 3k dartag panel and construction of genetic map. supplemental table 3. distribution of european atlantic salmon array-based markers on salmon genome supplemental table 4. sample and marker missing data from two batches of dartag genotyping. supplemental table 5. physical position, genetic distance and identification of the 2,642 snps included in the linkage map. author contributions dz, gg, yp and mjs contributed to experimental design and planning. gg and yp selected the diversity panel for wgs. sm contributed to writing and editing the paper. rl and mp collected and prepared all fin clip materials used in the study. dz performed all the snp database creation, filtering pipelines, and quality control analyses to create the 3k panel. khu managed the panel creation at diversity arrays technology. dz, as, jcv, ams and cht executed the data analyses and genetic mapping. dz, ams, jcv and mjs wrote the initial draft of the manuscript. cb managed experiments and communication among all authors involved. all authors contributed to reviewing the manuscript. acknowledgments and funding the authors extend their thoughts to their late colleague, guangtu gao, with whom they had the pleasure of working on this study, and who passed away too soon. the authors also thank dr alex casa for her careful review of this manuscript and her valuable feedback. breeding insight (rrid:scr_026645) was funded through cooperative agreements between usda-ars and cornell (project numbers: 8062-21000-043-004-a,  8062-21000-052-002-a, and 8062-21000-052-003-a). this study was also supported by the usda agricultural research service in-house project numbers 8030-31000-004/005 and 8082-31000-012/013. conflict of interest statement the authors declare no conflicts of interest. data availability statement the genotypic data in vcf format for the 1,013 individuals used to produce the linkage map 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husbandry: potentials under socio-economic conditions world conference center bonn, germany 15-16 october 2020 collection of abstracts © federal ministry of food and agriculture (bmel) germany this is an open access book distributed under the terms of the creative commons attribution license (cc by 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. special issue genetic resources (2021), 2 (s1) 1-114 doi: 10.46265/genresj.quld1720 https://www.genresj.org issn: 2708-3764 https://doi.org/10.46265/genresj.quld1720 https://www.genresj.org/ special issue editorial team: organizer christian gerlinger (bmel, germany) genetic resources etienne verrier (agroparistech, france) sandra goritschnig (ecpgr secretariat, italy) editorial office: ecpgr secretariat c/o bioversity international via dei tre denari 472a 00054 maccarese (rm), italy submissions to www.genresj.org the designations employed, and the presentation of material in the periodical, and in maps which appear herein, do not imply the expression of any opinion whatsoever on the part of ecpgr or bmel concerning the legal status of any country, territory, city or area or its authorities, or concerning the delimitation of its frontiers or boundaries. similarly, the views expressed are those of the authors and do not necessarily reflect the views of ecpgr or bmel. focus and scope of genetic resources genetic resources is an open access journal disseminating global knowledge and tools used by the community of practitioners of plant, animal and forest genetic resources involved in monitoring, collecting, maintaining, conserving, characterizing and using genetic resources for food, agriculture and forestry. genetic resources publishes original research as well as methods, strategies, guidelines, case studies or reviews on a variety of topics of interest on the present and future use of genetic resources. these may include the documentation, conservation, management, assessment, characterization and evaluation of genetic resources and their link to broader biodiversity, socioeconomic practices, policy guidelines or similar, serving stakeholders within and across sectors. occasionally, genetic resources publishes special issues with a focus on selected topics of interest for the genetic resources community. the journal welcomes contributions from all world regions. this special issue has been produced with the support of the federal ministry of food and agriculture (bmel), germany and in the frame of genres bridge, a project which has received funding from the european union’s horizon 2020 research and innovation programme under grant agreement no 817580 http://www.genresj.org/ http://www.genresbridge.eu/ international congress on sheep and goats – farmers and scientists create future 15-16 october 2020, bonn, germany table of contents scientific program group 1 editorial 2 congress summary 3 abstracts: block 1+2: animal breeding and genetics: what will sheep and goat breeding look like in the future? oral presentations 7 poster abstracts 18 block 3: economy: how can the profitability of sheep and goat farming be improved? keynote presentation 32 oral presentations 33 poster presentations 38 block 4: environmental performance and climate change: what do sheep and goats contribute to climate change mitigation? oral presentations 40 block 5: animal genetic resources: diversity and characterization keynote presentation 44 oral presentations 45 poster presentations 49 block 6: animal genetic resources: implementation of policies and support oral presentations 50 block 7: management and animal welfare: keynote presentation 54 oral presentations 56 poster presentations 61 block 8: other topics keynote presentation 68 oral presentations 69 poster presentations 72 block 9: animal health: parasitology oral presentations 78 block 10: animal health: locomotive apparatus and monitoring oral presentations 83 block 11: animal health: reproduction oral presentations 89 poster presentations (all animal health subtopics) 94 block 12: structures of and prospects for sheep and goat farming, sustainability oral presentations 105 poster presentations 109 scientific program group international congress on sheep and goats genetic resources (2021) s1 abstractbook 1 head of program group dr. bernhard polten (bundesministerium für ernährung und landwirtschaft, bmel) animal breeding and genetics prof. dr. gesine lühken (justus-liebig-universität giessen) dr. christian mendel (bayerische landesanstalt für landwirtschaft) economy prof. dr. stanislaus von korn (hochschule für wirtschaft und umwelt nürtingen geislingen) janine bruser (landesverband schleswig-holsteiner schafund ziegenzüchter e.v.) fides marie lenz (schafzuchtverband nordrhein-westfalen e.v.) environmental performance and climate change prof. dr. thomas döring (universität bonn) michael gertenbach (landwirtschaftskammer niedersachsen) animal genetic resources dr. roswitha baumung (food and agriculture organization of the united nations, fao) christian gerlinger (bundesministerium für ernährung und landwirtschaft, bmel) management and animal welfare dr. heiko georg (thünen institut) pd dr. pera herold (landesamt für geoinformation und landentwicklung badenwürttemberg) gerhard schuh (thüringer landesamt für landwirtschaft und ländlichen raum) other topics prof. dr. claudia klein (friedrich-loeffler-institut, fli) dr. bettina bongartz (deutsche gesellschaft für züchtungskunde e.v., dgfz) animal health prof. dr. martin ganter (stiftung tierärztliche hochschule hannover) pd dr. esther humann-ziehank (labvetcon) dr. henrik wagner (universität gießen) structures of and prospects for sheep and goat farming, sustainability dr. reinhard reents (vereinigte informationssysteme tierhaltung w.v., vit) dr. jens wilkens (vereinigte informationssysteme tierhaltung w.v., vit) session summaries and results prof. dr. bernt guldbrandtsen (universität bonn) editorial genetic resources (2021) s1 international congress on sheep and goats 2 abstract book the future of a sustainable sheep and goat breeding bernhard polten federal ministry for food and agriculture (bmel), rochusstr. 1, 53123 bonn, germany due to their manifold possible uses, sheep and goats are among the oldest domestic animals. there are plenty of good reasons to keep sheep and goats. approximately one billion sheep and 600 million goats are kept worldwide. they are used for landscape management purposes, e.g.: to keep heathlands open or to maintain the dykes along coasts and rivers. sheep and goats thus also contribute to the maintenance of biotopes. they promote biodiversity and sustainability. with the help of sheep and goats, valuable products can be gained from grass and, in the case of goats, also from leaves in grassland areas: milk, meat and wool. they therefore contribute to the income of sheep and goat keepers. sheep and goats can adapt to changing environments. yet, greater adaptive capacity is more important now than ever before. infectious diseases can spread quickly throughout the world. environmental changes are often perceived as a result of climate change with different manifestations. this international congress on the breeding of sheep and goats intends, for example, • to give an impression of sheep and goat keeping at global level, • to translate new research results into practice, • to identify the real-world need for research, and • to contribute to networking. the global covid-19 pandemic has posed great challenges to all parties involved in the congress. until only a few months ago, the congress had been planned as a pure face-to-face event. but due to the coronavirus, the congress had to be re-organised as a hybrid event at short notice. this has called for a great amount of extra time and technical efforts from all actors. but we – and especially the organisation team, the programme group and the scientific programme group, all people involved on and off stage, as well as all participants in this event – hope that all those efforts will be rewarded by an interesting congress and many new contacts and that the new technologies used may be able to outweigh at least some of the disadvantages caused by the coronavirus crisis. congress summary international congress on sheep and goats genetic resources (2021) s1 abstractbook 3 abridged press release on the “international congress on the breeding of sheep and goats” at the wccb in bonn on 15 and 16 october 2020, published in german in: züchtungskunde, 92, (6) pp. 452-459, 2020, issn 0044-5401, verlag eugen ulmer, stuttgart sheep and goat keepers worldwide discussed the future of breeding and husbandry bettina bongartz1 and christian gerlinger2 1 deutsche gesellschaft für züchtungskunde e.v. (dgfz – german society for animal production), adenauerallee 174, d-53113 bonn 2 bundesministerium für ernährung und landwirtschaft (bmel – german federal ministry of food and agriculture), rochusstraße 1, d-53123 bonn contact: [deutsche gesellschaft für züchtungskunde e.v. (dgfz), adenauerallee 174, 53113 bonn, info@dgfz-bonn.de] the “international congress on the breeding of sheep and goats”, held in bonn from 1516 october 2020 as a hybrid event, brought together researchers and professionals from the sheep and goat breeding sector across all borders. the federal ministry of food and agriculture (bmel) invited experts to the first international congress on the breeding of sheep and goats in bonn on 15 and 16 october 2020 under the title “farmers and scientists create future”. the conference provided a platform for researchers, professional associations, practitioners and non-governmental organisations to deepen their exchange on topical issues of sheep and goat breeding and husbandry. they discussed different current issues ranging from climate change to resilience with regard to animal breeding. more than 300 participants from all around the world joined the congress online or on site. the state-of-the-art world conference center bonn (wccb) provided the perfect framework for hosting a safe event in times of covid-19. nearly 150 contributions from approximately 40 countries were submitted, which made for a particularly interesting international programme. experts delivered more than 60 presentations, mainly in english, which were translated simultaneously into german. during the breaks and the evening event, the participants had the opportunity to talk to and network with other experts. throughout the entire conference, all participants had the chance to contemplate more than 50 posters. exhibitors were present in the foyer to round off the congress. the german society for animal production (dgfz) also provided information on their responsibilities, activities and services in english and german. due to the covid-19 pandemic, the event took place in a hybrid set-up, which involved considerable technical and organisational effort. the presentations were live-streamed and the posters were made available online, allowing all virtual participants to take part and join the discussion at all times. what an experience! even some facilitators successfully ran their sessions via live stream. impressed by what is possible thanks to technology, both speakers and mailto:info@dgfz-bonn.de congress summary genetic resources (2021) s1 international congress on sheep and goats 4 abstract book participants on site clearly enjoyed coming together after a six month conference break. “i am truly delighted to see you face-to-face again after such a long time,” said one speaker before delivering his presentation. while planning the event, all relevant parties met regularly to coordinate it together, assessing the ever-changing conditions and regulations in place regarding the feasibility of an international congress. at the same time, different technical options as well as the participant management had to be evaluated and considered against the backdrop of strict hygiene measures. the dgfz provided crucial assistance with their experience in event management. dr bettina bongartz, managing director of the dgfz, provided her full support in the preparations of the event. she is also a member of the scientific programme group. the bmel took a courageous decision by hosting a hybrid event and hence two events running simultaneously. this also meant double the amount of work and a budget twice as high. the kick-off event and the plenary meeting took place in the venerable plenary hall of the former german bundestag. state secretary hans-joachim fuchtel delivered an address outlining both the challenges and the achievements of the joint commitment by the federal government, the länder and the sheep and goat farmers. he also urged all participants to remain aware of their key responsibility for biodiversity conservation and climate stewardship. “this congress is to make a contribution to this end. it aims to take stock of sheep and goat breeding at global level and to communicate the outcomes, while also translating new research results into practice, identifying where global research is actually necessary and contributing to the exchange of ideas.” mr alfons gimber and mr bernd merscher, the respective chairs of the confederation of german sheep breeding associations (vdl) and the federation of german goat breeders (bdz), would not want to miss out on the opportunity to also deliver an address to the audience. “it is key that we recognise the global importance of sheep and goats as well as their economic relevance and their services to society,” said both of them. to kick off the plenary session, prof. dr. susanne crewell from the institute of geophysics and meteorology at the university of cologne and prof. dr. daniela jacob, director of the climate service center germany (gerics), showed impressive scenarios and research outcomes regarding climate change, which will also severely affect animal husbandry. impressive timelapse videos and expected changes showed the participants how urgently necessary global action is. mohammed bengoumi from the fao subregional office for north africa then gave a presentation on the role of small ruminants in poverty reduction and rural development, outlining the major positive impacts of optimising the breeding and husbandry of sheep and goats in africa. his presentation took the audience to another world, which faces different challenges and conditions. he gave an insight into the types of husbandry systems on different continents as well as the people, the specific needs they have and requirements they face. dr joanne conington from scotland’s rural college in edinburgh gave a presentation on the genomic selection in sheep and goats, which has already been carried out and outlined the differences compared to other animal species. as in bovine and porcine animals, the application of genomic selection results in an increased progress in breeding also in small ruminants. congress summary international congress on sheep and goats genetic resources (2021) s1 abstractbook 5 however, genomic selection has not yet achieved a breakthrough success in sheep and goat breeding due to structures which are different from the ones in cattle breeding. nonetheless, researchers and practitioners have made solid progress over the past years in order to reap the full potential of this breeding method in the stocks, as dr. conington explained. the diverse topics covered by the congress were grouped into eight fields, which were presented in three parallel sessions: ▪ animal breeding and genetics ▪ animal genetic resources ▪ animal health ▪ economy ▪ management and animal welfare ▪ environmental performance and climate change ▪ other topics ▪ structures of and prospects for sheep and goat farming, sustainability the issues of breeding, economy, management and animal genetic resources were of particular concern to the participants, resulting in diverse debates that even continued in the breaks. as for the economic dimension, examples from goat milk production were used to illustrate the economic variables in the output and cost estimation which are relevant to farmers, to what extent these variables are economically viable and what could be improved. another speaker presented an analysis of the current assessment of viability in sheep farming which is representative for the whole of germany. an impressive keynote address in the session on “other topics” proposed an entirely new way of earning money with sheep while at the same time improving the reputation of sheep farming. coaches and pastoralists can help conduct hands-on training courses for managers and teambuilding seminars for small and large companies. one training session can last between 3 and 4 hours. however, certain criteria need to be fulfilled. pastoralists, for instance, must have a minimum stock of 500 sheep and goats in an attractive landscape. examples like this prove that it is always worth thinking outside the box and exploring different opportunities to create an additional source of income. breeding plays a special role in developing sustainable sheep and goat farming as it critically paves the way for economic success. solid data collection, storage and processing play a key role here. animal genetic resources are of utmost significance for countries in asia and africa. the conference gave participants the opportunity to listen to various presentations and to contemplate numerous posters from various countries. but in germany, too, there are commitments and strategies for the conservation and use of animal genetic resources (angr). the national programme for the conservation and sustainable use of animal genetic resources in germany has proven to be advantageous in order to increase or at least maintain congress summary genetic resources (2021) s1 international congress on sheep and goats 6 abstract book the breeding stocks for endangered breeds and thereby maintain angr as the basis for adaptability in livestock production. “we fully achieved our common goal to further strengthen networks of experts and practitioners working in the area of sheep and goat breeding, to foster the exchange on latest research findings, and to promote the communication on innovations,” explained dr. bettina bongartz, managing director of the dgfz. the president of the dgfz, dr. erwin hasenpusch, was equally delighted about the conference, though it took place under such trying circumstances. “the federal ministry has demonstrated that it is possible to host events like this which are safe for all those involved. i am truly impressed by the commitment of everyone involved.” “congratulations to all parties involved and in particular to dr. bernhard polten, head of the division for animals and technology, for the great success that this congress has been, under difficult conditions due to covid-19. mr christian gerlinger (bmel) deserves many thanks for taking care of the very complex organisational and technical arrangements of the event. this unique conference in such trying times has showcased the considerable importance animal breeding has for the sustainable development of livestock farming and thereby for agriculture across the globe,” said the dgfz. the international congress delivered valuable information, incentives and insights thanks to the distinguished speakers and the very dedicated audience. the international line-up of the event, in particular, highlighted the multi-faceted importance of small ruminants across the world. those involved agreed that work on open questions and the food for thought provided should be continued and that the dialogue should be upheld in order to make headway regarding the burning issues in the field of breeding, animal welfare and environmental affairs together with a view to the practical implementation. animal breeding and genetics what will sheep and goat breeding look like in the future? oral presentations international congress on sheep and goats genetic resources (2021) s1 abstractbook 7 central information processing in german sheep and goat breeding jens wilkens vereinigte informationssysteme tierhaltung w.v., heinrich-schröder-weg 1, 27283 verden / aller, germany contact: jens.wilkens@vit.de abstract: german sheep and goat breeding has a distinctive federal structure. the breed societies work closely together under the umbrella of vdl/bdz. information processing with serv.it ovicap (https://service.vit.de/ovicap/) in the field of herd bookkeeping was established with vit (vereinigte informationssysteme tierhaltung) as the vdl service partner. serv.it ovicap has been an integral part of breeding work in germany for 10 years now. by working together in a central information system, synergy effects can be used, which contribute to cost reduction and optimization of functionality and data quality. the breed societies in the association are effectively enabled to functionally administer information on the identification of sheep and goats as well as their pedigrees and breeding relevant characteristics. this consolidated information base (4.884.539 animals in total, 1.104.314 animals registered in herdbook, 61.705 farmer adresses) is the basis for nationwide population analyses and breeding value estimations. use by the breeding farms is directly integrated into serv.it ovicap. 70% of active farms (2.900 breeders) use serv.it ovicap to retrieve information about animals and make recordings (e.g. lambing – e.g. 64.774 lambings in 2019). a breeding planner can be used by the breeding farmers to estimate inbreeding degrees in mating decisions. the nationwide pedigree is essential for calculating the inbreeding degrees. for breeders of all sheep and goat breeds, serv.it ovicap is the information medium for their own breeding work. working together with and on serv.it ovicap is an essential contribution to the maintenance and further development of sheep and goat breeding. with regard to future developments, a good, expandable basis has been created i.a. in the area of advancing digitalization and implementation of genomic methods of animal breeding. mailto:jens.wilkens@vit.de https://service.vit.de/ovicap/ animal breeding and genetics what will sheep and goat breeding look like in the future? oral presentations genetic resources (2021) s1 international congress on sheep and goats 8 abstract book genetic evaluation for sheep in germany dr. wolfgang ruten and dr. dierck segelke it-solutions for animal production, verden, germany contact: heinrich-schröder-weg 1, 27283 verden, wolfgang.ruten@vit.de abstract: the vdl (vereinigung deutscher landesschafzuchtverbände e. v.), as the umbrella organization of the german state sheep breeding associations, is the client for the sheep breeding value assessment. a consolidation of the pedigree and performance data during the development of the common herd book system at vit (serv.it ovicap) allowed the establishment and introduction of a nationwide breeding value estimation for sheep. since 2014 routine evaluation is executed once a year in july for 25 different sheep breeds. all known pedigree and performance data from the field test (fertility, fattening and slaughter performance characteristics, exterior assessment for herd book recording or licensing as well as maternity or mammalian performance) are taken into account for the breeding value estimation. table 1 shows the number of animals across all breeds in performance and in pedigree data: * repeated performance a total of 772.772 sheep received breeding values in the run 2020/07. various statistical models are used within the individual trait complexes: a blup single-trait repeatability model is used for fertility, and blup multi-trait models are used for all other feature complexes. the breeding values are expressed as relative breeding values with an average of 100 points and a genetic standard deviation of 20 points. with the introduction and establishment of the nationwide breeding value estimation for sheep, breeders and also the regional breeding associations have access to an innovative tool for breeding selection. furthermore, the objective breeding values are comparable across the regions. by using this source of information consistently, the breeding value estimation can and will contribute to productivity increase in sheep breeding. mailto:wolfgang.ruten@vit.de animal breeding and genetics what will sheep and goat breeding look like in the future? oral presentations international congress on sheep and goats genetic resources (2021) s1 abstractbook 9 mobps for sheep and goats – an innovative tool to design and optimize breeding programs henner simianer, lisa büttgen and torsten pook animal breeding and genetics group, center for integrated breeding research, university of goettingen, germany contact: henner simianer, animal breeding and genetics group, center for integrated breeding research, albrecht-thaer-weg 3, 37075 goettingen, germany, hsimian@gwdg.de abstract: designing efficient breeding programs with the objective of generating maximum genetic progress in a cost-efficient way while maintaining genetic diversity, sustainability and animal welfare is a major challenge. we have developed an r-package termed modular breeding program simulator (mobps; pook et al. 2020) allowing a flexible representation and a powerful simulation of current-time breeding programs. the basic conceptual assumption is that any breeding program can be represented by a network of nodes (typically cohorts of animals) and edges (typically breeding activities like selection or reproduction). a web-based graphical user interface (www.mobps.de) offers an intuitive approach to design breeding programs in a most flexible way. once such a breeding program is entered, it can be simulated with the r-package, providing predictions for the genetic trend in all traits as well as the development of inbreeding rate. by comparing alternative designs the tool can be used to optimize the breeding program. we will illustrate the usefulness and the functionality of the mobps package with various applications from sheep and goat breeding. it will also be demonstrated to what extent innovative technologies like gene-editing could potentially affect practical breeding in small ruminants. templates will be provided for major breeding program designs in sheep and goat breeding, including sensible pre-settings of genetic parameters and links to widely used resources, like the major species-specific snp genotyping arrays available. both the r-package and the web interface are free to use and provide a platform for the improvement of sheep and goat breeding programs worldwide. references: pook, t., m. schlather, and h. simianer. 2020. mobps modular breeding program simulator. g3; genes|genomes|genetics 10 (6): 1915–18. https://doi.org/10.1534/g3.120.401193 mailto:hsimian@gwdg.de http://www.mobps.de/ animal breeding and genetics what will sheep and goat breeding look like in the future? oral presentations genetic resources (2021) s1 international congress on sheep and goats 10 abstract book early data from a community-based breeding program indicates opportunity for genetic gain in black bengal goats owned by underprivileged rural women in bihar state of india chanda nimbkar1, tinni sawhney2, rubab azam2, rumani chakraborty2 and peter amer3 1 nimbkar agricultural research institute (nari), phaltan, india 2 aga khan foundation (akf), new delhi, india 3 abacusbio ltd., dunedin, new zealand contact: nari animal husbandry division, phaltan-lonand road, phaltan 415 523, dist. satara, maharashtra, india, chanda.nimbkar@gmail.com abstract: supported by the gates foundation, akf implemented a programme in muzaffarpur district of bihar state, india to transform the lives of rural poor through improving local black bengal breed goat production. project mesha strives to promote better goat management practices through trained women community workers. with advisory support from nari and abacusbio, a pilot breeding programme was established in four villages (~2000 goats) in 2018 for withinbreed genetic improvement by selection of male kids for breeding. based on discussions with rearers, twinning (but not litter sizes larger than twins) and early fast growth (weight at 100 days of age) were decided as selection criteria. data from october 2018 to march 2020 was analysed using the echidna software. the average daily gains (adg) of kids that were weighed >three times up to the age of 120 days, were estimated with a regression of weight on age for each kid. fixed models were fitted to analyse kid weights (59, 119 and 38 records for birth, 3 and 6 months’ weights respectively) and adg (199 records). there were 11% singles, 46% twins, 37% triplets and 6% quadruplets. birth type was significant for birth, 3 month weights and adg. sex was significant for birth weight. village was significant for 3 and 6 months’ weights and adg. the least squares mean (lsm) birth, 3 and 6 months’ weights were 1.7±0.1 kg, 5.9±0.2 kg and 11.0±0.5 kg respectively while lsm adg was 48.9±1.9 g. substantial variation in analysed traits indicates the opportunity to achieve genetic gain. mailto:chanda.nimbkar@gmail.com animal breeding and genetics what will sheep and goat breeding look like in the future? oral presentations international congress on sheep and goats genetic resources (2021) s1 abstractbook 11 development of a sustainable breeding programme in german sheep farming by using multi-live cover (munasch) nina ossowski1,2, jens wilkens3, christian mendel4, ingolf ruß5, henrik wagner², axel wehrend2 and georg thaller1 1 institute of animal breeding and husbandry christian-albrechts-university, kiel, germany 2 clinic for obstetrics, gynaecology and andrology of large and small animals with veterinary ambulance – justus-liebig-university, giessen, germany 3 united livestock information systems, verden/aller, germany 4 institute of animal breeding, bavarian state research center for agriculture, poing-grub 5tierzuchtforschung e.v., münchen, germany contact: nina ossowski, institute of animal breeding and husbandry, christian-albrechts-university kiel, nossowski@tierzucht.uni-kiel.de abstract: the project munasch (‘multi-natursprung schaf‘) launched to address scientific questions when using more than three rams contemporarily within mating seasons. eartag-tissue-samples from one complete lambing season were collected from 17 herdbook-breeders located in bavaria, baden-wuerttemberg and thuringia including the breeds merinoland-, merinolangwoll-, rhoenand dorperschaf. the results of paternity testing based on 19 microsatellite-markers were registered in the central data base serv.it ovicap (german herdbook-system for sheep). according to the mating register and the corresponding lambing list the most likely father among the potential candidates was assigned. major aims of this innovative project were: (1) determining the preference for particular rams, (2) effects on fertility at herd level, (3) performance of breeding animals and lambs, (4) impact on genetic variability, (5) improvement of breeding value estimation, (6) reduction of birth losses due to shortened lambing seasons. first analysis of 150 merinolandschaf ewes bred to three rams mated for four weeks verified the hypothesis of a ram-dominance. half of the lambs born stemmed from ram a, whereas only 40% descended from ram b and 10% from ram c. regarding the multiples, by half of the siblings had more than one father within a litter. currently, the maker-set gets reduced by regarding allele frequency, polymorphic information content, expected and observed heterozygosity to ensure financial efficiency. the shepherds’ feedback was unexceptional positive. with the help of this project, the profitability of herdbook-breeders should increase in spite of the tensed situation and help to withstand the current difficulties. mailto:nossowski@tierzucht.uni-kiel.de animal breeding and genetics what will sheep and goat breeding look like in the future? oral presentations genetic resources (2021) s1 international congress on sheep and goats 12 abstract book genomics to breed sheep resistant to footrot monia lara budnik, julia storms, anna wirth and ottmar distl university of veterinary medicine hannover, institute of animal breeding & genetics, hannover, germany contact: monia.budnik@tiho-hannover.de, ottmar.distl@tiho-hannover.de abstract: ovine footrot is a complex disease caused by dichelobacter nodosus and clinically characterized by interdigital dermatitis and under-running footrot. the objectives of our research project are to elucidate genomics of resistence to footrot within and across sheep breeds. in a large number of flocks in germany, we recorded prevalences of footrot using clinical data and the load of benign and virulent strains of d. nodosus. on farm data recording in more than 200 flocks comprising >30,000 sheep was done using a mobile electronic hand-held system for individual animal ear tags and data input. we employed qrt-pcr to differentiate benign and virulent d. nodosus strains for classification of the footrot status of flocks. based on these data, we were able to distinguish resistant, tolerant and susceptible animals. genotyping was done for approximately 4000 sheep on ovine snp50 and ovine infinium hd snp beadchips in merinos, leine, suffolk and east friesian for 250-650 animals each and across several breeds for at least 50-100 animals including bentheim, dorper, grey heath, forest sheep, romney marsh, texel, white p/h heath and pomeranian coarsewool. we found heritabilities for resistance to footrot using mixed models with genomic relationship matrices at h2=0.4-0.7 for the different breeds. genome wide association studies with mixed models showed significant associated regions within breeds. whole genome sequencing data from 120 individuals were employed to filter for highly associated variants. on whole genome sequences, imputed genotype data allowed us to validate highly associated regions and variants within and across breeds. mailto:monia.budnik@tiho-hannover.de mailto:ottmar.distl@tiho-hannover.de animal breeding and genetics what will sheep and goat breeding look like in the future? oral presentations international congress on sheep and goats genetic resources (2021) s1 abstractbook 13 high-resolution analysis of a qtl influencing sheep resistance to gastrointestinal nematode infection through whole-genome resequencing of a segregating trio beatriz gutiérrez-gil1, praveen k. chitneedi1,2, c. esteban-blanco1 and juan-josé arranz1 1 universidad de león, león, spain 2 leibniz institute for farm animal biology, dummerstorf, germany contact: dpto. animal production, faculty of veterinary sciences, campus de vegazana, universidad de león, 24071, león, spain, abstract: gastrointestinal nematode (gin) infections are one of the major health issues for grazing sheep populations. in the present study, we have used the imputed 50k-chip to high density (hd) chip (600k) genotypes obtained in a commercial population of 532 churra adult ewes to refine qtl previously identified in this population for two indicator traits of gin resistance (atlija et al. 2016). for the most promising qtl region previously reported on chromosome 6 (oar6) for faecal egg count (lfec), we selected a segregating trio including the qq sire and two daughters with extremely divergent phenotypes for the fec trait in concordance with their qtl inferred genotypes, qq and qq. the trio dna samples were subjected to whole genome resequencing (wgr) using the paired-end illumina technology. after applying a variant calling bioinformatic workflow, the variants identified within the refined confidence interval of the lfec oar6 qtl were filtered for concordance with the qtl segregation pattern. based on a variant functional annotation analysis, we identified a list of variants that based on their biological impact and their harboring gene could be considered as potential candidate causal variants of the qtl under study. future research efforts should confirm the role of the candidate genes and mutations highlighted by this study. this study represents a new forward step towards increasing our knowledge on the genetic basis of genetic resistance to gastrointestinal nematodes in sheep and goat commercial populations. references: atlija, m., arranz, j.j., martinez-valladares, m., gutiérrez-gil, b. (2016). detection and replication of qtl underlying resistance to gastrointestinal nematodes in adult sheep using the ovine 50k snp array. genet sel evol. 48, 4. doi: 10.1186/s12711-0160182-4 mailto:beatriz.gutierrez@unileon.es animal breeding and genetics what will sheep and goat breeding look like in the future? oral presentations genetic resources (2021) s1 international congress on sheep and goats 14 abstract book no doubt anymore: a simple genetic test to facilitate breeding for polledness in goats r. simon1, c. drögemüller² and g. lühken1 1 institute of animal breeding and genetics, justus liebig university, giessen 35390, germany ² institute of genetics, vetsuisse faculty, university of bern, bern 3012, switzerland contact: rebecca simon, institute of animal breeding and genetics, justus liebig university giessen, giessen 35390, germany, rebecca.simon@agrar.uni-giessen.de abstract: breeding for polledness in goats is more complicated than in cattle as polledness in goats is associated with disorders in sexual development. the incidence is also known as polled intersex syndrome. it is characterized by homozygous polled and genetically female (xx) individuals being infertile due to phenotypically diverse intersexuality. external genitals in affected intersexual goats range from true female to apparently male and all possible intermediate stages. this makes the identification of such cases in practice difficult and points the breeding on polledness in goats into its limits. previously, a large deletion was postulated as the causal variant for pis (pailhoux et al. 2001). however, so far the development of genetic testing for the polled genotypes was not successful. resequencing of the whole genomes of two genetically female (xx) goats, one pis-affected and a horned control revealed an additional complex structural variant, which is combined with the already published variant (simon et al. 2020). our data allowed the development of a diagnostic pcr to proof both, the individual horn genotype and the genetic sex of goats simultaneously, for the first time. the genetic test was validated with more than 1000 goats of different breeds confirming that all analyzed pissuspicious goats were homozygous polled and indeed female (xx). thereby, the identification of phenotypically questionable intersexes is possible now. the new genetic testing offers a tool to facilitate breeding for polledness in goats. references: simon, r., lischer, h.e.l., pieńkowska-schelling, a., keller, i., häfliger, i.m., letko, a., schelling, c., lühken, g., drögemüller, c. (2020) new genomic features of the polled intersex syndrome variant in goats unraveled by long-read whole-genome sequencing. anim genet. 2020 feb 14. doi: 10.1111/age.12918. [epub ahead of print]. pailhoux, e., vigier, b., chaffaux, s., servel, n., taourit, s., furet, j.p., fellous, m., grosclaude, f., cribiu, e.p., cotinot, c., vaiman, d. (2001) a 11.7-kb deletion triggers intersexuality and polledness in goats. nat genet. 29(4), 453-458. doi:10.1038/ng769. mailto:rebecca.simon@agrar.uni-giessen.de animal breeding and genetics what will sheep and goat breeding look like in the future? oral presentations international congress on sheep and goats genetic resources (2021) s1 abstractbook 15 analysis of candidate genes for growth and milk performance traits in the egyptian barki sheep ibrahim abousoliman1,2, henry reyer1, michael oster1, eduard muráni1, mosaad mourad3, mohamed abdel-salam rashed3, ismail mohamed2 and klaus wimmers1,4 1 leibniz institute for farm animal biology (fbn), dummerstorf, germany 2 desert research center, cairo, egypt 3 ain shams university, cairo, egypt 4 university of rostock, rostock, germany contact: abstract: indigenous sheep breeds in egypt, including ossimi, rahmani, and barki, are of great importance for meat production. the barki sheep is well adapted to desert hard condition like high temperature and shortage of food and water. animal growth traits like birth weight (bw), weaning weight (ww) and average daily gain (adg) are important traits due to their impacts on the viability of livestock raised for meat production. milk production has a very important role in ensuring adequate supplies of new-born lambs and ultimately reducing the mortality rate due to starvation. in this study, data from 251 barki ewes and lambs were used to investigate the association between genotypes at eight genes (lep, igf1, dgat1, stat5a, prl, csn1s2, ghr, and ghrhr) and production phenotypes. for each gene, one representative single nucleotide polymorphism (snp) located in the coding region was selected for genotyping using kompetitive allele specific pcr. lep (rs420693815) was significantly associated with weaning weight and average daily gain (p < 0.1). homozygous carriers of the tt genotype had a lower weaning weight and a lower average daily gain compared to the other genotypes. in ewes, significant effects on milk yield and composition were obtained for lep (rs420693815) (milk yield, fat %), stat5a (rs161082816) (lactose %), prl (rs422713690) (milk yield), and ghrhr (rs414991449) (protein%, total solids %), while igf1, dgat1, csn1s2, and ghr genes showed no significant associations. the results indicated that lep, stat5a, prl, and ghrhr genes might be considered as interesting candidates to improve growth and milk performance in barki sheep. references: abousoliman, i., reyer, h., oster, m., muráni, e., mourad, m., abdel-salam rashed, m., mohamed, i., wimmers, k. (2020). analysis of candidate genes for growth and milk performance traits in the egyptian barki sheep. animals 10, 197. sallam, a.m., galal, s., rashed, m.a., alsheikh, s.m. (2012). genetic diversity in barki sheep breed in its native tract in egypt. egyp. j. anim. prod. 49, 19–28. mailto:abou-soliman@fbn-dummerstorf.de animal breeding and genetics what will sheep and goat breeding look like in the future? oral presentations genetic resources (2021) s1 international congress on sheep and goats 16 abstract book assisted reproductive techniques in sheep and goats – chances and limits of alternative ways in breeding johanna maria meilwes and martin ganter clinic for swine, small ruminants and forensic medicine and ambulatory service, university of veterinary medicine hannover, foundation; hannover, germany contact: johanna maria meilwes, clinic for swine, small ruminants and forensic medicine and ambulatory service, university of veterinary medicine hannover, foundation, bischofsholer damm 15, 30173 hannover abstract: aim of this contribution is to give an overview about the opportunities of assisted reproductive techniques in small ruminants and their use and situation in germany. unlike in other countries, artificial reproductive medicine like artificial insemination (ai) and embryo transfer (et) in sheep and goats is not common in germany. there are no official data about the use of reproductive techniques in these species. compared to cattle, requirements for artificial techniques are much more complex. two breeding centers for semen collecting in small ruminants are registered in germany. in addition to research activities, the federal research institute for animal health, the friedrich-löffler-institute (fli) in mariensee is keeping genetic reserves of different sheep and goat breeds as “german gene bank for farm animals”. at the insemination center of clinic for small ruminants, university of veterinary medicine hannover semen of different sheep breeds is collected, stored and available. ai is performed but due to a low demand by sheep breeders rarely carried out. to expand and offer opportunities that could improve sheep breeding, a national and international exchange of knowledge, experience and not least genetic material is necessary. there is an international request for german sheep genetic resources, for example east frisian milk sheep, which could give the chance to an international exchange. ai and et could be a chance to improve the genetic diversity and breeding values. flock health status could be improved by genetic and health tested animals. many breeds are defined as rare breeds. genetic reserves can ensure future preservation mailto:johanna.maria.meilwes@tiho-hannover.de animal breeding and genetics what will sheep and goat breeding look like in the future? oral presentations international congress on sheep and goats genetic resources (2021) s1 abstractbook 17 the use of homologous seminal plasma before and during deep vaginal timed artificial insemination in sheep monitored by thermography and electrical impedance of vaginal mucous. beste cil1, koray tekin1, kumsal duzgun1 and calogero stelletta1,2 1 department of animal reproduction and artificial insemination, faculty of veterinary medicine, ankara university, turkey 2 department of animal medicine, production and health, university of padova, italy contact: beste cil, department of animal reproduction and artificial insemination, faculty of veterinary medicine, ankara university, turkey, +90 555 7318338, email: cil.beste@gmail.com abstract: artificial insemination in sheep is an intriguing technique because the species-specific cervix conformation does not allow intrauterine deposit and consequently, the inseminating doses are not yet optimized. the variability of the ovulation time following the estrous synchronization remains the main constraint to increase the accuracy and precision of the technique regarding the success rate. the aim of this work was to apply a timed artificial insemination (tai) protocol considering a deep vaginal deposition of inseminating doses, testing the effects of homologous seminal plasma (sp) and to simulate the application of “on-field” monitoring system based on thermography (irt) and vaginal electrical impedance (vei). a total of 109 young ewes were synchronized with 9-11 days of progestogen releasing device (p) and 300 iu of ecg at p removal in non-breeding season. ewes were divided into three groups: 1st as control (c), deep vaginally inseminated with 3.2 ml of chilled semen dosed as 100x106 motile sperm/ml, 2nd treated with seminal plasma (0.4 ml sp) before tai (sp24h) and 3rd treated with sp at tai (sptai). irt and vei were applied at the time of p removal and the tai. lamb rates were 33.33% (10/31), 32.36% (16/48) and 36.67% (11/30) for c, sp24h and sptai respectively. the monitoring system showed a significant (p<0.05) drop of vulvar temperature and vaginal electrical impedance in pregnant sp24h. irt and vei give the possibility to identify the timing of insemination and the animals answering to the hormonal synchronization. sp seems to synchronize the fertilization time and influence vaginal and thermal patterns. references: cil, b., and akcay, e. (2017). the importance of seminal plasma proteins in bulls in terms of cryopreservation and fertility. lalahan hayvancilik arastirma enstitusu dergisi, 57, 118-126. stelletta, c., tekin, k., tirpan, m. b., alemdar, h., cil, b., stelletta, f. o., olgac, k. t., inanc, m. e., daskin, a. (2017). vulvar thermal pattern following synchronization of estrus is linked to fertility after timed artificial insemination in goat. theriogenology, 103, 137-142. doi: 10.1016/j.theriogenology.2017.07.038 juyena, n. s., and stelletta, c. (2012). seminal plasma: an essential attribute to spermatozoa. journal of andrology, 33, 536551. doi: 10.2164/jandrol.110.012583 mailto:cil.beste@gmail.com animal breeding and genetics what will sheep and goat breeding look like in the future? poster presentations genetic resources (2021) s1 international congress on sheep and goats 18 abstract book blood levels of progesterone and cortisol in murciano granadina goats after artificial insemination begoña peinado, laura almela, ángel poto and sonia galián murcian institute for agricultural and food research and development (imida), la alberca-murcia, spain contact: begoña peinado, murcian institute for agricultural and food research and development (imida), la alberca-murcia, spain, email: begona.peinado@carm.es, telephone: +34 968 366756 abstract: cortisol is used to assess stress. early pregnancy detection by blood progesterone determination is important in reproductive programs. in a group of 11 adult murciano granadina goats wich underwent artificial insemination, a study has been carried out to determine blood levels of progesterone and cortisol as possible biomarkers for early prediction of gestation diagnosis. three measurements were taken to determine progesterone levels and two for cortisol. the frecuency of sampling was 21 days, with one measurement coinciding with the day of the artificial insemination. an automatic immunofluorescence analyser was used to measure both parameters. statgraphics centurion program was used for the statistical analysis. among the results obtained, we highlight that for pregnant females (n=4) the average progesterone values obtained, on the day of artificial insemination and 21 days later, were always higher compared to females that were not pregnant (n=7): 12.63 ± 1.59 ng / ml and 15.26 ± 2.3 ng /ml respectively, and compared to 8.63 ± 1.66 ng /ml and 10.74 ± 2.55 ng /ml respectively. similar results were obtained for the average cortisol values, obtaining 58.74 ± 31.06 nmol / l on the day of the artificial insemination in the females that became pregnant, compared to 90.08 ± 44.40 nmol /l in the females that did not become pregnant. these results indicate the usefulness of measuring blood progesterone and cortisol values as an early indicator of expected fertility after artificial insemination. animal breeding and genetics what will sheep and goat breeding look like in the future? poster presentations international congress on sheep and goats genetic resources (2021) s1 abstractbook 19 transcript abundance of candidate genes in different body tissues of egyptian barki sheep as an indicator of growth performance nasser ghanem1, mohamed zayed2, mohamed radwan1, mona mohmady2 and mohamed shehata2 1 animal production department, faculty of agriculture, cairo university, giza, egypt 2 animal and poultry division, desert research center, cairo, egypt contact: faculty of agriculture, cairo university, el-gamaa street, 12613 giza, egypt, abstract: sheep is considered one of the main animal genetic resources in egypt as contributing in meat production while surviving harsh desert conditions and consuming low quality of forage. the aim of this study was to link the expression profile of selected candidate genes with growth performance of egyptian barki sheep. barki sheep lambs (n=26) were kept and fed individually after weaning. growth performance traits as well as carcass characteristics were recorded after slaughtering. samples from different body tissues (muscle, liver, fat) were taken and stored in rna latter until rna isolation. real-time pcr was used to profile selected candidate genes (rpl7, ctp1, fabp4, adipoq and capn3) and gapdh was used as a housekeeping gene. our data indicated that, heavier final body weight in the fast growing sheep lambs (52.4 kg) than both medium (41.4 kg) and slow growing animals (31.7 kg) 6 months after weaning. genes involved in protein biosynthesis (rpl7), fatty acid oxidation (cpt1) and lipolysis (fabp4) were up-regulated in fast and medium growing animals in all studied tissues. while, gene regulating lipogenesis (adipoq) was expressed similarly in fat and liver tissues but increased its expression in muscle of fast and medium growing sheep. expression of capn3 was increased in fast and medium growing compared to slow growing animals. this study clearly indicated the transcriptional profile of cpt1, fabp4 rpl7 and capn3 is linked with growth performance of sheep lambs, providing an evidence for the importance of these genes. mailto:nassergo@agr.cu.edu.eg animal breeding and genetics what will sheep and goat breeding look like in the future? poster presentations genetic resources (2021) s1 international congress on sheep and goats 20 abstract book usefulness of the genetic variant tmem154 e35k for breeding against maedi visna susceptibility in the german sheep population gesine lühken institute of animal breeding and genetics, justus liebig university, giessen, germany contact: gesine lühken, institute of animal breeding and genetics, justus liebig university giessen, giessen 35390, germany, gesine.luehken@agrar.uni-giessen.de abstract: maedi visna, a disease caused by small ruminant lentiviruses, is present in sheep populations of many countries. the disease ends always fatal and cannot be prevented by vaccination. developed eradication programs including testing and culling may be helpful but are neither cost-effective nor sustainable. breeding against maedi visna susceptibility could solve or at least reduce these problems. at first in north american sheep populations, an amino acid substitution (e>k) at position 35 of the transmembrane protein 154 (tmem154) was observed to be associated with susceptibility to maedi visna. in our study, we tested this association in 21 maedi visna-affected sheep flocks with different breed background and from different regions in germany. for this purpose, sheep aged three years or older were tested serologically for maedi visna status by elisa and genotyped for e35k. secondly, we determined the e35k allele and genotype frequencies in unrelated sheep of 12 breeds kept in germany in order to estimate their genetic maedi visna susceptibility status. in most of the analyzed maedi visna-affected flocks, the 35e allele was significantly associated with seropositivity. however, an association was missing in two merinoland sheep flocks, therefore other, maybe breed-specific factors may additionally influence maedi visna antibody titers. frequencies of the susceptible 35e allele ranged among the 12 analyzed breeds from 2 % (german grey heath) to 93 % (cameroonian sheep), reflecting their genetic susceptibility status and different starting situations for potential breeding programs. mailto:gesine.luehken@agrar.uni-giessen.de animal breeding and genetics what will sheep and goat breeding look like in the future? poster presentations international congress on sheep and goats genetic resources (2021) s1 abstractbook 21 antibody titer after vaccination as potential phenotype for breeding against footrot in sheep: first results annabell amend, o. o. adeniyi and gesine lühken institute of animal breeding and genetics, justus liebig university, giessen, germany contact: gesine lühken, institute of animal breeding and genetics, justus liebig university giessen, giessen 35390, germany, gesine.luehken@agrar.uni-giessen.de abstract: footrot is a highly contagious disease causing lameness and occurs worldwide mainly in sheep and goats. the causal agent is the bacterium dichelobacter nodosus. in some few countries, selection against footrot susceptibility based on footrot scores is in progress or already established. however, footrot scores are difficult to collect as well as to interpret, and heavily influenced by weather and ground conditions. therefore, the aim of the current project was to establish and validate another method for phenotyping footrot susceptibility, which is the reaction of the animal’s immune response after contact with antigens of the pathogen. an enzyme-linked immunosorbent assay (elisa) including d. nodosus antigens extracted from a commercial vaccine was developed. with this elisa, antibody titers were measured in blood samples collected at several time points after vaccination from female merinoland and rhoen sheep. the animals were vaccinated with 5-8 months of age and a second time 4 weeks later. antibody titer data from 202 merinoland sheep, belonging to 7 half-sib groups, were included in the presented preliminary analyses. in all sheep, antibody titers increased after vaccination to a peak at week 6, followed by a slower decrease. between some half-sib groups, antibody titers were significantly different for several time points after vaccination. estimated heritabilities for antibody titers, e.g. at 4 months after vaccination (animal model: 0.22; sire model: 0.42) are promising for a potential use of this phenotype for breeding towards lower footrot susceptibility and/or better effect of vaccination. mailto:gesine.luehken@agrar.uni-giessen.de animal breeding and genetics what will sheep and goat breeding look like in the future? poster presentations genetic resources (2021) s1 international congress on sheep and goats 22 abstract book efficiency of estrus synchronization protocols and artificial insemination technologies in abergelle goat at station and on farm conditions of waghemira zone, ethiopia bekahegn wondim1,2, mengistie taye1,3, kefyalew alemayehu1, aynalem haile4 and mourad rekik5 1 animal production and technology department, college of agriculture and environmental science, bahir dar university, po. box 5501, bahir dar, ethiopia 2 sekota dryland agriculture research center (sdarc), po. box 62, sekota, ethiopia 3 biotechnology research institute, bahir dar university, pobox 79, bahir dar, ethiopia 4 international center for agricultural research in dry areas (icarda), po.box 5689, addis ababa, ethiopia 5 international center for agricultural research in dry areas (icarda), po.box 950764, aman, jordan contact: bewondim23@gmail.com abstract reproductive biotechnological tools are important to suit kidding time with better forage availability and accelerate improved genetics in the goat breeding programs. the study was conducted during 2019/2020 to investigate the effect of different estrus synchronization protocols on estrus response and conception rate of abergelle goats following fixed time artificial insemination. three estrus synchronization protocols: 1) pregnant mare serum gonadatropins with enzaprost® as separate injection, 2) single injection of prostaglandin, and 3) double injection of prostaglandin were evaluated during the experiment. a total of 278 does for the treatment group and 57 does were used as control group. for semen collection, 23 bucks were used. for the first treatment groups, progesterone impregnated vaginal sponge was inserted at day1 and stayed for 11 days inside, 48 hour before sponge removal, 1ml (600iu) gonadatropins followed by 1ml enzaprost® were administered. for the second treatment groups, 1ml of enzaprost® was administered at day 1. for the third treatment groups, 1ml of enzaprost® was administered at day 1 followed by the second 1ml injection of enzaprost® at day 11. insemination was performed after 48 hours of estrus follow up. the control groups were allowed to mate as usual. the study revealed, that overall estrus response of 87.6%, 61.4% and 53% were investigated from first, second and last treatments, respectively. relatively higher proportion of the conception rate (p<0.05) resulted from double injection of prostaglandin f2α (78.8%). it was concluded that double injection of prostaglandin f2α was selected as the best protocol for its efficiency. mailto:bewondim23@gmail.com animal breeding and genetics what will sheep and goat breeding look like in the future? poster presentations international congress on sheep and goats genetic resources (2021) s1 abstractbook 23 wool fiber density and other characteristics from birth to weaning in junin lambs e.c. quispe1,2, w.h. grabiel3, j. aliaga1, m.d. quispe4 and r. quispe3 1 animal production department, universidad nacional agraria la molina, lima, perú, jaliaga@lamolina.edu.pe 2 laboratory of animal fibers, natural fiber´s tech sac. lima, perú, edgarquispe62@gmail.com 3 animal production department, universidad nacional del centro del perú, huancayo, perú, rolandoquispe@outlook.com 4 engineering department, maxcorp technologies sac. lima, perú, maxdavid22@gmail.com contact: edgarquispe62@gmail.com abstract: this research had the objectives to evaluate the body weight and some characteristics of the skin and wool fibers of junín lambs monthly from birth to weaning (120 days) and to analyze relationships between them in order to assess the potential of fiber and duct density as a new selection criterion for improving wool quality. data on live weight, skin surface, monthly fiber growth (mfg), fiber density (fibden), ducts density (ductden), fibden/initially tattooed area (ita), ductden/ita, belonging to 24 junin lambs, were considered. additionally, the percentage of ducts with one, two, three, or four fibers (pd1f, pd2f, pd3f or pd4f, respectively) and average fiber diameter were considered also. live weight and surface skin were recorded with weight scale and graduated ruler, respectively. fibden and ductden were objectively determined using the device and methodology called fiber den. afd was measured with fiber ec instrument. it was found that the fibden and ductden increase monthly almost in proportion to the increase of the skin surface from birth to 120 days, with averages of 34.5 and 31.01 fibers and ducts per mm2. the direct relationship between afd and the number of cuts (shearing) of the fiber, and fibden with mfg into the period of study were found. besides, the indirect relationship between afd and fibden and ductden was also found, with sufficient statistical evidence. it is concluded that fibden and ductden could be considered as novel selection criteria for simultaneous improvement of wool quality and quantity because fibden and ductden have a relation with quality and quantity characteristics. mailto:edgarquispe62@gmail.com mailto:maxdavid22@gmail.com mailto:edgarquispe62@gmail.com animal breeding and genetics what will sheep and goat breeding look like in the future? poster presentations genetic resources (2021) s1 international congress on sheep and goats 24 abstract book relationship between seminal vesicles echogenicity and semen quality in rams of endangered venetian sheep breeds calogero stelletta department of animal medicine, production and health, university of padova contact: prof. calogero stelletta, department of animal medicine, production and health, university of padova, viale dell’universita 16, 35020, legnaro (pd), italy, email: calogero.stelletta@unipd.it abstract: seminal vesicles are the main producers of seminal plasma (sp) in rams. their secretions can greatly influence the quality of sperm suspended on them. sheep endangered breeds (seb) need more attention on semen treatment after collection because the genetic materials should be maintained efficiently to enhance the biodiversity sustainability. aim of this work was to investigate the relationship among testosteronemia under gnrh challenge test (t), echogenicity of seminal vesicles (low, medium, high) and testicles (vencato et al., 2014), sp composition (p, ca, mg, pt, alp, ldh, cholesterol, triglycerides) and spermatic post-thawed kinetic parameters (total motility, progressive motility, vap, vsl, vcl, alh, bcf, str, lin) in 4 venetian seb (alpagota, brogna, foza, lamon). a total of 22 rams (n=5, brogna, n=7 lamon, n=5 foza e n=5 alpagota), 1-5 years old, were used as breeders submitted to a preservation program for extinction threatened breeds and collected two times with electroejaculator. data analysis showed significant relationships among scrotal circumference, t and semen kinetic parameters. negative correlations indices (p<0.05) resulted between testicular parenchymal echogenicity and semen volume as well as among seminal vesicles echogenicity, semen volume (-0.76) and seminal plasma total proteins (-0.82). moreover, lower levels of calcium (2.15 ± 0.3 mg/dl) and cholesterol (28.83 ± 3.37 mg/dl) concentrations than reference levels (juyena and stelletta, 2012) were revealed in all breeds. clinical biochemistry can be considered routinely to check the quality of the sp helping the eventual make-up of the extenders for chilled or frozen inseminating doses. references: juyena, n.s., stelletta, c. (2012) seminal plasma: an essential attribute to spermatozoa. journal of andrology, 33, 536-551. doi: 10.2164/jandrol.110.012583 vencato, j., romagnoli, s., stelletta, c. (2014) trans-scrotal ultrasonography and testicular fine-needle aspiration cytology in the evaluation of ram sperm production. small ruminant research, 120, 112-115. doi: 10.1016/j.smallrumres.2014.05.005 mailto:calogero.stelletta@unipd.it animal breeding and genetics what will sheep and goat breeding look like in the future? poster presentations international congress on sheep and goats genetic resources (2021) s1 abstractbook 25 echotexture characterization of accessory glands in buck calogero stelletta1,2 and koray tekin2 1 department of animal medicine, production and health, university of padova, italy 2 department of animal reproduction and artificial insemination, faculty of veterinary medicine, ankara university, turkey contact: prof. calogero stelletta, department of animal medicine, production and health, university of padova, viale dell’universita 16, 35020, legnaro (pd), italy, email: calogero.stelletta@unipd.it abstract: accessory glands (ags) are the producers of seminal plasma (sp) which have strong influence on the quality of the ejaculates and the suspension of spermatozoa in it. ags can be classified in epididymis, deferential ampullas, seminal vesicles, prostate and bulbo-urethral glands. each ag is able to give specific compounds that are extremely important for the functionality of sperm within the female reproductive tract, other than the regulation of the resistance to chilling or freezing procedures. ags high resolute ultrasonography (us -10 mhz) can be considered as a monitoring tool to optimize the accuracy and precision of small ruminant male evaluation, thanks to the possibility of pixel grey intensity (pgi) calculation of regions of interest (rois) of us images. the aim of this work was to evaluate the variability of difference among the pgi accessory glands us images in buck. twenty adult and fertile bucks were used to measure the pgi of accessory glands by using an image analysis software (image j). epididymal tails presented the lower pgi (62.13 ± 20.67) with highest variability due to the presence of anechoic areas within the roi. deferential ampullas, seminal vesicles and bulbourethral glands showed different echotextures with 141.2 ± 14.4, 141.11 ± 19.26 and 171.7 ± 12.68 pgi respectively. echotexture index of accessory glands can give useful indications of edematous parenchyma and/or sclerotic glandular conditions and it could be used as tool for male health and reproductive management. mailto:calogero.stelletta@unipd.it animal breeding and genetics what will sheep and goat breeding look like in the future? poster presentations genetic resources (2021) s1 international congress on sheep and goats 26 abstract book genetic polymorphism in the tlr4 gene and its association with milk traits in egyptian sheep ahmed m. sallam animal and poultry production division, desert research center, cairo, egypt contact: 1st mathaf el-mataryia, 11735, cairo, egypt, abstract: the egyptian dairy production industry is predominantly based on cows and buffalos, with little care for sheep and goats. however, sheep contributes about 5% of the total whole milk produced annually in egypt and contributes a substantial part of their livelihood of a large proportion of the egyptians. information available regarding the genetics of sheep milk is scarce in the egyptian breeds. the objective of this study was to identify genetic variants in the tlr4 gene for milk traits in barki sheep as one of the three major sheep breeds in egypt. records were available for about 300 ewes and were genotyped using polymerase chain reaction (pcr)single strand conformation polymorphism (sscp) protocol. two distinctive conformation patterns (named g and t) were observed in the investigated region. the genotypic frequencies were 44.6%, 35.7% and 19.7% for gg, gt and tt genotypes, respectively. the direct sequencing identified a missense mutation located in the coding sequence of exon3 of the gene (c.1710c>a), which changes the amino acid sequence of the resulted protein (p.asn570lys). the association analyses suggested that the identified polymorphism had a significant effect (p-value<0.05) on the daily milk yield, fat percentage and protein percentage. summarizing, the tlr4 is suggested as candidate gene to improve milk traits in the egyptian sheep. mailto:ahmedsallam2@gmail.com animal breeding and genetics what will sheep and goat breeding look like in the future? poster presentations international congress on sheep and goats genetic resources (2021) s1 abstractbook 27 influence of lipoprotein lipase (lpl) gene polymorphism on production traits in dairy goats evaldas šlyžius1, birutė šlyžienė1 and renata bižienė2 1 lithuanian university of health sciences, department of animal breeding, kaunas, lithuania 2 lithuanian university of health sciences, institute of biology systems and genetics, kaunas, lithuania contact: evaldas šlyžius, lithuanian university of health sciences, department of animal breeding, kaunas, lithuania, email: evaldas.slyzius@lsmuni.lt abstract: the objective of this study was to investigate the influence of lpl gene polymorphism on production traits in dairy goats. the polymorphism of the goat was analysed in 272 unrelated individuals belonging to three pure goat breeds (saanen, n=104; anglo nubian, n=88 and alpine, n=80) in lithuania. the statistical characteristics were calculated using ibm spss statistics, version 20. in the examined herd, the genotype cc of lpl gene was present in 24.3% of goats, 19.9% of goats had heterozygotic cg genotype and 55.8% of goats had gg genotype. the results showed that goat lpl gene with cc genotype has a higher milk yield (0.14 kg higher compared to gg and 0.72 kg higher compared to cg genotype, p<0.01). the goats with gg genotype have a higher fat content (0.09% higher compared to cc, 0.23% higher compared to cg genotype, p<0.05), while the protein content was higher in goats with cg genotype (0.06% higher compared to cc, 0.03% higher compared to gg genotype, p<0.05). we also estimated, that goats with cc genotype had higher lactose (4.29 %) and lower scc log10 (4.506) value (p<0.01). the analysis of the goat milk production parameters showed the significant mean differences between the breeds (p<0.01). the highest milk yield was estimated in alpine goats, while the lowest scc and the highest milk fat and protein content in anglo nubian goats (p<0.01). the results of our research show that examined lpl gene polymorphisms seem to be the valuable biomarkers of the goat selection process. mailto:evaldas.slyzius@lsmuni.lt animal breeding and genetics what will sheep and goat breeding look like in the future? poster presentations genetic resources (2021) s1 international congress on sheep and goats 28 abstract book the nolana project breeding high-performance hair sheep for germany rolf minhorst nolana-netzwerk deutschland e.v., registered association, 56745 weibern, germany, https://www.nolana-schafe.de contact: dr. rolf minhorst, eichertstr. 48, 56745 weibern, germany, tel: ++48-(0)2655-962912 abstract: the price of raw wool in germany has been falling since the end of world war ii. today, the production costs of wool far exceed the gross income from the sale of wool. in view of this precarious situation, the author proposed in 1997 to breed a new synthetic hair breed called nolana. the project immediately enjoyed broad approval from many sheep breeders, some research institutions and animal husbandry administration. due to the eu import restrictions, no genetic material could be imported from overseas. so it was decided to follow the example of the synthetic or composite breeds that are widespread in the united states, relying only on the rules of quantitative genetics. it was supposed to be an open breeding program in which new breeds could be included at any time without a predetermined percentage of genes. the flocks present in the participating farms, primarily the merino landrace, suffolk or german blackhead, served as mother base. the wiltshire horn breed was used as the sire line. in later years, dorper and barbados rams were added. it was worked through simple back-crossing up to r2 and subsequent genetic consolidation. the 15th fattening and slaughter performance test for sheep in the köllitsch testing station in 2010, demonstrated that the performance parameters of the new nolana breed are in the good average of the existing dual-purpose breeds. the project was taken up by many breeders and supported by the sheep breeding associations of the federal states by setting up herd books. two new synthetic hair breeds have emerged from the nolana project: the white nolana meat sheep (nol) and the brown hair sheep (bhs), both of which were officially recognized by the vdl on november 01, 2018. actually, the nolana-network has 86 members. the nolana herdbook-stock includes 52 rams and 745 ewes, the brown hairsheep herdbook-stock includes 41 rams and 401 ewes. besides that, there is also an unknown number of breeders and animals that are not listed in the herdbook. a conservative estimate, for example on the basis of facebook ads, would result in at least five times the number of nolana and brown hairsheep existing at present in germany. https://www.nolana-schafe.de/ mailto:rolf.minhorst@t-online.de animal breeding and genetics what will sheep and goat breeding look like in the future? poster presentations international congress on sheep and goats genetic resources (2021) s1 abstractbook 29 new genomic tools for sheep breeding in germany monia budnik, julia storms, anna wirth, ann-kathrin struck and ottmar distl university of veterinary medicine hannover, institute of animal breeding & genetics, hannover, germany contact: abstract: new genomic tools became available through ovine genome projects and the development of genome-wide single nucleotide polymorphisms (snp) arrays. we employed the newly developed ovine ggp 50 k beadchip for >3000 sheep in a wide spectrum of sheep breeds in germany. the objectives were to test the suitability of this new tool for genotyping mendelian traits including haplotypes for scrapie resistance, genetic variants for muscularity, fecundity, microphthalmia, entropion, susceptibility for maedi-visna, parentage testing, determination of sex and genomic evaluations for quantitative traits. for genotyping, we used edta-blood samples on fta cards. all samples passed quality checks with a genotyping rate of 98%. most mendelian genotypes were accurately genotyped. we found alleles associated with spider lamb, microphthalmia and yellow fat phenotype in very few cases. the tmem154 k/k genotype associated with lower risk to maedi-visna showed a frequency of 46%. the myostatin allele causing muscular hypertrophy (mstn:g.6223g>a) was at a frequency of 0.16. more than 60% of the sheep were homozygous for the arr haplotype. we employed genome-wide snp genotypes to estimate heritabilities for growth traits recorded in field and stationary tests. in addition, we estimated genomic diversity measures for management the endangerment of particular breeds. size of training sets and blending of breeds were tested to determine increase in prediction accuracies under different scenarios for german sheep breeds. imputation of genotyping data on whole genome sequencing data seems to be advantageous for qtl detection. single-step methods should improve accuracy of prediction by 15-35% compared with other methods. mailto:monia.budnik@tiho-hannover.de mailto:ottmar.distl@tiho-hannover.de animal breeding and genetics what will sheep and goat breeding look like in the future? poster presentations genetic resources (2021) s1 international congress on sheep and goats 30 abstract book aortic connective tissue abnormalities resembling marfan syndrome in goats karianne lievaart-peterson1, liesbeth harkema1, rene van den brom1, reinie dijkman1, eveline dijkstra1, mark van der heijden2 and piet vellema1 1 royal gd, deventer, the netherlands 2 universitaire landbouwhuisdieren praktijk, harmelen, the netherlands contact: karianne lievaart-peterson, royal gd, po box 9, 7400 aa deventer, the netherlands telephone +31 (0)570-660478, k.lievaart-peterson@gdanimalhealth.com abstract: three cases of sudden death in adult dairy goats revealed a hemoabdomen due to rupture of the cranial abdominal aorta or cranial mesenteric artery at post mortem examination. histologically, there was evidence of impaired vessel wall architecture with loss and fragmentation of elastin fibres, proliferation of the lamina intima and fibrosis. similar microscopic lesions have been described in marfan syndrome. marfan syndrome is a genetic (autosomal dominant) disorder that affects the connective tissue, named after french paediatrician antoine marfan, who first described it in 1896 in children. clinical manifestations involve the cardiovascular, ocular and skeletal system. it is caused by mutations in the gene encoding fibrillin‐1, the major component of extracellular microfibrils that support elastin fibre deposition (coelho and almeida, 2020). marfan-like syndrome has since been suggested or described in dogs, cattle (hirano et al. 2012), sheep, and horses. multiple connective tissue disorders in man and animals are caused by heritable genetic defects and may share similar features. a genetic defect cannot be ruled out since the dutch dairy goat population share a small genetic background. occasionally rupture of the uterine artery is observed, but without similar vessel wall lesions. there is a need for further research including larger numbers of cases, further typing of vessel wall lesions as well as genetic typing. to the best of our knowledge, this is the first description of morphologic connective tissue abnormalities resembling features of marfan syndrome in goats. references: coelho, s. g., almeida, a. g. (2020). marfan syndrome revisited: from genetics to the clinic. rev port cardiol, 39(4), 215-226. doi:10.1016/j.repc.2019.09.008 hirano, t., matsuhashi, t., kobayashi, n., watanabe, t., sugimoto, y. (2012). identification of an fbn1 mutation in bovine marfan syndrome-like disease. anim genet, 43(1), 11-17. doi:10.1111/j.1365-2052.2011.02209.x mailto:k.lievaart-peterson@gdanimalhealth.com animal breeding and genetics what will sheep and goat breeding look like in the future? poster presentations international congress on sheep and goats genetic resources (2021) s1 abstractbook 31 prion protein gene (prnp) polymorphism in latvian native breed goats kristine piliena1, lasma zelca2 and daina jonkus2 1 latvian goat breeding association, talsi region, latvia 2 latvia university of life sciences and technologies, jelgava, latvia contact: berzi, vandzene, vandzenes municipal, talsi region, latvia, abstract: scrapie is a lethal, neurodegenerative disease that is affecting sheep and goats. it is a disease that belongs to the group of transmissible spongiform encephalopathies (tses). this disease causes economic loss to herds, where animals are affected by it. the classical scrapie in sheep and goats has been affected by prion protein (prnp) gene polymorphisms (goldmann et al., 2011). the disease is hereditary and can be limited or eliminated by a thoughtful animal breeding by the selection of scrapie-resistant animals. latvian native breed goats are dairy goats and their population is small. the aim of the study was to explain the polymorphism of the latvian native goat breed population according to codons 146 and 222 of the prnp gene. genetic analyzes were performed in a laboratory by eurofins medigenomix gmbh. in total 397 samples were analyzed. it has been found, that in latvia bred goats, the nn146 genotype is dominated after the codon 146. the frequency of nn146 genotype was 97.7%. the genotype ns146 of heterozygous animals was only 2.3%. only 9 animals or 1.1% are found with the resistant s146 allele. a larger polymorphism was observed at codon 222, where the frequency of the resistant k222 allele was 11.5%. the homozygotes kk222 genotype was in 3 animals or 1.0%, the heterozygotes qk222 genotype was in 21.2% and the qq222 genotype was in 77.8%. although the number of animals with resistant alleles is small, in further breeding process it is crucial to increase the number of animals with necessary genotypes. acknowledgements: research was conducted with support from lr ministry of agriculture. contract number 20-100-20-1.8.000011. references: goldmann w, ryan k, stewart p, parnham d, xicohtencatl r, fernandez n, foster j. (2011). caprine prion gene polymorphisms are associated with decreased incidence of classical scrapie in goat herds in the united kingdom. veterinary research 42(1):110. doi.org/10.1186/1297-9716-42-110 mailto:piliena@inbox.lv economics: how can the profitability of sheep and goat farming be improved? keynote presentation genetic resources (2021) s1 international congress on sheep and goats 32 abstract book sheep and goats in the world and the importance of economy stanislaus von korn hochschule für wirtschaft und umwelt nürtingen-geislingen (hfwu), germany contact: prof. dr. stanislaus von korn, institut für angewandte agrarforschung (iaaf), hochschule für wirtschaft und umwelt nürtingen-geislingen, 72622 nürtingen, stanislaus.korn@hfwu.de abstract: with the increasing number of sheep and goats in the world, the importance of small ruminants grew over the past 18 years. such increases can be observed especially in africa and asia, while sheep stocks in americas, europe and oceania had declined. sheep and goats mainly contribute to food and income security by producing of food goods. the small ruminants fulfill these functions in different systems in accordance with the respective site conditions: in subsistence farming, e.g. in africa and asia, as well as in profitoriented farms, e.g. in europe. favoured und less favoured areas are used, which often have no alternative use. however, prerequisites for sensible sheep and goat husbandry are adequate biological performance (good productivity in subsistence farming) or adequate profitability through the production of marketable products. both parameters (productivity and profitability) always determine the importance of small ruminants. in this context, it is the goal of the session economy to analyze positive as well as negative influencing factors. mailto:stanislaus.korn@hfwu.de economics: how can the profitability of sheep and goat farming be improved? oral presentations international congress on sheep and goats genetic resources (2021) s1 abstractbook 33 assessment of the production of goat milk by means of performance cost accounting in terms of economic efficiency and influence of stock size isabel sand1, christina gaio1, wilfried hartmann1, fides marie lenz2 and yvonne kranch3 1 kuratorium für technik und bauwesen in der landwirtschaft (ktbl), darmstadt, germany 2 landwirtschaftskammer nordrhein-westfalen, lippstadt, germany 3 profuma spezialfutterwerke gmbh & co kg, dormagen, germany contact: isabel sand, i.sand@ktbl.de abstract: purpose: to evaluate the performance and costs of goat milk production, if it is economical, and whether animal places have an influence on performance cost accounting. methods: we modelled three production processes of dairy goat farming. these only differed in stock size with the area per animal kept constant. this resulted in 125, 245, and 490 animal places. we also kept the production characteristics constant. with these assumptions, a performancecost calculation was carried out. it included a list of performance and direct costs, investment requirements and annual building costs, operating costs and performances, costs and success factors. the data we use are from our own data collection and research. results: animal places had no influence on the direct costs and direct cost free performance. however, animal places had an impact on variable costs. these increase with a decrease in animal places and thus decrease the profit margin. direct and operating free and single-free performance increase with increasing animal places. in the same way, the operating productivity increases and the unit costs are positively affected while the price per litre goat milk decreases. for all three scenarios, the gross margin was still positive, but the direct and operating cost free and individual cost free performance was negative. conclusion: all three models of production processes turned out as non-economical. to cover all costs, the milk price of € 0.75 per litre would have to increase by around 50 % in the largest barn. to increase profitability, direct marketing of goat's milk products could be added to milk production. good kid marketing would also increase profitability. relevance: we were able to show which economic variables in the performance and cost calculation arise for the farmer in goat milk production, whether it is economical in the planning examples and where there is room for improvement. mailto:i.sand@ktbl.de economics: how can the profitability of sheep and goat farming be improved? oral presentations genetic resources (2021) s1 international congress on sheep and goats 34 abstract book an assessment of economic efficiency in german sheep farming a nationwide analysis stanislaus von korn1, stefan völl2 and arbeitskreis wirtschaftlichkeit2 1 hochschule für wirtschaft und umwelt (hfwu), nürtingen-geislingen, germany 2 vereinigung deutscher landesschafzuchtverbände (vdl), berlin, germany contact: prof. dr. stanislaus von korn, hochschule für wirtschaft und umwelt nürtingen-geislingen, institut für angewandte agrarforschung (iaaf), 72622 nürtingen, stanislaus.korn@hfwu.de abstract: in view of the declining herd population (v. korn 2019), the analysis of the economic situation in sheep farming is of central importance for the establishment of suitable control measures in order to be able to maintain sheep farming. against this background, the aim of the project funded by the landwirtschaftliche rentenbank was to carry out a nationwide representative analysis of the current assessment of profitability in sheep farming. for this purpose, all available data from consulting initiatives as well as from accounting and testfarm results have been researched, merged and adjusted. thus, the most extensive data basis on sheep profitability was created with approximately 700 farm surveys from recent years. the results show large variations between regions, based mainly on different herd structures and location conditions. the deviations between the years are primarily determined by the fluctuating lamb prices. overall, it was found that approximately 60% of total income from sheep farming (290 €/ewe+year) comes from public donations. however, under the current price and cost relationships, full costs cannot be covered despite the comparatively high proportion of public funds. this means that if sheep farming is to be preserved in its multifunctionality for landscape, nature conservation, society and rural structures, suitable support instruments must also be installed in the future (eu 2018), but also consulting, training and commitment in sheep farms will be required. references: eu (2018). bericht über die derzeitige lage und die zukunftsperspektiven der schafund ziegenhaltung in der eu. europäisches parlament, a8-0064/2018, märz 2018 von korn, stanislaus (2019). strukturen und perspektiven der schafhaltung, workshop „zucht und haltung von schaf und ziege in deutschland“, okt. 2019 berlin mailto:stanislaus.korn@hfwu.de economics: how can the profitability of sheep and goat farming be improved? oral presentations international congress on sheep and goats genetic resources (2021) s1 abstractbook 35 the thuringian sheep and goat premium adriana schwarz1 and henryk baumbach2 1 thuringian state office for environment, mining and nature conservation, weimar, germany 2 thuringian ministry for environment, energy and nature conservation, erfurt, germany contact: adriana schwarz, thuringian state office for environment, mining and nature conservation, harry-graf-kessler-straße 1, 99423 weimar, tel: +49 361 57 3943 595, adriana.schwarz@tlubn.thueringen.de abstract: since 1990, the sheep population in thuringia has decreased significantly. the total number of sheep has shrunken to less than 1/4, compared to 1989 (542,253). the fundamental change of agricultural structures after the unification and the abolition of coupled payments for sheep and goats at federal level in 2005 led to this dramatic decline. therefore, thuringia has supported and initiated some initiatives to reintroduce a premium for grazing animals. thuringia introduced its own premium for sheep and goats in 2019 (limited until 2021), which is fully financed from state resources and granted as de-minimis aid. the premium is supposed to counter the decrease of animal population, which is essential to conserve high nature value grassland in the medium term. furthermore, the initiative aims to reintroduce coupled payments as appropriate instrument at federal level with the new cap period. thuringia supports farmers with 25 € per animal and year. due to the de-minimis rules, the total annual amount is limited to 6,666 € per agricultural holding. in 2019, the premium was paid to 321 farms for 47,000 animals. the total number of dams kept by these farms was about twice this number. however, due to the aid ceiling the premium was capped. in 2019 the sheep population in thuringia started to rise again slightly by 2 % (121,900 vs. 119,500 in 2018). with this concept, thuringia has a pioneering role in germany, which other federal states have picked up. however, a fundamental solution has to be found at the federal level. mailto:adriana.schwarz@tlubn.thueringen.de economics: how can the profitability of sheep and goat farming be improved? oral presentations genetic resources (2021) s1 international congress on sheep and goats 36 abstract book weidewonne – bringing together lamb marketing and landscape conservation stefanie schröter naturstiftung david, projekt weidewonne, brau, deutschland contact: heidelbergstr. 1, 06577 braunsroda, germany, stefanie.schroeter@naturstiftung-david.de abstract today, the main threat for the valuable dry grasslands is under-grazing, which results in matting and shrub encroachment. after german reunification, the sheep stock decreased especially in eastern germany. on average, 58 % of the shepherd’s income in thuringia results from subsidies. however, the german lamb market is challenged by the cost-effective imports from new zealand. the applying project weidewonne aims at supporting shepherds, who are also engaged in landscape conservation, by an increase of the value lamb caused by implementing direct marketing. the brand “weidewonne” was created for the marketing of landscape conservation lamb in the thuringian basin. since 2011, quality criteria and marketing channels were developed within an eu life project. since 2017, the project is managed on behalf of the thuringian ministry for environment, energy and nature conservation by the foundation “naturstiftung david” and funded by eler. currently, 16 shepherds participating in the project, along with butchers, who stock “weidewonne lamb”. regular marketing campaigns involve partners from slaughter and distribution. in march 2020, an online shop was launched. now, customers, who have no access to “weidewonne butchers”, are able to order fresh lamb online. the project shows that nowadays shepherds not only have issues of economical nature but also of social acceptance, bureaucracy and farm succession. in future, the weidewonne project gains to increase its activities by fully supporting shepherds throughout thuringia. then it will be possible to evaluate the improvement on a statistical basis. mailto:stefanie.schroeter@naturstiftung-david.de economics: how can the profitability of sheep and goat farming be improved? oral presentations international congress on sheep and goats genetic resources (2021) s1 abstractbook 37 ways to improve the profitability of sheep farming with rare indigenous sheep breeds nathalie ketterle1 and caroline ketterle2 1 kollektion der vielfalt, zell, germany 2 university of applied science nürtingen-geislingen (hfwu), nürtingen, germany contact: nathalie ketterle, boßlerstr. 1, d-73119 zell u.a., nathalie.ketterle@kollektion-der-vielfalt.de abstract: the main goal is the preservation of rare indigenous sheep breeds by using their typical and natural coloured wool in traditional and modern ways, creating sustainable woollen products and selling them directly from farmgate to the consumers and therefore increase the profitability of sheep farming and preserve biodiversity. when we started 2004, the renewable research wool was regarded as waste and it had no use at all. instead of increasing the income of farmers, wool costs them and shearing their sheep is only done for animal welfare reasons. instead of throwing away an eco-friendly and sustainable fibre, we found use for the wool of our indigenous sheep breeds thereby avoiding microplastic pollution since wool is reusable and 100% biodegradable, compared to synthetical fibre. since 2009, we consult and support projects all over europe to develop a whole value chain for wool and collaboratively create a local brand for woollen products from rare indigenous sheep breeds. by selling high quality woollen products directly from "sheep to shop", the whole value chain is maxed out, increase the income of the breeders, supporting preservation of rare indigenous sheep breeds and maintaining and sustaining biodiversity and cultural heritage. another benefit, 5 % of the weight of wool is pure organic carbon. when stored in wool, there is less carbon in the atmosphere and this mitigates climate change. our work contributed to agriculture and landscape conservation as well as to the promotion of old and endangered sheep breeds and the preservation of their genetic diversity. mailto:nathalie.ketterle@kollektion-der-vielfalt.de economics: how can the profitability of sheep and goat farming be improved? poster presentations genetic resources (2021) s1 international congress on sheep and goats 38 abstract book place of goat meat in human consumption in the chlef region in algeria mohamed sadoud and yassine sadoud h. benbouali chlef university, bp 151, chlef, algeria contact: m_sadoud@yahoo.fr abstract: according to fao statistics, algeria had around 5 million goat heads and produced 7500 tonnes of goat meat in 2017. according to madani (2000), the goat breeds existing in algeria are of the traditional type, the majority of which are subject only to natural selection. they are composed by animals of the local population with generally nubian blood. in addition to local populations, there are also introduced populations, and crossed populations. the survey took place during 2019. the questionnaire was sent to 200 heads of households. the surveys comprising 24 questions targeted the usual consumers of goat meat, who were previously selected according to the difficult piedmonts and mountain areas of the chlef region. consumer preferences are very complex and heterogeneous and depend not only on the sensory properties of the meat, but also on psychological and socio-demographic factors. thus, they look for freshness considered as a factor in the appreciation of the quality of a meat and for this they pay great attention to the color of the meat, which is used as an indicator of the age of the animal (oury et al., 2009). they consider juiciness and taste as very important criteria in the choice of meat. references: fao. (2017). food and agriculture data, http://www.fao.org/faostat/en/ madani t., 2000. goat farming in north-eastern algeria. gruner l and chabert y (ed). inra and institut de l'elevage pub, tours 2000. proceedings of the 7th conference international on goats, tours (france) 15-21 / 05 / 00,351-353 oury, m.p., picard, b., briand, m., dransfield, e., blanquet, j. p., dumont, r. (2009). interrelationships between meat quality traits, texture measurements and physicochemical characteristics of m. rectus abdominis from charolais heifers. meat science, 83, 293-301 mailto:m_sadoud@yahoo.fr http://www.fao.org/faostat/en/ economics: how can the profitability of sheep and goat farming be improved? poster presentations international congress on sheep and goats genetic resources (2021) s1 abstractbook 39 impact of direct ewe support on the cost-income situation of the sheep farm bence jávor1, andrás nábrádi2 and sándor kukovics3 1 hódmezőgazda closed corporation, hódmezővásárhely, hungary 2 university of debrecen, debrecen, hungary 3 sheep and goat products’ board and inter-professional organization, várpalota, hungary contact: bence jávor, arany sándor u. 17. debrecen 4014 hungary; javorbence0804@gmail.com abstract: the basic condition for the survival of the sheep sector is that it is worth keeping sheep. this requires, in addition to the revenue generated by the products manufactured under the given condition scheme, a level of support for the services provided to the community. to analyse the situation of domestic sheep farming, the economic database of the sector was subjected to a sensitivity analysis. database contained data on existing sheep farms in test farm system (fadn), data from central statistical office, and analyses of sectoral organizations made during the period between 2003 and 2018. in examinations, the production value per ewe, revenues (animals, wool, milk), direct ewe support, costs (wages, feed, services, overheads), the margin contribution, and the sectoral result were analysed. data were processed using microsoft excel 2016 software package and correlation studies (pearson's correlation calculation, covariance analysis, r2 test) were performed. the results show that production costs increased at a faster rate than revenues during the period under review, and direct ewe support plays a very important role in shaping the sectoral result. in the process of this since 2012, with the increase of this support, the sectoral income per ewe has increased, which has yielded a positive result since 2014. in conclusion, we found that at the current level of production, only direct ewe support allows for economical sheep farming. without this, the utilised progeny rate should be improved from 85% to 150%. in addition, area-based direct payments represent the real income of a given sheep farm. references: kukovics s. sándor and jávor, andrás (2010): a fejlesztés lehetőségei a juh ágazatban, juhinnov platform /development possibilities in sheep sector, juhinnov platform/, k-ovi-cap bt. és debreceni egyetem agtc, érd-debrecen, isbn 978-96308-0624-4, 365 pages bence jávor, andrás nábrádi, sándor kukovics (2018): subsidies are potential sources of profitable management – their payment between 2010 and 2016; apstract vol. 12. number 1-2. 2018.pages 97-120. issn 1789-7874 mailto:javorbence0804@gmail.com environmental performance and climate change: what do sheep and goats contribute to climate change mitigation? oral presentations genetic resources (2021) s1 international congress on sheep and goats 40 abstract book adaptation to saline drinking water in goats rukhsana amin runa1, alexander riek2, lea brinkmann3 and martina gerken3 1 department of surgery and obstetrics, bangladesh agricultural university, mymensingh, bangladesh 2 3department of animal sciences, university of göttingen, göttingen, germany contact: dr. alexander riek, institute of animal welfare and animal husbandry, friedrich-loefflerinstitut, dörnbergstr. 25/27, 29223 celle, tel: +49 5141 3846 160, email: alexander.riek@fli.de abstract: in the context of global warming, salinization of groundwater and soil is a prevalent global issue with serious consequences on animal health and production. therefore, we investigated the capacity of goats to adjust their salt intake from saline drinking water in a free choice system. twelve non-pregnant boer goats were kept in individual pens for 4 weeks. in the control phase (1 week), only fresh water was supplied in five identical buckets for each pen. during the subsequent treatment phase (3 weeks), fresh tap water and four different concentrations (0.75, 1.0, 1.25, and 1.5% nacl) of saline water were offered simultaneously in a free choice system. hay, water and a mineral lick were provided ad libitum. dry matter intake, total water intake and total sodium intake were significantly (p<0.001) higher during the treatment phase. all goats had a significant preference for fresh (0% nacl) over saline water. at the beginning of the simultaneous choice situation, animals did not differentiate between salt concentration of 0.75% and 1.0%. however, with successive treatment, animals distinguished more sensitively between saline water concentrations and preferred the 0.75% salt concentration. the total sodium intake of goats ranged between 0.37-0.55 g/kg bm0.75 per day during the treatment phase, being 8 to 11-fold higher than the daily requirements of sodium for body maintenance. the results suggest that goats are able to differentiate between saline water concentrations and adjust their sodium intake by quick adjustments in selfselection in a free choice system. mailto:alexander.riek@fli.de environmental performance and climate change: what do sheep and goats contribute to climate change mitigation? oral presentations international congress on sheep and goats genetic resources (2021) s1 abstractbook 41 anaplasma phagocytophilum in cattle, sheep and goats in germany – results of a systematic review of the literature matthias g. wagener and martin ganter university of veterinary medicine hannover, foundation, clinic for swine and small ruminants, hannover, germany contact: university of veterinary medicine hannover, foundation, clinic for swine and small ruminants, bischofsholer damm 15, 30173 hannover, germany, matthias.gerhard.wagener@tiho-hannover.de abstract: anaplasma phagocytophilum, formerly called ehrlichia phagocytophila, is a gram-negative, obligate intracellular bacterium that has a broad host range. because it is transmitted by ticks and enters the neutrophil granulocytes, clinical pictures are called granulocytic anaplasmosis in dogs or humans, or tick-borne fever in ruminants. clinical signs in small ruminants include an acute febrile reaction, abortion, depressed appetite and respiratory symptoms. due to secondary infections a broad range of other clinical signs is also observed. tick-borne fever in small ruminants typically played a role in northern europe, and higher-altitude regions in southern europe. but there are also high seroprevalences in dogs or wild ruminants in germany. positive samples of patients show that there is also a clinical impact of the infection in german small ruminants. a systematic literature search in three scientific databases was performed. search terms were “anaplasma phagocytophilum” and “germany” and (“sheep” or “goat” or “cattle”) as well as synonyms or german translations of the terms. 161 sources (without replicates) from 1920 to 2020 were found. 13 sources were identified as suitable in providing information about the status of a. phagocytophilum in cattle or small ruminants in germany. except one source all were from the last decade, 9 of them dealt with cattle, 2 with sheep, 2 with goats, 2 with mouflons. all sources containing small ruminants included more than one host species. these data show a lack of knowledge concerning this pathogen in small ruminants in germany. tick-borne fever in ruminants might be currently underdiagnosed and even might get a higher impact on flocks in germany due to the climate change and the accompanying better conditions for ticks as a vector of a. phagocytophilum. references: huhn, c., winter, c., wolfsperger, t., wüppenhorst, n., smrdel, k. s., skuballa, j., pfäffle, m., petney, t., silaghi, c., dyachenko, v., pantchev, n., straubinger, r. k., schaarschmidt-kiener, d., ganter, m., aardema, m. l. and loewenich, f. d. von (2014). analysis of the population structure of anaplasma phagocytophilum using multilocus sequence typing. plos one, 9(4), e93725-e93725. tegtmeyer, p., ganter, m., von loewenich, f.d. (2019). simutaneous infection of cattle with different anaplasma phagocytophilum variants. ticks and tick-borne diseases 10, 1051-1056. woldehiwet, z. (2007). tick-borne diseases. in: aitken i.d. ed. diseases of sheep. 4th ed. oxford, uk: blackwell publishing, 347355. mailto:matthias.gerhard.wagener@tiho-hannover.de environmental performance and climate change: what do sheep and goats contribute to climate change mitigation? oral presentations genetic resources (2021) s1 international congress on sheep and goats 42 abstract book opportunities to mitigate ghg emissions from sheep and goat farming in indonesia mohammad ikhsan shiddieqy1, bess tiesnamurti1 and yeni widiawati2 1 indonesian center for animal research and development (icard), bogor, indonesia 2 indonesian research institute for animal production, (iriap), ciawi, indonesia contact: jalan raya pajajaran kav. e-59, bogor 16151, indonesia, (+62) 251 8322185, m.ikhsan.shiddieqy@litbang.pertanian.go.id abstract: sheep and goat farming play an important role in the livelihoods of the farmers in indonesia. small ruminants farming in indonesia is identified with limited ownership by farmers (2-7 heads/household). farming management systems are dynamic in response to the availability of resources. indonesia’s ministry of agriculture is trying to boost the small ruminant population. however, an increase in small ruminant population is related to the contribution of sheep and goat to greenhouse gas (ghg) emission. the small ruminant production system is affected by climate change and contributes to global warming with ghg emissions (marino et al., 2015). since goats are considered more climate resilient than other ruminant species (pragna et al., 2018), their contribution to ghg emissions is more important to highlight. this paper aims to provide an integrated overview on the opportunities to mitigate ghg emissions from sheep and goat farming in indonesia. beef cattle are the highest contributor of enteric ch4 (18.04 gg co2-e or 65,12%) among livestock’s ghg (tier 1) in the country, while goat and sheep are 2.043 gg co2-e (8.47%) and 1.509 gg co2-e (6,26%) in 2014, respectively. however, ghg emissions from small ruminants are significant since the population are growing 10.1% per year. ghg emissions (enteric ch4, feces ch4 and feces n2o) from sheep using tier 2 method are 1.693 gg co2-e in 2014 (widiawati and tiesnamurti, 2019). among the various ghg mitigating strategies, our review suggests that the most effective mitigation strategies in indonesia is through nutritional intervention, such as utilization of tropical legumes and palm kernel cake as small ruminants feed which reduce enteric ch4 production. references: pragna, p., chauhan, s.s., sejian, v., leury, b.j., dunshea, f.r. (2018). climate change and goat production: enteric methane emission and its mitigation. animals 2018, 8(12), 235. https://doi.org/10.3390/ani8120235 marino, r., atzori, a.s., d’andreac, m., iovaned, g., trabalza-marinuccie, m., rinaldi, l. (2015). climate change: production performance, health issues, greenhouse gas emissions and mitigation strategies in sheep and goat farming. small ruminant res. volume 135, february 2016, pages 50-59. https://dx.doi.org/10.1016/j.smallrumres.2015.12.012 widiawati, y., and tiesnamurti, b. (2019). “sumbangan emisi gas rumah kaca dari domba di indonesia,” in emisi gas rumah kaca dari peternakan di indonesia dengan tier 2 ipcc, ed. e. aldrian, s. puspowardoyo, and b. haryanto (jakarta, lipi press), 57-70. mailto:m.ikhsan.shiddieqy@litbang.pertanian.go.id https://doi.org/10.3390/ani8120235 https://dx.doi.org/10.1016/j.smallrumres.2015.12.012 environmental performance and climate change: what do sheep and goats contribute to climate change mitigation? oral presentations international congress on sheep and goats genetic resources (2021) s1 abstractbook 43 the impact and breeding perspectives of maternal energy metabolism profiles preand postpartum on lamb body weight development with special regard to ewe methane emission traits jessica reintke1, kerstin brügemann1, tong yin1, petra engel1, henrik wagner2, axel wehrend2, anja müller3 and sven könig1 1 institute of animal breeding and genetics, university of giessen, ludwig st. 21b, 35390 giessen, germany 2 clinic for obstetrics, gynaecology and andrology of large and small animals with veterinary ambulance, university of giessen, frankfurter st. 106, 35392 giessen, germany 3 idexx laboratories, vet med labor gmbh, im moltengraben 65, 70806 kornwestheim, germany contact: jessica reintke, institute of animal breeding and genetics, university of giessen, ludwig st. 21b, 35390 giessen, germany, jessica.reintke@agrar.uni-giessen.de abstract: the profitability of sheep production strongly depends on the maternal health and feed efficiency status, and on lamb weaning performance. this study focused on intergenerational aspects and associations between maternal energy metabolism profiles with lamb body weight development. data included repeated observations for body condition and methane (recorded via laser methane detector) traits from 330 ewes (253 merinolandsheep, 77 rhönsheep) postpartum, and macro-, microelement and metabolic status (blood samples) from 46 ewes preand postpartum. additionally, the body weight of 629 lambs was recorded. in merino ewes, a maternal serum magnesium level > 1.0 mmol/l at weaning was associated with an increase of 13 % in lamb body weight, compared to offspring from ewes with lower serum magnesium concentration. furthermore, copper positively affected ewe body condition at weaning in both breeds. in rhönsheep, a sufficient selenium supply was important to optimize body condition. moreover, high zinc level during lactation was associated with reduced methane emissions in merino ewes and a low ß-hydroxybutyrate level contributed to decreased methane emissions in both breeds. large methane levels of an ewe were associated with reduced lamb body weight and reduced ewe body condition. heritabilities for methane traits were small (h2 ≤ 0.03) and genetic correlations between methane emissions, ewe body condition traits and lamb body weight were mostly negative. results indicate that the maternal mineral and metabolic status affects ewe body condition, methane emissions and lamb body weight. furthermore, breeding on reduced methane emissions also contributes to genetic improvements of lamb weaning performance. mailto:jessica.reintke@agrar.uni-giessen.de animal genetic resources keynote presentation genetic resources (2021) s1 international congress on sheep and goats 44 abstract book eugena: the european genebank network for animal genetic resources fernando tejerina ampudia1, coralie danchin-burge2 and sipke joost hiemstra3 1 ministerio de agricultura, pesca y alimentación. subdirección general de medios de producción ganadera. c, almagro 33, cp28010, madrid, spain 2 institut de l’elevage, 149 rue de bercy, f-75595, paris 12, france 3 centre for genetic resources (cgn), wageningen university & research, wageningen, netherlands abstract: conservation of within and across breed genetic diversity in genebanks (ex situ) is a complementary strategy of the in situ conservation to maintain a broad genetic base for future breeding and research. it is important to safeguard farm animal genetic diversity (both local and mainstream breeds) for future generations, in particular in the global context of food and nutrition security and climate change. with reference to the achievement of sustainable development goal target 2.5, the food and agricultural organization of the united nations is monitoring the number of animal genetic resources for food and agriculture secured in medium or long term conservation facilities (genebanks) (indicator 2.5.1.b). in a recent survey developed by the ex situ conservation working group of the european regional focal point for animal genetic resources (erfp, https://www.animalgeneticresources.net/), a total of 125 genebanks (reproductive and/or genomic) were identified in 26 european countries. the most frequent objective as indicated by these facilities in the survey was the conservation of animal genetic resources, followed by research activities and commercial use of the stored material. the results of the survey show the high interest in the establishment and development of collections for the ex situ conservation of animal genetic resources and for use in breeding, conservation and research. the european genebank network for angr (eugena) is a network of nationally recognized member genebanks in european countries with the aim to support the medium/long term ex situ conservation and sustainable use of angr, and to facilitate the implementation of the fao global plan of action at national and pan-european level. exchange of knowledge and experiences as well as facilitating access to information about genebank collections in europe are the main objectives of eugena. development of eugena was initiated by erfp in 2016, and since then the network has grown constantly in terms of number of countries, genebanks members and information about genebank collections. currently, eugena is composed of 8 genebanks from 6 different countries, which store 865.324 samples (semen, embryos, hair and blood) from 10 different species. eugena gathers information from 59.774 samples (59.549 semen and 226 blood) from 23 goat breeds, allocated in 4 different genebanks, and 113.805 samples (113.151 semen, 23 wool and 631 blood) from 40 sheep breeds, allocated in 4 different genebanks. you can find more information about eugena at https://www.eugena-erfp.net/en/. https://www.eugena-erfp.net/en/ animal genetic resources: diversity and characterization? oral presentations international congress on sheep and goats genetic resources (2021) s1 abstractbook 45 genomic characterization of selection signatures: what is the future for sustainable utilization of ugandan goat breeds robert b. onzima1,2,4,5, maulik r. upadhyay1,3, harmen p. doekes1, mirte bosse1, martien a. m. groenen1 and richard p. m. a. crooijmans1 1 animal breeding and genomics, wageningen university and research, wageningen, netherlands 2 department of agricultural sciences, faculty of agriculture and environmental sciences (faes), muni university, arua, uganda 3 department of veterinarysciences agricultural population genomics, ludwig-maximilians-university munich, germany 4 department of agricultural sciences, faculty of agriculture and environmental sciences (faes), kabale university, kabale, uganda 5 national agricultural research organization (naro) p o box 295, entebbe, uganda contact: department of agricultural sciences, faculty of agriculture and environmental sciences (faes), muni university, p.o. box 725, arua, uganda, abstract: both natural and artificial selection are among the main drivers shaping genetic variation across the genome of livestock species. selection typically leaves signatures in the genome, which are often characterized by high genetic differentiation across breeds and/or a strong reduction in genetic diversity in regions associated with traits under intense selection pressure. we evaluated selection signatures in six ugandan goat breeds: boer (n=13), and the indigenous breeds karamojong (n=15), kigezi (n=29), mubende (n=29), small east african (n=29) and sebei (n=29). after genotyping quality control, 45,294 autosomal single nucleotide polymorphisms (snps) remained for further analyses. a total of 394 and 6 breed-specific putative selection signatures were identified across all breeds, based on marker-specific fixation index (𝐹st-values) and haplotype differentiation (hapflk), respectively. these regions were enriched with genes involved in signalling pathways associated directly or indirectly with environmental adaptation, such as immune response (e.g. il10rb and il23a), growth and fatty acid composition (e.g. fgf9 and igf1), and thermo-tolerance (e.g. mtor and mapk3). the study revealed little overlap between breeds in genomic regions under selection and generally did not display the typical classic selection signatures as expected due to the complex nature of the traits. in the boer, candidate genes associated with production traits, such as body size and growth (e.g. gjb2 and gja3) were identified. this study provides insights into the effects of long-term selection in boer and indigenous ugandan goat breeds, which are relevant for implementation of breeding programs and conservation of genetic resources, and their sustainable use and management. mailto:robertonzima@gmail.com mailto:r.onzima@muni.ac.ug animal genetic resources: diversity and characterization oral presentations genetic resources (2021) s1 international congress on sheep and goats 46 abstract book morphological variability and geographical distribution of goat populations from benin (west africa) habib r. v. whannou1, ulriche c. afatondji1, dominique demblon2, marcel r. b. houinato1 and luc h. dossa1 1 faculty of agricultural sciences, university of abomey-calavi, benin 2 higher education institute condorcet province of hainaut, belgium contact: faculty of agricultural sciences, university of abomey-calavi, email: raidvich27@gmail.com abstract: worldwide, insufficient knowledge of the genetic and phenotypic variabilities within and between local goat populations and of their suitability to diversified production environments hampers the development of programs for their rationale selection, use and management. this study explored the current geographical distribution of goat populations in benin based on their morphology, as a first step in their genetic molecular and phenotypic characterizations. from november 2019 to february 2020, ten qualitative and twenty-six linear body measurements were taken on 2114 adult female goats in the ten phytogeographic zones that compose the three vegetation areas of benin. fifteen ratios were generated from the quantitative variables. the data were analyzed using generalized linear model procedures followed by multiple comparison of least square means and multivariate analytical methods, including canonical discrimination analysis and hierarchical ascendant classification. all linear body measurements significantly varied (p<0.05) among zones. the highest mean values of height traits were recorded in the drier zones of the sudanian area and the lowest in humid guineo-congolese area. in the discriminant function analysis, only 61.80% of the measured individuals were correctly classified in their district of origin by 26 measured variables and 8 ratios. the cluster procedure analysis revealed four sub-populations within the three large known goat populations. these results confirm the spatial variation of goat populations according to production areas in benin, but also suggest that interbreeding may have taken place among animals from the different phytogeographic zones. mailto:raidvich27@gmail.com animal genetic resources: diversity and characterization? oral presentations international congress on sheep and goats genetic resources (2021) s1 abstractbook 47 discovery of casein variants in goats using capture sequencing siham a. rahmatalla1,2, danny arends1, ammar said ahmed1, lubna m. a. hassan3, stefan krebs4, monika resissmann1 and gudrun a. brockmann1 1 albrecht daniel thaer-institut für agrarund gartenbauwissenschaften, humboldt-universität zu berlin, invalidenstraße 42, d-10115 berlin, germany 2 department of dairy production, faculty of animal production, university of khartoum, shambat p.o. box 32, 13314 khartoum north, sudan 3 laboratory of functional genome analysis, gene center, university of munich (lmu), feodor-lynenstrasse 25, 81377 muenchen, germany contact: siham a. rahmatalla, albrecht daniel thaer-institut für agrarund gartenbauwissenschaften humboldt-universität zu berlin, invalidenstraße 42, rahmatas@hu-berlin.de abstract: genetic polymorphisms in casein genes (csn1s1, csn2, csn1s2, and csn3) are known to affect milk protein, milk composition, cheese processing properties and human digestibility (martin et al. 2002; selvaggi & tufarelli 2012; lad et al. 2017). in this study, we investigated sequence variants in coding regions of casein genes in four sudanese goat breeds (nubian, desert, nilotic, and taggar) and compared them with saanen goats, bezoar ibex, as well as alpine and nubian ibex. using high-density capture sequencing 22 non-synonymous and 13 synonymous snps were identified, among them 11 and 7 novel snps, respectively. in the csn1s1 gene, six non-synonymous (two novel) and seven synonymous snps (five novel), in the csn2 gene five non-synonymous (three novel) and one synonymous snp, in the csn1s2 gene, six non-synonymous (four novel), and in the csn3 gene five non-synonymous (two novel) and five synonymous (one novel) snps were detected. most novel snps in coding regions of the casein genes were detected only in the critically endangered nubian ibex. the identified markers for milk protein variants are additional useful tools for breed characterization, investigating biodiversity and phylogeny, and for the preservation of endangered breeds. further research is needed to characterize these new sequence variants with respect to allele frequencies in different breeds and consequences on milk properties for human nutrition. references: lad, s.s., aparnathi, k.d., mehta, b.m., suresh, v. (2017). goat milk in human nutrition and health – a review. international journal of current microbiology and applied sciences 6, 1781-92. doi: 10.20546/ijcmas.2017.605.194 martin, p., szymanowska, m., zwierzchowski, l. & leroux c. (2002). the impact of genetic polymorphisms on the protein composition of ruminant milks. reprod nutr dev. 42, 433-59. doi:10.1051/rnd:2002036 selvaggi, m., tufarelli, v. (2012). "caseins of goat and sheep milk: analytical and technological aspects," in casein: production, uses and health effects, ed. a. m. ventimiglia, j. m. birkenhäger (nova publishers), 1-26. mailto:rahmatas@hu-berlin.de animal genetic resources: diversity and characterization oral presentations genetic resources (2021) s1 international congress on sheep and goats 48 abstract book runs of homozygosity islands across 100 sheep and 96 goat populations reveal selection signatures wim gorssen, roel meyermans, steven janssens and nadine buys department of biosystems livestock genetics ku leuven, leuven, belgium contact: +32 16 37 78 24, abstract: runs of homozygosity (roh) are long stretches of homozygosity which are mainly caused by inbreeding. these roh can be used for the detection of selection signatures by computing/defining roh islands. although there are numerous goat and sheep snp datatesets available online, many of these data were never investigated for roh (islands) and an overview of known roh islands in sheep and goats is currently missing. in this study, we analysed roh inferred from medium density snp datasets in goats (96 populations; 4327 animals; colli et al., 2018) and sheep (100 populations, 3490 animals; sempéré et al., 2015). roh analyses were performed using plink and graphically presented per population. next, results on roh islands per population were summarised per species. we defined roh islands as snps with a p-value for roh incidence larger than 0.999 (using standard normal z-scores), and an incidence of minimum 30%. a number of known roh islands was confirmed, for example selection signatures for the myostatin gene in texel sheep on oar2. however, we also identified several roh islands which were common in many populations, but with yet unknown, underlying biological mechanism. for example, fifteen sheep populations showed an roh island on oar6 at 37-38 mb and six goat populations showed an roh island on ars12 at 35mb. these findings may direct future studies or can serve as a reference for results in other populations or other investigations. therefore, these results can be a valuable tool in future genetic research in sheep and goats. references: colli l, milanesi m, talenti a, bertolini f, chen m, crisà a, et al. (2018). genome-wide snp profiling of worldwide goat populations reveals strong partitioning of diversity and highlights mailto:wim.gorssen@kuleuven.be animal genetic resources: diversity and characterization poster presentations international congress on sheep and goats genetic resources (2021) s1 abstractbook 49 genetic diversity of algerian and turkish native sheep breeds abdelkader ameur ameur1, onur yilmaz2, nezih ata2, ibrahim cemal2 and semir bechir suheil gaouar3 1 agronomic sciences department, university of tlemcen, tlemcen, algeria 2 department of animal science, adnan menderes university, aydın, turkey 3 department of biology. university of tlemcen, tlemcen, algeria contact: ameur ameur a., university of tlemcen, department of agronomic sciences, and faculty life and nature sciences, email ameurabdelkader@gmail.com, tel: +213 657 052 803 orcid: https://orcid.org/0000-0002-7450-4311 abstract: the present study was conducted to investigate the genetic diversity and relationship of some native sheep breeds reared in turkey and algeria. a set of fourteen microsatellite markers was used for genotyping 240 animals from four algerian (hamra, ouled djellal, sidaou and tazegzawt) and four turkish (white karaman, south karaman, karacabey merino and kıvırcık) sheep breeds. a total of 340 alleles were observed. the mean number of alleles (na) and the effective alleles (ne) were, respectively, 24.29 and 10.99 with an average polymorphic information content (pic) of 0.90. hight genetic diversity level was inferred by observed (0.90) and expected heterozygosity (0.76). all fis (inbreeding coefficient) values were obtained as positive and significant in all sheep breeds studied except white karaman and sidaou breeds. mean value of dst, gst and ht values were found as 0.054, 0.060 and 0.91, respectively. the mean global coefficient of gene differentiation (gst) showed that approximately 94.0% of the genetic variation was within-population. the highest number of private alleles with frequency above 5% was observed in ouled djellal sheep. as a result, the eight sheep breeds studied show a distinction between them. nevertheless, only the algerian breed ouled djellal which is located near the turkish ovine breeds in the dendrogram, this breed which introduced by the tribes of arabia "benou hilal" coming from the middle east. however, the other breeds (hamra, tazagzawet and sidaou) are native breeds of north africa. in addition, such information provides an overview of the effect of conservation activities on breeding farms, suggesting that we should take some measures to avoid further losses of genetic diversity and minimize inbreeding represented by these breeds as soon as possible. keywords: polymorphism, native sheep breeds, turkey, algeria, microsatellite. mailto:ameurabdelkader@gmail.com https://orcid.org/0000-0002-7450-4311 animal genetic resources: implementation of policies and support oral presentations genetic resources (2021) s1 international congress on sheep and goats 50 abstract book the implementation of international commitments for animal genetic resources in europe and germany holger göderz1 1 federal office for agriculture and food, bonn, germany contact: holger göderz, federal office for agriculture and food, deichmanns aue 29, de-53179 bonn, germany, holger.goederz@ble.de abstract: countries have agreed international and national commitments and strategies for the conservation and use of animal genetic resources (angr). information is provided on the most relevant international commitments: the global plan of action for angr (gpa), the convention on biological diversity and the sustainable development goals with a focus on the situation in germany. in germany, a programme for angr has been adopted in the year 2003. after the adoption of the gpa in the year 2007, this programme has been revised. important actions are a continuous monitoring of all livestock breeds in germany, the establishment of a national gene bank, the providing of public payments for the breeding of endangered breeds and project funding. in the year 2013, the national risk classification system in germany has been revised. since then, the number of breeds that are strongly endangered could be reduced. the stocks of the 19 endangered native sheep breeds increased. one of the success stories is the alpines steinschaf, which has been also facilitated by a wool marketing initiative. its stock of breeding animals increased from 465 (2011) to 1,265 (2019). the stocks of the three native goat breeds have increased by approximately ten percent in this period. the national programme for the conservation and sustainable use of animal genetic resources in germany has proven to be suitable to increase or at least to maintain breeding stocks of endangered breeds and thus to maintain angr as a basis for the adaptability of livestock production. mailto:holger.goederz@ble.de animal genetic resources: implementation of policies and support oral presentations international congress on sheep and goats genetic resources (2021) s1 abstractbook 51 the krainer steinschaf in austria – in situ conservation of an endangered breed beate berger1 and barbara soritz2 1 arec raumberg-gumpenstein, thalheim, austria 2 arche austria, austria contact: beate berger, arec raumberg-gumpenstein, institute of organic farming and biodiversity of farm animals, austrasse 10, 4601 thalheim, austria. +43(0)72424701122, abstract: the austrian conservation breeding program supports 29 breeds of farm animals. the example of the krainer steinschaf shows the network and different approaches necessary to analyze, rebuild and use a highly endangered breed. the krainer steinschaf is a small framed, nonseasonally fertile dairy sheep. the breed belongs to the cluster of rough wool breeds stemming from the medieval zaupelschaf and is well separated from the other endangered austrian sheep breeds. the flock book population increased from 160 animals (1997) to 4071 animals (2020). due to a strictly controlled mating plan based on pedigree analysis, the inbreeding rate in the active breeding population was 0,45% for 2007 and 0,4% for 2020. the breeding organization is responsible for the breeding goals and registration of breeding animals on a genetic basis as broad as possible. a farmer driven marketing organization production systems and quality marketing schemes. pasturing without concentrates is the main production system. on pasture, milk yield drops about two months after lambing with the onset of estrus. twinning rate is not an issue as the ewes can lamb twice a year. for eight years, a quality program of a big food retailer brought up to 1000 lambs per year to market. unfortunately, the program ceased because of reasons beyond control of the breeders. now, on farm marketing is the main distribution channel. breeding goals and marketing issues to improve the conservation of the breed are discussed. mailto:beate.berger@raumberg-gumpenstein.at mailto:beate.berger@raumberg-gumpenstein.at animal genetic resources: implementation of policies and support oral presentations genetic resources (2021) s1 international congress on sheep and goats 52 abstract book is the german white-headed mutton sheep an endangered breed? sowah addo1, stefanie klingel2, georg thaller3 and dirk hinrichs1 1 university of kassel, witzenhausen, germany 2 arche warder zentrum für alte hausund nutztierrassen e.v., warder, germany 3 christian-albrecht university, kiel, germany contact: animal breeding department, university of kassel, nordbahnhofstr. 1a, 37213 witzenhausen, germany; uk069108@uni-kassel.de abstract: german white-headed mutton is a meat type sheep classified as a monitoring population, meaning that the effective population size is between 200 and 1000, and a semen cryoconservation programme should be initiated as soon as the number of adult male animals falls below 100. effective population size estimation for this classification is based on the number of flock book animals, being 1,912 ewes and 102 rams in 2019. in light of the availability of pedigree and marker information, the present study aimed at elucidating the diversity status of german white-headed mutton from pedigree and genome perspectives. our data consisted of pedigree information on 19,000 animals, and on 46 individuals genotyped at 40,753 single nucleotide polymorphism markers. pedigree-based inbreeding coefficient and effective population size estimates were 1.02% and 132 for the whole population, and 3.50% and 99 for a reference subpopulation of animals born between 2012 and 2015, respectively. estimate of average generation interval was 3.24 years. runs of homozygosity-based inbreeding coefficient estimates varied with approximate number of ancestral generations (g) i.e., 1.36% (g = 2), 4.47% (g = 8) and 11.30% (g = 64). a linkage disequilibrium-based effective population size estimate was 53 at 5 generations ago. our estimates of effective population size are above the commonly accepted minimum value of 50 (fao, 1998). however, values below 200 imply, that the german risk classification system could be revised considering both, the method/data to estimate effective population size and the thresholds for assigning breeds to certain risk classes. reference: fao (1998). secondary guidelines for development of national farm animal genetic resources management plans: management of small populations at risk. url: http://www.fao.org/3/a-w9361e.pdf mailto:uk069108@uni-kassel.de http://www.fao.org/3/a-w9361e.pdf animal genetic resources: implementation of policies and support oral presentations international congress on sheep and goats genetic resources (2021) s1 abstractbook 53 marketing situation and concepts for products of endangered local breeds of sheep and goats in germany antje feldmann and katrin dorkewitz gesellschaft zur erhaltung alter und gefährdeter haustierrassen e.v. (geh), witzenhausen, deutschland contact: antje feldmann, gesellschaft zur erhaltung alter und gefährdeter haustierrassen e.v. (geh); walburger str. 2, 37213 witzenhausen, e-mail: feldmann@g-e-h.de, tel.:+49 (0)5542-1864 abstract: many local breeds of sheep and goats are on the red list of endangered livestock breeds in germany (ble, 2019). the project "marketing concepts for products of endangered breeds of farm animals" (menger et al., 2020) analyzed the current marketing situation of endangered breeds with the focus on food retailing. on the basis of questionnaires from 182 farms and an online research it became clear that the majority of the farmers sell their products via direct marketing (81 %). wild fluctuations were found, especially in pricing. the marketing situation was mostly balanced (51.5 %) or demand exceeded supply (33.8 %). sales tests in food retailing showed that consumers are willing to pay premium prices. sales promotion measures are able to stimulate business. products from endangered breeds can be sold very well, especially in urban areas. markups of 65 % on goat's cheese from the thuringian forest goat did not show negative effects on sales. after a period of communication methods the sales figures almost doubled, even without negative effects on the sale of other goat cheese products. there was neither a substitution nor a cannibalization effect. the establishment of products from endangered breeds of farm animals in the food retail trade supports sustainable conservation in agricultural use. the project results enable sheepand goat-breeders and retailers to gain an overview of the communication with the customer, the requirements of the trade and the needs of the farmer and helps them to transfer this knowledge to their own work. references: bundesanstalt für landwirtschaft und ernährung (ble) (2019): einheimische nutztierrassen in deutschland und rote liste gefährdeter nutztierrassen 2019. online verfügbar unter https://www.ble.de/shareddocs/downloads/de/landwirtschaft/biologische-vielfalt/roteliste.html, zuletzt geprüft am 30.04.2020 menger, k., feldmann, a., dorkewitz, k., hamm, u. (2020): vermarktungskonzepte für produkte gefährdeter nutztierrassen. online verfügbar unter: https://orgprints.org/37582/1/37582-15na168-15na028-geh-unikassel-feldmann-hamm-2020vermarktung-gefaehrdeter-nutztierrassen.pdf, zuletzt geprüft am 30.04.20 mailto:feldmann@g-e-h.de management and animal welfare keynote presentation genetic resources (2021) s1 international congress on sheep and goats 54 abstract book keynote: sheep production in ireland – key factors influencing profitability t.w.j keady teagasc, athenry, co galway, ireland. contact: tim.keady@teagasc.ie abstract: ireland has a population of 4.7 million people and produces enough food to feed 40 million; consequently 85% of its agricultural production is exported. sheep production in ireland is grass-based and lambing generally coincides with the initiation of grass growth in early march. ireland is the fourth largest exporter, worldwide, of sheep meat and the largest net exporter in the eu. the eu is the second largest importer of sheep meat in the world, is only 85% selfsufficient in sheep meat production, and accounts for 88% of irish sheep meat exports. the following are some key factors impacting sheep farm productivity and profitability: 1. use of prolific ewe genotypes (e.g., belclare x) increases annual ewe productivity by up to 0.35 lambs reared per ewe joined. 2. mating flock replacements to lamb at 1 year of age has no negative effect on ewe productivity at 2 years of age and significantly increases ewe lifetime performance. 3. each 0.5 kg increase in lamb body weight (bw) at birth increases weaning weight (100 days) by 1.5 kg and thus reduces age at slaughter by approximately 10 days. optimum birth bw is the highest weight that is compatible with a live, unassisted birth that survives. the optimum birth bw for lambs born as twins and triplets is 0.93 and 0.78 that of singles. 4. each 5-percentage-point increase in the digestibility of the grass silage offered during mid and late pregnancy increases lamb bw at birth by 0.25 kg and ewe bw at lambing by 6.5 kg. soybean meal as the dominant protein source in concentrate offered during late gestation increases lamb bw at birth by 0.3 kg. 5. shearing ewes at housing (mid-december) rather than early summer (may/june) increases lamb bw at birth by 0.7 kg. 6. leaving male lambs entire increases bw at weaning by 1.8 kg and reduces age at slaughter by 16 days without any negative impact on meat eating quality characteristics. 7. lambs managed on a grazed-grass only diet can be consistently finished prior to the end of the grazing season. in a 12-year study (in which concentrate, maximum of 300 g/day, was only offered to lambs being reared as triplets) the mean bw gain pre weaning was 330, 271 and 279 g/day for lambs born and reared as singles, twins and triplets, respectively; corresponding bw weight gains from weaning to drafting for slaughter were 183, 178 and 163 g/day. 8. increasing the feed value of the silage offered to lambs being finished on ensiled forage diets increases bw gain or reduces the level of concentrate supplementation required to achieve a give level performance. 9. many swards are marginal or deficient in minerals (e.g., cobalt). need to identify deficiency based on laboratory analyses and/or veterinary guidance. mailto:tim.keady@teagasc.ie management and animal welfare keynote presentation international congress on sheep and goats genetic resources (2021) s1 abstractbook 55 10. there are a number of eu-funded thematic networks (e.g., sheepnet, eurosheep) that use a multi-actor approach to engage farmers, scientists, advisors/consultants, veterinarians etc. these networks demonstrate that solutions to many producer needs are available, either locally or worldwide, and compile reservoirs of solutions (e.g., www.sheepnet.network, www.eurosheep.network). sheep productivity and performance can be improved dramatically by using existing technologies and information resources, thus potentially increasing farm profitability. http://www.sheepnet.network/ http://www.eurosheep.network/ management and animal welfare oral presentations genetic resources (2021) s1 international congress on sheep and goats 56 abstract book development of an animal-friendly feeding system for horned goats – preliminary results bianca greiner1, stanislaus von korn1, katrin sporkmann2, heiko georg2, andreas kern3, charlotte lutz1 and maren bernau1 1 hochschule für wirtschaft und umwelt nürtingen-geislingen, nürtingen, deutschland 2 thünen-institut für ökologischen landbau, westerau, deutschland 3 bioland erzeugerring bayern e.v., augsburg, deutschland contact: bianca greiner, hochschule für wirtschaft und umwelt nürtingen-geislingen, institut für angewandte agrarforschung, hechingerstraße 12, 72622 nürtingen, phone: 07022 201-271, bianca.greiner@hfwu.de abstract: in germany, particularly in federal state baden-württemberg, dairy goat farming is becoming increasingly important as an alternative source of income (statistisches bundesamt, 2017). in feeding dairy goats there is a need for developing concentrate feeding systems adapted for horned goats, since dehorning is forbidden in germany (animal welfare act, 2019). due to their species-specific behavior, agonistic interactions and horn-induced injuries are often found, especially while competition during feed intake. injuries affect animal well-being, animal health, and result in economic losses (leitner et al., 2007). the project aimed to develop a functional and safe feeding system for horned goats. for further development of concentrated feeding systems, two different systems that are available for hornless goats were selected: a) the lamking double box (lb) (wasserbauer gmbh) and b) the capra box (cb) (dedden/hanskamp). one of each system was installed on two different farms in germany (together 320 goats). both systems differ fundamentally in the way they work. lb works with a sideways swinging door, whereas cb works as a walk through. to evaluate the feeding system, body condition scoring as well as incidence and type of udder and body injuries were evaluated before and after installation. a first result is the functionality of the optimized feeding system in each farm, which will be presented during the talk. data evaluation is still ongoing to be able to give sufficient information about both feeding systems in terms of animal well-being, animal health and economics. funding: the project is funded within the framework of the european innovation partnership "landwirtschaftliche produktivität und nachhaltigkeit" (eip-agri). the funding measure is a measure of the "maßnahmenund entwicklungsplan ländlicher raum baden-württemberg 2014-2020" (mepl iii). the project is funded by the state of baden-württemberg and the "europäischer landwirtschaftsfond für die entwicklung des ländlichen raums (eler)". references: animal welfare act (2019). tierschutzgesetz in der fassung der bekanntmachung vom 18. mai 2006 (bgbl. i s. 1206, 1313), zuletzt geändert durch art. 101 g v. 20.11.2019. leitner, g., silanikove, n., merin, u. (2007). estimate of milk and curd yield loss of sheep and goats with and intrammamary infection and its relation to somatic cell count. small ruminant research 74 (2008), 221-225. doi:10.1016/j.smallrumres.2007.02.009 statistisches bundesamt (hg.) (2017) landund forstwirtschaft, fischerei viehhaltung der betriebe agrarstrukturerhebung 2016, fachserie 3, reihe 2.1.3 mailto:bianca.greiner@hfwu.de management and animal welfare oral presentations international congress on sheep and goats genetic resources (2021) s1 abstractbook 57 influence of housing and management on claw health of swiss dairy goats lisa-marie sailer1, joan-bryce burla1, beat wechsler1, mirjam holinger2, patrik zanolari3 and katharina friedli1 1 federal food safety and veterinary office (fsvo), centre for proper housing of ruminants and pigs, tänikon, switzerland 2 department of livestock sciences fibl, frick, switzerland 3 university of bern, vetsuisse faculty, clinic for ruminants, bern, switzerland contact: lisa-marie sailer, tänikon 1, 8356 tänikon, schweiz, +49 151 59216779 , lisa-marie.sailer@agroscope.admin.ch abstract: dairy goats are commonly housed on deep bedding, at least during winter periods. due to the lack of claw horn wear on abrasive surfaces, goats have a high prevalence of overgrown wall horn. as a result, claw health and locomotion behaviour could be impaired. to evaluate claw conditions and claw lesions, data was collected on 28 dairy goat farms all over switzerland in autumn 2018 and spring 2019. locomotion activity and lying behaviour were recorded by means of msr-loggers. furthermore, management data concerning housing and claw trimming was gathered on all farms. linear and generalized linear mixed-effects models were designed to analyze data. almost all claws showed at least moderate or even severe wall horn overgrowth. horn separation and sole hemorrhages turned out to be the most frequent lesions. severe pathologies and infectious claw diseases did not occur. with severely overgrown claws the risk of developing sole hemorrhages was nearly twice as high as with moderate overgrowth. for horn separation, we could show a positive effect of education of the trimmer and of pasture at the time of data collection. furthermore, season had a significant effect on locomotion activity and on the number of lying bouts per 24 hours. in conclusion, the goats did not seem to be impaired in their locomotion by wall horn overgrowth. however, severe wall horn overgrowth was associated with an increase in the proportion of claws with sole hemorrhages. therefore, regular, frequent and skilled claw trimming should be propagated. references: christodoulopoulos g. foot lameness in dairy goats. research in veterinary science. 2009;86(2):281-4 hill pp, murphy pe, nelson aj, mouttotou n, green le, morgan kl. lameness and foot lesions in adult british dairy goats. the veterinary record. 1997;141:412–6. mailto:lisa-marie.sailer@agroscope.admin.ch management and animal welfare oral presentations genetic resources (2021) s1 international congress on sheep and goats 58 abstract book welfare effects of introducing and separating/reintroducing individual goats antonia patt1, lorenz gygax2, beat wechsler3, edna hillmann2 and nina m. keil3 1 institute of animal welfare and animal husbandry, friedrich-loeffler-institut, celle, germany 2 animal husbandry and ethology, albrecht daniel thaer-insitute of agricultural and horticultural sciences, humboldt-universität zu berlin, berlin, germany 3 centre for proper housing of ruminants and pigs, federal food safety and veterinary office, agroscope, ettenhausen, switzerland contact: antonia patt, dörnbergstr. 25/27, 29223 celle, +49 5141 3846 150, antonia.patt@fli.de abstract: in goat husbandry, several management procedures are associated with regrouping of animals, e.g. introduction of unfamiliar goats or temporary separation and subsequent reintegration. in two experiments, we thus aimed to quantify the effects of a) introducing (for five days) an unfamiliar goat into an established herd using both horned and hornless groups, and of b) separating (two days) and reintegrating (three days) individual goats. in both experiments, we collected data on social interactions, lying and feeding behavior, and concentrations of fecal cortisol metabolites. data were analyzed using linear mixed-effects models. in the first experiment, introduced goats showed substantially longer lying times, shorter feeding times and elevated concentrations of fecal cortisol metabolites. further, introduced goats received most agonistic interactions on the first day. these changes were found in all groups, but were more pronounced in horned goats. during the second experiment, we investigated whether an increased level of contact (visual and tactile) with the original group during separation could reduce the negative effects of separation and reintegration compared to only allowing for acoustic contact with the group. separated goats showed shorter feeding times during separation and higher fecal cortisol metabolites concentrations during both periods. increased contact during separation mitigated these effects. both the introduction of an unfamiliar individual into established groups and the temporary separation of individuals from the rest of the groups led to clear stress responses and should thus be avoided whenever possible. if separation is unavoidable, visual and tactile contact should be permitted to mitigate adverse effects. mailto:antonia.patt@fli.de management and animal welfare oral presentations international congress on sheep and goats genetic resources (2021) s1 abstractbook 59 goat commercialization through increasing productivity of homestead herds in south africa rauri alcock1 and marisia geraci2 1 mdukatshani rural development project, hilton, south africa 2 heifer project south africa, hillcrest, south africa contact: khonya@yebo.co.za abstract: the south africa goat agribusiness project (gap) is a project aimed at commercialising goats owned by african communal farmers in rural areas in south africa. gap has been working for 5 years to pilot interventions to support smallholder farmers focusing on goats as this sector of the economy has never been considered commercial. it is estimated that south africa imports a million goats from its neighbours worth 67 million euro annually. the entire market for goats in south africa is for live sacrifice rather than for meat. even with an urbanising and growing middle class, the demand for these animals and related cultural practices is increasing. through a process of innovation support and learning, gap has facilitated and captured in field work through farmer exchanges and distributed these successes to scale up the productivity across the country. bush encroachment is destroying many grazing areas in the country and linked to successive droughts, making goat farming a successful alternative to livestock farmers in an environment where cattle are on the decline. the gap innovations and interventions focused on low productivity experienced by smallholder women goat farmers left in charge of homes through a legacy of migrant labour system. research in field showed that kid mortality was the biggest production loss, with losses ranging from 60-80% of kids. a pilot involving building enclosures and creep feeding kids from 2 weeks to 3 months reduced mortality to 5%. locally harvested plant materials were processed to feed the kids 100 grams a day. gap also developed an energy block, with 15% protein, that farmers mix and process themselves. a health management and disease control program carried out by 260 community animal health workers who were locally recruited, unemployed youth. they were extensively trained and equipped as an independent business to support communities and their livestock. mailto:khonya@yebo.co.za management and animal welfare oral presentations genetic resources (2021) s1 international congress on sheep and goats 60 abstract book a topical anaesthestic wound formulation diminishes pain responses and improves wound healing of lambs at tail-docking ferrer, l.m.1, lacasta, d.1, ramos, j.j.1, ortín, a.1, tejedor, m.t.2, castells, e.3, ruiz de arcaute, m.1, pérez, m.1, rubira, i.4 and windsor, p.a.5 1 animal pathology department, instituto agroalimentario de aragón-ia2 (universidad de zaragozacita), veterinary faculty of zaragoza, c/miguel servet 177. 50013 zaragoza, spain 2 anatomy, embryology and animal genetics department, ciber cv (universidad de zaragoza-iis), veterinary faculty of zaragoza, c/miguel servet 177. 50013 zaragoza, spain 3 centro clínico veterinario, c/ madre genoveva torres morales, 8, 50006 zaragoza, spain 4 gabinete técnico veterinario s.l. c/ isla conejera s/n. 50013 zaragoza, spain 5 sydney school of veterinary science, the university of sydney, camden, nsw | 2570, australia contact: dlacasta@unizar.es abstract: tail docking is a painful husbandry practice performed routinely in sheep production worldwide. we examined two different procedures for surgical tail docking; with and without general anaesthesia (ga), including the use of a topical wound gel formulation to alleviate pain and improve healing after surgery, containing local anaesthetics lignocaine and bupivacaine, with cetrimide and adrenalin (tri-solfen®; ts). forty-four female lambs with similar weights were recruited into four equal cohorts: groups a and c, the tail was excised with a scalpel without anaesthesia and groups b and d, the tail was surgically excised and stitched under ga. c and d groups were immediately treated with ts. pain-related behaviour was assessed using a numerical rating scale (nrs) developed previously. a trained scientist blinded to treatment observed the lambs immediately (t0), 2.5h (t1) and 5h (t2) after tail docking. following the procedure, the animals were examined daily for 15 days, with lesions photographed. this enabled analysis of wound healing after taildocking and the detection of secondary infections. behavioural observations identified that groups without ga (a & c) displayed significantly less pain-related behaviours immediately after the procedure, especially if treated with ts (c). similarly, the mean of days when animals showed no signs of wound infection was longer in the groups not undergoing ga, especially in those treated with ts. surgical tail-docking without ga but where wounds are immediately sprayed with ts is an affordable and welfare appropriate method of conducting tail docking in lambs, avoiding the unnecessary use of antibiotics. references: lomax, s., sheil, m. and windsor, p.a., 2008. impact of topical anaesthesia on pain alleviation and wound healing in lambs after mulesing. australian veterinary journal 86, 159–168. doi: 10.1111/j.1751-0813.2008.00285.x. lomax, s., dickson, h., sheil, m. and windsor, p.a., 2010. topical anaesthesia alleviates short-term pain of castration and tail docking in lambs. australian veterinary journal 88, 67–74. doi: 10.1111/j.1751-0813.2009.00546.x. orihuela. a. and ungerfeld. r., 2019. tail docking in sheep (ovis aries): a review on the arguments for and against the procedure, advantages/disadvantages, methods, and new evidence to revisit the topic. livestock science 230, 103837. doi: 10.1016/j.livsci.2019.103837. mailto:dlacasta@unizar.es management and animal welfare poster presentations international congress on sheep and goats genetic resources (2021) s1 abstractbook 61 evaluation of behavior during grazing for crossbred hair sheep in the dry caribbean clara rúa-bustamante1, sandra c perdomo ayola1, juan zambrano-ortiz1 and lorena aguayo2 1 corporación colombiana de investigación agropecuaria – agrosavia, ci. motilonia, codazzi-cesar, colombia 2 corporación colombiana de investigación agropecuaria – agrosavia, ci turipaná. cereté –córdoba, colombia. contact: abstract: the aim was to evaluate the behavior of crossbreed hair sheep, grazing under dry caribbean agro-ecological conditions from north of colombia. the behavior of nine males (300d) in a pasture composed of native trees, shrubs, weeds and different tropical grasses, was recorded. during four consecutive days, an instantaneous sampling was performed between 8:00 and 15:30, every 15 min. the behavioural events recorded were grazing, eat fruit (mango), browse, resting, walking, ruminating, drinking, others. the temperature (t) and relative humidity (h) were stored in dataloggers. the ith index was calculated. for the statistical analysis, three blocks were formed: morning, noon and afternoon, using sas 9.4 statistical software. the general behavioural budget was 56.4% grazing, 12.4% eating fruit, 9.8% browsing, 7.6% rest, 6.4% walking, 5% ruminating, 0.1% drinking, 0.1% others. most grazing was during afternoon (44.9%) , then morning (39.8%) and noon (15%), where the ith index was respectively 74.8 (t=23.7 ± 0.31°c, rh= 100%), 74.1 (t= 23.3 ± 0.1°c, rh 100%) and 73,7 (t= 23.1 ± 0.17°c, rh 100%). this could suggest that grazing frequency does not depend on this indicator. the consumption of mango (49.2%) and browsing (49%), was mainly in the morning. the frequency of behaviours appears to not be limited by the ith-related variables rated within normal values. more studies are necessary to determine if the intensity of ultraviolet radiation is a factor that may be influencing the behaviour of sheep in general, and consumption specifically. mailto:crua@agrosavia.co management and animal welfare poster presentations genetic resources (2021) s1 international congress on sheep and goats 62 abstract book sheep farming in marginal lands of central italy: recognition of common failures during shearing sebastian alessandro mignacca1, claudio forte1, laura vieceli2, nigel thompson3, luca schillaci4, chiara francesca magistrali1 and marco antonini5 1 istituto zooprofilattico sperimentale dell’umbria e delle marche ‘togo rosati’, perugia, italy 2 department of veterinary medicine, university of perugia, perugia, italy 3 biella the wool company, biella, italy 4 gran sasso e monti della laga park, l'aquila, italy 5 italian national agency for new technology, energy and sustainable economic development (enea) roma, italy contact: [via gb giuliano, catenanuova (enna) 94010, italy, sebastian.mignacca80@gmail.com] abstract: europe has a worldwide leading position in environmental and animal welfare legislation. among other good practice recommendations, guidelines describing the recommended management of sheep shearing have been defined. “woolfair” is a project enrolling 95 farms (approximately 18,000 heads) aimed at addressing resilience, animal welfare and competitiveness of sheep farming in marginal lands in central italy. major errors observed during the shearing season of 2019 in 25 farms (approximately 5900 heads) are reported in this work. most of the farms showed the peculiarity of starting the shearing in march, in order to transhumate with a minimum of fleece for the highest pastures. errors were detected in all the shearing phases. during pre-shearing, inadequate protection of animals against weather conditions was recorded in 3 out of 25 farms, non-fasted animals in 15 farms, overcrowding in 5 farms, and inadequate restraint and manipulation in 15 farms. shearing phase revealed inadequate restraint and manipulation in 15 out of 25 farms, excessive presence of wounds in 8 farms, excessive time of shearing in 7 farms. post-shearing management resulted in inadequate protection of animals against weather conditions in 3 farms while inadequate nutrition supplementation was recorded in 10 farms. due to the local peculiar environment and farming systems, it does not result easy to adapt shearing good practices. during the onfarm visits, improvements to the shearing management and the link between animal welfare and wool quality have been discussed with stakeholders and easily applicable guidelines for a correct shearing procedure have been provided. mailto:sebastian.mignacca80@gmail.com management and animal welfare poster presentations international congress on sheep and goats genetic resources (2021) s1 abstractbook 63 handling sheep without docking of the tail demonstration project animal welfare martin steffens hesse department of agriculture affairs (landesbetrieb landwirtschaft hessen), wetzlar, germany contact: schanzenfeldstr. 8, 35578 wetzlar, +49 64419289-372, martin.steffens@llh.hessen.de abstract: the german federal ministry of food and agriculture supports the “demonstration project animal welfare” through the federal office of agriculture and food to give famers the opportunity to change husbandry conditions in order to improve welfare to the sheep. in total 6 farmers were chosen to keep sheep with long tails. the participants started in 2017. the docking is done in particular for hygienic reasons. most of the breeds of sheep bred in germany have long, woolly tails that can be very dirty. the contaminated areas can be used by various types of flies to lay their eggs or larvae and lead to so called myiasis (fly maggot infestation) in affected sheep. by shortening the tail, the above-mentioned fecal contamination can be reduced. although scientific studies have not unanimously linked tail length and the likelihood of myiasis, blowfly infestation is one of the main reasons for tail cropping in sheep. the keeping of uncrossed long-tailed breeds is hardly practiced in germany. throughout germany, up to six companies were sought for the thematic network “no tail docking in sheep lambs” who would like to participate in this pilot project as demonstration companies over a period of two and a half years. to handle this challenge, the improvement of all areas in the environment of the sheep e.g. food, stable management, genetics, parasite management, and much more is necessary. the hesse department of agriculture affairs, represented by martin steffens, was instructed by the federal office of agriculture and food to perform the advising part for these farmers and can show the results and options to handle sheep with long tails. mailto:martin.steffens@llh.hessen.de management and animal welfare poster presentations genetic resources (2021) s1 international congress on sheep and goats 64 abstract book comparison of concentrate and flour mixture digestibility results in purebred latvian dark-head lambs liga senfelde1, daina kairisa1and dace barzdina1 1 latvia university of life sciences and technologies, faculty of agriculture, institute of animal science, street liela 2, lv-3001 jelgava, latvia contact: latvia university of life sciences and technologies, faculty of agriculture, institute of animal science, street liela 2, lv-3001 jelgava, latvia, abstract: research has been conducted to compare the efficiency of concentrate and flour mixture digestibility in lambs during fattening. purebred latvian dark-head ram lambs were used in the research. feed were provided ad libitum for two groups: con (concentrate and hay) and bnf (hay and flour mixture consisting of 50% beans, 25% barley and 25% oats). the mean live weight of the lambs at the start of research was 24.6 kg (con) and 25.6 kg (bnf), with a mean age of 83 ± 1.4 days (con; p <0.05) and 75 ± 1.6 days (bnf). feed digestibility data were recorded in three periods each for five days in week 3 (f), 6 (s) and 9 (t) of the fattening when the lambs were placed in cages with a slatted wooden floor and a container with a grate under it for faecal and urine production. the average daily consumption of concentrate for con were higher and ranged from 1.25 ± 0.11 kg (f) to 1.75 ± 0.09 kg (t). the average daily hay consumption were higher for bnf and ranged from 0.23 ± 0.03 kg (f) to 0.18 ± 0.03kg (t). higher faecal production per lamb were collected in bnf (0.85 ± 0.05 kg (f) to 0.96 ± 0.07 kg (t)). the proportion of digested dry matter during the research ranged from 76.3% to 80.2% (con) and from 63.1% to 77.9% (bnf). a faecal fraction was soft (con) and solid (bnf). mailto:shenfeldel@gmail.com management and animal welfare poster presentations international congress on sheep and goats genetic resources (2021) s1 abstractbook 65 problems with the back teeth! a relevance for breeding sheep and goats in germany? philip christian tegtmeyer tierarztpraxis tegtmeyer sheep and goat health consultancy – langenhagen, germany contact: dr. med. vet. philip c. tegtmeyer, resser str. 1a, 30855 langenhagen, germany, mail@tierarztpraxis-tegtmeyer.de abstract: teeth problems, particulary the defective position of molar teeth might have a considerable importance for malnutrition of sheep and goats in germany. it is recognised in elder animals mainly. it is generally known that the set of teeth of small ruminants wears down over the years. this might be influenced by the type of feed. because of a following defective position of back teeth and loss of teeth sheep and goats often cannot eat adequately and fast enough. loss of incisor teeth is a common finding in german sheep flocks. in most cases, the animals do not suffer from the loss of incisors but they are often culled. in the author`s experience we have to look closer at the back teeth instead of the common practice of controlling incisors. what kind of teeth problems can be detected? comparable with horses, small ruminants develop sharp edges on the molar teeth caused by the chewing process. these edges often overtop the other teeth and cause mechanical irritations when animals are chewing. often, the corresponding teeth of the other side are damaged by these edges. the prevalence of back teeth disorders is not known. in order to get some numbers 18 skulls of preselected old ewes were examined for teeth disorders after slaughter. eight ewes had back teeth problems which caused functional damage. in postmortem examinations and inspections of the oral cavity often massive failures of the denture can be detected. back teeth disorders can also be detected in younger sheep. problems can become present for example under second dentition of gimmers. the defective positions of molar teeth may advance the hypothesis that these problems are heritable. first of all, a prevalence study is needed for a real confirmation of the hypothesis. furthermore, research on the aspect of the hereditability has to be conducted. references: erjavec, v., crossley,d. (2010). initial observations of cheek tooth abnormalities in sheep in slovenia. veterinary record 167, 134-137 laws, aj., baker, rl., aitken, wm. (1993). the heritability of gingival crevice depths in sheep. res vet sci. may;54(3):379-83. kaulfuß, k.h., hoffmann, b., (2004). erkrankungen der kiefer und zähne beim schaf (übersichtsreferat). literaturübersicht und ergebnisse eines zuchtversuchs zur brachygnathia inferior beim ostfriesischen milchschaf. tierärztl. umschau 59, 380-387. mailto:mail@tierarztpraxis-tegtmeyer.de management and animal welfare poster presentations genetic resources (2021) s1 international congress on sheep and goats 66 abstract book effect of a topical anaesthetic formulation on viral load in lambs naturally infected with orf virus lacasta, d.1, reina, r.2, ramos, j.j.1, ferrer, l.m.1, benito, a.a.3, tejedor, m.t.4, martínez, s.1, ruiz, h.1, echeverria, i.2 and windsor, p.5 1 animal pathology department, instituto agroalimentario de aragón-ia2 (universidad de zaragozacita), veterinary faculty of zaragoza, c/miguel servet 177. 50013 zaragoza, spain 2 instituto de agrobiotecnología (csic-gobierno de navarra), av. pamplona, 123, 31192 mutilva baja, navarra, spain 3 laboratorios exopol diagnóstico y autovacunas, polígono san mateo de gállego, spain 4 anatomy, embryology and animal genetics department, ciber cv (universidad de zaragoza-iis), veterinary faculty of zaragoza, c/miguel servet 177. 50013 zaragoza, spain 5 sydney school of veterinary science, the university of sydney, camden, nsw | 2570, australia contact: dlacasta@unizar.es abstract: orf is a highly contagious eruptive skin condition of sheep and goats. vaccination with live orf virus is the preferred option for disease control, despite this vaccine is unavailable in many countries. treatment of orf lesions involves standard hygiene and management of presumptive secondary infections with antibiotics. the wound dressing formulation, tri-solfen® (animal ethics pty ltd, australia) offers advantages over current therapies, providing pain relief and potentially, more rapid healing of lesions. the formulation contains two local anaesthetics (lignocaine and bupivacaine), adrenalin and an antiseptic (cetramide) in a gel formulation, and creates a ph of ~2.7 that is potentially viricidal. fourteen one-month-old lambs, naturally infected with orf, were recruited from a farm during an outbreak of orf disease. the animals were selected at the early stages of the infection and divided into two cohorts: group a (n=11) treated with tri-solfen® and group b (n=3), a control group without treatment. swabs were obtained before treatment (t0) and days 1 (t1), 3 (t2) and 5 (t3) post-treatment, then submitted to direct dna extraction and real-time pcr quantification (exopol) or to incubation with primary tissue cultures from ovine skin fibroblasts (osf) and t-immortalized goat embryonic fibroblasts (tigef). in the study conducted using quantitative pcr, no significant differences were found (p=0.722). however, when the viral load was assessed in osf cell cultures, there was a significant difference (p<0.05) in reduction between both groups between t0 and t3. these findings suggest that treatment of orf lesions with tri-solfen® reduces the viral load present in lesions. references: lomax, s., sheil, m. and windsor, p.a., 2008. impact of topical anaesthesia on pain alleviation and wound healing in lambs after mulesing. australian veterinary journal 86, 159–168. doi: 10.1111/j.1751-0813.2008.00285.x. roberts, c.d. and windsor p.a., 2019. innovative pain management solutions in animals may provide improved wound pain reduction during debridement in humans: an opinion informed by veterinary literature. international wound journal 16 (2) doi: https://doi.org/10.1111/iwj.13129. spyroua, v., valiakosb, g., 2017. orf virus infection in sheep and goats. veterinary microbiology 181 (2015) 178–182. doi: http://dx.doi.org/10.1016/j.vetmic.2015.08.010. mailto:dlacasta@unizar.es https://doi.org/10.1111/iwj.13129 http://dx.doi.org/10.1016/j.vetmic.2015.08.010 management and animal welfare poster presentations international congress on sheep and goats genetic resources (2021) s1 abstractbook 67 management and animal welfare poster presentations genetic resources (2021) s1 international congress on sheep and goats 68 abstract book effect of a topical wound anaesthesia formulation on the cortisol and the acute phase responses of lambs undergoing tail docking. ortín, a.1, borobia, m.1 ramos, j.j.1, lacasta, d.1, ferrer, l.m.1, tejedor, m.t.2, ruiz, h.1, jimenez, c.3,4 and windsor, p.a.1 1 animal pathology department, instituto agroalimentario de aragón-ia2 (universidad de zaragozacita), veterinary faculty of zaragoza, zaragoza, spain 2 anatomy, embryology and animal genetics department, ciber cv (universidad de zaragoza-iis), (universidad de zaragoza-cita), veterinary faculty of zaragoza, zaragoza, spain 3 gabinete técnico veterinario s.l. c/ isla conejera s/n. 50013 zaragoza, spain 4 sydney school of veterinary science, the university of sydney, sydney, australia contact: aurora ortín, aortin@unizar.es abstract: in this study we evaluated the effect of a topical wound gel formulation containing local anaesthetics lignocaine and bupivacaine, with cetrimide and adrenalin (tri-solfen®; ts) on the concentrations of serum cortisol (sc) and the acute phase protein serum amyloid a (saa) in tail-docked lambs. forty-four female lambs with similar weights were recruited into four equal cohorts: groups a and c, the tail was excised with a scalpel without anaesthesia and groups b and d, the tail was surgically excised and stitched under general anaesthesia (ga). c and d groups were immediately sprayed with ts. blood samples were collected before tail docking and at different time intervals post-tail excision. concentra-tions of sc and saa were determined using elisa assays (salivary cortisol elisa slv-2930, drg diagnostics, marburg, germany; phase tm serum amyloid a assay, tridelta development ltd., maynooth, ireland). statistical analysis was preformed using ibm spss statistics version 26 (2019) software (ibm, armonk, ny, usa). sc concentration did not change significantly over time in cohorts tail-docked under ga (b & d), but peaked at 30 min post tail removing without anaesthesia (a & c), and treatment with ts (c) appeared to reduce this cortisol response. in cohorts b & d , saa concentrations increased significantly 48 hours after tail docking, as it is expected after a noxious stimulus, but treatment with ts avoided the elevation of saa at this time point in lambs where the tail was excised without anaesthesia. these results appear to indicate that treatment with ts reduces cortisol and saa responses in lambs tail docked without anaesthesia, although further research is needed to corroborate these findings. references: marini, d., colditz, i.g., hinch, g., petherick, j.c., lee, c., 2017. self-administration by consumption of flunixin in feed alleviates the pain and inflammation associated with castration and tail docking of lambs. appl. anim. behav. sci. 188, 26–33. doi: 10.1016/j.applanim.2016.12.008. paull, d.r., lee, c., colditz, i.g., atkinson, s.j., fisher, a.d., 2009. the effect of a topical anaesthetic formulation, systemic flunixin and carprofen, singly or in combination, on cortisol and behavioural responses of merino lambs to mulesing. aust. vet. j. 85, 98–106. doi: 10.1111 /j.1 751 -0813.2009.00429.x. tothova c, nagy o, kovac g. 2014. acute phase proteins and their use in the diagnosis of diseases in ruminants: a review. vet. med. 59, 163-168. doi: 10.17221/7478-vetmed. mailto:aortin@unizar.es other topics keynote presentation international congress on sheep and goats genetic resources (2021) s1 abstractbook 69 keynote: team sheep vs. team corporate team building and leadership training as additional income source for shepherds hans-peter etzold natuerlichteambuilding.de, andernach, germany contact: im rosental 23, d-56626 andernach, peter@leadalpha.eu, spitzenplatz@gmail.com https://www.natuerlichteambuilding.de/ abstract: team building activities and leadership training are additional income sources for professional shepherds. the innovative activity combines the experience of nature with immediate feedback regarding leadership and teamwork styles. with the help of a management coach and under supervision of the shepherd corporate teams lead and drive a herd of sheep and goats from a to b in a touristically attractive area. while the sheep and goats are grazing during breaks, the teams reflect upon their performance and discuss the lessons learnt related to the herding of the sheep, but also their leadership styles and teamwork. the author is the pioneer of sheep herding as a team building and leadership training activity and offers this kind of activity in cooperation with local shepherds to sme and global corporations. since 2015 he has served approx. 350 clients. shepherds are required to have at least 500 sheep and goats in an attractive landscape preferably within one hour’s drive of a major city. the shepherds are also required to consider themselves as service providers to professionals from outside the primary sector. references: https://www.natuerlichteambuilding.de/ https://programm.ard.de/tv/programm/sender/?sendung=28106372186471 kevin leman, william pentak: das hirtenprinzip, sieben erfolgsrezepte guter menschenführung, 2010. mailto:peter@leadalpha.eu mailto:spitzenplatz@gmail.com https://www.natuerlichteambuilding.de/ https://www.natuerlichteambuilding.de/ https://programm.ard.de/tv/programm/sender/?sendung=28106372186471 https://www.amazon.de/das-hirtenprinzip-sieben-erfolgsrezepte-menschenf%c3%bchrung/dp/3442172187 other topics oral presentations genetic resources (2021) s1 international congress on sheep and goats 70 abstract book zoonotic diseases in small ruminants: risks and opportunities benjamin bauer1, martin runge2, annika wolf1, louise prüfer2, laura wiesner3, imke steffen3, cornelia silaghi4, matthias wagener1, wibke rubel1 and martin ganter1 1 university of veterinary medicine hannover, foundation, clinic for swine and small ruminants, hannover, germany 2 lower saxony state office for consumer protection and food safety (laves), food and veterinary institute braunschweig/hannover, hannover, germany 3 university of veterinary medicine hannover, foundation, institute for biochemistry and research center for emerging infections and zoonoses, hannover, germany 4 friedrich-loeffler-institut, institute of infectiology, greifswald – isle of riems, germany contact: university of veterinary medicine hannover, foundation, clinic for swine and small ruminants, bischofsholer damm 15, 30173 hannover, germany; benjamin.bauer@tiho-hannover.de abstract: the awareness of zoonotic diseases in small ruminants has increased within the last two decades. most of the zoonotic pathogens cause abortion in sheep and goats (ganter 2015). for instance, the intracellular bacteria coxiella burnetii affected more than 4,000 people in the netherlands from 2007 until 2011. large dairy goat farms were responsible for this q fever epidemic. in contrast, lambing sheep are responsible for small-scale q fever epidemics across germany. a recent study revealed a prevalence between 31.3% and 33.8% in german sheep flocks (wolf et al. 2020). main risk factors are purchases of sheep and goats and year-round lambing. an active monitoring and surveillance system followed by strategic vaccination programs in small ruminant flocks are suitable to prevent q fever in humans. furthermore, there are several zoonotic vector-borne diseases harming small ruminants and humans alike. these are mainly transmitted either by ticks or by insects. sheep and goats have a limited home range, are available in large numbers, are well dispersed in the environment and show a long-lasting antibody response after natural infections (gerth et al. 1995). in consequence, small ruminants seem to be ideal sentinels for surveillance of zoonotic vectorborne pathogens like tick-borne encephalitis virus (tbev), west nile virus, crimean-congo hemorrhagic fever virus and anaplasma phagocytophilum. for instance, recent examinations of small ruminant sera determined a tbev intra-herd prevalence of 12.5% in a goat flock. this flock is located in a non-tbev risk area in northern germany and a potential new endemic area of tbev was identified. according to the one-health approach, it is necessary to implement efficient measures to control zoonotic livestock diseases. however, it is also essential to increase the awareness and preparedness of the public health sector for zoonotic vector-borne diseases using small ruminants as sentinel animals. references: ganter, m. (2015). zoonotic risks from small ruminants. vet microbiol 181, 53-65. doi: 10.1016/j.vetmic.2015.07.015. gerth, h.-j., grimshandl, d., stage, b., döller, g., kunz, c. (1995). roe deer as sentinels for endemicity of tick-borne encephalitis virus. epidemiol infect 115, 355-365. doi: 10.1017/s0950268800058477 wolf, a., prüfer, t. l., schoneberg, c., campe, a., runge, m., ganter, m., bauer b. u. (2020). prevalence of coxiella burnetii in german sheep flocks and evaluation of a novel approach to detect an infection via preputial swabs at herd-level. epidemiol infect 148, e75. doi: 10.1017/s0950268820000679 mailto:benjamin.bauer@tiho-hannover.de other topics oral presentations international congress on sheep and goats genetic resources (2021) s1 abstractbook 71 comparative fattening and slaughter performance of four sheep breeds under practical farming conditions in northern germany regina roessler1, stefanie klingel2, nina ossowski3, georg thaller3 and dirk hinrichs1 1 university of kassel, section animal breeding, nordbahnhofstr. 1a, 37213 witzenhausen, germany 2 arche warder zentrum für alte hausund nutztierrassen e.v., langwedeler weg 11, 24646 warder, germany 3 christian-albrechts-universität zu kiel, section animal breeding and genetics, hermann-rodewaldstraße 6, 24118 kiel, germany contact: head of section animal breeding, university of kassel, nordbahnhofstr. 1a, 37213 witzenhausen, dhinrichs@agrar.uni-kassel.de abstract: the production of lamb meat has become more important than wool production in the past decades due to changing market conditions. therefore, the influence of breed on fattening and slaughter performance was studied in 20 charollais, 40 german white-headed mutton, 17 suffolk and 13 texel male lambs in a pasture feeding trial in eiderstedt over a 2-month period. the aim was to identify animals that perform well under the practical farming conditions in northern germany. analysis of variance or kruskal-wallis tests were performed in r 3.6.1. tukey-hsd test was used for pairwise comparisons of the corrected mean values. corrections were made for the age, the liveweight or the cold carcass weight. a significant effect of breed on the fattening and slaughter performance was observed. initial liveweight was highest in suffolk (46.7 kg) and lowest in german white-headed mutton (40.4 kg). the highest final liveweight (61.3 kg), carcass weight (28.1 kg warm, 26.7 kg cold) and daily weight gain (271 g) were observed for charollais. suffolk had the lowest daily weight gain (161 g/d), and their carcass weight was nearly 2 kg lighter than for charollais. however, no breed differences were observed for net daily gain. the german white-headed mutton were genetically inferior with regard the subcutaneous fat (0.57 cm) and ultrasonic muscle thickness (26.3 mm), while texel had similar slaughter performance than charollais. the carcass examination showed no breed differences in the incidence of pulmonary pneumonia. in conclusion, no sheep breed was clearly superior in all the studied performance traits. mailto:dhinrichs@agrar.uni-kassel.de other topics oral presentations genetic resources (2021) s1 international congress on sheep and goats 72 abstract book udder health in dairy goats and dairy sheep in hesse irene noll regional council of gießen, wetzlar, germany contact: irene noll, schanzenfeldstr. 8, dezernat 51.2, 35578 wetzlar, abstract: since milk of small ruminants, unlike dairy cows, is largely processed on the farm itself, often as raw milk, the increased microbiological risk requires special care. the occurrence of individual, mostly serious clinical udder diseases contrasts with a multiple of subclinical udder diseases. in order to detect subclinical diseases, the content of somatic cells in milk can be estimated using the california mastitis test (cmt). in case of a cumulative or increasing number of half-differences, half-milk samples are taken. these milk samples are sent to a recognized laboratory for cytobacteriological analysis. in subclinical mastitis in small ruminants, coagulase-negative staphylococci are mostly found. in contrast, staphylococcus aureus (s. aureus) is usually the triggering agent in clinical mastitis, occasionally pasteurella multocida, mannheimia haemolytica, escherichia coli and pseudomonads can be found. since s. aureus can also often be detected in wounds, greater attention should also be paid to teat and udder wounds as part of a rehabilitation, as these can represent a constant reservoir of pathogens. this mainly happens when the lambs/ fawns have not yet been weaned and the dams are already being milked. as part of the official milk hygiene monitoring, cytobacteriological total stock examinations are regularly carried out on dairy goat and dairy sheep farms in hesse. the results are presented. mailto:irene.noll@rpgi.hessen.de other topics poster presentations international congress on sheep and goats genetic resources (2021) s1 abstractbook 73 goat production systems characteristics and management practices in zambia kolawole odubote university of zambia, lusaka, zambia contact: department of biomedical sciences, school of veterinary sciences, university of zambia, email: kola.odubote@gmail.com abstract: this study was undertaken to describe goat production system characteristics and management practices in zambia. the data collected during the zambia 2017/2018 livestock and aquaculture census were analyzed using both, qualitative and quantitative methods. the main production system was essentially traditional and extensive with free range being the dominant feeding practices. the management practices were minimal although main purpose of keeping goats were for sale and income. the constraints to goat production were mostly on the inadequacies of the production systems and limited management practices such as disease control, management of grazing lands, feeds and feeding, theft, livestock extension services, access to water, distance to dipping facilities, access to quality breeding stock, record keeping, access to credit and market development amongst others. the study also revealed a wide array of genetic resources of local breeds, exotic breeds and crosses between and among them. however, the local-exotic crosses represented the largest single genetic group, which was a reflection of government and development organizations programmes and the farmers’ desire to improve productivity through crossbreeding although indiscriminately carried out. it is therefore imperative that a holistic system approach is required to develop the goat sector in zambia. a community based goat breeding programme to optimally utilize available genetic resources and livestock extension services to reach farmers with husbandry skills that include crop livestock integration, promotion of pasture production with high yielding fodders, management of communal grazing lands, hygienic practices, disease control and market development would be key steps to improve production and productivity. mailto:kola.odubote@gmail.com other topics poster presentations genetic resources (2021) s1 international congress on sheep and goats 74 abstract book gender roles in goat production in ogbomoso region, southwestern nigeria david aderemi ajayi, saheed adeyemi busari, comfort oluwabori durosinmi, sunday olufemi osewa, olusola jolaiya and oluwasogo dammy abegunrin national centre for genetic resources and biotechnology, moor plantation, ibadan, nigeria. contact: animal genetic resources department, national centre for genetic resources and biotechnology, pmb 5382moor plantation, ibadan, nigeria. rhemiajayi@gmail.com tel: +2348034073868 abstract: women play important roles in livestock production in developing countries. information on their roles in goat production activities is however not sufficiently documented in literature and hence a study was conducted to document gender roles in goat production in ogbomoso region of southwestern nigeria. data was collected by using questionnaire administered to142 purposively selected farmers who kept goats on personal information and goat production related activities. about 51.10 % of the farmers interviewed were aged 41 years and above. the breed reared by 90 % of the respondents was west african dwarf goats. furthermore, 60 % of the farmers reared the animals for meat purpose while 36% kept goats to serve as a source of income. the major constraints in goat production enterprises included lack of capital, diseases and thefts of the animals. majority of the farmers agreed that the roles of selection of foundation stock, pen construction, caring for sick animals, slaughtering and processing of slaughtered animals are played by men while women played leading roles in feeding, caring for young animals, cleaning of the pens and marketing of goats. in conclusion, men play more roles than women especially in those activities requiring more energy in goat production in ogbomoso region of southwestern nigeria. mailto:rhemiajayi@gmail.com other topics poster presentations international congress on sheep and goats genetic resources (2021) s1 abstractbook 75 comparative study of some physiochemical properties in milk from murciano granadina goats which had, or not, been treated with antibiotics laura almela1, begoña peinado1, sonia galián1, ángel poto1, elisa escudero2 and pedro marín2 1 murcian institute for agricultural and food research and development (imida), la alberca-murcia, spain 2 department of pharmacology, murcia region, spain contact: laura almela, murcian institute for agricultural and food research and development (imida), la alberca-murcia, spain. email: laura.almela@carm.es, telephone: +34 968 366756 abstract: some physicochemical properties have been compared in the milk from murciano-granadina (m-g) goats which had been given antibiotics (a) and milk from untreated goats (na), to find out if the addition of antibiotics makes any difference in the processing of the milk. of the total goats in the study, 20 goats were not treated at all and 6 goats were given antibiotics. the milk collection was daily, separating milk a from milk na. thirty-six samples (18 a and 18 na), from different days over 2 months, were analysed. milk a and milk na were subjected to a pasteurization process, and once cooled to 40ºc commercial rennet was added (enzymatic coagulation). the coagulation point was timed and measured using the “buttonhole technique”, checking the coagulation status every 5 seconds. the rennet and whey were separated and the following parameters were measured: whey amount, rennet weight, whey and rennet ph, and rennet colour. the results showed a milk a curdling time of three minutes and fourteen seconds, compared to a milk na curdling time of three minutes and four seconds. according to the statistical comparison study, there are no significant differences between them for this variable, nor for the other variables studied. the results indicate that after the pasteurisation process there are no significant differences between the milk from those goats treated with antibiotics and those which weren’t. neither are there any observable alterations in the milk processing process. other topics poster presentations genetic resources (2021) s1 international congress on sheep and goats 76 abstract book goat semen collection with two different sexual stimuli begoña peinado1, ángel poto1, laura almela1 and sonia galián1 1 instituto murciano de investigación y desarrollo agrario y alimentario, la alberca, murcia, spain contact: dra. begoña peinado, equipo de mejora genética animal, instituto murciano de investigación y desarrollo agrario y alimentario, 30150 la alberca, murcia, spain abstract: the aim of this work was to know if it was possible to perform semen collection from bucks of the murciano-granadina breed, previously trained in semen collection with females of the breed, using another male as a sexual stimulus and to determine if there are differences in their sexual behaviour, libido and seminal quality with respect to that obtained when the stimulus is a female. we compared the data from the training period of 8 bucks under one year of age with those obtained in the same bucks when they were sexually stimulated by a female or another buck. we assess the reaction time, ejaculate volume, total sperm concentration, individual sperm motility and percentage of motile sperm in each ejaculate. it was also compared if the number of rides in a morning was different. after statistical analysis of the results, we observed that the reaction time was lower once the males had been trained, but it did not differ whether the lure was another male or a female. seminal quality was better after the training period due to the older age of the bucks, but did not show large differences in any of the parameters evaluated when using a female or a male and the number of services performed by the bucks in one morning was also the same. we conclude that the semen collection from bucks can be done using another male as a stimulus, which would avoid the repeated use of hormones to females, improving animal welfare. other topics poster presentations international congress on sheep and goats genetic resources (2021) s1 abstractbook 77 sira smart scale, a milk lamb weight scale that helps to introduce maternal index in extensive meat breed breeding productivity marta gómez segura1, jesús ochoa2, manuel quinteiro3 and juan altarriba4 1 arana, navarra, spain 2 intia, navarra, spain 3 compañía de instrumentación y control, alcobendas, spain 4 zaragoza veterinary university, zaragoza, spain contact: marta gomez segura, arana, ainziburu s/n, 31170 iza, navarra (spain), mgomez@arana.centroiza.com abstract raza navarra is a north spain extensive meat breed. after 30 years breeding to select the most prolificably maternal lines we are trying to introduce maternal index in order to improve productivity and profit. handling to weight milk lambs in an extensive breed means lots of problems farmers are not willing to assume. nowadays, the sheep sector lacks financial return, it is aged and farmers are discouraged, but even then, it needs imaginative technical improvements to try to increase its profits. selecting ewes by their maternal index could reduce the time lambs need to reach their optimal weight to go to the market, which means costs are likely to be lower. sira smart scale is a milk lamb weight scale which registers individual lamb weight without farmer interaction. it includes a camera that discriminates between one or more lambs on the scale and if they have fully stepped on it. lambs are identified by means of an electronic ear tag so the scale registers their individual number and the date they have been weighed. with this poster we would like to show the material, method and results obtained with sira smart scale during 2019-2020 in several raza navarra herds. results: during 2019, 12,642 lambs have been weighed. data shown below are from a single herd, where 1762 lambs where weighed (they mean 90% of lambs born during 2019) on average, the lambs had 37 days of life the day of weighing. results shown the average daily weight gain is 0.211 grams per day. the ewe age does not seem to interfere on lamb weight. female lambs weight nearly a kilogram less than males at day 37 of birth. on average, at day 37 of birth, single born lambs weigh 1.257 kg more than doubles and 1.730 kg more than triples, mailto:mgomez@arana.centroiza.com other topics poster presentations genetic resources (2021) s1 international congress on sheep and goats 78 abstract book feeding dairy sheep and goats george christodoulopoulos clinical veterinary medicine dept, faculty of veterinary science, university of thessaly, karditsa, greece contact: p.o. box 199, karditsa, gr-43131, greece; tel: 00306942061708; email: gc@vet.uth.gr abstract: on the farm, dairy sheep and goats are usually fed a mixture of concentrates as well as a kind of roughage. often, the advice given by the practitioners on what quantities they should be fed is empirical. here are our calculations which determined what quantities they should be fed. our calculations are intended for mixtures of concentrates containing 15-17% crude proteins and 20-25% digestive fibers; therefore the mixtures are “total mixed rations”, as they are meant to feed each head over 0.5 kg per day (thonney and hogue, 2013; ahdb, 2018). in a dairy ewe farm, the quantities of roughages (x) and concentrate mixture (y) that should be fed can be calculated using the formulas: x = 7.1l+0.085w−1.2 bw k +2.85 a− bq k and y = 1.2 w k q k x, where x and y are given in kg of dry matter; l is the daily milk yield (kg), w the live weight of ewe (kg), k and q the percentage of neutral detergent fibers and a and b the amount of metabolisable energy (me) in the concentrates and roughage correspondently. since the me of the concentrates is greater than that of the roughage, if the animals consume more roughage than the quantity calculated their me needs will not be met. however, if the required quantities of roughage are not available, any available amount may be fed and the missing quantity may be replaced by a quantity of concentrates equivalent in me to meet the livestock’s needs. references: thonney, m.l., and hogue, d.e. (2013) fermentable fiber for diet formulation. in proceedings of the cornell nutrition conference, ithaca. ahdb (2018). feeding the ewe. a manual for consultants, vets and producers. (united kingdom: better returns programme), 64 p. mailto:gc@vet.uth.gr animal health: parasitology oral presentations international congress on sheep and goats genetic resources (2021) s1 abstractbook 79 potentials of using milk performance data as indicator for targeted selective treatment in lacaune dairy sheep in switzerland katharina schwarz1,2, beat bapst3, mirjam holinger1, inga schleip2 and steffen werne1 1 research institute of organic agriculture (fibl), po box, 5070 frick, switzerland 2 eberswalde university for sustainable development, schicklerstrasse 5, 16225 eberswalde, germany 3 qualitas ag, chamerstrasse 56, 6300 zug, switzerland contact: steffen werne, ackerstrasse 113, 5070 frick, switzerland, email: steffen.werne@fibl.org abstract: anthelmintic resistance is a major threat in small ruminant farming worldwide. one approach to slow down the development of anthelmintic resistance is targeted selective treatment (tst), where a part of animals is left unexposed to anthelmintic treatment and thus providing refugia for susceptible parasites. closely linked to the successful implementation of tst is the identification of animals in need of treatment. the aim of this study was therefore to investigate the general relation between milk yield and gastrointestinal nematode infections in a swiss lacaune dairy sheep subpopulation and, based on this, to evaluate milk yield data as a potential tst indicator in dairy sheep. on 15 swiss farms nematode egg excretion per gram faeces (epg) and individual milk performance of 1159 lacaune ewes were obtained between august and december 2019. faeces collection and individual milk performance data were closely timerelated. coprocultures of pooled samples were conducted to determine the proportion of haemonchus contortus on farm level. a linear mixed model was fitted, using log-transformed epg as dependent variable and milk yield, milk protein content, days in milk, lactation number and the proportion of h. contortus as fixed effects. the farm was included as random effect. the results revealed a significant relation between milk yield and epg (p < 0,01), which was most pronounced in the earlier stage of lactation, indicating high yielding ewes to be less resistant to gin infections than low yielding ewes. the results suggest therefore the possibility of using milk yield data as tst indicator in dairy sheep. mailto:steffen.werne@fibl.org animal health: parasitology oral presentations genetic resources (2021) s1 international congress on sheep and goats 80 abstract book data of targeted selective treatment based on live weight gain should be used for breeding for resistance against gastrointestinal nematodes martin ganter1, bernd möller2 and inga nolte1 1 university of veterinary medicine hannover, foundation, clinic for swine and small ruminants, hannover, germany 2 institute of farm animal genetics. friedrich-loeffler-institute. neustadt-mariensee, germany. contact: university of veterinary medicine hannover, foundation, clinic for swine and small ruminants, bischofsholer damm 15, 30173 hannover, germany; martin.ganter@tiho-hannover.de abstract: the development of the famacha© system (van wyk and bath, 2002) was a milestone in the use of targeted selective treatment (tst). the transfer for non-hematophagic endoparasite populations remains a challenge (kenyon and jackson, 2012). in a study on adapting tst to a farm with non-hematophagic endoparasite populations daily weight gain was used as parameter for treatment of individual lambs within a group of 76 bleackheaded mouton lambs. lambs who did not achieve the target weight were treated with ivermectine (2 mg/kg body weight). target weights were evaluated in the grazing season before in the same flock on the same pastures. individual samples for faecal egg count were taken every four weeks. by reaching 45 kg b. wt. ram lambs were slaughtered and the gut examined for gastrointestinal nematodes (gin). compared to a regular deworming every four weeks the number of deworming was reduced by 66.9% in male lambs and 76.6% in female lambs respectively. high rates of not treated lambs turned out. in total 36 lambs (18 = 41.9% female + 18=47.4% male) remained untreated over the whole grazing season and achieved the target weights. clinical endoparasitosis was not present. positive effects of some lamb’s treatment on gastrointestinal nematodes (gin) on fec in the feces of untreated lambs were observed (nolte, 2019). the comparison with the two previous years (trapp, 2013; schöwerling, 2016) showed a high variability in gin populations in the gut of the slaughter lambs. lambs who need no anthelmintic treatment over the entire grazing season, but meet the target weight gain, should be selected as replacements. by using this as a selection criterion for breeding, an indirect selection for resistance against gin could be achieved. references: kenyon, f.& f. jackson (2012): targeted flock/herd and individual ruminant treatment approaches. veterinary parasitology 186, 10-17. nolte, i. (2019): erprobung des „targeted selective treatments“ auf der basis der gewichtsentwicklung bzw. klinischer scores mit makrozyklischen laktonen zur endoparasitenbekämpfung bei lämmern. tierärztliche hochschule hannover, diss. schöwerling, j. (2016): erprobung des "targeted selective treatment" mit levamisol zur endoparasitenbekämpfung bei lämmern. tierärztliche hochschule hannover, diss. trapp, c. (2014): erprobung des ,targeted selective treatment´ zur endoparasitenbekämpfung bei lämmern. tierärztliche hochschule hannover, diss. van wyk, j.a. u. g.f. bath (2002): the famacha system for managing haemonchosis in sheep and goats by clinically identifying individual animals for treatment. veterinary research 33, 509-529. mailto:martin.ganter@tiho-hannover.de animal health: parasitology oral presentations international congress on sheep and goats genetic resources (2021) s1 abstractbook 81 selfmedicative behavior and tanniferous fodder plants: alteration in taste perception and feed preferences of gin-infected boer goats marvin heuduck1, christina strube2, katharina raue2, eva schlecht1 and martina gerken1 1 georg-august-university, goettingen, germany 2 university of veterinary medicine, hanover, germany contact: marvin heuduck, department of animal sciences, albrecht-thaer-weg 3, 37075 goettingen, tel.: +49-(0)551-3925779, @uni-goettingen.de abstract: nematode infections are a common thread in ruminant livestock and excessive usage of conventional anthelmintics led to emergence of resistant nematode populations. this underlines the relevance of paradigm shift towards a sustainable control approach of nematode infections. in this study, possible changes in taste perception and in feed preferences of goats were scrutinized to prove ascertained higher feed intake of tanniferous plants by goats in case of nematode-infection. feed preferences of 18 boer goats were analyzed via cafeteria-trial (12 weeks) regarding influence of changes in health status from non-infected to infected. goats were divided in different groups: i) non-infected + feeding-trial ii) infected + feeding-trial iii) infected without feeding-trial. the cafeteria-trial was conceptualized with pellets of tanniferous plants (leaves of sainfoin, willow, walnut, blackberry) of various tannin-contents and tannin-free hay pellets. after four weeks a mixed nematode-infection was administered to group ii) and iii). besides feed intake and selection procedure, blood parameters, saliva composition and feces were analyzed on weekly basis in order to assess the course of infection and potential shifts in feed preferences due to changes of taste perception. analysis of trial data revealed an alteration from tannin-free (hay) and low tannin-containing feed (sainfoin) to higher tannin-contents (walnut, blackberry) in the course of infection. results of blood, saliva and feces parameters have not yet been completely evaluated to give a more detailed overview about the effects on parasite burden and a potential altered taste perception. mailto:marvin.heuduck@uni-goettingen.de mailto:marvin.heuduck@uni-goettingen.de animal health: parasitology oral presentations genetic resources (2021) s1 international congress on sheep and goats 82 abstract book sainfoin pellets for preventive parasite control and improved protein efficiency in dairy goats steffen werne1, nadine arnold2, erika perler1 and florian leiber1 1 research institute of organic agriculture fibl, 5070 frick, switzerland 2 zhaw life sciences und facility management, 8820 waedenswil, switzerland contact: steffen werne, ackerstrasse 113, 5070 frick, switzerland, email: steffen.werne@fibl.org abstract: condensed tannins of the legume sainfoin (onobrychis viciifolia) are frequently associated with beneficial effects when offered as feed to ruminants. we hypothesised that offering sainfoin to dairy goats may improve the ruminal protein-efficiency indicated by lower urea and higher protein yields in goat milk and as well may lower nematode egg extcretion (epg). over a period of 7 weeks, in total 20 alpine goats were fed with 700g sainfoin (n=10) or 700g (n=10) alfalfa pellets daily. milk protein and urea yield as well as epg were measured regularly on individual basis. all animals were kept in one group, except for milking and pellet feeding and had access to pasture for approx. 5 hours daily. intake from pasture or ad libitum offered hay was not determined. the concentration of condensed tannins in sainfoin and alfalfa was 4% and 0.3%, respectively. crude protein content was 18.2% for sainfoin and 20.1% for alfalfa (corrected for 100% dry matter). repeated measurement analysis could not reveal significant differences (p=0.148) of the epg between groups, even though arithmetic mean of epg in the sainfoin group was 18% lower compared to the control group. also the total daily milk protein (p=0.700) and total daily urea milk content (p=0.410) per animal did not differ between treatments. other studies often report a reduced nematode egg excretion if sainfoin was administered ad libitum. as a dose dependent effect for condensed tannins is assumed, the total amount of condensed tannins in our trial might have been too low to provoke any effect. mailto:steffen.werne@fibl.org animal health: parasitology oral presentations international congress on sheep and goats genetic resources (2021) s1 abstractbook 83 coccidiosis in small ruminant’s farms case study in trás-os-montes (northeastern portugal) hélder quintas1, margarida afonso2, daniela silva3, luís cardoso2,5, delia lacasta4, luis miguel ferrer4 and ana patrícia lopes2,5 1 centro de investigac ̧a ̃o de montanha (cimo), instituto politécnico de braganc ̧a, campus de santa apolo ́nia, 5300-253 braganc ̧a, portugal 2 departamento de ciências veterina ́rias, escola de ciências agra ́rias e veterina ́rias, universidade de tra ́sos-montes e alto douro (utad), vila real, portugal 3 segalab, po ́voa de varzim, portugal 4 departamento de patología animal, facultad de veterinaria c/ miguel servet 177, 50013 zaragoza, spain 5 centro de ciência animal e veterina ́ria (cecav), utad, vila real, portugal contact: helder5tas@ipb.pt abstract: coccidiosis is one of the most important intestinal diseases in small ruminants and has a significant economic impact in farms over the world. the manifestations of the disease occurs mostly among young animals when submitted to stressful conditions. thus, in improving prophylactic plans, it is necessary to understand the risk factors associated with each production system in particular. this study was carried out in the region of trás-osmontes (portugal) in animals between 4 and 6 weeks of age from 46 different farms in two different years. several factors related to the management of farms were associated with the presence of strong infections (opg> 5 000) and isolation of pathogenic species. it is concluded that the eimeria species with the highest prevalence in sheep was e. ovinoidalis (68.70%) and the less prevalent e. intricata (13.30%). in the case of goats, e. ninakohlyakimovae was the species with the highest prevalence (100%) and e. alijevi (25.00%) with the lowest. in farms where it was possible to verify a high concentration of animals near drinking spots, there were significantly (p < 0.001) more strong infections. the likelihood of developing strong infections was almost 8 times higher (or = 7.677; ic 95%: 2.729 – 21.589) than the other farms. moreover, in farms where antiparasitic treatments were used, the likelihood of isolating oocysts from pathogenic species of eimeria (p = 0.0065) was 12,17 times lower (or = 12.167, ic 95%: 1.786 82.864) than in the other farms. mailto:helder5tas@ipb.pt animal health: locomotive apparatus and monitoring oral presentations genetic resources (2021) s1 international congress on sheep and goats 84 abstract book know how: the secret of successful lameness management heinz strobel schafpraxis, stoffenried, germany, contact: dr.heinz strobel, am hopfenberg 8, d-89352 ellzee, drheinzstrobel@t-online.de abstract: lameness still is a major problem with ecological, economical and ethical relevance on many farms (keywords: antibiotica, welfare, residues, manpower, productivity). during the last decade frustrating cycles of footrot could be broken on a fair number of farms by applying sustainable strategies. the goal was either dichelobacter nodosus pcr negative flocks or permanent footrot control on acceptable levels (< 3%). the evaluation of difficulties along the elimination process on farm and the results of the following monitoring clearly shows, that relapses and failures were mainly due to carelessness or wrong decisions by stocksmen in critical situations, e.g. when joining rams to the flock without quarantine or prophylactical treatment. as a consequence, the transfer of knowledge should be intensified and optimized along with the design of protocols tailor suited for individual farms and maintained during the following observation period. it is not sufficient to replace traditional and counterproductive opinions of owners by evidence based informations. it is essential to provide a common basic knowledge to all people involved. all staff in charge of handling sheep must be able to identify risks and take appropriate measures irrespective of language barriers or hierarchies. as a result of many presentations, consultancies and stable schools a set of didactical components was created. so knowledge on lameness can be transferred by webinars, faq lists, interactive courses and posters (demonstrated in this presentation). antibiotic stewardship, animal welfare und environmental aspects make sustainable strategies indispensable on the way to effective lameness management. references: strobel h, hilke j, spengler d, axt h, ganter m, voigt k. klaueninfektionen beim schaf – therapiemöglichkeiten in der tierärztlichen praxis unter besonderer berück sichtigung der moderhinkebekämpfung [claw infections in sheep treatment options in veterinary practice, with special emphasis on ovine footrot treatment. an update]. tierarztl prax ausg g grosstiere nutztiere. 2018 dec;46(6):385-398. german. doi: 10.15653/tpg-180285. epub 2019 jan 7. pmid: 30616280. http://www.schafpraxis.de/ animal health: locomotive apparatus and monitoring oral presentations international congress on sheep and goats genetic resources (2021) s1 abstractbook 85 evaluation of prevalence and risk factors associated with virulent and benign dichelobacter nodosus in german sheep breeds julia storms1, anna wirth1, monia lara budnik1, peter kuhnert2, jörg jores2 and ottmar distl1 1 university of veterinary medicine hannover, institute of animal breeding & genetics, hannover, germany 2 university of berne, institute of veterinary bacteriology, bern, switzerland contact: julia.storms@tiho-hannover.de abstract: footrot is a contagious disease of major welfare and economic concerns in sheep worldwide. the etiological agent of ovine footrot, dichelobacter nodosus, differs in virulence due to the subtilisin-like proteases. two proteases, aprv2 and aprb2, determine virulent and benign strains. the thermostable aprv2 protease is required for virulence. the objectives of our study were to screen a large number of sheep breeds for d. nodosus strains, their clinical status and associated risk factors. data and samples were collected from > 9000 sheep in 200 flocks with >20 different breeds from all over in germany. in addition, herd footrot history and potential risk factors for footrot were recorded. we used a competitive qrt-pcr method to detect the allelic differences ta/cg at nucleotide 661/662 in the aprv2 and aprb2 gene sequences. all sheep were examined and scored for foot and claw lesions and recorded along with breed, sex, age and previous footrot treatments. specifity of the qrt-pcr was >99%. sheep with underrunning footrot showed a detection rate of 96.3% for virulent strains and sheep with mild and moderate to severe interdigital dermatitis of 41.4% and 86.6%. low prevalences (< 20%) were seen in forest sheep, east friesian, and skudde and moderate to high prevalences in merinos, leine, bentheimer, texel, suffolk, grey and white heath (>40%). risk factors by breed, season, region, flock and footrot history were analysed and will be shown. breed and flock had the highest impact on d. nodosus prevalence. mailto:julia.storms@tiho-hannover.de animal health: locomotive apparatus and monitoring oral presentations genetic resources (2021) s1 international congress on sheep and goats 86 abstract book study of antibiotic susceptibility against the main pathogens involved in fattening lambs pathology in spain and portugal josé maría bello dronda1 and lorenzo fraile sauce2 1 nanta s.a. ronda de poniente,9, 28760 tres cantos (madrid), spain 2 departament de ciència animal, etsea, universitat de lleida, lleida, spain contact: jm.bello@nutreco.com abstract: the increase of antimicrobial resistance has become a global public health problem. in the eu, reduction policies have been implemented under the “one health” concept that implies human and animal health. the monitoring of resistance in different animal species has become a priority within the strategies of fighting against resistance. four hundred and twelve cases were analyzed from 2015 to 2019, coming from 121 lamb feedlot farms in spain and portugal. several studies were carried out to obtain practical knowledge in order to reduce the use of antimicrobials as much as possible. firstly, several comparative studies were carried out between samples from nasotracheal lavages and those obtained from lung tissue (“in farm examination necropsy”) in order to check the suitability of this type of samples in monitoring the susceptibilities of the pathogens (pasteurella multocida, mannehimia haemolitica and biberstenia threalosi) involved in respiratory problems. secondly, another study was carried out in order to know the evolution along the years of the susceptibilities to some antibiotics of the most important pathogens (p. multocida, m. haemolitica, b. threalosi and escherichia coli) involved both in respiratory and digestive diseases. finally, a “map” of the susceptibilities of pathogens to a significant number of antibiotics available in the market were obtained. similar results in susceptibility were found in most of the pathogens studied comparing lavages and tissue samples. besides, similar evolution in susceptibility of pathogens to most antibiotics checked was observed, with an increment during 2018 and 2019. in general, tetracycline (very used by oral supply) gave less susceptibility than others did. on the opposite way, amoxicillin could be an antibiotic available for further oral use. in conclusion, the nasotracheal lavages are an efficient and valid method of taking suitable respiratory samples in practical conditions of monitoring. besides, there is a tendency to the increment of the susceptibility of pathogens to a large part of antibiotics, in parallel to the reduction of the actual use of antimicrobials. finally, the maps of susceptibilities provide very useful epidemiological information to be used in the strategies of antibiotic pressure reduction. references: gonzález, jm et al (2016). lamb feedlot production in spain: most relevant health issues. small ruminant research. 142: 83:87. https://www.sciencedirect.com/science/article/abs/pii/s092144881630044xy eur-lex (2017). communication from the commission to the council and the european parliament. a european one health action plan against antimicrobial resistance (amr), com/2017/0339. https://eur-lex.europa.eu/legalcontent/en/txt/?uri=celex:52017dc0339 fraile, l (2013). antimicrobial therapy in swine. a practical approach. ed. servet. grupo asis biomedia s.l.pages 25-43. https://store.grupoasis.com/en/swine/235-antimicrobial-therapy-in-swine-practical-approach-9788494101496.html mailto:jm.bello@nutreco.com https://www.sciencedirect.com/science/article/abs/pii/s092144881630044xy https://eur-lex.europa.eu/legal-content/en/txt/?uri=celex:52017dc0339 https://eur-lex.europa.eu/legal-content/en/txt/?uri=celex:52017dc0339 https://store.grupoasis.com/en/swine/235-antimicrobial-therapy-in-swine-practical-approach-9788494101496.html animal health: locomotive apparatus and monitoring oral presentations international congress on sheep and goats genetic resources (2021) s1 abstractbook 87 evaluation of anthelmintic efficacy in gastrointestinal nematodes of goats raised under mountain farming conditions ioanna poulopoulou1, christian lambertz2, kunlayaphat wuthijaree3 and matthias gauly1 faculty of science and technology, free university of bolzano, piazza università 5, 39100 bolzano, italy 2 research institute of organic agriculture (fibl), kasseler straße 1a, 60486 frankfurt am main, germany 3the department of agricultural science, faculty of agriculture, natural resources and environment naresuan university, 65000, phitsanulok, thailand contact: ioanna poulopoulou, faculty of science and technology, free university of bolzano, piazza università 5, 39100 bolzano, italy, e-mail: ioanna.poulopoulou@unibz.it abstract: the frequent use of anthelmintics in mountain farms have led to the development of anthelmintic resistance (ar). the aim of the present study was to estimate the ar of gastrointestinal nematodes (gin) in goats in a mountain area. on eight farms (n = 143 animals) egg excretion (expressed as eggs per gram of faeces (epg)) before and after routine anthelmintic treatments were used for faecal egg count reduction (fecr) tests. either macrocyclic lactone (ml) or benzimidazole (bz) were used as anthelmintics. on five farms (n = 135 animals) fecr was estimated after controlled application of one of the following anthelmintics: ml, bz (partly in combination with salicylanilide (sa)) or a combination of imidazothiazole (it) and sa. ar was assumed if fecr and the upper confidence interval (ci) were < 95% and the 95% confidence level < 90%. average epg before anthelmintic application showed a large variation (epg = 751, sd = 1090). after treatments, 50% of the routinely ml-treated goat flocks showed adequate efficacy. fecr in all others ranged between 64 and 93%. under controlled anthelmintic treatments, ml had an adequate efficacy on four farms and a fecr of 88% on another one. bz was effective on all farms. on one farm, bz and sa had a fecr of 99%. on two farms, it + sa was effective and had a fecr of 91% on a third. low efficacy was observed in goat farms after routine and controlled treatments, which demand immediate actions to prevent further spread and development of ar. mailto:ioanna.poulopoulou@unibz.it animal health: locomotive apparatus and monitoring oral presentations genetic resources (2021) s1 international congress on sheep and goats 88 abstract book relevance of selenium imbalances in sheep flocks and implications for herd health management consultancy esther humann-ziehank labvetcon – laboratory veterinary consulting, burgdorf, germany contact: labvetcon, foehrenkamp 20, d-31303 burgdorf, mail@labvetcon.de abstract: adequate nutritional supplementation of selenium is indispensable for adequate selenoprotein expression and function in animals. germany was considered to provide low selenium in regional grown fodder causing selenium deficiency in sheep flocks as well as in wild ruminants as shown in roe deer (humann-ziehank et al., 2008). marginally selenium supplemented sheep underwent ongoing selenium deprivation. vital organs are affected by selenium deficiency attenuating functional parts of the antioxidative system. improvement of selenium supplementation to an adequate level strongly alters serum and liver selenium concentration within 10 and 20 days, respectively, followed by a plateau (humann-ziehank et al., 2013a). the occurrence of selenium deficiency was demonstrated in an epidemiological study including 150 german sheep flocks. selenium deficiency was found to be widespread in german sheep flocks. more than one third of the flocks showed relevant selenium deficiency in serum indicating the strong need to optimise the nutritional management. as it is impossible to estimate the final forage se concentration by environmental factors only, there is a need for validation at animal level (humann-ziehank et al., 2013b). the best metabolic biomarker of selenium deprivation and nutritional selenium upgrade, respectively, was selenium in serum. moreover, hepatic selenium concentrations reliably reflected the upgrade of selenium supply within days (humann-ziehank et al., 2013a). factors raising suspicion of selenium imbalances are large flocks and transhumance. stationary flocks had constantly higher mean serum selenium concentrations during the breeding, lambing and grazing period, whereas flocks practising transhumance had significant lower selenium status except during lambing (humann-ziehank et al., 2013b). mailto:mail@labvetcon.de animal health: locomotive apparatus and monitoring oral presentations international congress on sheep and goats genetic resources (2021) s1 abstractbook 89 exploiting scanning surveillance data to assess the impact of different initiatives and inform future strategies to control sheep scab eilidh geddes1, sibylle mohr2, sian mitchell3, sara robertson4, anna brzozowska4, stewart burgess5 and valentina busin1 1 school of veterinary medicine, university of glasgow, 464 bearsden road, glasgow, united kingdom 2 boyd orr centre for population and ecosystem health, college of medical, veterinary and life sciences, university of glasgow, glasgow, united kingdom 3 carmarthen veterinary investigation centre, animal and plant health agency, carmarthen, united kingdom 4 surveillance intelligence unit, animal and plant health agency, weybridge, surrey, united kingdom 5 moredun research institute, pentlands science park, bush loan, edinburgh, united kingdom contact: valentina busin, school of veterinary medicine, university of glasgow, 464 bearsden road, glasgow, united kingdom, valentina.busin@glasgow.ac.uk abstract: veterinary surveillance is an essential tool designed to aid decision making and a fundamental concept in food security, public health and international trade (hoinville et al., 2013). this study aimed to investigate the use of existing scanning surveillance data for sheep scab to assess the impact of different initiatives and inform future control strategies. the data analysed were submissions with a positive sheep scab diagnosis confirmed through identification of psoroptes ovis mites in skin scraping in great britain for 2003-2018. information were also collected on all known knowledge transfer, skills training, free testing and legislative actions (initiatives) designed to improve surveillance or decrease disease within the study period for sheep scab. first, a spatial analysis to highlight areas of concern was carried out, followed by an analysis of the effect of past initiatives on temporal patterns (tongue et al., 2019). a total of 2,401 positive skin scrapes were recorded within the study period. the yearly distribution showed a significant downward trend in positive cases, from a peak of 277 in 2004, to 55 cases in 2015. in the study period nine initiatives occurred. three of these initiatives had a significant effect on the number of positive cases diagnosed and this type of scheme did evoke the intended response. in conclusion, the analysis of an existing scanning surveillance source enhanced our knowledge of sheep scab by identification of areas for targeted control and offered a framework to measure the impact of future initiatives. references: hoinville, l.j., alban, l., drewe, j.a., gibbens, j.c., gustafson, l., häsler, b., saegerman, c., salman, m., stärk, k.d.c. (2013). animal health surveillance terminology final report from pre-icahs workshop july 2013. tongue, s. c., eze, j. i., correia-gomes, c., brulisauer, f., gunn, g. j. (2019). improving the utility of voluntary ovine fallen stock collection and laboratory diagnostic submission data for animal health surveillance purposes: a development cycle. front. vet. sci. 6: 487. mailto:valentina.busin@glasgow.ac.uk animal health: reproduction oral presentations genetic resources (2021) s1 international congress on sheep and goats 90 abstract book the trouble with the reproduction – chosen problems of the small ruminants henrik wagner and axel wehrend clinic for obstetrics, gynaecology and andrology for large and small animals with veterinary ambulance, justus-liebig-university giessen contact: dr. henrik wagner, frankfurter straße 106, 35392 gießen, phone: +49 641 9938703 mail: abstract: in breeding and keeping small ruminants, successful and safe reproduction is the basis for the financial security of the farm. in the course of the lecture, four problem areas of reproduction are to be worked out and case studies are given, which are very common in animal husbandry. 1. low lambing rate 2. extended lamb time 3. weakness lambs 4. abortions the ram are of great importance to the clinical examination, which is unfortunately too often forgotten. as a further investigation, the ultrasonographic display of the testicles is a simple and quick diagnostic tool to better classify the breeding ability of the bucks. this technique is used too rarely at the licensing events and performance tests to assess the quality of the bucks in relation to their reproductive ability. quarantine measures for new purchases and correct operational management (vaccination, deworming, serological examinations) are explained and their necessity discussed. abortions continue to play a major role in small ruminants. due to the mostly seasonal lambing, high losses occur quickly. what exactly has to be done with an abortion is with regard to securing and curtailing a possible transmission, especially in the case of zoonoses, and the possible future avoidance should be discussed. the neonatal loss rate is always used as an index for animal welfare-related violations. this aspect is discussed in the lecture and the importance of minimizing such losses is emphasized. the veterinary care of the herds must also be critically evaluated. the density of specialist veterinarians for small ruminants can still be optimized. mailto:henrik.w.wagner@vetmed.uni-giessen.de animal health: reproduction oral presentations international congress on sheep and goats genetic resources (2021) s1 abstractbook 91 coxiella burnetii on dutch dairy sheep farms between 2006 and 2020 rené van den brom, carlijn ter bogt-kappert, erik van engelen, marian aalberts and piet vellema royal gd, deventer, netherlands contact: r.vd.brom@gdanimalheath.com abstract: q fever is a zoonotic disease caused by the intracellular bacterium coxiella burnetii, which is able to infect several animal species, as well as humans. domestic ruminants are the main animal reservoir. in small ruminants, infections mostly don’t give clinical symptoms. however, abortions and stillbirths can occur, mainly during late pregnancy. shedding of c. burnetii occurs in faeces, milk and mostly in placental membranes and birth fluids. during parturition of infected small ruminants, bacteria from birth products become aerosolized. transmission to humans mainly happens through inhalation of contaminated aerosols (maurin and raoult, 1999). in 2005, c. burnetii was diagnosed for the first time as cause of abortion on a dairy goat farm in the netherlands, and between 2005 and 2010, this diagnosis was confirmed on in total 28 dairy goat and two dairy sheep farms (van den brom and vellema, 2009). during this period, more than 4000 people became infected. a large package of preventive measures was implemented, mainly for dairy goat and dairy sheep farms, with the aim to reduce shedding, subsequent environmental contamination and thus human exposure to c. burnetii (van den brom et al., 2015), and compulsory vaccination was the most important one. monthly bulk tank milk (btm) surveillance, using a c. burnetii pcr, is functioning since october 2009, and has been an effective and useful tool to distinguish between c. burnetii excreting and nonexcreting small ruminant farms. vaccination of dairy sheep with phase 1 vaccine coxevac®, at a dose of 1 ml as prescribed for sheep since the start of compulsory vaccination, seems very effective in preventing abortion and reduction of shedding of c. burnetii, since no cases of abortion caused by c. burnetii have been confirmed after vaccination, and no c. burnetii shedding has been detected on dairy sheep farms after 2013 in the btm surveillance. antibody levels against c. burnetii have been high to very high since vaccination started. we briefly present the history of dutch dairy sheep farming, and will discuss results of implemented measures as notification, vaccination, hygiene, and surveillance, on dutch dairy sheep farms between 2006 and 2020. references: maurin m., raoult d., 1999. q fever. clin. microbiol. rev. 12 (4), 518–553. van den brom r., and vellema p., 2009. q fever outbreaks in small ruminants and people in the netherlands. small rumin res. 86, 74-79. van den brom r., van engelen e., roest h.i.j., van der hoek w., vellema p., 2015. coxiella burnetii infections in sheep and goats; an opionated review. vet microbiol. 2015 jul 15. pii: s0378-1135(15)00273-4. doi: 10.1016/j.vetmic.2015.07.011. mailto:r.vd.brom@gdanimalheath.com animal health: reproduction oral presentations genetic resources (2021) s1 international congress on sheep and goats 92 abstract book investigation of abortions in small ruminants in greece due to chlamydia abortus giadinis n.d.1, kiossis e.1, chochlakis d.2, lafi s.q.3, psaroulaki a.2, petridou e.j.4, filippopoulos l.k.5, t. calvo gonzalez-valerio5 and massimiliano baratelli5 1 faculty of veterinary medicine, thessaloniki, greece 2medical school, heraklion, greece 3faculty of veterinary medicine, irbid, jordan 4faculty of veterinary medicine, thessaloniki, greece 5laboratorios hipra s.a., girona, spain contact: nektarios giadinis, clinic of farm animals, faculty of veterinary medicine, aristotle university of thessaloniki, st. voutyra 11, 54627, thessaloniki, greece. e-mail: ngiadini@vet.auth.gr abstract: chlamydiosis due to chlamydia abortus is one of the most common causes of abortion in small ruminant flocks worldwide. although the causative agent is zoonotic, chlamydiosis is not a reportable disease. sixty abortion cases originating from different sheep flocks in greece were investigated for chlamydia abortus. fetal stomach content, fetal liver, placenta or vaginal swabs were used for examinations. pcr for chlamydia abortus gene 16s was positive in 25 out of 60 cases, while examination for gene 23s was positive for 12 of them. three samples were found doubtful. moreover, serological investigation for chlamydia abortus was conducted. blood samples from 26 randomly selected sheep flocks not vaccinated against chlamydiosis were collected. from each flock, 15 to 20 blood samples were taken from adult female sheep. in total 464 blood samples were examined. one hundred and six samples were found positive (22.8%), while 24 samples (5.24%) were found doubtful. also, at farm level, from the total number of 26 farms, 18 of them had positive animals (69.2%). in conclusion, chlamydiosis is considered a highly likely cause of sheep abortion in greece. therefore, vaccinations of the sheep flocks should be recommended for the prevention and control of the disease. references: longbottom d. and coulter l.j. (2003) journal of comparative pathology 128 (217-244). longbottom d. et al. (2005) the veterinary journal (171) 263-275. mailto:ngiadini@vet.auth.gr animal health: reproduction oral presentations international congress on sheep and goats genetic resources (2021) s1 abstractbook 93 ultrasound findings of common genital pathologies in small ruminants mário balaro, isabel cosentino, felipe leal, lucas barbosa, fernanda gonçalves, ana beatriz carvalho, bruno vieira, mirella dias, paula cortat de souza and felipe brandão fluminense federal university, niteroi, rj, brazil contact: department of pathology and clinical veterinary, faculty of veterinary, fluminense federal university, rua vital brasil filho, 64, zip code: 24320-340, niteroi, rj, brazil, e-mail address: mariobalaro@id.uff.br (mfa balaro). abstract: this study reports the prevalence of some reproductive tract pathologies of small ruminants from september 2012 to february 2020 at rio de janeiro state, brazil (16 extensive sheep and 10 intensive dairy goat flocks). an ultrasound device (sonoscape s6®, sonoscape, yizhe building, yuquan road, shenzhen, china) coupled to a linear transducer of 7.5 mhz (transrectal) or 5 mhz convex (transabdominal) were used; b-mode and color doppler-mode ultrasound tapes were recorded and evaluated by the same operator (mb). the chi-square test was adopted for frequency comparison (p<0.05). reproductive disorders were detected in 6.5% (216/3331) female’s and 47.0% (32/68) male’s examinations. in females: hydrometra (2.5%; 83/3331), aseptic resorption of the embryo/fetus (0.9%; 29/3331), recent embryonary/fetal death detected by the lack of heartbeat (0.7%; 23/3331), cystic endometrial hyperplasia (0.6%; 19/3331), follicular cyst (0.5%; 16/3331), hydrosalpinx (0.3%; 9/3331), luteal cyst (0.2%; 7/3331), pyometra (0.1%; 4/3331), retained placenta (0.1%; 4/3331), septic resorption (0.09%; 3/3331), endometritis (0.06%; 2/3331), macerated fetus (0.06%; 2/3331), visceral cysticercosis (0.06%%; 2/3331) and single cases of ovarian tumor, mummified fetus, cervicitis and upper or infra-cervical abscess (0.03%; 1/3331). ewes had significantly fewer reproductive disorders than does (3.9%; 65/1647 vs. 8.9%; 151/1684 p<0.05). in males: testicular microlithiasis (33.8%; 23/68), testicular degeneration (4.4%; 3/68), varicocele (2.9%; 2/68) and single cases of testicular tumor, hydrocele, cryptorchidism and inguinal hernia (1.5%; 1/68). no differences between rams (43.4%; 23/53) and bucks (60.0%; 9/15) were found. in conclusion, does have significantly more reproductive tract disorders than ewes. ultrasonography provides clinically useful information relating to diagnosis, prognosis, and therapeutics. references: descôteaux, l.; gnemmi, g.; colloton, j. (2010). practical atlas of ruminant and camelid reproductive ultrasonography (united states of america: wiley-blackwell), 244p. gouletsou, p.g. (2017). ultrasonographic examination of the scrotal contents in rams. small ruminant research 152, 100–106. doi: 10.1016/j.smallrumres.2016.12.022 scott, p. (2016). practical use of ultrasound scan in small ruminant medicine and surgery. veterinary clinics of north america: food animal practice 32,181205. doi: 10.1016/j.cvfa.2015.09.008 mailto:mariobalaro@id.uff.br animal health: reproduction oral presentations genetic resources (2021) s1 international congress on sheep and goats 94 abstract book chronic mycotoxicosis in a dairy goat farm in brazil paula renata cortat¹, felipe seabra cardoso leal¹, helena gomes ferreira pinto¹, caroline brito dos santos², mirela balistrieri dias¹, bruno ribeiro vieira¹, felipe zandonadi brandão¹, claudia del fava², luiz antonio moura keller¹ and mário felipe alvarez balaro¹ 1 fluminense federal university, niteroi, rj, brazil. 2 pathological anatomy laboratory, biological institute, são paulo, sp, brazil contact: department of pathology and clinical veterinary, faculty of veterinary, fluminense federal university, rua vital brasil filho, 64, zip code: 24320-340, niteroi, rj, brazil, e-mail address: mariobalaro@id.uff.br (mfa balaro) abstract: mycotoxicosis is a disease caused by distinct mycotoxins. this study aimed to describe a case of chronic mycotoxicosis in a dairy goat flock kept under an intensive system in minas gerais, brazil. lactating goats presented history of drop in milk production and weight loss in the past six months with abortion and premature births. twelve goats showed thinness (bcs = 2), pale mucous membranes and mild dehydration at clinical examination. blood samples and feces were collected for the whole blood count, biochemistry and coproparasitological exam. due to the poor quality of cornmeal, samples were collected for mycotoxicological analysis. goats showed mild anemia (hb ~22 %), platelet aggregate, thrombocytosis (~1.2x105/μl), reversal of the neutrophil/lymphocyte ratio (~1.2), hyperproteinemia (~7.5 g/dl) with hypoalbuminemia (~2.7 g/dl) and hyperglobulinemia (~4.8 g/dl). the average egg per gram of feces count was low (~625 e.p.g.). hemoparasites were not detected. in cornmeal samples were detected average of 354.5 μg/kg aflatoxins (afs), 2300 μg/kg fumonisins (fumo), 2165 μg/kg zearalenone (zea) and 3325 μg/kg deoxynivalenol (don). from an euthanized goat, it was found anemia, generalized anasarca, severe ascites, and decreased liver size in the necropsy exam. histopathological report revealed lobular center degeneration and liver necrosis with nephrosis and cerebral cortical spongiosis discreet. microscopic lesions were suggestive of fumonisin action. in conclusion, the chronic mycotoxicosis diagnosis was based in clinical, pathological, and toxicological findings. therefore, the importance of purchase ingredients from reputable suppliers with quality assurance for the feed formulation is emphasized to avoid mycotoxicosis and other disorders. references: gallo, a., giuberti, g., frisvad, j., bertuzzi, t., & nielsen, k. (2015). review on mycotoxin issues in ruminants: occurrence in forages, effects of mycotoxin ingestion on health status and animal performance and practical strategies to counteract their negative effects. toxins 7(8), 3057–3111. doi:10.3390/toxins7083057. gutleb, a. c., caloni, f., giraud, f., cortinovis, c., pizzo, f., hoffmann, l., bohn, t., pasquali, m. (2015). detection of multiple mycotoxin occurrences in soy animal feed by traditional mycological identification combined with molecular species identification. toxicology reports 2, 275–279.doi:10.1016/j.toxrep.2015.01.006 völkel, i, merker e. s., czerny, c. (2011). the carry-over of mycotoxins in products of animal origin with special regard to its implications for the european food safety legislation. food and nutrition sciences. 02. 852-867. doi:10.4236/fns.2011.28117 mailto:mariobalaro@id.uff.br animal health: poster presentations international congress on sheep and goats genetic resources (2021) s1 abstractbook 95 outbreak of abomasal bloat in kid goats due to clostridium ventriculi and clostridium perfringens type a fernanda martins gonçalves1, felipe seabra cardoso leal1, isabel oliveira cosentino1, júlia alves vignoli1, nathalia xavier da silva1, alessandra figueiredo de castro nassar2, simone miyashiro2, nathalie costa da cunha1, claudia del fava3 and mario felipe alvarez balaro1 1 fluminense federal university, niteroi, rj, brazil 2 general bacteriology laboratory, biological institute, são paulo, sp, brazil 3 pathological anatomy laboratory, biological institute, são paulo, sp, brazil contact: department of pathology and clinical veterinary, faculty of veterinary, fluminense federal university, rua vital brasil filho, 64, zip code: 24320-340, niteroi, rj, brazil, e-mail address: mariobalaro@id.uff.br (mfa balaro) abstract: this study aimed to describe an outbreak of abomasal bloat in kid goats and its clinical, pathological, microbiological, molecular, and epidemiological characteristics. the kidding season presented an increased mortality of kid goats with a history of abdominal bloating, dullness, and death. clinical examinations were carried out, and biological samples from necropsied kids (n = 11) were collected for pathological, microbiological, and molecular diagnosis. likewise, an epidemiological survey was carried out to verify possible associated factors related to the disorder. the main necropsy findings were dehydration, pale mucosa, ascites, abomasal and intestinal meteorism and congestion, emphysematous abomasitis, and consolidation of lung’s cranial areas. through staining techniques for cytological evaluations of the abomasum, it was possible to identify gram positive bacteria, coccoid, with a cuboid shape suggestive of clostridium ventriculi, gram positive bacilli suggestive of clostridium perfringens and ovoid basophilic yeasts compatible with saccharomyces cerevisiae. by anaerobic culture and molecular tests, c. ventriculi and c. perfringens type a were confirmed. the main histopathological findings were cholangiohepatitis, nephrosis, emphysematous abomasitis, hyalinization of the gastric and intestinal walls, gastroenteritis, intestinal thromboembolism, pulmonary edema, and non-purulent pneumonia, overall suggesting a systemic enterotoxaemia picture. there was a final mortality rate of 24.4% (20/82). regarding the possible associated factors, the erroneous use of the milk replacer associated with inadequate kid management was verified. among the prophylactic measures, hygiene care, proper use of milk replacer, vaccination plan containing c. perfringens alpha toxoid associated with a good colostrum management were suggested. references: burgstaller, j.; wittek, t.; smith, g.w. (2017). invited review: abomasal emptying in calves and its potential influence on gastrointestinal disease. journal of dairy science 100, 17–35. https://doi.org/10.3168/jds.2016-10949. debey, b.m.; blanchard, p.c.; durfee, p.t. (1996). abomasal bloat associated with sarcina-like bacteria in goat kids. journal of the american veterinary medical association 209, 1468–1469. vatn, s.; tranulis, m.a.; hofshagen, m. (2000). sarcina-like bacteria, clostridium fallax and clostridium sordellii in lambs with abomasal bloat, haemorrhage and ulcers. journal of comparative pathology 122, 193–200. mailto:mariobalaro@id.uff.br animal health: poster presentations genetic resources (2021) s1 international congress on sheep and goats 96 abstract book teaching vet students on sheep and goat farming: brazilian experience mário balaro, lucas barbosa, fernanda gonçalves, ana beatriz carvalho, marta maria da costa, bruno vieira, bruna figueiredo, mirella dias, paula cortat de souza and felipe brandão fluminense federal university, niterói, rj, brazil contact: department of pathology and clinical veterinary, faculty of veterinary, fluminense federal university, rua vital brasil filho, 64, zip code: 24320-340, niteroi, rj, brazil, e-mail address: mariobalaro@id.uff.br (mfa balaro) abstract: the group of study, research and extension in goats and sheep (gepeco) aims to provide training for vet students in sheep and goat farming and to boost the consolidation and profitability of the small ruminant sector in brazil. to reach such goals, the gepeco is composed by undergraduate and postgraduate vet students, coordinated by lectures from the veterinary school of the universidade federal fluminense. students develop initial skills in the sheep and goat unit of the farm school and later they practice their learning along the extension actions in private farms assisted by the group. throughout the semester, gepeco holds meetings at the farm school and, at each one, a new subject is presented to vet students. during this period of time, they are taught and debate about learned techniques. in each semester, the subjects are (1) facilities, ambience and animal welfare; (2) sustainable parasite management and vaccination protocols; (3) nutrition and pasture management; (4) reproductive management; (5) animal health and main surgical procedures; (6) data control and animal improvement; and (7) breeding and profitability. prior to visiting a private farm, the group discusses about the procedures to be performed. all visits are guided by a postgraduate vet student or lecture that deals with the farmer and performs the activities with all students involved. in conclusion, the gepeco join forces in skills development and professional insertion for future vet practitioners in sheep and goat farming and promoting profitability to farmers, as well as keeping people in rural zones. references: bachynsky, e.a, dale, v.h.m., kinnison, t., gazzard, j., baillie, s. a. (2013). survey of the opinions of recent veterinary graduates and employers regarding early career business skills. veterinary record 172, 604. doi: 10.1136/vr.101376. collins, h. (1998). veterinary education and role-based learning. synerg. pollott g.; wilson, r.t. (2009). sheep and goats for diverse products and profits. rome: food and agriculture organization of united nations – fao diversification booklet n. 9, 42p. mailto:mariobalaro@id.uff.br animal health: poster presentations international congress on sheep and goats genetic resources (2021) s1 abstractbook 97 computed tomography and magnetic resonance imaging in the diagnosis and follow-up of chronic coenurosis in sheep (with spontaneous remission of clinical signs) pérez, m.1, climent, m.2, de miguel, r.1, ortíz, m.t.1, magdalena, a.1, castells, e.3, escudero, a.1 and ferrer, l.m.1 1 animal pathology department, instituto agroalimentario de aragón-ia2 (universidad de zaragozacita), veterinary faculty of zaragoza, c/miguel servet 177, 50013 zaragoza, spain 2 anatomy, embryology and animal genetics department, veterinary faculty of zaragoza, c/miguel servet 177, 50013 zaragoza, spain 3 centro clínico veterinario, c/ madre genoveva torres morales, 8, 50006 zaragoza, spain contact: mariaperezb3@hotmail.com abstract: coenurosis is a condition caused by coenurus cerebralis, the larval stage of taenia multiceps, and causes enormous economic losses in husbandry production. c. cerebralis develops in the central nervous system of sheep and usually have an acute and chronic form of the disease, being the former caused by larval migration and the later by intracranial parasitic cyst development. five adult young sheep presenting neurological clinical signs were referred to the clinical ruminant service of the university of zaragoza. a clinical examination revealed circling, frequent bleating, separation from the flock and visual impairment. computed tomography (ct) scan evinced the presence of intracranial single fluid-filled cystic structures. diagnosis of chronic coenurosis was stablished and confirmed at the postmortem evaluation. one of the referred animals, being pregnant, was hospitalized until she raised the lamb. surprisingly, the symptoms remitted spontaneously in the absence of any specific treatment. ct demonstrated a marked retraction and increased consistency of the parasitic cyst. additionally, magnetic resonance imaging (mri) was carried out in order to study the type of tissue that had replaced the cyst fluid and revealed a pericystic infiltration of encephalic tissue, characterized by histopathology as fibroblastic tissue with a diffuse lymphoplasmacytic infiltrate. ct and mri allow proper localisation and measurements of the larval cyst as well as nondeniable information on the damage of adjacent nervous structures, which can be really useful in the development of new therapeutical approaches for this and other intracranial conditions of sheep. references: gonzalo-orden, j.m., díez, a., altónaga, j.r., gonzalo, j.m., orden, m. a.,2005. computed tomographic findings in ovine coenurosis. veterinary radiology & ultrasound. 40, 441-444. doi: 10.1111/j.1740-8261.1999.tb00372.x sanna, e., careddu, g. m., manunta, m. l., masala, g., columbano, n., muzzetto, p., muzzeto, p., 2007. cerebral cenurosis in sheep: an even present pathology. veterinary research communications. 31, 331-333. doi: 10.1007/s11259-007-0106-4 tena, a., de miguel, r., castells, e., escudero, a., lacasta, d., 2020. chronic coenurosis in sheep: spontaneous remission of clinical signs and role of ct and mri in the diagnosis and follow-up. veterinary record case reports. 8:e001092, 1-3. doi: 10.1136/vetreccr-2020-001092 mailto:mariaperezb3@hotmail.com https://doi.org/10.1111/j.1740-8261.1999.tb00372.x https://www.researchgate.net/deref/http%3a%2f%2fdx.doi.org%2f10.1007%2fs11259-007-0106-4 animal health: poster presentations genetic resources (2021) s1 international congress on sheep and goats 98 abstract book vitamin e in the diet of lactating goats: bioavailability and influence on the passive immunity of kids mercedes roncero-díaz1, begoña panea2, maría de guía córdoba3, anastasio argüello4 and maría j. alcalde1 1 university de sevilla, seville, spain 2 centro de investigación y tecnología agroalimentaria de aragón (cita), instituto agroalimentario de aragón – ia2 (cita-universidad de zaragoza), zaragoza, spain 3 university of extremadura, badajoz, spain 4 university of las palmas de gran canaria, las palmas (canarias), spain contact: maria j. alcalde: department of agricultural and forestry science, university de seville, ctra, utrera km. 1, 41013 seville, spain, email: abstract: a total of 30 male single-birth kids from the payoya breed were used. during the trial, kids were only fed natural milk without any dietetic complement and remained stabled. two batches of fifteen animals each were established according to the feeding systems of their dams: cultivated meadow (cm) and total mixed ration (tmr). the immunoregulatory potential of the different forms of vitamin e (natural/synthetic) provided to the kid through maternal diets (cm and tmr, respectively) was evaluated during lactation. then, α-tocopherol was quantified in the plasma of goats and their kids using high-performance liquid chromatography (hplc). additionally, the kidney fat of the kids was weighed. correlation analysis and anova with maternal diet as the principal effect were performed using the spss statistical package. the health of a new-born is influenced by its nutritional level and hence influences visceral fat (gall 1982). therefore, kidney fat can be a good indicator of a kid's health and immunity. colostrum and milk provide vitamin e that stimulates the immune system and is essential for the health of the new-born (przybylska et al. 2007). positive correlations are noted between the plasma concentrations of α-tocopherol in goats and their kids and between the plasma α-tocopherol in kids and the weight of kidney fat (r=0.606, p˂0.001; r=0.335, p=0.013, respectively). the increased bioavailability of natural vitamin e (debier et al., 2005) from the diet of cm goats influences the health status of their kids [more kidney fat (p˂0.001), and compared with tmr kids, results in a higher plasma tocopherol concentration (p˂0.001)]. references: debier c.; larondelle y. (2005). vitamins a and e: metabolism, roles and transfer to offspring. british journal of nutrition, 93 (2): 153–174. gall, cf (1982). carcass composition. in third international conference on goat production and disease. dairy goat journal publication pgs. 472-487. przybylska, j, albera, e and kankofer, m (2007). antioxidants in bovine colostrum. reproduction in domestic animals 42, pgs 402–409. mailto:aldea@us.es animal health: poster presentations international congress on sheep and goats genetic resources (2021) s1 abstractbook 99 use of thermography for the diagnosis of chronic proliferative rhinitis in sheep and its application in the differential diagnosis of the first case affecting the dorsal and medial turbinate ceresuela, c.1, lópez-tamayo, s.1, bueso, j.p.2, alzuguren, o.3, rodríguez, a.1, figueras, l4 and ferrer, l.m1 1 animal pathology department, instituto agroalimentario de aragón-ia2 (universidad de zaragozacita), veterinary faculty of zaragoza, c/miguel servet 177. 50013 zaragoza, spain 2 laboratorio agroambiental de aragón, montañana, zaragoza, spain 3 laboratorios exopol diagnóstico y atovacunas s.l., polígono de san mateo de gállego, zaragoza, spain 4 gabinete técnico veterinario s.l., c/ isla conejera s/n, zaragoza, spain contact: abstract: chronic proliferative rhinitis (cpr) is an upper respiratory tract disease of sheep associated with salmonella enterica subsp. diarizonae serotype 61:k:1:5:(7). it affects the ventral nasal turbinate and sometimes the proliferative tissue can be seen emerging from the nostril. in the following case, the animal affected was an ewe with a severely bilateral inflammatory process of the upper respiratory tract. the thermal imaging camera revealed an increase in the temperature of both nasal cavities and the hottest area in the middle part of the nose, which corresponded with the area of the swollen dorsal turbinate. the medial localization of the affection, exhibited by thermography, directed the diagnosis towards cpr and enzootic nasal adenocarcinoma (a tumoral disease which affects the ethmoidal area). the post-mortem examination confirmed the cpr diagnosis and revealed inflammation of the ventral nasal turbinates, and, for the first time, of the dorsal and medial ones of both nasal cavities, coinciding with the images obtained by thermography. to date, in all the descriptions made of cpr the ventral turbinate was always affected and only minor changes were observed in dorsal turbinate or ethmoidal area. the affection of the dorsal and medial turbinates here described shows how this bacterium could deepen and damage more internal structures of the respiratory tract. this was easily observed by thermography, which helped to locate the affection and facilitated the diagnosis, proving to be a useful resource for clinical diagnosis of upper respiratory tract diseases of sheep. references: lópez-tamayo, s., rubira, i., de las heras, m., castells, e., lacasta, d. (2020) use of thermography for the diagnosis of chronic proliferative rhinitis in sheep and its application in the differential diagnosis of the first case affecting the dorsal turbinate. veterinary record case reports 8: e001070. doi: 10.1136/vetreccr-2020-001070 lacasta, d., et al. (2012). chronic proliferative rhinitis associated with salmonella enterica subspecies diarizonae serovar 61:k:1,5, (7) in sheep in spain. elsevier vol. 147, 406-409. doi: 10.1016/j.jepa.2012.03.004. lacasta, d., et al. (2017). experimental infection with salmonella enterica subsp. diarizonae serotype 61:k:1,5,(7) in sheep: study of cell mediated immune response. small ruminant research 149 (28-33). doi: 0.1016/j.smallrumres.2017.01.011. mailto:vet.ceresuela@gmail.com animal health: poster presentations genetic resources (2021) s1 international congress on sheep and goats 100 abstract book valvular endocarditis in small ruminants: case reports ana beatriz da silva carvalho¹, paula renata cortat¹, bruno ribeiro vieira¹, felipe seabra cardoso leal¹, isabel oliveira cosentino¹, caroline brito dos santos², fernanda martins gonçalves¹, mirela balistrieri dias¹, claudia del fava² and mário felipe alvarez balaro¹ 1 fluminense federal university, niteroi, rj, brazil 2 pathological anatomy laboratory, biological institute, são paulo, sp, brazil contact: department of pathology and clinical veterinary, faculty of veterinary, fluminense federal university, rua vital brasil filho, 64, zip code: 24320-340, niteroi, rj, brazil, e-mail address: mariobalaro@id.uff.br (mfa balaro) abstract: endocarditis is the inflammation of the endocardium, which might be located on the heart wall and/or on the valve. this study aimed to describe two cases of valvular endocarditis in small ruminants located in rio de janeiro, brazil. the first case occurred in a saanen lactating goat (five-years-old), kept under intensive system. at the ultrasound scan for pregnancy diagnosis, a large amount of free anechoic fluid was found. the animal underwent a daily treatment with furosemide (2.5 mg/kg) but died two weeks later due to an acute respiratory distress. postmortem examination revealed yellowish friable masses adhered to leaflets that compound the tricuspid valve, as well as intense ascites, hepatomegaly, congestion and pulmonary edema. histopathological report revealed in the mass adhered to the valve, there was a focus of neutrophilic infiltration and splendore-hoeppli phenomenon, compatible with bacterial and mycotic valvular endocarditis. the second case occurred in a crossbred hair sheep (three-yearsold) kept under semi-intensive system that died under suspicion of previous fight and cervical trauma. at necropsy, a friable mass was found in the tricuspid, diffuse whitish lesions on the epicardium and myocardium (suggestive of infarction), pale ocular conjunctiva, great amount of haemonchus sp. within the abomasum, enlarged lung with diffuse congestion and whitish multifocal lesions on kidneys. in both cases, endocarditis may have been secondary to chronic bacteremia, such as pneumonia, subacute ruminal acidosis (sara), periodontitis and others. therefore, the diagnosis of valvular endocarditis is important to relate to other primary causes that can affect flock productivity and establish prophylactic measures. references: buczinski, s., tsuka, t., tharwat, m. (2012). the diagnostic criteria used in bovine bacterial endocarditis: a meta-analysis of 460 published cases from 1973 to 2011. veterinary journal, 193, 349–357. doi: 10.1016/j.tvjl.2012.02.012 radostitis, o.m., gay, c.c., hinchcliff, k.w., constable, p.d. (2016). veterinary medicine — a textbook of the diseases of cattle, horses, sheep, pigs and goats. (london: saunders ltd.). 2278 p. mailto:mariobalaro@id.uff.br animal health: poster presentations international congress on sheep and goats genetic resources (2021) s1 abstractbook 101 malignant neoplasms in sheep and goats in sicily sebastian alessandro mignacca1, sandro bevacqua2, flavia stassi2, benedetta amato3, colombino elena4, vincenzo di marco lo presti3 and maria teresa capucchio4 1veterinary practitioner, enna, italy 2veterinary practitioner, palermo, italy 3istituto zooprofilattico sperimentale della sicilia “a.mirri”, barcellona p.g., italy 4department of veterinary sciences, university of torino, turin, italy contact: via gb giuliano, catenanuova (enna) 94010, italy, sebastian.mignacca80@gmail.com abstract: tumors in small ruminants are generally rare, even if their incidence is quite different in the literature. viruses (papillomavirus and jaagsiekte sheep retrovirus), chemical and physical agents are considered potential etiological agents. the authors reported the tumors observed during their diagnostic activity in sicily (italy) from january 2009 to may 2020. neoplasms were collected at the slaughterhouse, during necropsies or surgeries from adult animals of both sexes mainly belonged to local breeds. gross and histological investigations were performed. a total of 226 malignant neoplasms were detected (sheep: 159; goats: 67). particularly, in sheep 122 squamous cell carcinomas (scc), 27 ovine pulmonary carcinomas (opa), 4 intestinal adenocarcinomas, 2 lymphomas, 1 melanoma, 1 mesothelioma, 1 cholangiocarcinoma and 1 anaplastic carcinoma were observed. in goats 59 scc, 6 melanomas, and 2 sarcomas were reported. scc were most frequently reported followed by opa. the high incidence of scc permits to hypothesize that papillomavirus could play a role in the carcinogenesis. however, its role remains to be clarified as its presence is inconstant. in authors' opinion small ruminants tumours are underestimated and an increased surveillance is recommended because these species could represent interesting animal models in comparative medicine and valid environmental bio-indicators. references: mignacca s.a., zoccola r., monnier m., costa m., colombino e., goria m., capucchio m.t. (2018) papillomavirus (oapv3) in tumori cutanei ovini in sicilia: lesioni anatomo-patologiche e indagine biomolecolare. in proceedings of xviii national congress s.i.di.l.v., perugia (italy). 115-116 mignacca s.a., biasibetti e., spuria l., amato b., di marco lo presti v., and capucchio m.t. (2016). cutaneous tumors in small ruminants: uv ray exposure and papillomavirus infection as potential risk factors. in proceedings of ix world cancer congress, shanghai (china). 366 mignacca s.a., capucchio m.t, biasibetti e., guarneri g., milone s., marchisotta a., amato b., and di marco lo presti v. (2015). three cases of melanoma in small ruminants: clinical symptoms and pathological results. small rumin res. 126, 25-27 mailto:sebastian.mignacca80@gmail.com animal health: poster presentations genetic resources (2021) s1 international congress on sheep and goats 102 abstract book efficacy of a stabulogen vaccine for streptococcus dysgalactiae-induced dermatitis in sheep sebastian alessandro mignacca1, lorella ciccarello2, salvatore di bella2, vincenzo di marco lo presti3 and gavino marogna4 1veterinary practitioner, enna, italy 2 azienda sanitaria provinciale palermo, italy 3istituto zooprofilattico sperimentale della sicilia “a.mirri”, barcellona p.g., italy 4 istituto zooprofilattico sperimentale della sardegna "g. pegreffi”, sassari, italy contact: via gb giuliano, catenanuova (enna) 94010, italy, sebastian.mignacca80@gmail.com abstract: streptococcus dysgalactiae has been reported to be the cause of several infections in farm animals, fishes and humans. in small ruminants, it has been associated with bacteremia, meningoencephalitis, mastitis, omphalophlebitis, polyarthritis and dermatitis. s. dysgalactiae has also been isolated from reproductive tract discharges and tonsils of healthy ruminants. the authors described outbreaks of dermatitis in sheep by s. dysgalactiae and its treatment with a stabulogen vaccine in flocks in sicily (italy). outbreaks occur during winter season, a couple of weeks after a new purchase of ewes, in 10% of animals of 4 semi-extensively flocks (1300 adults in total) reared in the same pastures. the animals showed hemorrhagic-necrotic dermatitis to the udder, limbs and head. swabs from lesions were collected and submitted for conventional bacteriological and virological tests. samples tested positive only for s. dysgalactiae. a stabulogen inactivated vaccine was set up following the national guidelines. in particular, colonies were inactivated with formaldehyde, and aluminum hydroxide was used as adjuvant. it was calculated that the suspension contained around 109 ufc/ml of s. dysgalactiae. sheep were subcutaneously inoculated with 2ml of vaccine, without showing adverse effects after administration, and treatments were repeated after 1, 6, 12, 18 and 24 months. ameliorative effects had already been registrated in over 40% of the affected animals after the first inoculation, and complete recovery in the third year was observed. authors want to point out that the use of stabulogen vaccine represents a valid, quick and safe procedure for many diseases in farm animals. in the meantime, its use is authorized in symptomatic animals decreasing the use of antibiotics. references: abdelsalam m., asheg a., eissa a.e. (2013) streptococcus dysgalactiae: an emerging pathogen of fishes and mammals. international journal of veterinary science and medicine, 1, 1-6. agnello s., stancanelli a., amato b., campo f., di marco lo presti v., mignacca s.a. (2016) artrite da streptococcus dysgalactiae in capretti in sicilia: descrizione di un focolaio. in proceedings of xxii national congress s.i.p.a.o.c., cuneo (italy). 132. lacasta d., ferrer l.m., ramos j.j., loste a., bueso j.p. (2008) digestive pathway of infection in streptococcus dysgalactiae polyarthritis in lambs. small. rum. res., 78, 202-205. mailto:sebastian.mignacca80@gmail.com animal health: poster presentations international congress on sheep and goats genetic resources (2021) s1 abstractbook 103 study of the use of bronchoalveolar lavage as an in vivo diagnostic method to detect clinical pulmonary maedi visna magdalena, a.1, menjón, a.1, de las heras, m.1, borobia, m.1, arnal, jl.2, elorza, j.1 and lacasta, d.1 animal pathology department, instituto agroalimentario de aragón-ia2 (universidad de zaragozacita), veterinary faculty of zaragoza, zaragoza, spain exopol diagnóstico y autovacunas, s.l. polígono san mateo de gállego, zaragoza, spain contact: albimag@yahoo.es abstract: maedi-visna virus (mvv) is a lentivirus that infects sheep causing a multi-systemic and slow progressive syndrome, inducing a chronic inflammation of the lung, mammary gland, central nervous system and joints. it causes relevant economic losses worldwide, and due to the lack of vaccines and treatments, a suitable diagnosis is decisive to develop control programs. the main objective of this study was to evaluate the usefulness of the molecular study of bronchoalveolar lavages using pcr techniques to improve the in vivo diagnosis of the pulmonary form of mvv. in order to investigate the efficacy of this diagnostic method, the mv compatible lung lesions found after the necropsy of the studied animals were analyzed, and lung tissue samples were also collected to perform a molecular diagnosis of the mvv. one hundred and fifty-five culled sheep were analyzed at the ruminant clinic service of the university of zaragoza, spain. at necropsies, 19 of the lungs presented compatible mv lesions (12.25%). the lesions associated with the disease were lung enlargement, general greyish discolouration, increased size of mediastinal lymph nodes and grey subpleural dots. regarding bronchoalveolar lavages, 15 of the 19 lungs with mv compatible lesions (78.94%) were mvv-positive by pcr. therefore, this technique is revealed as a suitable, little invasive and innovative in vivo diagnostic method of mv, with a positive predictive value of 82.40% and a negative predictive value of 96.00%, both related to the presence of macroscopic lesions. references: blacklaws, b. a., 2012. small ruminant lentiviruses: immunopathogenesis of visna-maedi and caprine arthritis and encephalitis virus. comparative immunology, microbiology and infectious diseases, 35(3), 259-269. doi: 10.1016/j.cimid.2011.12.003. herrmann-hoesing, l. m. (2010). diagnostic assays used to control small ruminant lentiviruses. journal of veterinary diagnostic investigation, 22(6), 843-855. doi: 10.1177/104063871002200602. luján, l., pérez, m., de andrés, d., and reina, r. (2019). pulmonary lentivirus infection in sheep. small ruminant research, 181, 87-90. doi: 10.1016/j.smallrumres.2019.05.006. mailto:albimag@yahoo.es animal health: poster presentations genetic resources (2021) s1 international congress on sheep and goats 104 abstract book abdominal perforation in ewes due to fetal maceration sebastian alessandro mignacca1, benedetta amato2, vincenzo di marco lo presti2, gaetano guarneri3 and maria costa1 1veterinary practitioner, enna, italy 2istituto zooprofilattico sperimentale della sicilia “a.mirri”, barcellona p.g., italy 3azienda sanitaria provinciale, palermo, italy contact: via gb giuliano, catenanuova (enna) 94010, italy, sebastian.mignacca80@gmail.com abstract: fetus retention is considered a complication of pregnancy and has been described both in domestic animals and in human. mummification and maceration of the fetus are the two typical conditions of this pathology which can lead to death of the dam. when the mother remains alive for a longer period, the macerated fetal bones can perforate the uterus generating also peritonitis and fistulas with other organs. the authors describe the clinical symptoms and pathological findings in two ewes with fetal retention that caused uterine fistula with perforation of the abdominal wall. both animals were cross-bread and were reared in two different small farms. ewes were judged pregnant by owners and had a pregnancy extension of several months without showing any apparent previous signs of dystocia. they appeared in good general condition but with progressive weight loss. the bottom part of the abdomen was swollen and with some small holes that let out necrotic fragments of macerated fetus. no vaginal discharges were observed and palpation confirmed the presence of a hard and cold mass in the abdomen. due to the poor prognosis, ewes were culled. fistulous channels from the uterus were opened to the outside, perimetrium adhered to the surrounding organs through extended fibrous connections, and severe chronic endometritis with two retained macerated fetuses were observed in each sheep. this paper increases the clinical records in this species, and the authors add that veterinary practitioners should take into account that these conditions, although exceptional, can occur. references: drost m. (2007) complication during gestation in the cow. theriogenology. 68, 487-491. lefebvre r.c. (2015) fetal mummification in the major domestic species: current perspectives on causes and management. veterinary medicine: research and reports. 6 233-244. mailto:sebastian.mignacca80@gmail.com animal health: poster presentations international congress on sheep and goats genetic resources (2021) s1 abstractbook 105 ozonized oil can be used for topical treatment of sheep affected by contagious ecthyma helena gomes ferreira pinto¹, lucas de figueiredo cardoso barbosa¹, mariana santos ribeiro¹, bruna ramalho rigaud de figueiredo¹, marta maria campos pereira da costa¹, caroline brito dos santos², claudia del fava² and mário felipe alvarez balaro¹ 1fluminense federal university, niteroi, rj, brazil 2biological institute, são paulo, sp, brazil contact:department of pathology and clinical veterinary, faculty of veterinary, fluminense federal university, rua vital brasil filho, 64, zip code: 24320-340, niteroi, rj, brazil, e-mail address: mariobalaro@id.uff.br (mfa balaro) abstract: sunflower oil is ozonized directly by the flow of the gas (ozone), which reacts with the linoleic acid present forming hydrogen peroxide (antimicrobial, anti-inflammatory). thus, the objective of the study was to test ozonized oil in cases of contagious ecthyma, in sheep located in brazil’s farm. a total of four adults santa ines ewes (2.9 ± 0.5 years old, bcs: 3.0 ± 0.2) with similar pattern of lesion were organized in two groups to compare different topical treatments: g1 treated with 1% iodine, and g2 treated with ozonated sunflower oil, daily, for 2 consecutive weeks. from each ewe, skin biopsies were collected for histopathology evaluation and bacteriological and fungal culture, before, during and after treatments. first cultures performed showed the presence of opportunist infection by candida sp., staphylococcus coagulase negative and yersinia sp., not shown in other collections. in the first biopsy, all tissues had ulcers and inflammation. in the second biopsy, both groups presented half of animals with complete tissue re-epithelialization and the other half in an intermediate process. in the third biopsy, all animals had tissues totally re-epithelialized. in the clinical analysis, the g1 still had crusts after one week of treatment, differently from g2, which no longer formed crusts at this moment. it can be concluded that both treatments were effective for the treatment. nevertheless, ozonized oil facilitated the employee's work by the lower formation of crusts over the healing period, as well as avoiding tissue dryness (different from iodine), with greater safety for ocular and oral regions. references: jandui e.; nóbrega jr.; et al. (2008). contagious ecthyma in sheep and goats in the semiarid of paraiba, brazil. pesquisa veterinária brasileira 28, 135-139. salles, m.w.s.; et al. (1992). contagiousecthyma (pustulardermatitis) ofsheep. ciência rural 22, 319-324. travagli, v.; zanardi, i.; valacchi, g.; bocci, v. (2010). ozone and ozonated oils in skin diseases: a review. mediators of inflammation. https://doi.org/10.1155/2010/610418 mailto:mariobalaro@id.uff.br structures of and prospects for sheep and goat farming, sustainability oral presentations genetic resources (2021) s1 international congress on sheep and goats 106 abstract book sustainability assessment of sheep and goat production systems in periurban areas of southern benin (west africa) bossima ivan koura1, esteban henoc medenou2, fifame panine yassegoungbe2, ulriche cossi afatondji2 and luc hippolyte dossa2 1 school of animal production, national university of agriculture, porto-novo, benin 2 faculty of agricultural science, university of abomey-calavi, abomey-calavi, benin contact: school of animal production, national university of agriculture, bp 43 ketou, benin, e-mail: kouraivan@gmail.com abstract: a study was carried out in southern benin with the aims to understand sheep and goat farmers’ resilience strategies face to climate change and urbanization, to assess the sustainability of their production systems and assist them in increasing their animals’ performances. individual interviews were conducted with 125 small ruminant farmers, using a questionnaire which included questions on their socioeconomic characteristics and farming practices. the farms were typified using the categorical principal component analysis followed by a two-step cluster analysis. subsequently, the french idea (farm sustainability indicators) method was adapted to local production conditions and used to assess the agro-ecological, socio‐territorial and economic sustainability of the obtained farm types. six small ruminant farm types were identified as follows: goat only (36.80%), sheep only (12%), sheep-goat (8.80%), mixed sheep-goat-crop (12.80%), mixed sheep-crop (5.60%) and mixed goat-crop (24%). as perceived by the respondents, rapid urbanization, agricultural expansion and climate variability affect negatively the availability of grazing areas (76%), biomass production in grasslands (98%), animals’ access to forage (97%) and animal growth. farmers’ coping strategies included adoption of free scavenging systems (94%) and feed supplementation with agro-processing by-products (82%) or tree fodders (64%). the agro-ecological and economic sustainability components scores varied (p ≤ 0.05) across farm types. the mixed sheep-goatcrop farm type obtained the highest (p ≤ 0.001) total sustainability score (149.88) whereas the lowest score (122.09) was obtained by goat only farm type. these results highlight the importance of farm activities diversification in enhancing the sustainability of smallholder small ruminant farms. key words: climate variability, diversification, resilience, small ruminant, smallholder, urbanization mailto:kouraivan@gmail.com structures of and prospects for sheep and goat farming, sustainability oral presentations international congress on sheep and goats genetic resources (2021) s1 abstractbook 107 characteristics of goat transhumance and its benefits for environmental sustainability sezen ocak yetişgin1 and hasan alp şahin1 1ondokuz mayıs university, department of animal science, samsun, turkey contact: sezen ocak yetişgin, ondokuz mayıs university, faculty of agriculture, department of animal science55139 samsun, turkey; ; +905322151989 abstract: transhumanceseasonal migration of livestock and human, is a dynamic production strategy between spatial and temporal environments in many parts of the world. beyond their benefits to the ecosystem, they face various challenges at different scales around the globe. the origins of transhumance in turkey dates back to the neolithic period, and todaytwo different transhumance systems are present in turkey (sheep and goat transhumance). this type of production is an historical and traditional strategy to the climatic changes and inaccessibility. in addition to its cultural significance they have an important role on maintaining cultural landscapes and ecosystem services. transhumance practice in turkey has been affected by many factors resulting with a dramatic decline of the herders although they shaped for more than 1000 years the ecosystem in the mountainous areas and contributed economically to the livelihoods. in this paper we will focus on goat transhumance, an ancient transhumance system where turkish herders for centuries move with their goats from mediterranean lowlands where they winter, to the central anatolian highlands for rich summer grazing areas. for this aim, a qualitative research approach with in depth-face to face interviews was used to interview 26 transhumant families from the same community named” sarıkeçili”. participant observation was conducted in the research area during one year by spending a week /month in order to observe the daily routines. the aim was to characterize their spatial-temporal movement patterns, contemporary production practices and ecosystem services they provide. land use/cover information for 1990 and 2018 was analyzed by geographic information systems (gis) and remote sensing (ua) systems. according to the results, shrublands and natural grasslands have decreased during the study period, %13.9 and 8.2, respectively while urban & industrial areas and agriculture lands increased %87 and 10, respectively. number of herders were decreased 134,7% in the last 30 years. transhumance abandonment caused a significant reduction in shrublands and natural grasslands. considering biodiversity and ecosystem sustainability, it is already a critical issue in the mediterranean region of turkey and affecting a whole system. mailto:sezenocak1@gmail.com structures of and prospects for sheep and goat farming, sustainability oral presentations genetic resources (2021) s1 international congress on sheep and goats 108 abstract book what policy and future do sheep and goats need in iran? farhad mirzaei animal science research institute of iran, karaj, iran contact: farmir2001@gmail.com abstract: livestock agriculture in iran covers over 40% of the agricultural activities. sheep and goat production is one of the oldest productions in the country as it is considered the origin sheep and goat domestication in the world. currently, more than 57% of the available animal units in the country are sheep and goats (mostly sheep and goats are together). desertification, deforestation, water shortage, erosion, low efficiency, output, and illiterate small farmers are the typical iranian shepherds’ obstacles. currently it has more than 44 million sheep and 19 million goats. iranian sheep and goat are providing an important part of the demand for red meat at about 321000 tons and 93000 tons in 2018 respectively as a main product, but considerable production of milk, wool and hair too. utilization of pastures, crops' post-harvest and agricultural wastes in small ruminants' feed, which are not mainly used for other livestock, and the taste of sheep and goats meat, and various delicious food made of their products are a part of the necessity of their production, development and stability across the country. more than 20 native breeds of these small ruminants are going to be threatened by socio – ecological issues in addition to economic challenges that it is discussed in this paper. it also covers different production systems and their problems solving how the present situation can be overcome to maintain and sustain sheep and goat driven hundred thousand livelihoods presently. another purpose of this study is to analyze the factors that influence and determine accessible needs and appropriate policies for the sustainable future of sheep and goat production systems. the results of the study will explain that the condition factors (natural resources, human resources, infrastructure and technology) were the most important factors in increasing of the hopefulness of nomads and villagers to prevent critical effects on animalbased food security, if they move on their jobs. therefore, more attention is required from the government and non-governmental organizations for handling this trend to the well-managed and right direction. references: valizadeh,r.(2010). iranian sheep and goat industry at a glance, ferdowsi university of mashhad, p.o. box: 91775-1163, mashhad, iran. kamalzadeh, a., rajabbaigy, m. & kiasat, a. (2008). livestock production systems and trends in livestock industry in iran. journal of agriculture & social sciences. 04: 183–88. statistical center of iran, annual report. (2019). mailto:farmir2001@gmail.com structures of and prospects for sheep and goat farming, sustainability oral presentations international congress on sheep and goats genetic resources (2021) s1 abstractbook 109 evaluation of dried citrus pulp based total mixed ration (tmr) for mutton (goat) production abdur rahman1, huma kalsoom1, sadia khanum1 and muhammad sajid2 1department of animal sciences, university of veterinary and animal sciences, jhang campus, 35200, pakistan 2department of pathobiology, university of veterinary and animal sciences, jhang campus, 35200, pakistan contact: , tel: +92-346-4607300 abstract: pakistan contributes about 7% of mutton production, ranked 3rd in goat rearing countries in the world after china and india. in pakistan per capita consumption of animal protein is 17grams compared to 27grams in developed countries. pakistan rank at 30th number among 163 nations with food security. mutton need and demand will be overwhelmingly increasing in coming years and it needs attention to enhance its efficient production. the major factor in cost effective production is feeding. the use of non-conventional feed resources, like citrus pulp waste as a replacement of energy and protein feeds may support the economical farming. in current study, a total of 12 bucks were divided into 4 groups each having 3 animals and fed total mixed ration for 90 days including 7 days as adaptation. rations were formulated having dried citrus pulp at 0% (control), 10%, 15% and 20% replacing corn, respectively. performance parameters including feed intake, body weight gain, feed conversion ratio, hematological and serological parameters were evaluated. results showed non-significant differences between all the treatment groups as compared to control group, which revealed that dried citrus pulp have equal potential to corn grains as an energy rich concentrate. in conclusion, dried citrus pulp has shown no adverse impact on buck health, proving that dried citrus pulp can replace corn grains in goat feeding and disposed in better way avoiding environmental pollution. keywords: mutton production, citrus pulp, non-conventional feed resources mailto:abdurrehman@uvas.edu.pk structures of and prospects for sheep and goat farming, sustainability poster presentations genetic resources (2021) s1 international congress on sheep and goats 110 abstract book growth response of west african dwarf sheep fed detoxified neem (azadirachta indica) seed cake diets david aderemi ajayi1 and micheal kolawole adewumi2 1national centre for genetic resources and biotechnology, moor plantation, ibadan, nigeria 2department of animal science, university of ibadan, ibadan, nigeria contact: department of animal genetic resources, national centre for genetic resources and biotechnology, pmb 5382moor plantation, ibadan, nigeria, rhemiajayi@gmail.com, +2348034073868 abstract: neem seed cake has potential to replace expensive soyabean meal in livestock diets but requires the removal of toxic bitter triterpenoids. utilisation of detoxified neem seed cake in ruminant feeding has not been adequately documented, hence, performance of west african dwarf sheep fed detoxified neem seed cake-based diets were assessed. the neem seed cake was subjected to be either water-washing, sodium hydroxide-soaking, ammoniation or sun-curing. six diets were formulated: i (20% soyabean meal, control), ii (raw neem seed cake), while the detoxified cakes were used to replace soyabean meal in four concentrate diets: iii (water-washed), iv (sodium hydroxide-soaked), v (ammoniated) and vi (sun-cured). in a completely randomised design, thirty-six west african dwarf sheep were randomly allotted to the six treatments with six replicates per treatment and were fed basal guinea grass and experimental diets in ratio 70:30 for 105 days. dry matter intake, crude protein intake, feed conversion ratio and daily weight changes were recorded. data were analysed using anova at α0.05. the dry matter intake ranged from 218.14±29.23 (ii) to 330.69±7.74 g/day (v). the feed conversion ratio in sheep fed treated neem seed cake diets were similar to the control and better than raw neem seed cake diet, while daily weight changes in treatments i (54.95 g/day) and v (51.28 g/day) were higher than others. the crude protein intake was 39.00, 25.46, 37.06, 35.16, 32.27 and 31.39 g/day for treatments i to vi respectively. ammoniated neem seed cake supplemented diet supports best performance in sheep growth response. mailto:rhemiajayi@gmail.com structures of and prospects for sheep and goat farming, sustainability poster presentations international congress on sheep and goats genetic resources (2021) s1 abstractbook 111 precision in shoats selection using telomeres mercy mulandi, rawlynce bett and joseph jung’a university of nairobi, nairobi, kenya contact: +254 700649328 abstract: time wastage in the farm due to 'unseen' genetic and health disorders contributes to economic losses and high levels of methane, this occurs due to unprecise selection criteria. the heritable nature, correlation between lifespan, genetic diseases to telomere length is of great impact to the selection of suitable sheep in early life stages. sheep and goat telomere length is heritable making it a possible parameter for selection. telomeres are protective structures at the end of chromosomes that maintain the integrity of the genome by ensuring that chromosomes are not recognised as sites of dna damage. this study followed the telomere measurement guidelines as described by cawthon, 2002. we conducted two seperate pcrs simultaneously, a telomere pcr and a single copy gene (beta-2-microglobulin (b2m) pcr for all samples. this was as such in order to get the ratio between the two as the average telomere length is usually presented as the amount of single copy gene, which is a constant to the amount of telomeric dna. b2m was used as it has been previously used in soay sheep and freshian cattle telomere dynamics experiments and has shown stable qpcr results. the fluorescence thresholds were different across all samples. the single gene copy had an average of 2.081 while the tel gene had an average of 2.123. the lengths are shorter for animals with lower growth rates, lower milk production, more disease manifestations and more reproductive disorders like abortions. the telomeres are longer for animals described to have a better quality life. references: blackburn eh (2005) telomeres and telomerase:their mechanisms of action and the effects of altering their functions. blackburn, e. & gall, j. a (1978).tandemly repeated sequence at the termini of extrachromosomal ribbosomal rna genes in tetrahymena. blackburn, e. h. (1991). structure and function of telomeres. mailto:mercykabondo@gmail.com structures of and prospects for sheep and goat farming, sustainability poster presentations genetic resources (2021) s1 international congress on sheep and goats 112 abstract book milk composition of chusca goat (capra aeagrus hircus) in husbandry extensive environment in the dry forest of the southern of ecuador edgar lenin aguirre1, ramiro armijos1, miriam puchaicela2, belén gahona2, priscila pineda2 and juan pablo riofrio2 1agricultural faculty, veterinary medicine and zootechnics school, national university of loja-ecuador 2graduation students in veterinary medicine and zootechnics, national university of loja-ecuador contact: av. pio jaramillo, ciudadela “la argelia”, loja city, ecuador-south america, telf. +593986448562, edgar.aguirre@unl.edu.ec, leninaguirrer@yahoo.es abstract: the goat “chusca lojana”, which is found in the tropical zone of the dry forest of the province of loja-ecuador, where 73% of the goat population of the nationwide is found in this place. is important to study the productive characteristics and the milk quality of this population, because this genetic resource represents a valuable socio-economic element for the habitants of this region. production and milk quality of 198 goats managed extensively and from different herds were evaluated. for this, 4 periodic visits were made, where production was recorded at the different stages of lactation and milk samples were taken for analysis using lactoscan milk analizer. it was determined that 64% has a globular udder, 93% have pigmented nipples, the productive life curve increases in third calving and reaches its peak in fourth, and then declines (r2=0.82). lactation duration is 140±20 days, lactation curve follows a two period mobile trend, showing two peaks, one higher (30 days) and another of less intensity between 120-150 days coinciding with the post-weaning period, the mean production day is 390±231 ml. both production and composition of milk, it is statistically affected by the place husbandry, calving number, lactation stage (p<0.05) and the biotype (p<0.1). the milk composition of this goat presents 5.5±1.9% fat, 8.2±0.83% nfs, 13.7±2.1% total solids, 4.5±0.47% lactose, 3±0.3% protein, 0.68±0.07% mineral salts and 6.7±0.26 ph. with the information obtained from this study, those animals with a tendency to produce quality milk can be standardized for the purposes of selection, management and multiplication. references: djebly, i., ameur-ameur, a., gaouar, s. (2020). general characteristics of goat milk cheese (feta) in the region of tlemcen, algeria. genet. biodiv. j, 4(2), 60-73. available at: http://ojs.univ-tlemcen.dz/index.php/gabj/article/view/760. mailto:edgar.aguirre@unl.edu.ec mailto:leninaguirrer@yahoo.es http://ojs.univ-tlemcen.dz/index.php/gabj/article/view/760 structures of and prospects for sheep and goat farming, sustainability poster presentations international congress on sheep and goats genetic resources (2021) s1 abstractbook 113 in vitro assessment of the larval exsheathment inhibition of extracts of lentinula edodes (shiitake) against haemonchus contortus (l3) tania maría rodríguez-barrera1,2, jetzabelt ambrosio-bautista3, maura téllez-téllez2, josé e. sánchez4, gloria sarahí castañeda-ramírez1, maría de lourdes acosta-urdapilleta2 and liliana aguilar-marcelino1 1centro nacional de investigación disciplinaria en salud animal e inocuidad, inifap, km 11 carretera federal cuernavaca-cuautla, no. 8534, col. progreso, jiutepec, morelos, méxico, c.p. 62550 2centro de investigaciones biológicas, universidad autónoma del estado de morelos, av. universidad no. 1001, col chamilpa cuernavaca, morelos, méxico, c.p. 62209 3facultad de medicina veterinaria y zootecnia, universidad mesoamericana, privada de la soledad no. 311, puebla de zaragoza, méxico, c.p. 45575 4el colegio de la frontera sur, apdo. postal 36, tapachula, chiapas, méxico, c.p. 30700 contact: dr liliana aguilar-marcelino, aguilar.liliana@inifap.gob.mx, phone: +52 777 319 28 60 ext. 121 abstract: livestock is one of the most important economic activities in mexico, is a profitable sector that guarantees the production and supply of food. this sector is affected by parasites such as gastrointestinal nematodes. one of the main ones is the nematode h. contortus, this etiological agent is considered the cause of diseases that afflict small ruminants. an alternative to this problem is the edible mushroom l. edodes, which is a natural antagonist of parasitic nematodes of sheep (al-ani et al., 2020; pineda-alegría et al., 2020). the objective of the present study was the determination of anthelmintic activity in vitro was carried out with the larval unsheath inhibition test. this study used the larval exsheathment test, which is based on measuring the inhibitory capacity of unsheathing using l3 h. contortus to interrupt the life cycle. statistical analysis based on determining the percentage of inhibition of the unsheathing larvae l3 h. contortus with the formula: percentage of unsheathing = (l3 larvae without sheath)/(larvae with sheath + larvae without sheath) x 100. the results of the five concentrations of the extract (150, 312.5, 625, 1250 and 2500 µg/ml) were evaluated with one-way anova comparing means. the results show that a l. edodes obtained a percentage of 100% inhibition of larval unsheathing at a concentration of 2500 µg/ml. in conclusion, the extract of l. edodes can be used as a sustainable integral control method in the livestock area. the present study was financed by the national problems, conacyt, project number 9342634372. references: al-ani, l. khalil, t., aguilar-marcelino, l., fiorotti, j., sharma, s., sharif m., furtado e.l., wijayawardene n.n., herrera-estrella a. (2020). biological control agents and their importance for plant health. in: microbial services in restoration ecology. (eds) singh j.s., raj vimal s. elsevier. u.s.a. p 42. doi.org/10.1016/b978-0-12-819978-7.00002-6 pineda-alegría, j.a., sánchez, j.e., gonzález-cortázar, m., von son-de fernex, e., gonzález-garduño, r., mendoza-de gives, p., zamilpa a., aguilar-marcelino, l. (2020). in vitro nematocidal activity of commercial fatty acids and β-sitosterol against haemonchus contortus. journal of helminthology, 94(135): 1-4. doi.org/10.1017/s0022149x20000152 mailto:aguilar.liliana@inifap.gob.mx https://doi.org/10.1016/b978-0-12-819978-7.00002-6 structures of and prospects for sheep and goat farming, sustainability poster presentations genetic resources (2021) s1 international congress on sheep and goats 114 abstract book in vitro assessment of the larval exsheathment inhibition of extracts of edible mushroom pleurotus djamor against haemonchus contortus (l3) jaime cristóbal hernández-rodríguez1,2, jetzabelt ambrosio-bautista4, ivan flores-pérez2, juan felipe de jesús torres-acosta3, gloria sarahí castañeda-ramírez1, claudia hallalcalleros and liliana aguilar-marcelino1 1centro nacional de investigación disciplinaria en salud animal e inocuidad, inifap, km 11 carretera federal cuernavaca-cuautla, no. 8534, col. progreso, jiutepec, morelos, méxico, c.p. 62550 2facultad de ciencias agropecuarias, universidad autónoma del estado de morelos, av. universidad 1001, col. chamilpa, cuernavaca, morelos, mexico, c.p. 62209 3facultad de medicina veterinaria y zootecnia, universidad autónoma de yucatán, km 15.5 carretera mérida-xmatkuil, cp 97100, mérida, yucatán, méxico 4facultad de medicina veterinaria y zootecnia, universidad mesoamericana, privada de la soledad no. 311, puebla de zaragoza, méxico, c.p. 45575 contact: dr liliana aguilar-marcelino, aguilar.liliana@inifap.gob.mx, phone: +52 777 319 28 60 ext. 121 abstract: in the present study, it was carried out in vitro assessment of the larval exsheathment inhibition of extracts of pleurotus djamor against haemonchus contortus (l3). they were used in series 6 (n=4). series 1, (control group) contained pbs ph 7.4; series 2) 150, 3) 312.5, 4) 625, 5) 1250, 6) 2500 µg/ml of extract. the chlorine concentration was determined to induce the larvae exsheathment of an extra series of pbs (control group), the concentrations of chlorine (sodium hypochlorite (4.0 to 6.0%) and sodium hydroxide (0.002-0.10%)) were: 20, 30, 40, 45 and 60 µl (chan et al., 2017). it is taken in 50 µl of each concentration of the extract and placed on the slide. to add chlorine solution to induce larvae, select and the number of sheathed and unsheathed larvae that were observed at each concentration (0, 20, 40, and 60 minutes). the following formula is used to determine the inhibition percentage of exsheathment (id) at 60 min for each extract: exsheathment % = (larvae l3 without sheath) / (larvae with sheath + larvae without sheath) x 100. id% = 100exsheathment. anova and the media comparison test were performed. the percentage of inhibition of larval exsheathment presented by the p. djamor extract was 100% at a concentration of 2,500 µg/ml, 99.2% at the concentration of 1,250 µg/ml, 60.4% at a concentration of 625 µg / ml, 8.2 with a concentration of 312.5 µg / ml, 2.6 with a concentration of 150 µg / ml. the present study was financed by the national problems, conacyt, project number 9342634372. references: chan-pérez, j.i., torres-acosta, j.f.j., sandoval-castro, c.a., castañedaramírez, g.s., vilarem g., mathieu, c., hoste, h. 2017. susceptibility of ten haemonchus contortus isolates from different geographical origins towards acetone:water extracts of polyphenolrich plants. part 2: infective larvae. vet parasitol 240:11–16. doi: 10.1016/j.vetpar.2017.04.023 mailto:aguilar.liliana@inifap.gob.mx original article genetic resources (2024), 5 (10), 126–138 doi: 10.46265/genresj.nyfm1739 https://www.genresj.org issn: 2708-3764 analysis of passport data of sechium spp. from the mexican chayote genebank in huatusco, veracruz jorge cadena-iñiguez a,b, luis a barrera-guzmán *,c,b, v́ıctor m cisneros-solano c,b, carlos h avendaño-arrazated, ma. de lourdes c. arévalo-galarza e,b, kazuo n watanabe f and jorge d cadena-zamudiod,b a colegio de postgraduados campus san luis potośı, salinas de hidalgo 78600, san luis potośı, méxico b interdisciplinary research group of sechium edule in méxico (gisem), 56160, texcoco, estado de méxico c universidad autónoma chapingo, centro regional universitario oriente, carretera federal huatusco-xalapa km. 6.5, c.p. 94100, veracruz, méxico d instituto nacional de investigaciones forestales, agŕıcolas y pecuarias, centro nacional de recursos genéticos, recursos genéticos. boulevard de la biodiversidad 400 tepatitlán de morelos, jalisco, c. p. 47600, méxico e colegio de postgraduados campus montecillo, km 36.5 carr. méxico-texcoco, montecillo 56230, méxico f tsukuba plant innovation research center, japan abstract: chayote (sechium spp.) (cucurbitaceae) is a mesoamerican genus, with mexico being the primary centre of biodiversity for four species: sechium compositum, s. chinantlense, s. hintonii and s. edule. mexico also hosts the only chayote genebank in the world, which follows a taxonomic arrangement for its intraspecific variants. descriptive and multivariate techniques were used to analyze the passport data of the accessions conserved ex situ aiming to identify associations and similarity patterns to allow efficient management and origin traceability, stakeholder engagement, consumer preferences, destination, use and conservation practices. results showed that 23% of the accessions belong to s. edule var. virens levis, 62.8% come from backyards, and 58.9% of the s. edule variants are kept and preserved by women. interestingly, about 70.8% of the accessions are used for trading, and the rest for self-consumption. multiple correspondence analyses showed that 27.24% of the first two components variation and the variables with the greatest phenotypic contribution were fruit flavour (sweet and bitter), condition of the populations (forest, ruderal-wild, orchard), fruit colour (yellow, light green), small size and flattened shape. the morphological boundaries of s. edule, s. compositum and s. chinantlense variants are defined by the fruit morphology. these findings from passport data analysis support the development of strategies for replacement, regeneration, distinction, genetic improvement, conservation and bioprospective studies. citation: cadena-iñiguez, j., barrera-guzmán, l. a., cisneros-solano, v. m., avendaño-arrazate, c. h., arévalo-galarza, m. d. l. c., watanabe, k. n., cadena-zamudio, j. d. (2024). analysis of passport data of sechium spp. from the mexican chayote genebank in huatusco, veracruz. genetic resources 5 (10), 126–138. doi: 10.46265/genresj.nyfm1739. © copyright 2024 the authors. this is an open access article distributed under the terms of the creative commons attribution license (cc by 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. introduction plant genetic resources for food and agriculture are the basis of human nutrition, industrial inputs and pharmacologically active ingredients. cereals, fruits, roots and vegetables contribute significantly to agrobiodiversity (bellon et al, 2009). the ex situ conservation of underutilized landraces and wild relatives has ∗corresponding author: luis a barrera-guzmán (luisangelbg@gmail.com) become a relevant source of unexplored genes, enriching germplasm banks and their use in breeding. mexico is a megadiverse country, and rural communities manage a wide range of domesticated and semi-domesticated biological variants, cultivated and wild relatives which significantly increase agrobiodiversity (casas and vallejo, 2019). field collections of species with recalcitrant seeds, such as chayotes (genus sechium p. br.), are among the strategies for ex situ conservation of agrobiodiversity. chayote is an increasingly important vegetable in international markets, and morphotypes of the received: 27.08.2024 accepted: 17.10.2024 published online: 21.11.2024 https://www.genresj.org https://www.doi.org/10.46265/genresj.nyfm1739 https://www.genresj.org https://www.doi.org/10.46265/genresj.nyfm1739 mailto:luisangelbg@gmail.com genetic resources (2024), 5 (10), 126–138 mexican chayote collection 127 mexican clade have gradually been collected (barreraguzmán et al, 2021a). in mexico, sechium compositum (donn. sm.) c. jeffrey, s. chinantlense lira & f. chiang, s. hintonii (paul g. wilson) c. jeffrey and s. edule (jacq.) sw have been recorded as endemic species. among these, only s. edule is edible since the other three species have bitter-tasting fruits. s. edule, particularly the smooth green var. virens levis variant, is distributed on all continents (cadena-iñiguez, 2005). its success as a vegetable has transcended local markets, becoming an export product (cadena-iñiguez and arévalo-galarza, 2011). different studies highlight s. edule as a species with notable intraspecific diversity, contributing to the feeding and rural local economy (aguirre-medina et al, 2021). however, most published research only mentions s. edule without specifying the biological variant. for example, dire et al (2003), setzer and setzer (2003), ordoñez et al (2006), loizzo et al (2016), vieira et al (2019), among others, do not specify the variants studied, making it challenging to reproduce their findings. lira et al (1999) performed a grouping analysis of edible morphotypes of s. edule and another for a wild type, without distinguishing or specifying the variation. this makes conservation actions difficult since morphotypes must be identified by some taxonomic method to be included in genebanks. in this case, the popular common name is not appropriate. morphological, anatomical, biochemical and genetic studies, carried out by various authors (donato et al, 1994; cadena-iñiguez, 2005; cadena-iñiguez and arévalo-galarza, 2011; iñiguez et al, 2011; avendañoarrazate et al, 2012; machida-hirano et al, 2015; barrera-guzmán et al, 2021a,b; iñiguez-luna et al, 2021), have shown that there are stability, heritability and uniformity traits that allow the distinction of s. edule variants, which facilitate their conservation, management and research. these traits confer different and desirable characteristics for its use as food (cadenaiñiguez et al, 2013a; aguiñiga-sánchez et al, 2015, 2017; salazar-aguilar et al, 2017). genebanks are crucial to avoiding diversity loss and maintaining this agrobiodiversity. in 2005, the national germplasm bank for sechium edule (bangese) was founded to preserve endemic species and biological variants. to achieve this, fruits were collected from 12 mexican states, as well as from guatemala and costa rica. currently, the bangese safeguards more than 300 accessions in a field collection since sechium seeds are recalcitrant and do not respond to traditional conservation methods (ramı́rez-rodas et al, 2021). the accessions are conserved in a taxonomic arrangement following the principles by (stace, 1986) and adapted for s. edule. it integrates morphotypes as varietal groups that possess stable, heritable characteristics and fertile offspring. these varietal groups are albus minor, a. levis, a. dulcis and a. spinosum for plants with yellow, smooth and prickly fruits. for fruits with dark green epidermis with and without thorns are nigrum minor, n. conus, n. levis, n. xalapensis, n. spinosum and n. maxima. the morphotype virens levis, the most widely distributed in the world, reports light green fruit without spines, while the only representative with bitter fruits of s. edule is the amarus sylvestris morphotype. the above taxonomic nomenclature has facilitated the handling, distribution and distinction of accessions, allowing research, bioprospecting, breeding and preservation activities. as in many germplasm collections, bangese accessions come from direct collection from the areas of origin, distribution and domestication, as well as from donations by rural inhabitants who have locally safeguarded agrobiodiversity for a particular interest. this raises several important questions related to the management, design and regeneration strategies of these resources. for example: which of the sechium varietal groups have the broadest geographical distribution? which biological variants (morphotypes) are the most important in terms of consumer preferences and what characteristics drive this? what is the origin of the accessions, and what implications does this have for their conservation? which stakeholders are most engaged with conservation efforts? what are the primary uses of the fruits? how does the distribution of varietal groups vary with altitude? finally, what are the morphological traits of the fruits that facilitate their visual distinction? genebanks are long-term conservation centres of genetic resources, especially to preserve agrobiodiversity. however, without thorough characterization and documentation of the accessions, making informed decisions about conservation, research, genetic improvement and potential use is challenging (weise et al, 2020). since sechium is a cross-pollinated plant, asexual methods of multiplication are used to preserve the genetic identity of the accessions. the most successful are grafting, rooting of cuttings, and in vitro multiplication (figure 1). materials and methods location of the genebank and passport data the national germplasm bank of sechium edule (bangese) is located in huatusco, veracruz, mexico (19◦ 08’ 48” n, and 97◦ 57’ 00” w). the vegetation type is mountain cloud forest (altitude of 1,340 masl), with a mean annual temperature of 19–22◦c, 85–90% relative humidity, and 2,250mm mean annual precipitation. the soils are vitric luvisol, rich in organic matter, low calcium and high iron, manganese and zinc nutrients, with moderate fertility, coarse texture and volcanic glass fragments (ph 4.3–6.5). the current area of the genebank covers 3ha. because chayote is a climbing plant, accessions are kept in a metal netlike support structure at a 2.2m height. the accessions are introduced into the genebank as ±20cm plants, obtained by direct sampling, donation or purchase from rural inhabitants. sechium plants are cross-pollinated. to maintain the genetic identity of each accession, asexual 128 cadena-iñiguez et al genetic resources (2024), 5 (10), 126–138 figure 1. methods of regeneration and asexual multiplication of sechium spp. accessions. a) in vitro multiplication, b-c) grafting and c) callus emission, training sites and emission of roots from the use of regulators of growth. multiplication techniques such as grafting, rooting of three-node cuttings, and in vitro multiplication are employed. statistical analysis the passport data in bangese include georeferenced and taxonomic data, origin, morphological characteristics of fruits and seeds, biological status and ethnobotanical information, following the descriptor guide for s. edule varieties (cadena-iñiguez et al, 2017). passport details can be found at bangermex (https://bangerme x.snics.gob.mx/bancos). the information was analyzed with the rstudio software (r core team, 2023). out of a total of 309 accessions, only 231 were analyzed, since 78 were excluded being genetic segregants from the hybridization programme. graphic analysis was performed with tidyverse (wickham et al, 2019) to assess the proportion of varietal complexes regarding their origin, type of establishment (orchard, backyard, ruderal) and owner/donor. additionally, the central tendency and dispersion statistics of the altitude variable were evaluated to know any pattern related to the distribution ranges at which sechium varietal complexes develop. qualitative variables were studied by multiple correspondence analysis (mca) with factominer (lê et al, 2008) and factoextra (kassambara and mundt, 2020). to calculate the eigenvectors, eigenvalues and the graph of each variable’s contribution to the components, the same statistics used for the mca were employed. results descriptive analysis bangese contains 231 accessions representing the intraspecific variability of s. edule in 12 varietal complexes: albus dulcis, albus levis, albus minor, albus spinosum, amarus sylvestris, nigrum conus, nigrum levis, nigrum maxima, nigrum minor, nigrum spinosum, nigrum xalapensis and virens levis. also, there are four accessions of s. chinantlense, 12 of s. compositum and two of s. hintonii (the latter was not analyzed). the virens levis, n. spinosum and n. xalapensis varietal complexes are the most represented in the collection (figure 2a). the major diversity of s. edule in mexico is found in the state of veracruz, with 110 accessions (47.6% of the total) (figure 2b). of the 231 accessions, 62.8% were collected from backyards and 23.3% from commercial orchards, while s. compositum, s. chinantlense and s. edule var. amarus sylvestris came from ruderal and forest areas (figure 2c). some accessions of virens levis, n. spinosum and n. xalapensis were from costa rica (cr) and guatemala (guat) (figure 3). it is well known that women play a key role in vegetable cultivation and plant breeding in domestic orchards. our data showed that women managed 58.9% of the s. edule accessions, all of which are edible. wild accessions are outside the interest of men and women; however, their conservation in genebanks is important due to their phytochemical potential for pharmacological use and as sources of genes for genetic improvement in the face of new challenges. the varietal complexes virens levis, nigrum xalapensis, n. spinosum, n. levis and albus dulcis were identified as man-enterprise, due to their large-scale commercialization. the 70.8% of the varietal complexes are cultivated for commercial purposes, while 29.2% are used for self-consumption, mainly focusing on fruit. this is especially relevant for the lesser-known variants, highlighting their potential to open new markets. the virens and nigrum groups are mainly used for commercialization, while albus is used for self-consumption. bitter-flavoured varieties are generally not used by rural stakeholders (figure 4). sechium sp. can be found at altitudes from 1,200–3,376m, due to its plasticity, which allows it to adapt to conditions different from its original habitat in mesophyll forests (cadena-iñiguez et al, 2008). the highest average altitude for nigrum spinosum was 1,993m. outliers’ presence affects some central tendency measures such as the mean, and some populations of the varietal complexes appear far from their optimal ranges for each specific variety. multiple correspondence analysis (mca) for this analysis, the phenotypic variables of fruits were included (colour, presence/absence of thorns, size, genetic resources (2024), 5 (10), 126–138 mexican chayote collection 129 figure 2. composition of the sechium collection at bangese. (a) number of accessions per varietal group of s. edule, s. compositum and s. chinantlense; (b) geographical origin of the accessions; (c) sampling locations. flavour and shape). the first two principal components explained 27.24% of the total obtained variation. table 1 shows the eigenvalues and eigenvectors, respectively. for principal components 1 and 2 (pc1 and pc2), the variables contributing the most were fruit flavour, populations condition (forest, ruderal-wild and orchard), fruit colour (yellow and light green), small fruit size and flattened shape. the distribution of the accessions depended on the environmental conditions. accessions of s. compositum, s. chinantlense and s. edule var. amarus sylvestris were distributed in wild and ruderal-wild forms. the virens levis complex was primarily found in orchard conditions and is the most commercially important variety of s. edule. the yellow-fruited varietal complexes were generally found in backyards. ownership patterns indicate that the virens levis varietal group was more figure 3. proportion and distribution of sechium edule varietal groups in the bangese collection according to place of origin of sechium germplasm. cr, costa rica; guat, guatemala. often associated with male farmers, while female tended to maintain the albus and nigrum groups. regarding fruit colour, most accessions exhibited various shades of green, except for the albus group, which had yellow fruits (figure 5a). figure 5b shows that all accessions of amarus sylvestris (aw), nigrum spinosum (ns) and albus spinosum (as) had thorns on their fruits. this is relevant, considering that the wild ancestor s. edule (wild type) is dark green, bitter and densely spiny, suggesting that consumer preference is for smooth fruits and neutral or sweet flavour. the fruit size of the chayote is highly variable, ranging from very large (15–22cm), medium (6–10cm), and small (3–4cm) (figure 6a). regarding flavour, the ellipses show a very noticeable separation, revealing that the fruits of s. chinantlense, s. compositum and amarus sylvestris are bitter, a trait attributed to their tetracyclic triterpenes content, mainly cucurbitacins (aguiñigasánchez et al, 2015, 2017; salazar-aguilar et al, 2017). fruits of the albus group have a slightly sweet flavour (7.6–8.1◦brix), while nigrum (4.9–6.4◦brix) and virens groups have a neutral flavour (5.1◦brix) (figure 6b) (cadena-iñiguez et al, 2007; cadena-iñiguez and arévalo-galarza, 2011). cucurbitacins are secondary metabolites of triterpene origin that give a bitter taste to plants (root, guides, leaves, and fruits) and have been recorded in all sechium species in the bangese, although with notable differences in concentration and type of compound (aglycones and glycosides). for instance, the albus group contains one-hundredth of the triterpenes levels found in wild species, and ten times less than nigrum (uriostegui, 2014; iñiguez-luna et al, 2021). fruit shape is highly variable and it is difficult to establish a clear grouping pattern (figure 7); however, the commercially preferred shape is pyriform, which is the shape of virens levis and is the basis of the international standard codex-stan-83-993 (pyriform 130 cadena-iñiguez et al genetic resources (2024), 5 (10), 126–138 figure 4. relationship between stakeholders and conservation of sechium edule, s. chinantlense and s. compositum of the sechium germplasm collection at bangese; a) the conservation methods/users and b) the use of the accessions. table 1. eigenvalues and variance are explained for the first ten components of the mca. pc, principal component. component eigenvalue variance (%) cumulative variance (%) pc 1 0.49 15.50 15.50 pc 2 0.42 13.48 28.98 pc 3 0.30 9.48 38.46 pc 4 0.24 7.79 46.24 pc 5 0.20 6.49 52.74 pc 6 0.19 6.16 58.90 pc 7 0.18 5.82 64.72 pc 8 0.16 5.20 69.92 pc 9 0.15 4.82 74.73 pc 10 0.15 4.70 79.44 fruits, light green colour, smooth without thorns and grooves, 12–15cm long, 8–10cm equatorial width, 270–310g, neutral flavour, and free of pathogens). the pyriform shape may be a derivation of continued selection by cultivars, as the s. edule fruits of the wild type (ancestor) are small and ovate (figure 8). morphological boundaries establishing morphological limits in intraspecific complexes is relatively difficult, since it requires to determine whether distinctive traits are stable and heritable. variation in plants may be continuous (clinal) or discontinuous, consisting of distinct morphotypes or races (styles, 1986). in sechium, variation is clinal since it shows a gradient that allows any character to take values between two extremes (cadena-iñiguez, 2005). environmental factors, including numerous physical, chemical and biological sub-variables, induce this variation by exerting extra pressure on the differentiation process, enabling organisms to express a sufficiently satisfactory plasticity response for survival. for example, in s. edule and s. compositum, fruit shape and size vary according to altitude, with accessions from lower elevations (60m) producing round and small fruits, while those from higher elevations (200m) produce medium pyriform fruits in evergreen forests. figure 9 shows the morphological limits based on fruit shape, which helps to distinguish the varietal complexes of s. edule, s. chinantlense and s. compositum, based on the colour of the epidermis, spines, shape and size. discussion the preference in the consumption of chayote varietal groups is based on the physical and chemical characteristics of its fruits. in mexico, the most present in the markets are virens levis, nigrum xalapensis and nigrum spinosum, the latter recently introduced in the united states. for these markets, fruits are harvested at horticultural maturity (aung et al, 1990) and have a watery consistency, neutral flavour and little fibre content. in regional markets, fruit with higher starch content in the endocarp, such as albus, or physiologically mature green fruits, are preferred. this promotes unconscious conservation actions in backyards by women, who determine the type of chayote to be used in the diet. their decision to either conserve a single morphotype or diversify indirectly leads to reproductive isolation and inbreeding selection, which helps to fix characters and make them stable and heritable or may induce spontaneous crosses (cadena-iñiguez et al, 2010). genetic resources (2024), 5 (10), 126–138 mexican chayote collection 131 figure 5. multiple correspondence analysis for a) fruit colour, and b) fruit texture variables on sechium germplasm. al, albus levis; as, albus spinosum; aw, s. edule var. amarus sylvestris; nc, nigrum conus; nm, nigrum maxima; nm, nigrum minor; ns, nigrum spinosum; nx, nigrum xalapensis; sci, s. chinantlense; sco, s. compositum; vl, virens levis. pc, principal component. women play a key role in the conservation of plant genetic resources mainly through the management of family orchards, where these resources are used for food and sometimes for local sale. local marketing or exchange of backyard surpluses allows genetic diversity to flow at the regional level (watson and eyzaguirre, 2002). when phytogenetic resources are used for human consumption, strong selection pressures arise. for chayote, the preference tends to converge on green fruits with neutral or sweet flavour and low fibre, although preferences for fruits with starchy consistency (albus group) have also been identified (cadena-iñiguez et al, 2010). the first wild populations of s. edule evolved in mesophyll forests, producing dark green, spiny and bitter fruits (lira, 1996). this type of vegetation is conducive to intraspecific variation in s. edule (iñiguez et al, 2011). mesophilic forests, distributed across the highlands of the sierra madre oriental, sierra norte de chiapas and sierra madre del sur, range from 600–3,100m in altitude, with annual precipitation of 1,000-3,000mm, and temperatures ranging between 12–23◦c (ruiz-jiménez et al, 2012). along with natural and artificial selections, migratory routes also cause variations in fruit colour and flavour. when wild populations of s. edule spread to low subevergreen forests, greater irradiance led to a change in chlorophyll content (iñiguez et al, 2011), giving rise to some varietal complexes such as virens levis and those of the nigrum group. in the high plateaus and high valleys, it is common to find thorny green varietal complexes. in medium evergreen and low deciduous forest vegetation types, leaves and fruits began to change in colour and shape, later giving rise to varietal complexes of the albus group, where the fruits have low chlorophyll and cucurbitacin content but high carotenoid and ascorbic acid content (iñiguez et al, 2011), presumably for photoprotection, since these fruits have high stomata density (ramı́rez-rodas et al, 2021). also, the derivation of yellow fruits is strongly influenced by selection pressures. valenta et al (2018) indicate that fruit colour plays an important role in seed dispersal and can be considered an evolutionary signal in plants. a contrast between leaf and fruit colour is important for zoochory success. in this regard, iñiguez et al (2011) mention that yellow chayotes are considered the most evolved in terms of their environmental adaptation. mca of the fruit size shows that this variable tends to be dynamic. wild populations of s. edule, s. chinantlense and s. compositum have relatively small fruits (figure 9) and bitter taste. the latter is related to bi-1 and bt-1 genes, which confer bitter taste to leaves and 132 cadena-iñiguez et al genetic resources (2024), 5 (10), 126–138 figure 6. multiple correspondence analysis for the variables a) size, and b) flavour of fruit on sechium germplasm. sco, s. compositum; sci, s. chinantlense; aw, s. edule var. amarus sylvestris; vl, virens levis; nm, nigrum maxima; nm, nigrum minor; nx, nigrum xalapensis; nc, nigrum conus; ns, nigrum spinosum; al, albus levis; as, albus spinosum, pc, principal component. fruits (valenta et al, 2018). subsequently, with recurrent selection, this characteristic is reduced. in sechium spp., the bitter taste is influenced by the concentration of cucurbitacins, higher in wild populations, and has applications in pharmacology particularly in cancer treatment (cadena-iñiguez et al, 2013b; rosado-pérez et al, 2019). numerous examples illustrate the role of rural women in conserving plant diversity in home gardens for family food, which in turn supports broader biodiversity. in indonesia, elfrida et al (2020) recorded the conservation of 39 fruit plant species of 23 genera and 17 botanical families by women. other studies such as tefera and kim (2019) demonstrated the diversity of medicinal plants conserved by rural families in ethiopia (52 families and 96 genera). rural women are critical to conserving local knowledge, encompassing beliefs, medicine, food and economic livelihood. conservation behaviour often arises from culture rather than formal education. ondiba and matsui (2021) mention that, from a sample of rural women surveyed, 98% expressed motivation to obtain economic income through acts of conservation, and 88% expressed a commitment to environmental conservation. george and christopher (2020) recorded the high species diversity in home orchards in kerala, india; others, such as zhang et al (2020) documented the plants and their local function, highlighting that altitude could be the most important variable that determines the composition of home gardens in china. galluzzi et al (2010) reported the importance of home orchards in safeguarding agrobiodiversity, which reflects a complex structure. according to the passport data, the variability of sechium accessions in bangese is mainly attributed to the actions of rural women in home gardens or backyards, despite conservation not being their conscious objective. however, and for the reasons documented in the passport data, aspects such as self-consumption, local trade, medicinal and ancestry uses, have fostered a wide agrobiodiversity, mainly determined by these plants’ value as food. an important aspect that interacts with conservation, agrobiodiversity, bioprospective research, new applications, industry, rural players, home orchards and genebanks, are international treaties. the nagoya protocol (cbd, 2011) addresses several of these aspects and seeks, as far as possible, a fair and equitable distribution of benefits to the stakeholders that have safeguarded agrobiodiversity or have enriched collections in genebanks. in this regard, the analysis of passport data is a useful tool for identifying potential beneficiaries for bioprospective research and products over the genetic resources (2024), 5 (10), 126–138 mexican chayote collection 133 figure 7. multiple correspondence analysis for the fruit shape variable of sechium germplasm in the bangese collection. sco, s. compositum, sci, s. chinantlense; aw, s. edule var. amarus sylvestris; vl, virens levis; nm, nigrum maxima; nm, nigrum minor; nx, nigrum xalapensis; nc, nigrum conus; ns, nigrum spinosum; al, albus levis; as, albus spinosum, pc, principal component. figure 8. colour, shape and fruit size variation in sechium compositum (a and b, 6.0–8.0cm), s. chinantlense (c, 5.0–6.0cm), s. edule (wild type: d, 5.0–6.0cm) and s. edule var. amarus sylvestris (e, 6.5–7.0cm). medium or long term. varshney et al (2020) proposed a 5g approach to conserved agrobiodiversity: genome of each crop species, genomically and agronomically characterized germplasm, gene function identification, genomic breeding methodologies, and finally gene editing. this approach is a desirable strategy for germplasm collections; however, it does not consider the participation of rural stakeholders, whose ancestral knowledge and practices have helped maintain high agrobiodiversity. therefore, analyzing passport information is key to designing strategies for replacing and regenerating genotypes in ex situ collections, as well as for designing shared conservation models with rural stakeholders. such models could include morpho-anatomical, phytochemical, genomic and agronomic characterization lines, as well as bioprospective studies to explore new applications and benefits. these efforts could also support descriptor guides for legal registration of new plant varieties or facilitate compliance with international treaties and material transfer agreements. wild populations with small, dark green fruits, thorns and bitter taste were the promoters of the morphological diversity that is currently known of s. edule. however, there is also evidence of introgression and hybridization with the species s. compositum and s. chinantlense (barrera-guzmán et al, 2021a). conserving this valuable resource begins with evaluating and characterizing genetic variability through morphological and molecular markers, as well as establishing evolutionary relationships among wild, cultivated populations and related species (cadena-iñiguez et al, 2007). ecology studies in conjunction with ecological niche models also provide information on species adaptability, highlighting 134 cadena-iñiguez et al genetic resources (2024), 5 (10), 126–138 figure 9. colour, shape and fruit size variation in varietal complex of sechium edule; a) albus dulcis (6.2–7.0cm), b) albus levis (6.0–7.0cm), c) albus minor (3.0–3.5cm), d) albus spinosum (13–16cm), e) nigrum conus (5.5–7.5cm), f) nigrum levis (5.7–7.0cm), g) nigrum maxima (16–22cm), h) nigrum minor (3.0–3.5cm), i) nigrum spinosum (10–16.5cm), j) nigrum xalapensis (15–17.5cm) and k) virens levis (13–16cm). genetic resources (2024), 5 (10), 126–138 mexican chayote collection 135 potential areas for conservation and zones threatened by climate change. the study of biodiversity is also an incentive for participatory genetic improvement programmes of chayote, fostering collaboration and knowledge exchange between researchers and farmers. this encourages the continuous planting of native varieties or populations in traditional plots, and economic support and public policies are essential to maximize these efforts. additionally, awareness campaigns are needed to inform the population about the importance of chayote as a plant genetic resource (aguiñiga-sánchez et al, 2017). it is important to highlight that cytological information is missing or scarce in many accessions both in chromosome number and ploidy level as well as nuclear amount. the chromosomal level variegation such as translocation or inversion could explain species incompatibility (olvera-vazquez et al, 2019). conclusion the morphological diversity of s. edule lies mainly in fruit characters, where traits such as bitterness, the presence of thorns and a dark green colour are representative of wild populations. geographical and cultural richness also influence crop diversification, and the mountainous areas of veracruz offer the ideal habitat for growth and development of new varieties. women play a vital role in the conservation of native chayote populations through backyard cultivation. s. chinantlense and s. compositum are species related to s. edule, and from which important advances are being made in the production of cucurbitacins for the pharmaceutical sector. data availability statement to facilitate access to the data from our chayote germplasm collection, information on how interested researchers can get full access is provided below. the data is hosted in our online database, which is accessible upon request. researchers interested in accessing the full dataset can do so by contacting the corresponding author and providing a brief description of the intended use of the data. in addition, access requests will be reviewed to ensure proper use in accordance with established ethical guidelines for the conservation and research of plant genetic resources. our goal is to support collaborative research efforts and encourage studies that expand the knowledge and utilization of this important collection of germplasm. authors contribution jorge cadena-iñiguez y luis a. barrera-guzmán wrote the article and performed the statistical analysis of the database; v́ıctor m. cisneros-solano, the curator of the genebank, provided the information on the morphological aspects of the collection; carlos h. avendaño-arrazate, ma. de lourdes c. arévalo-galarza, kazuo n. watanabe and jorge d. cadena-zamudio, who have been developing the database over the years, also contributed to the discussion of this research. conflict of interest statement the authors have no conflicts of interest to report. references aguiñiga-sánchez, i., cadena-́iñiguez, j., santiagoosorio, e., gómez-garćıa, g., mendoza-núñez, v. m., rosado-pérez, j., rúız-ramos, m., cisneros-solano, v. m., ledesma-mart́ınez, e., de, j. d.-b. a., and sotohernández, r. m. 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(2019). welcome to the tidyverse. j open source softw 4(43), 1686–1686. doi: https://doi.org/10.21105/joss.01686 zhang, y., yang, l. x., li, m. x., guo, y. j., li, s., and wang, y. h. (2020). the best choices: the diversity and functions of the plants in the home gardens of the tsang-la (motuo menba) communities in yarlung tsangpo grand canyon, southwest china. j ethnobiol ethnomed 16(1), 50. doi: https://doi.org/10.1186/ s13002-020-00395-z https://doi.org/10.21105/joss.01686 https://doi.org/10.1186/s13002-020-00395-z https://doi.org/10.1186/s13002-020-00395-z introduction materials and methods location of the genebank and passport data statistical analysis results descriptive analysis multiple correspondence analysis (mca) morphological boundaries discussion conclusion data availability statement authors contribution conflict of interest statement review and position paper genetic resources (2025), (s2), 119–134 doi: 10.46265/genresj.luzj7324 https://www.genresj.org issn: 2708-3764 strengthening european research cooperation on plant genetic resources conservation and use sandra goritschnig *,a, stephan weise b, filippo guzzon a, lorenzo maggioni a, theo van hintum c, lise lykke steffensend, nils stein b,e and giovanni giuliano f a european cooperative programme for plant genetic resources (ecpgr), alliance of bioversity international and ciat, via di san domenico 1, 00153 rome, italy b leibniz institute of plant genetics and crop plant research (ipk), gatersleben, germany c centre for genetic resources, the netherlands (cgn), wageningen, the netherlands d nordic genetic resources centre (nordgen), alnarp, sweden e crop plant genetics, institute of agricultural and nutritional sciences, martin-luther-university of halle-wittenberg, halle (saale), germany f italian national agency for new technologies, energy and sustainable economic development (enea), via anguillarese 301, 00123 rome, italy abstract: plant genetic resources (pgr) are a vital research infrastructure and an important asset to increase the resiliency of agri-food systems, conserve agrobiodiversity and mitigate the effects of climate change. in the current scenarios of climate change and biodiversity loss, it becomes increasingly urgent to ensure the conservation of existing crop diversity and assure its availability for research and breeding to enable the development of new, adapted crops. throughout europe, more than 400 collections conserve pgr of over 6,500 genera, with more than 2 million accessions documented in the european search catalogue for plant genetic resources (eurisco). to make these resources available to breeders, more research investment in these collections is needed. here, we analyze the participation of european genebanks in collaborative projects within the eu horizon scheme as an indicator for the use of pgr collections in research. we highlight two horizon projects, agent and g2p-sol as well as the ecpgr initiative european evaluation network (eva), which have brought together genebanks and other stakeholders to create tools and knowledge on pgr. their experience could be translated into a dedicated, large european research infrastructure for pgr (grace-ri), suggested in the plant genetic resources strategy for europe and currently in the concept phase by the horizon europe project pro-grace. grace-ri will connect european research institutes involved in pgr conservation and research and will be key to ensuring access to well-documented and maintained pgr and methods for their characterization and utilization, preventing further loss of plant biodiversity which is increasingly threatening european agriculture and natural landscapes. keywords: plant genetic resources, genebank, research, breeding, documentation, infrastructure, biodiversity, conservation, sustainable use citation: goritschnig, s., weise, s., guzzon, f., maggioni, l., van hintum, t., steffensen, l. l., stein, n., giuliano, g. (2025). strengthening european research cooperation on plant genetic resources conservation and use. genetic resources (s2), 119– 134. doi: 10.46265/genresj.luzj7324. © copyright 2025 the authors. this is an open access article distributed under the terms of the creative commons attribution license (cc by 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. ∗corresponding author: sandra goritschnig (s.goritschnig@cgiar.org) introduction plant genetic resources (pgr) are important assets to address the challenges associated with climate change, sustainability in agriculture and food and nutrition security, as they provide the genetic diversity necessary to develop adapted crop cultivars for agriculture in received: 18.11.2024 accepted: 14.02.2025 published online: 21.03.2025 https://www.genresj.org https://www.doi.org/10.46265/genresj.luzj7324 https://www.genresj.org https://www.doi.org/10.46265/genresj.luzj7324 mailto:s.goritschnig@cgiar.org 120 goritschnig et al genetic resources (2025), (s2), 119–134 diverse and rapidly changing ecosystems (haussmann et al, 2004; mccouch et al, 2013; swarup et al, 2021). they are also an invaluable reservoir of genetic information that scientists can decipher thanks to modern technologies and the increasing understanding of the relationship between genomes and the metabolic functions of living organisms (bilbrey et al, 2021; derbyshire et al, 2022) an important prerequisite to ensure pgr availability is their active conservation and documentation (weise et al, 2020) with the involvement of all relevant stakeholders, including genebanks, in situ conservation sites, farmers, researchers and breeders (engels and ebert, 2024). pgr conservation falls under the responsibility of national programmes in each country, with international agreements such as the convention on biological diversity (cbd) and its nagoya protocol (cbd, 2011), as well as the international treaty for plant genetic resources for food and agriculture (itpgrfa, fao (2009)) providing the legal frameworks for conservation and facilitated access to germplasm and its associated information and the shared benefits deriving from their use. since the establishment of the first genebank collections in the 1950s, now more than 4.4 million accessions are documented in the worldwide data platform genesys (https: //www.genesys-pgr.org/). these diverse national pgr collections are becoming more and more relevant for research and breeding programmes as they provide the wild relatives and locally adapted landraces necessary for breeding improved varieties. therefore, ensuring the availability of these plant materials is of crucial importance (gullotta et al, 2023). adding genotypic and phenotypic information on accessions in the collections greatly facilitates their management and use, helping with identifying duplicates and selecting appropriate material for further studies and breeding activities (mccouch et al, 2013; mascher et al, 2019). accessing international funding through participation in collaborative projects and networks has been a way to improve knowledge about institutes’ pgr collections and their management, build capacity and strengthen connections among stakeholders. at the european level, since 1980 the european cooperative programme for plant genetic resources (ecpgr) has built a network for national pgr conservation programmes currently encompassing 36 countries, and has been instrumental in building capacity and developing tools for the use of the pgr community, including the european search catalogue for plant genetic resources (eurisco) (kotni et al, 2023) and the european genebank integrated system (aegis). the latter aims at standardizing quality management across participating genebanks and establishing a european collection of unique pgr accessions, promptly available under the same terms and conditions. still, overall funding is inadequate to effectively conserve, document and exploit the available pgr diversity, as outlined in the plant genetic resources strategy for europe (ecpgr, 2021). a worldwide survey conducted among stakeholders within itpgrfa contracting parties identified a number of bottlenecks in the pgr use system, including insufficient support and implementation of national policy systems, lacking personal and institutional capacities, insufficient involvement of important stakeholders such as farmers and breeders as well as lack of awareness of the importance of pgr conservation in the general public (kell et al, 2017). they also identified several important constraints for access to pgr and connected data, including insufficient characterization and evaluation across diverse gene pools, the fragmentation and inaccessibility of generated data and the difficulty to access material with specific traits. the survey further highlighted the value of collaboration, between public sector administrations and researchers as well as between the public and private sectors, in programmes towards sustainable use of pgr in research and breeding (kell et al, 2017). improving our knowledge of crop genetic diversity conserved across the world, in particular to promote coverage of genetic and phenotypic data on pgr as suggested by mccouch et al (2013) will enable focused research and breeding of well-adapted, nutritious and resilient crops. with the world’s biodiversity in decline (ipbes, 2019) and the loss of genetic diversity in our crops threatening food security (pilling et al, 2020), more decisive actions and policies are thus needed to ensure the conservation of pgr for their use in research and breeding. in the recently published plant genetic resources strategy for europe (ecpgr, 2021), issues and relevant gaps in conserving and facilitating pgr use across europe were highlighted, and the authors called for a transformative change whereby europe should reinforce its leading role in their conservation and sustainable use. the strategy’s objectives by 2030 include the expansion, improvement and consolidation of ex situ, in situ and on-farm conservation activities, strengthened data management and germplasm information systems, increased access and sustainable use of pgr and monitoring of progress in pgr conservation and use (ecpgr, 2021). in order to ensure the availability of well-documented pgr for use by future generations, the strategy proposed a number of actions to strengthen national programmes, build capacity of conservation actors and promote international collaborations, involving all relevant stakeholders including the general public. this requires an appropriate policy and legal framework, combined with secure and appropriate financing to strengthen national and regional programmes as well as institutional and human capacity. a recommended key step to enable the transition to a fully functional european system effectively supporting high-quality research is the establishment of an efficient research infrastructure dedicated to pgr conservation, documentation, research and sustainable use. in this paper, we provide an overview of european genebank collections documented in eurisco and analyze the current engagement status of european genetic resources (2025), (s2), 119–134 european cooperation as a cornerstone of pgr research 121 genebanks and other pgr-holding institutes in international projects funded by the eu as an indicator for the use of pgr collections in research. we provide examples of successful cooperation between research institutes, genebanks and other stakeholders in three european projects that highlight the usefulness of international collaboration in advancing research on pgr. finally, we propose a coordinated european approach to pgr research involving a dedicated research infrastructure (grace-ri) that will enable effective conservation and sustainable use of pgr through the provision of a suite of relevant technical services to facilitate adaptation of european agriculture to the climate emergency and food security issues. materials and methods to conduct a summary analysis of the information stored in eurisco to describe the current situation in terms of genebank documentation, data were extracted from eurisco on 27/01/2025 and filtered for size of collections with regard to level of safety duplication, biological status and genera represented. to map the involvement of european genebanks in such projects, we conducted a keyword search with relevant terms against the eu horizon project databases (fp7, https://data.europa.eu/data/datasets/cordisfp7 projects?locale=en; h2020, https://data.europa.eu/d ata/datasets/cordish2020projects?locale=en; horizon europe, https://data.europa.eu/data/datasets/cordiseu-research-projects-under-horizon-europe-2021-2027 ?locale=en; accessed on 10/09/2024). out of 35,386 projects granted within the horizon2020 framework, 424 projects contain the keywords ‘biodiversity’, ‘agriculture’, ‘breeding’, ‘crop wild relative’, ‘genetic resource’ or ‘plant’ in varying combinations in their titles and objective descriptions. this list was manually curated to eliminate projects that were not directly working on pgr or were involving only one institute, yielding 40 projects with multi-actor consortia working on pgr in the years 2014–2022 (supplemental table 1). a similar filtering approach for horizon europe projects yielded 40 pgr-related projects out of 13,215 total, which started in the years 2022–2024 and are ongoing (supplemental table 2). within the framework programme fp7 funding scheme, which was active between 2008 and 2018, we identified 33 multi-actor projects related to the above keywords (supplemental table 3). projects were analyzed and grouped according to the crops and pgr studied and the overarching topics of their research questions. we then compared the involved project partners against the eurisco institutes list (downloaded from eurisco: http://eurisco.ecpgr. org, accessed on 23/04/2024) since these officially conserve germplasm that is part of the european countries’ national inventories. some project partners could represent multiple holding institutes (e.g. inrae,1 csic2 or crea3); in these cases, the projects were analyzed to match the correct institute. in this way, we identified 76 institutions from 25 countries listed in eurisco that have participated in projects financed by the european commission since 2008 (supplemental tables 1–4). we contacted genebank curators and managers of the identified institutes with a short survey to collect additional information on their involvement in the listed projects, including whether or not they contributed pgr and how research materials produced in the projects were conserved and received responses from 35 institutes. the qualitative analysis of horizon2020 projects agent and g2psol as well as the ecpgr european evaluation network (eva) is based on data available to project partners among the authors. results overview of european genebank collections across europe, more than 400 institutes in 43 countries conserve pgr in ex situ and field collections, with at present more than 2 million accessions recorded in eurisco (kotni et al, 2023). these holding institutes vary greatly in size, capacity and mandate within national conservation programmes, with documented collections of between one (recorded by 22 holding institutes) and more than 680,000 accessions (by the nottingham arabidopsis stock centre nasc, figure 1a). figure 1b shows the number of genebanks conserving the most represented genera (excepting arabidopsis), with collections of more than 100 and 1,000 accessions emphasized, further highlighting the diversity in size of european genebanks. thirty-four countries have safetyduplicated parts of their collections in genebanks located in other countries or in the svalbard global seed vault, although overall, only 5.4% of european ex situ accessions have been added to the svalbard seed vault so far (figure 1c, asdal (2025)). the collections cover more than 6,500 different genera of crop species and their wild relatives (cwr) stored as seeds, in vitro, in cryo or field collections and provide long-term storage of the plant reproductive material. reviewing the biological status of material in the collections (alercia et al, 2015), wild or natural materials (code 1xx) represent around 13% of collections but have the greatest diversity with 6,185 genera (figure 1d). around 15% of the collections are traditional cultivars and landraces (code 300), with 599 genera represented. the largest part of the documented collections are 1 inrae, institut national de recherche pour l’agriculture, l’alimentation et l’environnement (france’s national research institute for agriculture, food and environment) 2 csic, consejo superior de investigaciones cient́ıficas (spanish national research council) 3 crea, consiglio per la ricerca in agricoltura e l’analisi dell’economia agraria (council for agricultural research and economics, italy) http://eurisco.ecpgr.org 122 goritschnig et al genetic resources (2025), (s2), 119–134 breeding materials (code 4xx) and advanced/improved cultivars (code 500) with together ~44% of the documented accessions, covering 473 and 549 genera, respectively. material of unknown status (999 or na) makes up ~27% of accessions, representing more than 1,600 different genera, highlighting the need to improve knowledge of the european genebank collections and their documentation. a coordinated system for in situ conservation of pgrs and its documentation is still in its infancy, with a recent extension of eurisco facilitating the documentation of in situ populations of cwr. information on in situ conserved pgr is more difficult to review as data are slowly being inserted into the public domain. survey of eu-funded research on plant genetic resources in the european research strategy, funding has been made available through the 7th framework programme and horizon schemes to support research on genetic resources and breeding, providing crucial innovation towards developing more sustainable agrosystems in the face of changing environments. research and innovation actions create platforms for collaborative research consortia that connect institutions from diverse countries and with complementary expertise to produce valuable outputs for the european research agenda. using a keyword search of all projects recorded in the eu cordis database we identified 33, 40 and 40 multiactor projects funded through the fp7, horizon2020 and horizon europe frameworks, respectively (supplemental tables 1–3). we focused our survey on these funding mechanisms because they offer frameworks for international cooperation, cover all eu and associated countries and provide the backbone of european research funding. additional analyses exploring other funding sources (national, bilateral, philanthropic, etc.) would complement our analysis. as per the project descriptions, these addressed various aspects of biodiversity research, agronomy, policy actions and development of research infrastructures. many of these projects are transdisciplinary, working on the development of methods and practices on a variety of crops and agricultural systems, rather than focusing on specific pgr genera. overall, the number of projects on biodiversity, agriculture and pgr funded through eu research actions have increased over time reflecting the increasing importance of related topics in the european research agenda in horizon europe (figure 2a), with an investment of more than 625 million euros. considering the main topics and crops covered by these projects, there is a clear tendency towards cross-cutting projects covering multiple crops or agrobiodiversity in general, with a focus on breeding (figure 2b). the main crops studied by these projects were cereals, legumes and solanaceae, while other vegetables, perennials and non-food crops were represented in fewer projects (figure 2c). survey of research involvement of european genebanks within the above-mentioned projects we analyzed the involved project partners to identify those who are holding institutes of pgr included in eurisco and thus potentially contributing to the projects by providing genebank materials. we identified 40, 28 and 25 projects in h2020, horizon europe and fp7, respectively, involving 76 organizations from 25 countries listed in eurisco (supplemental tables 1–4). some genebanks are embedded in larger organizations and universities (e.g. inrae, wur4, ipk5) and therefore their parent institutions may participate in projects in other capacities. table 1 shows the participation of institutes listed in eurisco in projects under the different funding schemes disaggregated by countries, highlighting that some countries and organizations are well represented in project consortia, as also reflected in the number of projects per institute (supplemental table 4). however, many countries and eurisco institutes take part in only one project at a time, suggesting limitations exist in organizations to fully participate in and benefit from international research opportunities. to better understand the role of eurisco-listed institutes in european research, we contacted genebank managers of the identified organizations to confirm whether they provided pgr accessions to the horizon or other european projects they participated in and received feedback from 35 institutes (table 2). of these, three did not contribute pgr to the projects they participated in, indicating that they played a different role in these projects. in total, respondents listed 29 projects funded by different mechanisms (e.g. horizon, prima or eranet as well as the ecpgr eva project) for which they had provided pgr accessions from their collections. nineteen genebanks indicated that they had incorporated material developed in 15 horizon projects back into their collections to make them available for further exploitation, mainly covering different cereals, legumes, solanaceae and brassica species. based on the identified involvement of their institutes in european projects (supplemental table 4), these responses suggest that while some organizations are very active in horizon projects, european genebanks and their collections are not systematically involved or effectively utilized in european pgr-related projects. in addition, european projects working on pgr appear to utilize plant germplasm that may not be documented within eurisco and included in european national collections, showing an obvious gap in pgr documentation. lastly, promising plant materials generated by european projects are not systematically introduced in genebank collections for further conservation and exploitation. it would be especially interesting to further research 4 wur, wageningen university & research, the netherlands 5 ipk, leibniz-institut für pflanzengenetik und kulturpflanzenforschung (leibniz institute of plant genetics and crop plant research, germany) genetic resources (2025), (s2), 119–134 123 figure 1. european plant genetic resources (pgr) collections documented in eurisco (data from 27/01/2025). a) european holding institutes grouped by size of their collections documented in the eurisco catalogue. in total, 432 institutes have deposited passport data for a total of 2,101,833 pgr conserved accessions in eurisco as of that date. b) distribution of main crop genera holdings among european holding institutes, with collections with more than 100 (>100) and 1,000 (>1,000) accessions indicated. the most represented genus arabidopsis is mainly conserved at the nottingham stock centre and not included in this analysis. c) percentage of safety duplication of ex situ national inventories. d) distribution of documented accessions according to biological status, with associated number of represented genera. numbers for biological status 1xx and 4xx include several subclasses, which were combined for ease of viewing (alercia et al, 2015). european cooperation as a cornerstone of pgr research 124 goritschnig et al genetic resources (2025), (s2), 119–134 figure 2. eu-funded multi-actor projects on plant genetic resources since 2008. a) number of projects on biodiversity and plant genetic resources funded through european funding schemes 7th framework programme (fp7), horizon 2020 (h2020) and horizon europe since 2008, listed by starting date. data extracted on 09/10/2024, hence additional projects may still start in 2024. b) main general topics and c) major crops studied in these projects. genetic resources (2025), (s2), 119–134 125 table 1. participation of 76 eurisco institutes from different countries in multi-actor projects on biodiversity and plant genetic resources funded by the european funding schemes 7th framework programme (fp7), horizon 2020 (h2020) and horizon europe between 2008 and 2024. details are included in supplemental table 4. country # institutes participation in eu projects fp7 h2020 horizon europe total austria 2 1 5 0 6 belgium 4 5 7 11 23 bosnia and herzegovina 2 0 1 1 2 bulgaria 1 0 1 1 2 croatia 1 0 0 1 1 czechia 2 0 3 2 5 france 9 15 25 15 55 germany 4 7 10 7 24 hungary 1 0 0 1 1 israel 3 1 7 1 9 italy 11 6 22 14 42 latvia 1 1 1 0 2 lithuania 1 0 1 1 2 netherlands 1 10 16 8 34 poland 4 2 6 0 8 portugal 2 3 1 3 7 romania 2 0 3 0 3 russia 1 0 2 0 2 serbia 2 1 4 5 10 slovakia 1 0 2 0 2 slovenia 2 0 3 4 7 spain 9 5 22 12 39 sweden 1 0 2 1 3 switzerland 2 5 10 10 25 united kingdom 7 14 12 12 38 the contributions of genebank collections in terms of the numbers of accessions provided and conserved for projects. examples of successful cooperations of genebanks in international projects agent in the h2020 project activated genebank network (agent, https://agent-project.eu/) running from 2020–2025, partners created a network of genebanks and bioinformatics institutes with the aim to improve genebank operations and data management through the development of standardized approaches to data curation and management, data integration and analysis pipelines. the goal was to apply the concept of ‘genebank genomics’ (mascher et al, 2019) to accession management, whereby genomics data would facilitate the identification of potential duplicate accessions and enable tracking the identity of accessions through regeneration cycles for quality management. in combination with relevant phenotypic data, genomics data would also allow prediction and association analyses to identify useful germplasm for further research and breeding. during the project, 12 genebank partners (11 of which are in eurisco) created precision collections (with an emphasis on unique material from the individual genebanks) and bridging collections (potential duplicates between different european genebanks) of the target crops wheat and barley. in total, 6,956 wheat and 5,315 barley accessions were genotyped and phenotyped for agronomic traits by the holding institutes. additional trials for biotic and abiotic stress traits on subsets of the collections complemented the phenotyping and provided data for association analyses. furthermore, genebanks digitized historical data collected during regenerations of their collections, in some cases going back almost 80 years. these historical datasets have proven useful for predicting performance in view of various stresses (gonzalez et al, 2021). european cooperation as a cornerstone of pgr research 126 g oritschnig et al g enetic resources (2025), (s2),119–134 table 2. eurisco institute involvement and plant genetic resource (pgr) provision to european projects. information represents survey responses received from genebank managers. #n/a, not applicable. wiews code institute, location involved in european projects provided pgr to projects conserved pgr derived from projects bgr001 institute for plant genetic resources ’k.malkov’, sadovo, plovdiv district, bulgaria pro-grace, agent, eva agent, eva agent che001 agroscope changins, nyon, switzerland agent, eva agent, eva #n/a che063 prospecierara, basel, switzerland genresbridge, bresov, farmers pride, diversifood, pro-grace bresov bresov cze122 gene bank, prague 6 ruzyne, czech republic agent, ecobreed, bresov, pro-grace, eva agent, ecobreed, bresov, eva agent, ecobreed deu146, deu159, deu271 genebank, leibniz institute of plant genetics and crop plant research, germany eucleg, farmer’s pride, genres bridge, g2p-sol, agent, increase, pro-grace, cousin, eva, legume generation eucleg, agent, increase, eva, legume generation #n/a esp004 centro nacional de recursos fitogenéticos, alcalá de henares. madrid, spain increase, agent pro grace, eva increase, agent, eva agent esp009 consejo superior de investigaciones cient́ıficas. misión biológica de galicia, pontevedra, spain pro-grace, eva eva, minelanddiv (suscrop-eranet 2022), dromamed (prima) #n/a esp026 generalidad valenciana. universidad politécnica de valencia. escuela técnica superior de ingenieros agrónomos. banco de germoplasma, valencia, spain traditom, bresov, farmers’ pride, g2p-sol, harnesstom, pro-grace traditom, bresov, g2p-sol, harnesstom #n/a esp032 principado de asturias. servicio regional de investigación y desarrollo agroalimentario, villaviciosa, spain bresov, increase, legumegeneration bresov, increase, legumegeneration bresov, increase, legumegeneration fra010 institut de génétique environnement et protection des plantes, plant biology and breeding, inrae ploudaniel, france g2p-sol, brasexplor, nem-emerge, pro-wild g2p-sol; nem-emerge; pro-wild brasexplor continued on next page g enetic resources (2025), (s2), 119–134 127 table 2 continued wiews code institute, location involved in european projects provided pgr to projects conserved pgr derived from projects fra065 plant biology and breeding, inrae versailles, france g2p-sol, traditom, genres bridge, harnesstom, agent, invite, genbecon, prowild, nem-emerge, innobreed, pji-facce grasslandscape, prima brasexplor, prima freeclimb, prima dromamed, suscrop eranet minelanddiv all projects except genres bridge, agent, invite brasexplor, innobreed; harnesstom fra250 institut agro rennes-angers irhs, angers, france optima, eva optima, eva #n/a gbr004 millennium seed bank royal botanic gardens kew pro-grace, agent #n/a #n/a gbr016 genetic resources unit, institute of biological, environmental & rural sciences, aberystwyth university, united kingdom eucleg, cropdiva, legume generation, legendary, eva eucleg, legume generation #n/a gbr140 nottingham arabidopsis stock centre, loughborough nottingham, united kingdom pro-grace, pgr secure any project that has requested them arabidopsis lines from various projects gbr247 germplasm resources unit, john innes centre, norwich, united kingdom legumegeneration, prowild, eva legato, legumegeneration, prowild, eva gediflux, legato gbr251 the james hutton institute, dundee, scotland, united kingdom g2p-sol, pro-grace, eva g2p-sol g2p-sol hrv041 faculty of agriculture, university of zagreb, croatia belis, geronimo, grass ceiling, pastinnova, resbios, todo, pro-grace, strenght2food, farmers pride, treasure, mendthegap, eva eva #n/a hun003 centre for plant diversity, tápiószele, hungary pro grace #n/a #n/a isr001 department of field and vegetable crops, hebrew university of jerusalem, rehovot, israel g2p-sol, eva g2p-sol g2p-sol isr002 israel gene bank for agricultural crops, agricultural research organisation, volcani center, bet dagan, israel g2p-sol g2p-sol g2p-sol continued on next page european cooperation as a cornerstone of pg r research 128 g oritschnig et al g enetic resources (2025), (s2),119–134 table 2 continued wiews code institute, location involved in european projects provided pgr to projects conserved pgr derived from projects ita331 facolta di agraria, università degli studi di catania, italy bresov, cousin bresov, cousin bresov ita363 dipartimento di chimica, biologia e biotecnologie, universitá degli studi perugia, italy farmers pride, pro-wild #n/a #n/a ita382 crea-centro di ricerca genomica e bioinformatica sede di fiorenzuola d’arda, italy neurice, best-crop, gp2-sol, pro-wild, pro-grace, eva gp2-sol, pro-wild, eva neurice, best-crop, gp2-sol, pro-wild ita383 crea-centro di ricerca cerealicoltura e colture industriali sede di vercelli, italy agent, pro-grace, eva agent, eva agent ita391 crea-centro di ricerca orticoltura e florovivaismo sede di pontecagnano, italy g2p-sol, pro-grace, eva g2p-sol, eva g2p-sol ita394 crea-centro di ricerca zootecnia e acquacoltura sede di lodi, italy belis, divinfood, intercropvalues, liveseeding, invite, liveseed, remix, eva eva #n/a ita436 istituto di bioscienze e biorisorse, consiglio nazionale delle ricerche, bari, italy pro-grace, eva eva #n/a lva024 institute of agricultural resources and economics, riga, latvia liveseed, eva eva #n/a nld037 centre for genetic resources, the netherlands, wageningen, netherlands pro-grace, g2p-sol, agent, prepactgr, framers pride, genres bridge, eva agent, g2p-sol, eva #n/a pol003 plant breeding and acclimatization institute, blonie, poland agent, increase, rustwatch, g2p-sol, ecobreed, detectiv increase, agent, g2p-sol agent, increase, g2p-sol prt001 banco português de germoplasma vegetal, braga, portugal farmer´s pride; liveseed, pro-grace, eva liveseed, eva liveseed srb062 institut for forage crops krusevac, krusevac, serbia eucleg, belis, eva eucleg, belis #n/a svk001 national agricultural and food centre (nppc), research institute of plant production (ripp), piestany, slovakia rustwatch, ecobreed, agent, eva ecobreed, agent, eva ecobreed svn019 crops and seed production department, agricultural institute of slovenia, ljubljana, slovenia ecobreed, medvitis, brasexplor, diversilience, increase, liveseeding, root2res, eva ecobreed, medvitis, brasexplor, diversilience #n/a genetic resources (2025), (s2), 119–134 129 to manage the data, the bioinformatics partners involved in the project (ipk, inrae, wur, rbgk6) created a range of tools, starting with standard data collection templates, an online data curation tool, a web application, rest interfaces using the breeding api specification (selby et al, 2019) and various data analysis pipelines, which allow the users (primarily genebank data managers) to effectively manage and exploit the data on their collections (beier et al, 2022; berkner et al, 2022, 2024; el-hanafi et al, 2023). several in-project training events and datathons were an opportunity for capacity building and four rounds of genebank peer reviews following the ecpgr aquas protocol have provided useful feedback on genebank operations and identified gaps and areas for improvements (https://www.ecpgr.org/aegis/aquas/pe er-visits). european evaluation network (eva) some of the tools developed by agent build on project outputs of the ecpgr initiative european evaluation network for pgr (eva, https://www.ecp gr.org/eva). this network brings together genebanks, public research institutes, private sector breeders and farmers’ organizations in public–private partnerships to jointly evaluate genebank accessions for relevant traits in multilocation trials across europe. activities are typically provided in-kind and are distributed among partners according to their expertise and capacity. these activities include provision of accessions, regeneration of material (where necessary as single-seed descent lines or crosses), evaluation in the field, lab or greenhouse, genotyping, data curation and data analysis. ecpgr funds, so far provided by the german ministry for food and agriculture, have been essential to kickstart activities that would be difficult to carry out as in-kind inputs, especially seed multiplication or genotyping. participation of breeding companies was incentivized by the agreement to enforce a 3-year embargo on the publication of evaluation data outside the consortium. the crop-specific networks have been operating since 2019 and currently have more than 120 partners including around 50 private breeding companies working on 15 crops. so far, the networks have generated phenotypic data on more than 5,000 accessions in almost 400 trials, where they have so far collected more than half a million datapoints on more than 200 different traits (table 3). the use of standard phenotyping protocols, along with standardized metadata and phenotypic data collection templates, was imperative for the integration of data collected by different partners in more than 100 locations. the eurisco-eva information system, a project-internal platform, developed using the same framework as the public eurisco catalogue, enabled effective data management and analysis (kumar et al, 2024) and provided the template for the agent 6 rbgk, royal botanic gardens, kew, united kingdom database structure. within their collaborative networks, eva partners have developed a new tool for the genotyping of lettuce (tripodi et al, 2023) and have investigated the genetic diversity of european carrot and maize accessions (goritschnig et al, 2023; balconi et al, 2024), generating valuable knowledge for the genebanks and research and breeding communities. the plant material exchange within the networks is governed by the rules of the itpgrfa multilateral system (fao, 2009), using their standard material transfer agreement (smta) also for crops not included in annex 1, thus facilitating access to pgrfa for further research and development activities by all partners. g2p-sol the horizon 2020 project g2p-sol (www.g2p-sol.eu) was active from 2016 to 2021 and involved 19 partners (including 13 genebanks of which nine are listed in eurisco) as well as 12 external collaborating institutions from three continents. g2p-sol focused on four major solanaceae crops (tomato, potato, eggplant, pepper), for which the partner genebanks held around 65,000 accessions, of which around 14,000 cwr (table 4). the project consisted in five phases: 1) inventory of the partner genebanks, in which the images and passport data from the different genebank information systems were collected, manually curated and published, 2) genotyping of a subset of approx. 40,000 accessions with 2,000–10,000 high-quality snps and analyses of population structure, duplicates and taxonomic classification (toppino et al, 2021), 3) establishment of core collections of around 350–400 accessions (both cultivated and wild) for each crop, representing the worldwide genetic and phenotypic diversity of each genepool (e.g. nankar et al (2020)), 4) phenotyping of the core collections in multiple locations for resistance to biotic and abiotic stresses, agronomic, and (limited to the three fruit crops) metabolic traits (gramazio et al, 2020), and 5) pre-breeding, in which markers for described and novel traits from cwrs were established and the traits pre-bred into the genetic background of each cultivated species (stefa´ et al, 2020). g2p-sol has been classified as a success story by the european commission, and its core collections are available under smta and with phytosanitary certificates from partner genebanks acting as distribution points. discussion benefits of international cooperation in pgr research in the three projects highlighted above, partners described the possibility of engaging in active networks and exchanging pre-competitive ideas and knowledge as primary benefits of participation. novel traits for a more sustainable agriculture have been identified and some partners have already applied approaches and european cooperation as a cornerstone of pgr research 130 goritschnig et al genetic resources (2025), (s2), 119–134 table 3. summary of partnership and outputs of five crop-specific ecpgr european evaluation network eva (data as of 30 june 2024). all networks eva wheat barley eva carrot eva lettuce eva pepper eva maize crops 8 3 1 2 1 1 accessions 5,092 3,608 67 367 183 867 partner institutes 89 47 14 12 15 18 countries of operation 33 25 8 8 13 9 experiment locations 119 58 14 6 11 30 traits 285 46 138 24 26 51 trials with available data (2020–2024) 384 265 27 14 15 63 phenotypic datapoints available 539,678 318,891 95,695 10,717 24,016 90,359 table 4. summary of solanaceae accessions included in the g2p-sol collection by participating genebanks. avrdc, the world vegetable center, taiwan (twn); ipk, leibniz institute of plant genetics and crop plant research, germany (deu); huji, the hebrew university of jerusalem, israel (isr); inrae, institut national de recherche pour l’agriculture, l’alimentation et l’environment, france (fra); cip, international potato center, peru (per); wur, wageningen university & research, the netherlands (nld); upv, universitat politecnica de valencia, spain (esp); jhi, james hutton institute, united kingdom (gbr). partner avrdc ipk huji inrae cip wur upv jhi all others total country twn deu isr fra per nld esp gbr tomato 8,260 3,840 8,100 1,600 0 1,332 2,220 0 390 25,352 of which wild 812 26 100 200 0 108 220 0 50 1,466 potato 0 6,020 0 1,500 6,000 1,446 0 2,300 390 17,266 of which wild 0 2,845 0 500 1,800 1,243 0 1,400 300 7,788 pepper 8,235 1,530 0 1,460 0 1,010 1,400 0 850 13,635 of which wild 464 63 0 46 0 783 80 0 56 1,436 eggplant 3,713 110 0 2,015 0 510 260 0 280 6,608 of which wild 1,499 0 0 1,120 0 373 70 0 40 3,062 all four crops 20,208 11,500 8,100 6,575 6,000 4,298 3,880 2,300 1,910 62,861 of which wild 2,775 2,934 100 1,866 1,800 2,507 370 1,400 446 13,752 tools developed by these projects to other species and in other projects. early career researchers in the agent and g2p-sol projects highlighted the opportunity to learn diverse aspects ranging from fieldwork to data curation and analysis, which was facilitated by mentors from different project partners. in general, the standardization approaches followed by both agent and eva are prime examples of how access to data can be facilitated according to fair (findable, accessible, interoperable, reusable) principles (wilkinson et al, 2016), providing pipelines for data curation and management that will allow their reuse in future investigations. however, while these projects have shown a possible new way of working for genebank data management and collaboration, its long-term adoption and implementation involving the majority of european genebanks and covering more crops would require significant investment and political commitment to provide the necessary framework. at present, the ecpgr secretariat is best positioned to disseminate standards and methodologies for genebank operations and crop evaluations throughout their european networks. an assessment of the long-term impact of eu-funded research projects overall is difficult to produce but should consider the availability of generated materials, tools and project data for further research and the level at which project outputs have been exploited after the end of the projects. a well-known issue with project data is that they are often stored in fragmented form, and websites or databases are only maintained for a limited period after projects end. all these considerations call for the need to operate within a more coherent framework that facilitates and promotes genetic resources conservation, documentation and sustainable use, as the plant genetic resources strategy for europe (ecpgr, 2021) is advocating and a suitable eu research infrastructure might implement. our preliminary survey of genebanks involved in eufunded horizon projects showed that some genebanks are very active in participating in european research, contributing both materials and relevant expertise in conservation, cultivation, breeding and documentation. on the other hand, a significant number of genebanks with relatively large collections do not participate in many european projects, limiting the use of these collections in international projects and the benefits to organizations from knowledge exchange. in addition, much of the funded research has been focusing on a few staple crops, e.g. cereals, legumes, solanaceae and brassicaceae, with minor crops only recently genetic resources (2025), (s2), 119–134 131 receiving more attention (figure 2, supplemental tables 1–3). the involvement of genebanks in crop improvement, especially in research and pre-breeding projects, should also ensure that the final products (in terms of germplasm materials) of these projects are either fully incorporated into their collections or otherwise made available for further distribution and exploitation (hanson et al, 2024). it should be noted that inclusion in a genebank collection implies conservation in perpetuity. for some material, such as breeding or research material, this might not always be the proper approach and novel solutions to making project materials available for future use will be needed. follow-up studies are also needed on the effective use of pgr in breeding and to assess the impact of publicly funded research on pgr. the survey analyzed in this paper highlights the importance of pgr use in a collaborative research framework at both the regional and international levels. this is believed to be the most promising in terms of exploitation of the immense public good that is maintained by genebanks, often with insufficient public funds but with a significant potential economic value (gollin, 2020). considering the growing threat of genetic erosion resulting from the changing climate and changing habitats it will also be important to mainstream in situ conservation activities coupled with the assurance that populations of cwr and wild food plants are made available for use in breeding and research (khoury et al, 2022). the experiences from the collaborative projects described in this paper, as well as reports from other successful projects at the european level, highlight the benefits to be gained from international cooperation of genetic resources institutes to increase our knowledge on pgr and make them accessible for further research and breeding. collaborative activities in pgr research and breeding, through the implementation of public–private partnerships and multi-actor projects are imperative to realizing the potential of european pgr in strengthening the bioeconomy along the whole agri-food value chain (vangheluwe et al, 2023). tools and partnership models have been developed, but they may remain ineffective without a permanent coordinated approach and support for the long-term exploitation of project outputs. similarly, a variety of technical challenges in information management as well as political and institutional considerations for access to pgr will need to be addressed in novel cooperative approaches to enhance innovation in pgr research (halewood et al, 2018). the plant genetic resources strategy for europe (ecpgr, 2021) identified the need for long-term european cooperation, which could be realized through the establishment of a european research infrastructure fully dedicated to pgr, currently in the concept development phase through the project pro-grace (https://www. grace-ri.eu/pro-grace). a european research infrastructure to facilitate consolidated research on pgr the horizon europe project pro-grace involves genebanks, research institutes, and non-governmental and stakeholder organizations in developing a framework for collaboration at the european level to assure availability and access to pgr for breeding and research. within this project, standards have been proposed to provide guidance on pgr documentation, enabling effective linkage between conserved accessions, associated multicrop passport descriptors (alercia et al, 2015), and associated phenotypic and genotypic data. surveys have established a baseline of full implementation of quality management systems (qms) in genebanks and informed deliverables developing recommendations for implementation of qms in genebanks and the establishment of a genebank certification system, ensuring reliable conservation of and access to pgr conserved in genebanks (van hintum and wijker, 2024). gap analyses and surveys have identified a clear lack of consolidated pgr research infrastructures within europe and provided evidence for a need to establish a europe-wide distributed research infrastructure that would effectively ensure high-quality conservation of pgr, as well as develop state-of-the-art methods, tools and services for their use in research, breeding and cultivation. this approach represents a great opportunity to further develop eurisco into a fair-compliant european pgr information system that is more closely linked to regional and global platforms and integrates previously untapped data from additional sources and further data domains. the concept for a future european research infrastructure on pgr (grace-ri) covers important aspects which support the implementation of the plant genetic resources strategy for europe and align with needs of stakeholders from conservation and use: 1) enhance all pgr collections across europe to accepted standards through implementation of quality management and upgrade of genebank procedures, 2) improve and consolidate pgr documentation through advances in data integration and management, 3) develop multi-omics and phytosanitary technologies for pgr characterization, enhancing their availability for breeding, 4) assure access to all pgr across europe through supporting genebanks in meeting legal and phytosanitary requirements, 5) mainstream in situ conservation of pgr, particularly of cwr, to facilitate their availability for research and breeding, and 6) develop scientific services destined to the scientific and breeding communities, as well as programmes for capacity building, training and education to achieve the abovementioned objectives. participating countries and institutes will thus create a relevant and well-defined service for the user community, with a significant reduction of redundancies and increased efficiency through the pooling of capacities and expertise. in contrast, continued inaction in this field would mean surrendering to the loss of agricultural and general plant biodiversity which is already threatening our agriculture and ecosystems. european cooperation as a cornerstone of pgr research 132 goritschnig et al genetic resources (2025), (s2), 119–134 conclusion and outlook proper management of pgr is becoming increasingly important due to the growing demand for access to pgr for breeding of new varieties and thanks to better breeding techniques using genebank accessions as raw materials. at the same time, the need to conserve is expanding due to climate change and increasing genetic erosion. against this backdrop, international research projects have provided genetic resource centres with the possibility to participate in collaborations, improving their capacity, gaining knowledge about their collections and thus adding value to their holdings. the creation of a dedicated research infrastructure for pgr, as proposed by pro-grace, will allow the european conservation, research and breeding communities working on pgr to better organize and monitor their activities, reduce redundancies, improve processes and conservation, develop novel methods, with the aim to ensure continued access to and utilization of high-quality pgr materials and related information. this will offer current and future researchers access to pgr and methods for their study and valorization, which are necessary for addressing the main challenges of the present time and ensuring a sustainable and biodiverse agriculture in europe in the future. providing a stable political and financial framework for international research collaboration on pgr will enable europe to firmly establish a primary role in developing science-based solutions to the challenges of the twenty-first century. acknowledgements the authors would like to thank eva, agent, g2p-sol and pro-grace project partners as well as genebank managers for their relevant input into surveys informing this paper. this work was supported by the german federal ministry of agriculture and food (project genr 2019-2 for the eva networks), the eu horizon projects agent (grant agreement 862613) and pro-grace (grant agreement 101094738). supplemental data supplemental table 1. eu-funded multi-actor projects on pgr within the h2020 funding scheme (active 2015–2027). supplemental table 2: eu-funded multi-actor projects on pgr within the horizon europe funding scheme, which have started by mid-2024. supplemental table 3: eu-funded multi-actor projects on pgr within the fp7 funding scheme (2008–2018). supplemental table 4: european genebanks participating in horizon multi-actor projects funded through the european funding 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(2025) “solanum wild relative species indicate varying ecological resilience to climate change in benin (west africa)”, genetic resources, 6(12), pp. 95–110. doi: 10.46265/genresj.zrci8675. © copyright 2025 the authors. this is an open access article distributed under the terms of the creative commons attribution license (cc by 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. ahuéfa mauricel kégbéa,c, rodrigue idohoub,c,*, birane diengd, gafarou agoundec, anthony egerue,f, kandioura nobad, achille ephrem assogbadjoa,c a laboratoire d’ecologie appliquée, faculté des sciences agronomiques, université d’abomey-calavi, 01 bp 526, cotonou, bénin b ecole de gestion et de production végétale et semencière, université nationale agriculture, bp 43, kétou, bénin c laboratoire de biomathématiques et d’estimations forestières, faculté des sciences agronomiques, université d’abomey-calavi, 04 bp 1525, cotonou, bénin d laboratory of botany-biodiversity, faculty of sciences and techniques, department of plant biology, cheikh anta diop university, p.o box 5005, dakar, fann, senegal e department of geography and climatic sciences, makerere university, po box 7062, kampala, uganda f regional universities forum for capacity building in agriculture (ruforum), p.o. box 16811, wandegeya, kampala, uganda * corresponding author: rodrigue idohou (rodrigidohou@gmail.com) introduction crop wild relatives (cwr) are wild plant species closely related to cultivated crops and possess valuable traits that can contribute to crop improvement and breeding efforts. found in natural habitats, these wild species have co-evolved with domesticated crops over millennia (maxted et al, 2006; maxted et al, 2012). the genetic diversity present in cwr is crucial for enhancing the resilience and adaptability of agricultural systems, particularly in response to changing environmental conditions and the emergence of new pests and diseases (maxted and magos brehm, 2023). however, habitat loss, climate change, and other human activities threaten the survival of these species (idohou et al, 2025), limiting their potential contribution to food security, nutrition and human health. despite these challenges, cwr have received and continue to receive increasing interest from scientists worldwide (pilling et al, 2020). https://doi.org/10.46265/genresj.zrci8675 mailto:https://doi.org/10.46265/genresj.apnr6909%0d?subject= https://www.genresj.org http://10.46265/genresj.zrci8675 mailto:rodrigidohou%40gmail.com?subject= genetic resources (2025), 6(12), 95–11096 kégbé et al the solanaceae family is highly diverse, comprising over 2,000 species across approximately 90 genera, including major crops, their wild relatives, and a wide range of perennial and herbaceous annual species (samuels, 2015; gebhardt, 2016). many species provide a wide range of goods and services (gebhardt, 2016), and are used for food, medicinal and ornamental purposes (samuels, 2015). in benin, wild progenitors of solanum are distributed throughout the country but are more abundant in the sudano-guinean and the guineo-congolian zones (akoègninou et al, 2006). in fact, wild solanum species also play an important role in the diet of the local population (sarma and sarma, 2011). several of such species are directly consumed as wild foods rich in micronutrients, fibre and antioxidants. they diversify diets and enhance local food security, especially in rural and marginalized communities. fruits and leaves are used for soups or sauces and have been shown to successfully contribute to improving human health (sarma and sarma, 2011; okokon et al, 2017). for instance, solanum species have been reported to be effective in the treatment of various diseases, including malaria, stomach aches, asthma and diabetes (sarma and sarma, 2011). despite their importance, wild relatives of cultivated solanum vegetables are currently facing severe threats (syfert et al, 2016) in their natural habitats due to habitat loss and conversion primarily from agriculture and grazing (idohou et al, 2017; lala et al, 2018) and more critically, climate change. climate change impacts all aspects of agriculture, which remains a key economic sector and a cornerstone of food security and nutrition in africa. according to the intergovernmental panel on climate change (ipcc), changes in temperature, precipitation patterns, and the frequency of extreme weather events have been observed since the 1950s (masson-delmotte et al, 2022). climate change is now considered one of the major threats to biodiversity and food production, causing widespread disruptions to human systems and indigenous livelihoods, particularly in west africa (ipcc, 2021). as conservation efforts increasingly adopt a ‘biodiversity for livelihoods’ approach, accurate information on plant distributions and ecosystem shifts under environmental change is urgently needed. in the face of this global change, it became urgent to assess the distribution patterns of solanum wild relatives under climate change. indeed, many wild relatives of solanum are found in both agricultural and protected areas (pa), which are currently experiencing high levels of disturbance (steffen et al, 2015). given the current distribution of these species and the potential impact of climate change in many ecosystems across africa, we hypothesized that such environmental changes would negatively affect the future distribution ranges of wild relatives of cultivated solanum leafy vegetables in benin. we also assumed that abiotic factors, namely rainfall, temperature and soil, are the primary drivers influencing both the current and future distribution of these species. ecological niche models (enm), also known as species distribution models (sdm) are powerful tools widely used to predict the species’ abiotic niches and forecast their future distributions under climate change scenarios (feng et al, 2020). various methods have been applied to assess species distributions (hijmans and elith, 2017); however, the choice of the method largely depends on the aim of the study and the availability of data. maximum entropy modelling (maxent) (phillips et al, 2006) is among the most frequently used sdm algorithms. it requires only presence data as inputs and estimates species’ relative occurrence rates (yackulic et al, 2013) by minimizing the relative entropy between the probability distributions of species presence and the background environment. in benin, several researchers have explored the effectiveness of maxent in predicting the potential impacts of climate change on species distribution (daï et al, 2023; hounsou-dindin et al, 2023). however, no studies have specifically focused on the wild relatives of cultivated solanum leafy vegetables to assess their adaptive responses to predicted climate variations. furthermore, the assessment of pa effectiveness in conserving genetic resources must be included in such studies, given their crucial role in safeguarding biodiversity from human disturbances (stolton et al, 2006). in addition, pa serve as natural refuges for many wild plants, although these species have long been overlooked within these conservation frameworks (vargas et al, 2004; burgess et al, 2005). given the limited occurrence of wild solanum species in pa, we hypothesized that these ecosystems are unlikely to serve as hotspots for them. this study examined the potential impact of climate change on wild relatives of cultivated leafy solanum vegetables in benin to inform appropriate conservation strategies. specifically, it aimed to (1) identify the abiotic factors influencing the distribution of the species and their hotspots, (2) evaluate both current and future shifts in the species distribution, (3) determine the conservation status of the target solanum species in benin based on the international union for conservation of nature (iucn) red list categories and criteria and (4) assess the effectiveness of pa in conserving species distributions. the research questions for this study are as follows: which factors influence the geographical distribution patterns of solanum wild relatives? what conservation strategies could mitigate these impacts and enhance the resilience of the species to climate change? materials and methods study area the study was carried out in the republic of benin located in west africa between 6°0’ and 12°50’n, and 1°0’ and 3°40’e (figure 1). three biogeographical zones are distinguishable, namely sudanian, sudano-guinean, and guineo-congolian (adomou, 2005; figure 1). in the guineo-congolian, the mean annual temperature is generally 25–29°c. the relative humidity is 69–97% with a bimodal annual rainfall of 1,200mm. the land cover types are mainly fallow land and agricultural fields with the highest human population density. as for the sudanian zone, it is the driest in the country. however, the rainfall is also unimodal, sometimes reaching 1,000mm per year (adomou, 2005). temperatures range from 24 to 31 °c with a relative humidity between 18 and 99%. the vegetation is characterized by dry forests, riparian forests, tree, shrub and herb savannahs and woodlands. the transition zone of the sudano-guinean region is dominated by bimodal rainfall with a tendency towards unimodality varying between 900 and 1,110mm per year (adomou, 2005). the annual temperature ranges from 25 to 29°c and the relative humidity varies from 31 to 98%. here, the vegetation is characterized by riparian forests, woodlands and dry forests. the flora of benin is estimated to comprise genetic resources (2025), 6(12), 95–110 solanum cwr in benin 97 2,807 plant species, grouped into 1,129 genera and 185 families (akoègninou et al, 2006). the species-rich families are leguminosae (14.8%), poaceae (9.3%), rubiaceae and cyperaceae (5% each), asteraceae (4.6%) and euphorbiaceae (4.3%) (akoègninou et al, 2006; adomou, 2005). agriculture contributes about 28,04% to the gross national product, and the main crops cultivated in benin are cereals, legumes, tubers, vegetables and industrial crops (maep, 2020). solanum species selection a previous study by idohou et al (2013) generated a list of priority cwr for benin for conservation. the list was used to extract the existing list of solanum wild relatives’ species in benin. the species names were cross-checked with those existing in the analytic flora of benin (akoègninou et al, 2006). the species considered in this study were: solanum anguivi herb.lamb. ex dunal, s. anomalum thonn., s. dasyphyllum schumach. & thonn., s. distichum schumach. & thonn., s. erianthum d.don, s. incanum ruiz & pav., wild forms of s. melongena ruiz & pav., s. nigrum vell., s. terminale forssk., and s. torvum buch.-ham. ex wall. the description, distribution, ecology, uses, threats and conservation status of each species are summarized in supplemental table 1. figure 1. location of the study area and occurrence records of solanum wild relative species poplations across benin occurrence data the occurrence records for the ten species were initially downloaded from open-access platforms such as the global biodiversity information facility (www.gbif.org) and rainbio (http://rs.tdwg.org/dwc/terms/). records with the same coordinates and those without identification information were removed with enmtools (www.enmtools.com) (warren et al, 2010). to minimize temporal bias and ensure compatibility with the climate datasets, records prior to 2000 were excluded (idohou et al, 2017). additionally, further occurrence data were collected during a field expedition conducted between 2019 and 2021 across the sudano-guinean and guineocongolian zones. these records encompass a diverse array of ecosystems including farms, fallows and semi-deciduous forests. to reduce spatial autocorrelation among occurrence records, we used the occurrence rarefy tool in arcgis v.10.8 spatial analyst tool (brown, 2014) and retained only one record per 1×1km grid cell. initially, 1,175 raw occurrences were compiled; after filtering for uniqueness and accuracy, 693 records remained, of which 636 were ultimately used in ecological niche modelling (supplemental table 2). the minimum number of georeferenced occurrence records was 20, aligning with the recommendations of wisz et al (2008), who advised 25 records when performing sdm for african species. http://www.gbif.org http://rs.tdwg.org/dwc/terms/ http://www.enmtools.com genetic resources (2025), 6(12), 95–11098 kégbé et al table 1. number of records of wild solanum species and their sources. gbif, global biodiversity information facility; rainbio, megadatabase of tropical african vascular plants distributions. species field gbif rainbio raw data used data solanum anguivi herb.lamb. ex dunal 30 98 0 128 20 solanum anomalum thonn. 10 22 9 41 25 solanum dasyphyllum schumach. & thonn. 14 101 14 129 43 solanum distichum schumach. & thonn. 52 52 11 115 22 solanum erianthum d.don 28 50 9 87 60 solanum incanum ruiz & pav. 0 90 27 117 15 solanum melongena ruiz & pav. 3 114 14 131 102 solanum nigrum vell. 30 106 6 142 115 solanum terminale forssk. 0 14 21 35 31 solanum torvum buch.-ham. ex wall. 45 205 0 250 203 total 212 852 111 1,175 636 modelling technique forty sdm were built per species (4 methods x 2 replication methods x 5 replicates). four machine learning (ml) models available in the sdm package version 1.2-46 (naimi and araujo, 2016) were used: boosted regression trees (brt) (friedman et al, 2000), random forests (rf) (breiman, 2001), support vector machines (meyer and wien, 2001) and maxent (phillips et al, 2006; phillips et al, 2017). this ensemble modelling approach enhances model accuracy and robustness compared to single-algorithm methods (ahmad et al, 2020). due to the lack of true absence data, we generated 104 pseudo-absences as recommended (barbet-massin et al, 2012) using the sdm package. the dataset was split into 70% for model training and 30% for testing, and model fitting was expedited using parallel processing across four ‘n-cores’ (naimi and araujo, 2016). model performance was evaluated using 5-fold cross-validation and bootstrapping replication methods. an ensemble function was applied to integrate predictions from all four machine learning models. performance metrics included the area under the receiver operating characteristic (roc) curve (auc) and the true skill statistic (tss). the auc indicates the probability that the predictive power of a model is better than random prediction (auc = 0.5) (ramírez-villegas et al, 2010; castañedaálvarez et al, 2015); a model with an auc value close to 1 (auc ≥ 0.75) is considered to have a good fit. the tss is a measure of the ability of the model to detect true presence (sensitivity) and true absence (specificity). it is expressed as sensitivity plus specificity -1, with tss > 0.5 indicating a good predictive power (zhang et al, 2015). finally, suitability layers representing the current and future distribution of each species were exported in binary raster tiff format using the average tss value from the five replications, where a value of 1 denotes suitable habitat and 0 denotes unsuitable habitat. suitability habitat mapping, dynamic of the suitable areas, species richness and gap analysis the output raster representing the habitat suitability of each species (i.e. suitable and unsuitable habitats) was imported into arcgis 10.8, and the current and future suitable areas were then mapped. environmental data current and future climatic data at 30 arcsec spatial resolution (~1km at the equator) were obtained from the chelsa website (www.chelsa-climate.org) (parviainen et al, 2008). the datasets included 19 bioclimatic variables (supplemental table 3) related to temperature and rainfall (karger et al, 2017) and represented a baseline condition (1979 to 2013) along with two future periods: 2055 (2041– 2070) and 2085 (2071–2100). future climate conditions were projected using the ipsl-cm6a-lr climate model (boucher et al, 2020), under two shared socioeconomic pathways (ssp): the middle-of-the-road scenario (ssp3-7.0) and the worstcase scenario (ssp5-8.5). these scenarios predict conditions likely for africa by 2055 and 2085 (williams and jackson, 2007). ssp3-7.0 predicts an additional radiative forcing of 7w/m2 by 2100 while ssp5-8.5 forecasts an additional radiative forcing of 8.5w/m2 by 2100. the two scenarios were used to represent the moderate and the worst-case impacts of climate change for wild solanum species in benin. in addition, edaphic variables were downloaded from the africa soil profiles database (https://www.isric.org) and processed for benin. the selected variables are summarized in supplemental table 3. elevation data were obtained from the worldclim database (www.worldclim.org). all these variables had a spatial resolution of 1km and were merged using the raster package in r 4.0.3 (r core team, 2023). to determine the environmental factors influencing the current distribution of each taxon, we followed a 3-step procedure. first, we excluded bio8, bio9, bio18, and bio19 because these variables frequently exhibited discontinuities between neighboring pixels across the african continent (montoya-jiménez et al, 2022). second, we performed a pearson correlation test using the usdm package (naimi, 2017) to select variables with low correlation, retaining those with an absolute correlation coefficient (|r|) below 0.7 (supplemental table 4). third, we conducted a jackknife test using the sdmtune package (vignali et al, 2020) to identify variables that contributed significantly to the models. the final set of environmental factors used in modelling the ecological niche of each species was the least correlated, which made a substantial contribution to the models, and was ecologically relevant. http://www.chelsa-climate.org http://www.worldclim.org genetic resources (2025), 6(12), 95–110 solanum cwr in benin 99 habitat dynamics were quantified using the spatial analyst tool in gis by counting the total number of pixels corresponding to the current and future distribution. the rate of change index (rci) was calculated using the following formula (coulibaly et al, 2021): fa corresponds to the future area (e.g. suitable) of a species in the target horizon under scenario i (here horizon = 2055 and i = ssp3-7.0 and ssp5-8.5); ca corresponds to the current distribution area (e.g. suitable); ∆ is the percentage of area gain (∆ > 0) or lost (∆ < 0) and stable ∆ = 0. a pattern of change was qualified as minor when values varied between (0–5%), (6–15%) indicated moderate decrease and the upper 21% major decrease. the dynamics of species richness over time were evaluated by summarizing the binary (1 = presence, 0 = absence) layers for the present and future distributions of the ten solanum species. in this context, species richness referred to the number of solanum species found in 1km2 area, based on the resolution of environmental variables used in the models. richness levels were then classified into five different categories based on the number of solanaceae species modelled: high (8–10), moderate (5–7), low (2–4), very low (1) and no species (0). gap analysis was conducted by overlaying the shapefile of benin's pa network (world database on protected areas, unep-wcmc 2023) with the habitat suitability and species richness maps. this analysis aimed to evaluate the effectiveness in covering suitable habitats and to identify potential priority areas for future conservation efforts. conservation status assessment of the species in this study, we assessed the conservation status of ten solanum species in benin using projections from sdm under future climate scenarios. although many studies traditionally apply iucn criterion b by estimating the extent of occurrence (eoo) and area of occupancy (aoo) (e.g. dassou et al, 2024), criterion a3(c) is more appropriate for sdm as it considers the expected reduction in population size inferred from the projected decline in habitat suitability. criterion a3(c) relates to a reduction in eoo, aoo or habitat quality up to a maximum of 100 years (iucn, 2024). according to iucn guidelines, a species is classified as extinct (ex) if it is projected to lose 100% of its suitable area, critically endangered (cr) with ≥ 80% reduction, endangered (en) with ≥ 50% and < 80%, vulnerable (vu) with ≥ 30% and < 50%, near threatened (nt) with < 30%, and least concern (lc) if its suitable area is stable or increasing. the methodology for calculating the percentage change in the suitable area and for assigning iucn categories was detailed in the previous section. in benin, threats to cwr, including solanum species, fall into three major categories: (1) agricultural expansion and urbanization, leading to habitat loss, (2) overharvesting, resulting in population decline, and (3) invasive species and climate change, also contributing to population decline (idohou et al, 2013). among these, agricultural encroachment has been identified as the most significant threat to solanum species nationwide, severely affecting their suitable habitats. consequently, the preliminary conservation status of each species was determined by combining the predicted reductions in suitable area from sdm with an understanding of ongoing threats. results variable contribution, model validation and performance evaluation a total of 21 non-correlated variables (figure 2) were identified as important in determining the modelled distribution of the ten solanum species. among these, temperatureand precipitation-related variables had a significantly greater influence on the sdm compared to soil and elevation variables. individually, isothermality (bio3) emerged as the most influential variable shaping the distribution of the solanum species. however, precipitation of the driest quarter (bio17) had the highest individual contribution, particularly for s. nigrum with contributions of 27.16% and 24.23%, respectively. s. melongena showed the highest dependence on bio3 (42%), followed by s. distichum (27.27%), s. terminale (20.87%), s. nigrum (20.11%), and s. dasyphyllum, s. erianthum and s. incanum, each with contributions around 16.5%. annual temperature (bio1) moderately influenced the distribution of s. anguivii and s. incanum, each with a contribution of around 7%. the mean diurnal range (bio2) was a major predictor for s. dasyphyllum (38.17%) and s. distichum (20.66%). temperature seasonality (bio4) and maximum temperature of the warmest month (bio5) were key variables for s. torvum (42.11%) and s. anomalum (16.04%), respectively. precipitation seasonality of (bio15) was the most influential factor for s. anguivii (47.74%), followed by moderate to low contributions for s. erianthum (33.37%), s. distichum (24.39%) and s. torvum (16.84%). s. terminale strongly depended on precipitation of the driest month (43.10%) (bio14) while s. anomalum and s. erianthum also responded significantly (33.5% and 37.5%, respectively). for s. incanum, however, bio14 had a relatively low contribution (13.66%). overall, soils characteristics showed a weak contribution for all the species. among those, exchangeable acidity in subsoil (eackcl_d1) and exchangeable magnesium (emgx_d2) had some effect on s. incanum, s. anomalum, s. nigrum, s. torvum and s. melongena. bulk density in topsoil (bld_d1) contributed 10.05% for s. distichum, while subsoil bulk density (bld_d5) contributed approximately 13% for both s. anguivi and s. dasyphyllum. subsoil aluminium concentration (alum3s_d1) had contributions ranging from 13% to 18% for s. anguivi, s. anomalum and s. nigrum. soil texture class at 45cm depth weakly affected s. terminale (19.64%), while deeper texture layers (texmht_d5) impacted s. dasyphyllum (17.11%), s. distichum (17.64%) and s. torvum (3.68%). silt fraction (sltppt_d1) influenced s. melongena (18.7%) and s. erianthum (7.6%). soil ph in the topsoil (phihox_d1) slightly influenced s. terminale (7.73%), and subsoil ph (phihox_d6) impacted s. erianthum (6.27%) and s. melongena (10.65%). elevation (dem) was a significant predictor only for s. incanum, contributing 29.86% to its distribution model. genetic resources (2025), 6(12), 95–110100 kégbé et al the results showed an average auc value of 0.89 across the ten solanum species (table 2), indicating that sdm performed well in predicting their potential distributions. similarly, the average tss value was 0.74, reflecting a strong agreement between observed and predicted occurrences. table 2. model performance based on the two metrics. auc, area under the curve; tss, true skill statistic. species auc tss s. anguivi 0.9 0.8 s. anomalum 0.9 0.81 s. dasyphyllum 0.83 0.63 s. distichum 0.83 0.69 s. erianthum 0.96 0.85 s. incanum 0.8 0.5 s. melongena 0.87 0.69 s. nigrum 0.92 0.78 s. terminale 0.91 0.82 s. torvum 0.94 0.81 geographic distribution patterns of the species under current and future climate conditions the categorization of habitats under current conditions showed a diverse range of suitable areas for all species except for s. anomalum (figure 3). the primarily suitable habitats for the studied species were predominantly located in the sudano-guinean and guineo-congolian zones. in figure 2. contribution (%) of the variables to the models. the models were built using an ensemble of four algorithms boosted regression trees (brt), random forests (rf), support vector machines (svm), and maxent with five replications each. variable contributions were averaged across algorithms and replications to obtain final estimates. contrast, a significant portion of the coastal region was unsuitable for some species, notably s. anguivii and s. incanum. for s. anomalum, suitable areas were limited and mostly concentrated along the eastern coast of the guineocongolian zone. a similar distribution pattern was observed for s. dasyphyllum, although this also showed a broader extent of suitable habitat extending into the sudanian zone. under future climate scenarios (ssp3-7.0 and ssp58.5) for the horizons 2055 and 2085, a general decline in suitable habitats was projected for many species. however, s. melongena and s. dasyphyllum showed a clear expansion of suitable habitats. the suitable areas for s. distichum, s. nigrum and s. torvum remained relatively stable across all scenarios and time horizons. in contrast, s. anomalum, s. incanum and s. terminale were projected to lose the suitable habitats by 2055. for s. incanum, the distribution remains narrow and relatively stable in the future compared to its initial distribution. s. erianthum showed a decline in suitability, with its distribution shifting southwards into the guineocongolian zone – this spatial trend was consistent across both horizons and scenarios. when overlaying habitat suitability maps with the pa network, varied patterns emerged. in the sudanian zone, parts of the suitable habitats for some species overlapped with existing pa except for s. melongena and s. dasyphyllum, which were largely unprotected. under future climatic conditions, some pa emerged as important conservation zones. for instance, the classified forests of kétou and dogo are projected to harbour significant hotspots of s. terminale. similarly, the lama forest appears to be a potential refuge for s. incanum. genetic resources (2025), 6(12), 95–110 solanum cwr in benin 101 dynamics of suitable habitats for solanum species across benin the projected distribution of wild relatives of cultivated leafy vegetables of solanum species, indicated that, under both present and future scenarios, the extent of suitable habitats will decrease for most species (supplemental table 5). overall, eight out of ten species are expected to experience a reduction in suitable areas, while only two are projected to see an expansion. species such as solanum anguivii, s. anomalum, s. distichum, s. erianthum, s. incanum, s. nigrum, s. terminale and s. torvum are projected to lose suitable habitats. under current conditions, s. torvum occupies a suitable area of 4,560.47km². by 2055, this area is projected to increase by 7.06% under the ssp3-7.0 scenario but to decrease by up to figure 3. suitable habitats for the ten solanum species in the current and under future conditions accounted for socioeconomic pathways ssp3-7.0 and ssp5-8.5 by 2055 and 2085. genetic resources (2025), 6(12), 95–110102 kégbé et al 8.41% under ssp5-8.5. other species, such as s. distichum and s. incanum, are predicted to experience relatively minor reductions in habitat suitability, with losses not exceeding 21%. the current suitable area of s. distichum is estimated at 2,601.14km², with projected reductions ranging from 11.09% to 20.49%, the largest occurring under ssp3-7.0 by 2085 (supplemental table 5). for s. incanum, the current suitable area is 743.28km², with projected losses of 17.74% under ssp3-7.0 and 14.06% under ssp5-8.5 by 2055. by 2085, this downward trend is expected to continue, with projected reductions of 14.06% and 14.06% under ssp3-7.0 and ssp5-8.5, respectively. moderate habitat losses, i.e. not exceeding 45%, were observed only for s. anguivii and s. nigrum. the current suitable areas for s. anguivii and s. nigrum were estimated at 1,583.58km² and 4,288.29km², respectively. however, the projected trends in habitat suitability are not stable for either species. for s. anguivii, a minor reduction of 15.09% is projected under the ssp5-8.5 scenario by 2055, whereas a more substantial reduction of 40.88% is expected under the same scenario by 2085. similarly, under ssp3-7.0, reductions of 26.75% and 38.80% are projected for 2055 and 2085, respectively. s. nigrum exhibits the same pattern, with relatively minor reductions of 7.17% and 16.47% under ssp3-7.0 for 2055-time horizon and ssp5-8.5 time horizon, respectively, increasing to 21.91% under ssp3-7.0-2085 and 27.33% under ssp5-8.5-2085. species with major loss included s. anomalum, s. erianthum, and s. terminale, each experiencing projected reductions in suitable habitat exceeding 60% under all future scenarios (supplemental table 5). for instance, s. anomalum, which currently occupies 282.00km², is expected to lose up to 64.91% of its suitable area by 2085 under ssp58.5. similarly, s. erianthum, with a current suitable area of 4,971.81km², is projected to decline by as much as 72.53% by 2055 under ssp3-7.0. s. terminale, currently distributed across 1381.48km², faces reductions up to 86.37% under the same scenario by 2085. these sharp contractions indicate a significant risk of habitat-driven population decline and potential local extirpation without targeted conservation efforts. in contrast, s. dasyphyllum and s. melongena are projected to expand their suitable habitats under all climate scenarios (supplemental table 5). s. dasyphyllum, currently found in 5,314.32km², may gain up to 57.15% by 2085 under ssp5-8.5, while s. melongena, with a current area of 10,717.61km², is projected to expand by 6.13% under the same scenario. despite these positive trends, their future distributions remain susceptible to uncertainties inherent in climate projections, highlighting the need for continued monitoring. these contrasting patterns underscore the importance of adopting species-specific strategies for effective conservation planning in the face of climate change. dynamic of suitable areas of solanum species within the protected areas network supplemental table 6 presents the dynamics of suitable areas for solanum species within the protected areas (pa) network. overall, two main trends were observed, mirroring those at the national level. eight out of ten species (s. anguivi, s. anomalum, s. distichum, s. erianthum, s. incanum, s. nigrum, s. terminale, and s. torvum) are projected to lose suitable area within pa, while only s. dasyphyllum and s. melongena are expected to gain. s. anguivi currently occupies 1,790.92km² within pa and is projected to decline by up to 58.89% under ssp3-7.0 by 2085, reducing its extent to 736.27km². s. anomalum, with a current extent of 47.26km², shows consistent losses of 63.48% across all future scenarios, resulting in a stable yet severely reduced extent of 17.26km². s. incanum presently covers 3,158.83km² and is projected to decline by roughly 50% across all scenarios, reaching 1,561.9km² under ssp58.5 by 2085. s. torvum has a current distribution of 964.46km² and is projected to undergo losses under all scenarios, with the most severe reduction (62.66%) under ssp3-7.0 by 2085, dropping to 360.16km². while it displays a negligible gain of 0.55% in 2055 under ssp3-7.0, this is not sustained in later scenarios. moderate losses were observed for s. distichum, currently distributed over 3,472.39km². the most substantial reduction is projected under ssp5-8.5 by 2085, where the extent declines to 1,077.04km², accounting for a 68.98% reduction. s. nigrum shows contrasting dynamics, with a projected gain of 36.83% under ssp5-8.5 by 2055, but this is followed by a sharp decline (62.73%) under ssp5-8.5 by 2085, reducing its suitable area to 2,816.51km². s. terminale follows a similar trajectory, dropping from 7,557.58km² to 2,816.51km² under ssp5-8.5 by 2085 (62.73%). s. erianthum faces the most severe reductions, shrinking from 7,993.3km² to just 1,187.15km² by 2055 under ssp3-7.0 (85.15%) and showing similarly drastic losses under all scenarios. by contrast, s. dasyphyllum and s. melongena are projected to expand within the pa network. s. dasyphyllum currently occupies 9,668.47km² and may increase its extent by up to 78.65% (17,272.45km²) by 2085 under ssp5-8.5. s. melongena, with the largest current extent (31,355.97km²), shows consistent expansion across all scenarios, peaking at 18.51% (37,159.88km²) under ssp5-8.5 by 2085. these contrasting dynamics emphasize the need for targeted conservation planning tailored to each species’ future trajectory within the protected areas network. solanum richness dynamic accounted for current and future distribution under current conditions, sdm indicate a decline in solanum species richness towards the southern regions (figure 4). the highest richness category comprising eight to ten species was predominantly concentrated in the guineocongolian and sudano-guinean zones. this richness class currently covers an estimated 681.11km2 (5.94% of the total area). however, projections under future climate scenarios suggest a reduction of approximately 4.5% in this richness category (supplemental table 7). moderate richness levels (5–7 species) were observed in the ouémé-boukou and agoua pa, which are located in the guineo-congolian and sudano-guinean zones, respectively (figure 4). compared to the current condition, the future richness of the wild solanum species is likely to decrease southwards (figure 4). the current distribution area of solanum species richness was estimated at 2,374.49km2 (20.69%) with a projected reduction ranging from 3.18 to 5.34% under future climate scenarios. however, our findings indicated an increase in the species richness (2–4 species class) in the sudanian zone. when we considered the richness class 2–4, an expansion towards the guineo-congolian and sudano-guinean zones genetic resources (2025), 6(12), 95–110 solanum cwr in benin 103 could occur. this class occupied 11.28% (1,294.75km2) of the total area (supplemental table 7). the increase in area ranged from 3.56–10.54% with the lowest value under ssp3-7.0 by 2055 and the highest value at ssp5-8.5 in 2085. as for the richness class (1 species), it occupied 16.39% (1,880.91km2). the increase ranged from 10.89– 25.25% with the highest percentage under ssp5-85 in 2085 in terms of pa. the pattern of species richness shows that higher richness classes (5–7 and 8–10 species) are generally located outside the pa network. however, notable exceptions include the kétou and dogo pa, situated in the transition zones between the sudano-guinean and guineo-congolian regions. under current conditions, richness classes with 0 and 1 species together account for approximately 66% of the richness within the pa network (supplemental table 8). meanwhile, the 5–7 and 8–10 species richness classes make up only 11.37% of the total pa coverage. the conservation status assessment of the solanum genus the conservation status of the ten solanum species in benin was based on the iucn criteria. the results revealed varying levels of climate-induced vulnerability overall. one species was classified as cr, two as en, three as vu, two as lc and two as nt. this was based on their major threats and distribution range (supplemental table 9), as well as the projected percentage changes in suitable habitat under the ssp3-7.0 and ssp5-8.5 scenarios for 2055 and 2085 (supplemental table 10). figure 4. species richness dynamics accounted for the current and future distribution. s. terminale is the most severely affected species, with a projected habitat loss of over 70% under all future scenarios, reaching 85.86% under the ssp5-8.5 scenario by 2055. coupled with its restricted distribution and high exposure to anthropogenic pressures, this supports its classification as cr. similarly, s. erianthum and s. anomalum show consistent declines of between -53.06% and -69.42%, which justifies their classification as en given their sensitivity to fire and habitat degradation. at the next level of severity, s. incanum, s. distichum and s. anguivi experience a moderate decline in suitable habitat, ranging from -14.10% to -40.88%. despite having broader ecological amplitudes, these species face significant local threats, such as overgrazing, soil disturbance and agricultural encroachment. this warrants their classification as vu. by contrast, s. dasyphyllum and s. melongena are projected to remain stable or even increase in number under future climate conditions, with changes ranging from -3.95% to +57.15%. their wide ecological tolerance and lower exposure to critical threats mean they are designated as lc. however, ongoing monitoring is advised to detect any future shifts in vulnerability. finally, s. nigrum and s. torvum exhibit variable responses across scenarios, with projected changes ranging from a decline of 27.33% to an increase of 2.93%. while their overall decline is less severe, uncertainties surrounding their true wild distribution and local extinction risks justify their classification as nt. genetic resources (2025), 6(12), 95–110104 kégbé et al discussion factors determining the distribution of solanum wild relatives the distribution and suitable habitat of the species studied are largely influenced by key abiotic factors such as rainfall, temperature, and soil properties. climate variables (e.g. temperature and precipitation) and edaphic conditions play a fundamental role in determining species presence in a given environment (lewis et al, 2017). however, the ability of these species to persist in complex environment also depends on their interactions with biotic factors – competition, parasitism, commensalism – and on dispersal constraints that were not included in our models. moreover, the models did not consider species’ phenotypic plasticity and evolutionary adaptation to changing environments (pidwirny, 2006). integrating these aspects in future modelling efforts could substantially improve the predictive accuracy and ecological realism of species distribution models, especially under changing climate scenarios. in this study, we found that the distribution patterns of the ten solanum species were primarily influenced by bioclimatic variables, with isothermality emerging as the most dominant factor. edaphic variables also played a role, albeit with a relatively smaller contribution to the distribution of each species. our findings align with those of manda et al (2022) who identified isothermality as the second most important factor when modelling the potential impact of climate change on vigna wild relatives. similar patterns in the influence of bioclimatic and soil variables have been reported for tree species such as balanites aegyptiaca (l.) delile (chérif et al, 2022) in chad, fontainea species (brunton et al, 2023) in australia and palm species in benin (idohou et al, 2017; salako et al, 2019). in the case of wild relatives of cultivated solanum leafy vegetables, their responses to soil characteristics varied among species. indeed, several studies have highlighted the high nutritional value of african indigenous vegetables, including solanum species, attributing their richness in nutrients to underlying edaphic conditions (chinedu et al, 2011; keatinge et al, 2011) . this relationship suggests that soil properties play a significant role in shaping the nutrient profile of these plants. additionally, the observed variability among species likely reflects inherent physiological traits that influence water requirements and nutrient uptake. since solanum seed germination is sensitive to environmental stress, favourable soil and moisture conditions are essential for successful germination and, consequently, for the longterm persistence of the species in a given habitat (stanton et al, 2012). these factors should be carefully considered when it comes to cultivation or conservation. understanding their responses to environmental changes is therefore essential for designing effective conservation plans. as reported by aksoy et al (2021), evaluating the impact of climate change is crucial for the conservation of s. tuberosum. similarly, ogundola et al (2023) reported that soil characteristics, particularly silty loam soils at a depth of 2cm, enhance the germination and viability of s. nigrum seeds. in our study, the most influential soil factors included exchangeable acidity, exchangeable magnesium, bulk density, silt content, and soil ph. however, further research is necessary to better understand how these specific edaphic parameters affect seed germination, viability and overall fitness of solanum species under both current and future environmental conditions. suitable areas of solanum wild relatives and climate change the suitable habitats of wild relatives of cultivated solanum leafy vegetables are likely to be largely unstable in the coming decades, reinforcing our first hypothesis that abiotic factors are the main drivers of the current and future distribution of wild solanum species. climate change is predicted to increase the temperature by 1.5–2°c by 2100 in west africa, and to cause an uneven distribution of precipitation (ipcc, 2021). based on these projections, the suitable habitats of the ten wild solanum species are likely to be severely altered by the 2055 and 2085time horizons under both ssp3-7.0 and ssp5-8.5 scenarios. as noted by guisan and thuiller (2005), several factors (population dynamics, migration, local adaptations, ecological interactions, disease prevalence and human intervention) can influence the distribution of a species. our results indicate substantial habitat losses for nearly all species by both the 2055 and 2085 horizons under the two scenarios. surprisingly, a rise in the number of suitable areas was observed for wild s. melongena and s. dasyphyllum. this suggests that climate change could have a positive impact on the potential distribution of these species. this expansion could imply an increase in the potential cultivation area for domesticated or improved forms of these species. however, further agronomic and socioeconomic studies would be necessary to confirm this. under the ssp5-8.5, the situation appears particularly critical across both time horizons, with predicted high habitat losses. these findings align with numerous other studies which reported shifts in the distribution of many cwr. for instance, manda et al (2022) reported major changes in the distribution of vigna wild relatives in benin. similarly, findings in southern africa indicated that the majority of regionally priority cwr are expected to be negatively impacted by climate change, threatening their survival (magos brehm et al, 2022). additionally, van treuren et al (2020) demonstrated a reduction of suitable areas for cwr in the netherlands. seed dispersal is a crucial ecological mechanism that could either hinder or facilitate plant species’ response to climate change (öckinger et al, 2010). according to the same author, species with limited dispersal ability would be the most affected by the environmental changes compared to those with strong dispersal capacity. solanum species are dispersed by mammals, bats, birds, human-induced actions, as well as wind and water flow (roberts and florentine, 2022), which are predicted to be highly disturbed by climate change, potentially disrupting natural dispersal processes. besides, in the sudano-guinean zone, habitat fragmentation driven by agricultural expansion and rapid population growth has already impeded natural dispersal pathways (neuenschwander & adomou, 2017; abdul aziz et al, 2024). consequently, natural dispersion may not be efficient with humans as a major dispersing agent. compounding this issue, invasive species pose a significant threat to native biodiversity, challenging both ecological resilience and conservation efforts. in benin, many studies showed the detrimental impacts of invasive species on native species particularly in pa (gbètoho et al, 2017) and, at the same time, models predicted their capacity to thrive genetic resources (2025), 6(12), 95–110 solanum cwr in benin 105 in the changing climate (fandohan et al, 2015). this is an important aspect to consider since most wild solanum species are herbaceous and, thus, highly susceptible to displacement by aggressive invaders such as chromolaena odorata (l.) r.m.king & h.rob. solanum wild relatives and their conservation within protected areas the results showed that, with a few exceptions in the sudano-guinean and guineo-congolian zones, most existing pa offer limited potential for conserving the target solanum species. future projections confirmed these patterns, although localised expansion or contraction may occur. the destruction of natural habitats on a large scale, combined with climate change, is one of the main threats to plant species (hudson et al, 2014), and this includes cwr (hunter et al, 2012; magos brehm et al, 2022; maxted et al, 2012). protected areas are often cited as refuges for threatened species (le saout et al, 2013); their ability to maintain populations within defined boundaries offers a valuable conservation mechanism, especially in regions undergoing rapid environmental change (le saout et al, 2013; mao et al, 2020). however, the mere presence of a species within a pa does not guarantee its protection. more often than not, this represents passive in situ conservation, whereby species may persist without any specific monitoring or management actions. in the context of accelerating climate change, there is a critical need for active in situ conservation, which involves the direct management, monitoring and support of populations over time, to ensure long-term effectiveness. our findings align with those of manda et al (2022), who reported that the existing pa network was inefficient in conserving wild vigna species, a trend also observed in several other countries (e.g. davis et al, 2019; ratnayake et al, 2021). conversely, idohou et al (2017) highlighted the potential of pa for conserving palm species in benin. however, whether such potential persists under future climate scenarios remains uncertain and warrants further investigation. despite being considered refuges for many plant species, our projections suggest that both the current and future distributions of the studied solanum species may fall outside the existing pa network. this supports the hypothesis that current pa may not be sufficient to conserve wild solanum habitats under climate change. therefore, it is urgent to identify and prioritize additional conservation areas that are more likely to remain suitable in the future. sub-saharan africa, particularly its arid and semi-arid zones, has been identified as one of the most climate-vulnerable regions in the world (sintayehu, 2018), with native species facing multiple threats. under such pressures, the distribution of most wild solanum species is expected to become increasingly unstable in future climate scenarios. as a result, proactive conservation actions are essential (schuster et al, 2023). we thus emphasize the need to conserve wild solanum not only within pa networks but also beyond them, through mechanisms such as the iucn’s other effective area-based conservation measures (oecms). in particular, agroforestry systems and home gardens represent promising complementary conservation environments. if properly designed and supported, these managed landscapes could host viable populations of wild relatives and provide additional resilience to climate stressors. implications for better management of solanum wild relatives in this study, we investigated the threats to the conservation of ten wild solanum species under different future climate scenarios. many studies demonstrated the great importance of cwr worldwide (e.g. ng'uni et al, 2019; tas et al, 2019; maxted and magos brehm, 2023). because of the key role they play in crop improvement and food production, there is a global call for the conservation and preservation of these resources (maxted et al, 2010; maxted et al, 2012). assessment of the current and future distribution of solanum species under climate change revealed a decline in suitable habitats for the majority of the species. furthermore, the assessment based on iucn red list categories and criteria indicated that most species in benin are threatened, underscoring their high risk of genetic erosion, a pattern similarly observed among numerous cwr taxa worldwide. our study identified priority areas for target solanum species conservation as suggested by maxted et al (2009). the target species to be prioritized for conservation include s. anguivi, s. erianthum, s. anomalum and s. terminale (vu, en and cr) since they are likely to continue losing suitable areas of distribution. monitoring population status and habitat conditions, restoring degraded ecosystems, and analyzing genetic diversity are all essential steps for the effective conservation of solanum species. monitoring enables the early detection of population declines, while restoration efforts help ensure that species can persist in their natural habitats. preserving a broad spectrum of genetic diversity is crucial for the long-term adaptability and survival of species. it also ensures that these genetic resources remain available to plant breeders, helping to improve crops and ensure food security. ecogeographic diversity, which integrates environmental and geographical variation, can serve as a useful proxy for capturing genetic variation (parraquijano et al, 2012). these species could also be protected in several sacred forests throughout the country. although they are limited in size, sacred forests are often better conserved than most classified forests (adomou et al, 2007), making them suitable refuges for the conservation of solanum taxa through introduction or reintroduction programmes. to minimize ecological risks, such introductions should occur within each species' historical distribution range. in parallel, ex situ conservation is a valuable complementary strategy, particularly for species with critically reduced habitats. however, the long-term success of these measures depends on a better understanding of the ecological requirements of each species, including their capacity to adapt and their productivity under changing environmental conditions. conservation in the wari-maro and belefoungou forests could be more effective than in the semi-deciduous forests of southern benin, such as the lama reserve or sacred forests, primarily because many of the solanum species studied are heliophilous, meaning they thrive in environments with high light availability. unlike in dense, closed-canopy forests, where light penetration is limited, the drier, more open structure of the wari-maro and belefoungou forests provides favourable microhabitats for these species that demand light. these conditions support germination, growth and reproductive success, thereby enhancing their long-term survival prospects in these forest systems. furthermore, classified forest reserves, including sacred forests, have decreased in recent decades, genetic resources (2025), 6(12), 95–110106 kégbé et al implying the loss and fragmentation of suitable habitats for many taxa in benin (alohou et al, 2017). according to pinto et al (2024), the continued loss of suitable areas may result in significant genomic erosion for metapopulation taxa. the impact of these losses on solanum species should also be investigated. finally, the occurrence of invasive species in natural habitats needs to be considered when defining these strategies. limitations of the approach in this study, we investigated the current distribution of ten wild solanum species in benin and made predictions for their future distribution. the model accuracy and robustness were assessed by computing the auc and tss values of the final maxent outputs (allouche et al, 2006). however, we did not include seed dispersal limitations or constraints that could hinder the spread of species across landscapes. furthermore, we did not account for physiological, phenological and morphological traits in our models (koebsch et al, 2019; agounde et al, 2025). although the sdm-based projections offer valuable insights into the potential impact of climate change on the distribution of wild solanum species in benin, it is important to acknowledge that our assessment is still preliminary. the absence of data on demographic trends and population viability may result in certain taxa being underestimated as vulnerable. consequently, these results should be interpreted as indicative rather than definitive. future assessments will require the integration of ecological and population-level data in order to better reflect extinction risk and guide conservation priorities. conclusion developing an effective conservation strategy for a given species requires accurate information on the species' distribution range as a fundamental basis. using sdm tools and iucn criteria, we assessed the potential distribution, habitat suitability and preliminary conservation status of ten wild solanum species under current and projected climatic conditions. overall, most of the species currently have a wide distribution in the sudano-guinean and guineo-congolian zones of benin. significant declines in potentially suitable habitats were found in the two scenarios for 2055 and 2085. however, for more accurate projections, we recommend additional distribution studies taking into account physiological, phenological and morphological data. the study demonstrated that the current protected area network is ineffective in ensuring the long-term survival of these species in the face of changing climatic conditions. therefore, conservation efforts should prioritize active in situ strategies, such as restoring degraded habitats, reinforcing natural populations within their historical ranges and integrating climate-smart management plans. given the limited coverage and effectiveness of existing mechanisms in benin, such actions are urgently needed. in parallel, ex situ conservation measures, including seedbanks and living collections, should complement in situ efforts to preserve the genetic diversity of these species and facilitate their potential use in future food security initiatives. acknowledgements this research work was supported by european commission directorate-general for international partnerships through the regional academic exchange for enhanced skills in fragile ecosystems management in africa (grant number nr 2017-2861/001-001) and the phd fellowship from the organization for women in science for the developing world (grant number: 3240314469) provided to ahuéfa mauricel kégbé. ahuéfa mauricel kégbé is very grateful to dr valère salako and dr ablaye ngom for their encouragement and support during the implementation of this research. rodrigue idohou acknowledges support for bitc research and training visits from the john roger seamans biodiversity foundation, which enabled collaboration on this paper. author contributions ahuéfa mauricel kégbé, rodrigue idohou: conceptualization, methodology, visualization, software, resources, formal analysis, writing original draft, writing, review and editing; birane dieng: writing original first draft; gafarou agounde: software, formal analysis, resources, writing original first draft; anthony egeru: review and editing; kandioura noba: review and editing; achille ephrem assogbadjo: supervision, software, writing, review and editing. all authors read and approved the final manuscript. conflict of interest statement the authors declare that they have no conflict of interest. supplemental data supplemental table 1. the ecology, potential use and conservation status of the solanum species supplemental table 2: species occurrences used in ecology niche modelling supplemental table 3. description of the environmental variables supplemental table 4. correlation analysis supplemental table 5. dynamic of the distribution of solanum species in the whole study area supplemental table 6. dynamic of the distribution of solanum species in protected areas supplemental table 7. spatial reduction of extent of richness class in the whole study area supplemental table 8. spatial reduction 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(2025). the inrae prairies genebank for ex situ conservation of forage and turf species in france. genetic resources (s2), 106–118. doi: 10.46265/genresj.cwlj2580. © copyright 2025 the authors. this is an open access article distributed under the terms of the creative commons attribution license (cc by 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. introduction sown temporary grasslands cover around 2.47 million hectares in france (huyghe et al, 2014). they provide grazed or cut forage for livestock but also various other ecosystemic services such as soil fertility improvement, carbon sequestration and biodiversity shelter (martin et al, 2020). sown grasslands are seeded with improved cultivars from various perennial grass and legume species. most of these species commonly grow as natural populations in permanent and natural grasslands covering 9.8 million hectares in france (huyghe et al, ∗corresponding author: jean paul sampoux (jean-paul.sampoux@inrae.fr) 2014) and exhibit a diversity of ecotypes adapted to various conditions of soil, climate and use. only few species, e.g. lucerne (medicago sativa l.) and italian ryegrass (lolium multiflorum lam.), were bred as landraces before the onset of modern plant breeding. natural populations and landraces were the starting material to develop continuously improved cultivars adapted to various pedoclimatic conditions and forage systems, e.g. sampoux et al (2011) for perennial ryegrass (lolium perenne l.). grown as grass–legume binary associations, or as mixtures involving several species (up to ten or more), temporary grasslands can provide a high yield of good quality forage without artificial nitrogen and herbicide inputs (surault et al, 2024). furthermore, some grass species have also been received: 03.11.2024 accepted: 24.01.2025 published online: 11.03.2025 https://www.genresj.org https://www.doi.org/10.46265/genresj.cwlj2580 https://www.genresj.org https://www.doi.org/10.46265/genresj.cwlj2580 mailto:jean-paul.sampoux@inrae.fr genetic resources (2025), (s2), 106–118 inrae prairies genebank for forage and turf 107 bred for small size, high density and slow growth to create turf cultivars improved for either sport or lawn requirements (e.g. sampoux et al (2012)). the breeding of turf-type genotypes of legume species, e.g. white clover (trifolium repens l.), lucerne (m. sativa), birdsfoot trefoil (lotus corniculatus l.), has also recently begun. finally, all grassland and lawn areas contribute to providing pleasant landscapes for recreational activities. the inrae multidisciplinary research unit for grasslands and forage species (ur p3f) in lusignan hosts the prairies genebank targeted to the conservation of genetic resources of the main grass and legume species sown in france for either forage or turf usage (figure 1). genetic materials maintained by the prairies genebank collections of natural populations from grass and legume species of the prairies genebank are the outcome of collection trips undertaken by scientists of inra (institut national de la recherche agronomique, former acronym of inrae) in france and other countries. some of these collection trips were undertaken by inra staff alone and others together with academic research institutes from other countries (e.g. spain, portugal, morocco, algeria, usa) or breeding companies. most of the seed-collecting trips were carried out from 1970 to 1990 to gather samples of genetic resources necessary to start or diversify breeding programmes. since then, the long-term conservation and characterization of collected accessions have been progressively implemented (prospéri and sampoux, 2005). a more recent collecting campaign carried out in the southern part of france in 2014 gave the opportunity to collect new accessions from the natural diversity of grass and legume species that were afterwards included in the genebank besides already existing collections. the genebank also maintains some lucerne landraces, as well as some grass and legume historical cultivars formerly bred by inrae or other breeders, all removed from national lists. as a matter of fact, in france and other european countries, the commercialization of new cultivars is allowed only after their registration on national lists, which depends on sufficient value for cultivation and use (vcu) and distinctiveness, uniformity and stability (dus) requirements. after a certain duration (usually ten years), cultivars are removed from national lists unless they have not been outpaced by more recent cultivars (see for examples rules in france at www.geves.fr/variety-seed-expertise/field-forage/re gistration-of-field-and-forage-varieties-in-the-french-cat alogue). collection of orchard grass natural populations a breeding programme for orchard grass (dactylis glomerata l.) started in 1962 at inra in lusignan. the programme focused on the improvement of forage performances in the temperate climate areas of europe. an early start of vegetative growth in spring and a relatively late spike emergence date were targeted, as they were expected to provide a long annual period of forage production. thus, collection campaigns from 1962 to 1982 were directed towards oceanic regions of europe with mild winter and cool summer conditions, i.e. brittany and cotentin in france, northwestern spain and northern portugal (mousset, 2000). populations from the widespread autotetraploid (4x = 28) taxon d. glomerata subsp. glomerata were collected in france, spain and portugal. populations of the rare diploid taxon (2x = 14) d. glomerata subsp. lusitanica stebbins & d. zohary were found in portugal and populations of galician type (ortiz and rodriguez-oubiña, 1993), either diploid or tetraploid, were found in spain and portugal. the autotetraploid materials thus collected were used by the inra plant breeding unit of lusignan to create several successive innovative cultivars: ‘lully’ (1977), ‘lude’ and ‘lutétia’ (1978), ‘lupré’ (1992), ‘ludac’ (1997), ‘luron’ and ‘ludovic’ (1998). many orchard grass cultivars currently available from private breeding companies for oceanic climates in europe still stem from these original collections. later, from 1987 to 1992, new collections were undertaken to collect mediterranean types of orchard grass offering some winter growth potential and more or less substantial summer dormancy in southern france, southern portugal, southern spain and algeria (mousset, 2000). thereby, populations were collected from the widespread autotetraploid taxon d. glomerata subsp. hispanica (roth) nyman and from the diploid taxa d. glomerata subsp. castellata borrill & parker and d. glomerata subsp. mairei stebbins & d. zohary. the autotetraploid collected materials were used by the inra plant breeding unit in montpellier to select the cultivar ‘medly’ (1996), which presents adaption to summer drought and heat stresses of mediterranean areas. a total of 702 orchard grass natural populations were collected between 1962 and 1992. a core collection of 172 populations sampling the variability within the different taxa was extracted from this large set and is currently available from the prairies genebank. the new collection campaign undertaken in 2014 in the southern part of france enabled the collection of 39 new natural populations of the autotraploid d. glomerata subsp. glomerata. these new entries added to the previously mentioned core collection to make the set of orchard grass natural accessions currently available from the prairies genebank (figure 2 and table 1). other genetic materials accessions publicly available from the genebank also include the d. glomerata subsp. glomerata cultivars ‘floréal’ (1957), ‘lully’ and ‘lutetia’, formerly bred by inra. 108 sampoux et al genetic resources (2025), (s2), 106–118 figure 1. implementation of different tasks in the facilities of the inrae prairies genebank. a, growing grass plantlets in the greenhouse prior to planting accessions in spaced spots in a triticale (x triticosecale)–rye (secale cereale l.) field; b, growing grass plants in trays prior to transfer in confined compartments of a partitioned greenhouse; c, grass accessions planted in spaced spots within a triticale–rye crop which acts as a pollen barrier; d, growing lucerne (m. sativa) plants in pots prior to planting under insect proof tents; e1, insect-proof tents used to perform legume cross-pollinations; e2, a lucerne accession under an insect-proof tent (pollen transport inside the tent is ensured by bumblebees); f, storage of active seed lots in a cold room (5◦c and 30% hygrometry); g, storage of safety seed lots in a freezer (-20◦c). collection of tall fescue natural populations several hundred natural populations of tall fescue (festuca arundinacea schreb.) were collected by scientists of inra lusignan from 1969 to 1992 (ghesquière and jadas-hécart, 1995). collection campaigns focused towards southern france and the mediterranean basin (northern africa, portugal). the collected populations were then characterized at inra lusignan from 1985 to 1995 and a core collection of 128 accessions sampling the diversity of the different taxa was drawn up. this core collection included accessions from the common allohexaploid (6x = 42) tall fescue (f. arundinacea subsp. arundinacea) collected in france, portugal and switzerland (77 accessions) and in northern africa (34 accessions). sources of festuca arundinacea subsp. arundinacea from temperate areas and the mediterranean part of france were discovered to give deeply sterile hybrids with mediterranean sources from southern portugal, southern spain and northern africa (see jadashécart and gillet (1978) for the production of temperate x mediterranean amphiploid cultivars). the core collection was also supplemented with accessions from two other northern african types, three from f. arundinacea var. atlantigena (st.-yves) auquier (octoploid, 8x = 56) and 14 from f. arundinacea var. letourneuxiana (st.-yves) torrecilla (decaploid, 10x = 70). five accessions of f. arundinacea var. glaucescens boiss. are also maintained in the genebank. they result from the pooling of various natural populations of this taxon collected around lake embrun in the french alps in 1982. this taxon, which is autotetraploid (4x = 28), has been recognized as one of the two progenitors of the hexaploid tall fescue f. arundinacea subsp. arundinacea together with the diploid (2x = 14) festuca pratensis huds. after natural amphiploidization (humphreys et al, 1995). f. arundinacea var. glaucescens was widely used by the inra plant breeding unit of lusignan from the 1980s onwards in hybridization with autotetraploid ryegrasses to create the first x festulolium varieties (ghesquière et al, 2010). the collection campaign carried out in france in 2014 provided 36 new natural populations of the tall fescue f. arundinacea subsp. arundinacea. the historical core collection of 128 accessions, supplemented by accessions from f. arundinacea var. glaucescens and accessions collected in 2014 make the set of tall fescue natural populations available from the prairies genebank (figure 3 and table 1). genetic resources (2025), (s2), 106–118 inrae prairies genebank for forage and turf 109 figure 2. geographical distribution of d. glomerata natural populations maintained by the prairies genebank. in the background, isothermality is the ratio ‘temperature diurnal range/temperature annual range’ in percentage. values are computed from 1989–2010 climate norms. high isothemality values are typical of oceanic temperate climate whereas low values are typical of continental climate. credit: fabien sampoux, project colnat fourrage+ (aac rpg 2018-2 ctps gnis) other genetic materials several x festulolium accessions created by the inrae plant breeding unit of lusignan are maintained. this includes three amphiploid cultivars created after hybridization between f. arundinacea var. glaucescens genotypes and lolium multiflorum tetraploid genotypes, namely ‘lueur’ (2007), ‘luxane’ (2008) and ‘lusilium’ (2008). this also includes several pools obtained from the backcross of amphiploid (f. arundinacea var. glaucescens × l. multiflorum) material into tetraploid l. multiflorum or lolium perenne materials. accessions publicly available from the genebank also include the f. arundinacea subsp. arundinacea cultivars ‘gloria’ (1976), ‘lubrette’ (1981) and ‘ludical’ (2002) bred by inra. collection of ryegrasses natural populations in 1983 and 1984, the inra plant breeding unit of clermont-ferrand, together with the breeding companies of acvf (association des créateurs de variétés fourragères – society of plant breeding companies involved in the breeding of forage and turf cultivars in france), undertook a dense and even collection of more than 500 natural populations (diploid, 2x = 14) of perennial ryegrass (l. perenne) across france. these populations were thoroughly characterized at inra clermontferrand during the following years (charmet et al, 1990), providing data to reveal informative relationships between characterization traits and ecogeographical variables at the sites of origin of the populations (balfourier and charmet, 1991). the collected materials provided the sources to create the cultivar ‘clerpin’ (1996) by the inra plant breeding unit of clermont-ferrand and were the root of a substantial leap in the release of perennial ryegrass cultivars with late heading date and improved crown rust resistance by private breeding companies. a core collection of 160 accessions was finally set up using a clustering based on characterization data combined with a geostatistics contiguity constraint (charmet and balfourier, 1995). in 2008, this core collection was transferred to the prairies genebank in lusignan which is maintaining it. the whole set of perennial ryegrass natural populations maintained by the genebank also includes 27 other natural populations collected in france at diverse times as well as 36 natural populations collected during the 2014 collection campaign in southern france (figure 4 and table 1). the 2014 collection campaign also gave the opportunity to collect seven spontaneous popula110 sampoux et al genetic resources (2025), (s2), 106–118 table 1. number of accessions per species made publicly available by the prairies genebank in 2024. ∗, the whole set of 852 accessions proposed for notification by france as contracting party to the multilateral system (mls) of the international treaty on plant genetic resources for food and agriculture (itpgrfa ) comprises three different subsets: 1) a subset of 531 accessions maintained by inrae (prairies genebank) and notified to the mls in 2014, 2) a subset of 146 accessions maintained by inrae (prairies genebank) and 3) a subset of 175 accessions maintained by inrae (prairies genebank) and acvf (society of companies breeding forage and turf species in france), 2) and 3) proposed in 2024 to the french ministry of agriculture for notification to the mls. the subsets of 531 and 175 accessions can already be browsed on the public databases eurisco, florilège and siregal. the subset of 146 accessions is currently in the process of addition to these databases. a, accessions whose occurrence data were forwarded to the global biodiversity information facility (gbif) database are a subset of the set of accessions proposed for notification to the mls of the itpgrfa. b, 52 perennial ryegrass cultivars, 27 phalaris accessions and 3 x festulolium cultivars are currently in the process of seed regeneration and will be afterwards added to the public databases siregal, florilège and eurisco. species in collection number of accessions latin name vernacular name natural pop. landraces cultivars eurisco florilège siregal proposed to mls of tirpaa* gbifa dactylis glomerata orchard grass 211 3 175 214 172 lolium perenne perennial ryegrass 223 54b 193 206 191 lolium multiflorum italian ryegrass 7 2 2 9 lolium hybridum 1 1 1 festuca arundinacea tall fescue 169 3 128 164 festuca pratensis meadow fescue 11 11 x festulolium 3b festuca rubra aggr. red fescues 139 3 142 142 139 festuca ovina aggr. sheep fescues 35 35 35 35 phleum pratense timothy 2 2 2 phalaris arundinacea phalaris 27b phalaris aquatica medicago sativa lucerne 12 17 25 41 48 trifolium pratense red clover 2 3 3 5 trifolium repens white clover 6 4 4 10 onobrychis viciifolia sainfoin 2 2 2 vicia sativa vetch 3 3 3 total 842 17 108 731 852 537 tions (diploid, 2x = 14) of italian ryegrass (l. multiflorum) in natural grasslands. other genetic materials accessions publicly available from the prairies genebank also include the perennial ryegrass cultivars ‘primevère’ (1957) and ‘clerpin’ and the italian ryegrass cultivars ‘lutil’ (1972) and ‘rina’ (early 1960s), all bred by inra. furthermore, a set of 52 perennial ryegrass cultivars from different breeding origins was gathered in 2005 to represent the history of modern breeding in this species for forage and turf usages. this set was used to experimentally assess the genetic improvement for these two usages (sampoux et al, 2011, 2012) and it is currently in the process of seed regeneration in order to make it publicly available. collection of fine-leaved fescues natural populations an extensive collection was performed for fine-leaved fescues by the inra plant breeding unit of lusignan and breeding companies of acvf in 1993 and 1994. more than 500 natural populations from red fescue (stace, 1980) and sheep fescue (wilkinson and stace, 1991) taxa were collected across france. during the following years, they were characterized, evaluated for turf performances in dense swards and regenerated. sampoux and huyghe (2009) showed that the summer water balance, soil texture and land use were the main environmental variables differentiating the realized niches of the inland taxa, i.e. the caespitose red fescue taxon festuca nigrescens lam. (hexaploid, 6x = 42), the strong creeping (abundant long rhizomes) red fescue taxa festuca rubra subsp. fallax (thuill.) nyman (octoploid, 8x = 56) and f. rubra l. subsp. rubra (hexaploid, 6x = 42) and various festuca ovina l. taxa with karyotype varying from diploids (2x = 14) to octoploids (8x = 56). sampoux and huyghe (2009) also highlighted that the differentiation of inland taxa for adaptive traits contributed more than their ploidy level variation to the diversity of their realized niches (hutchinson, 1957). coastal populations of red fescues were also collected along the channel coast. hexaploid (6x = 42) slender creeping (few short rhizomes) red fescues were found on salt marshes (green f. rubra subsp. litoralis (g.mey.) auquier) and genetic resources (2025), (s2), 106–118 inrae prairies genebank for forage and turf 111 figure 3. geographical distribution of f. arundinacea natural populations maintained by the prairies genebank. in the background, the water balance is the cumulated rainfall minus the cumulated evapotranspiration of june, july and august. values are computed from 1989–2010 climate norms. credit: fabien sampoux, project colnat fourrage+ (aac rpg 2018-2 ctps gnis) on calcareous cliffs (glaucous f. rubra subsp. pruinosa (hack.) piper), and an octoploid (8x = 56) strong creeping red fescue (f. rubra subsp. arenaria (osbeck) syme) was found on sand dunes. during the years after collection, the populations of inland and coastal fine-leaved fescue taxa were extensively used by breeding companies in france to select new cultivars for turf usage. a core collection of 170 accessions was identified by selecting populations sampling the environmental range of each taxon (figure 5) and it now makes the set of fine-leaved fescues natural populations available from the prairies genebank (table 1). other genetic materials accessions publicly available from the genebank also include the f. rubra subsp. fallax cultivars ‘ludivine’ (1981) and ‘milda’ (1975) and the f. rubra subsp. pruinosa cultivar ‘luciole’ (2002), all formerly bred by inra. collection of lucerne history of the collection the cultivated lucerne (medicago sativa subsp. sativa) is not present with natural populations in france, except some feral populations escaped from sown stands. however, some wild populations of the spontaneous subspecies medicago sativa subsp. falcata (l.) arcang. and medicago sativa subsp. glomerata (balb.) rouy can be found in northeastern and southeastern france, respectively (julier, 1996). lucerne as a forage crop (the subspecies sativa) was introduced in france in the 16th century from spain (michaud et al, 1988). since then, traditional breeding has been applied on farm in almost all regions of france, producing landraces. seed exchanges were frequent among different regions and with foreign countries (julier, 1996). hybridization between cultivated landraces of the subspecies sativa and wild populations of the subspecies falcata, naturally occurring in continental europe, conferred frost resistance and variegated flower colour to cultivated populations. the first collections of landraces were set up during the first half of the 20th century by the plant breeding station of versailles, a precursor of inra, which delivered an extensive phenotypic description of them (alabouvette and méneret, 1935; mayer et al, 1951). these studies and that of demarly (1957) concluded that six main types of cultivated lucerne were present in france: ‘provence’ in the south-east, ‘poitou’, ‘marais de luçon’ and ‘marais de challans’ in the centre-west, ‘flamande’ (or ‘flemish’) in the north and ‘ormelong’ in the south of the paris basin. these six types differentiated for traits like tap root, kidney-shaped seeds, pod coil numbers and frost resistance and for the frequency of plants with varie112 sampoux et al genetic resources (2025), (s2), 106–118 figure 4. geographical distribution of l. perenne natural populations maintained by the prairies genebank. in the background, isothermality is the ratio ‘temperature diurnal range/temperature annual range’ in percentage. values are computed from 1989–2010 climate norms. high isothemality values are typical of oceanic temperate climate whereas low values are typical of continental climate. credit: fabien sampoux, project colnat fourrage+ (aac rpg 2018-2 ctps gnis) gated flowers (julier, 1996). they were used to initiate breeding programmes and the very first registered cultivars (e.g. ‘du puits’) were produced after a brief selection in ‘flamande’. the six types of french landraces were transferred to the inra forage plant breeding unit in lusignan at its creation in 1960. in order to widen the genetic basis of its lucerne breeding programmes, the plant breeding unit of lusignan received various cultivars, landraces and also wild populations from different countries in europe, north africa and america. a few of them were included in the genetic resources made publicly available, in agreement with provider requirements and plant breeding rights. as a noticeable event, a collection trip was undertaken by the inra plant breeding unit of lusigan in 1985 in lorraine (east of france) after a witness mentioned the presence of some prostrate, yellow-flowered lucerne populations in this region. two populations, named ‘malzeville’ and ‘marron’, were collected and multiplied, and a smooth selection was applied to remove cultivated off-types. these populations turned out to be typical of the eurasian continental subspecies falcata. furthermore, from 1985 to 1987, scientists of the inra plant breeding unit of montpellier organized collection trips in spain and portugal to collect wild populations of lucerne, also named ’mielga’ populations (prosperi et al, 1989). these populations, mainly located on roadsides and lowinput grasslands, had a prostrate growth habit but their violet flower colour attested to their relatedness to the subspecies sativa. the phenotypic description of these wild populations showed various levels of introgression by cultivated materials (prosperi et al, 2006). some ‘mielga’ genetic materials are currently available in the prairies genebank (see below). several studies, carried out by the inrae plant breeding unit of lusignan, provided extended analyses of the phenotypic and genetic diversity of lucerne genetic resources, including cultivars, landraces and natural populations (julier et al, 1995; crochemore et al, 1998; herrmann et al, 2018; pégard et al, 2023a). genetic resources currently available the numbers of landraces, natural populations and cultivars maintained by the prairies genebank are reported in table 1. the landrace accessions include the six original french landrace types (see above) as well as several landraces from northern africa. the natural populations comprise five m. sativa subsp. falcata accessions including the ‘malzeville’ and ‘marron’ populations, one m. sativa subsp. glomerata accession as well as seven m. sativa subsp. sativa accessions including genetic resources (2025), (s2), 106–118 inrae prairies genebank for forage and turf 113 figure 5. geographical distribution of festuca rubra aggr. and festuca ovina aggr. natural populations maintained by the prairies genebank. in the background, the water balance is the cumulated rainfall minus the cumulated evapotranspiration of june, july and august. values are computed from 1989–2010 climate norms. credit: fabien sampoux, project colnat fourrage+ (aac rpg 2018-2 ctps gnis) five ’mielga’ populations from spain. note that a large set of ‘mielga’ accessions has recently been transferred from inrae montpellier to the prairies genebank in lusignan but these accessions are not currently ready for distribution. there are 25 m. sativa subsp. sativa cultivars maintained by the genebank. they notably include several historical cultivars formerly grown in france, i.e. cultivars ‘coussouls’ (1998), ‘janine’ (1974), ‘luciole’ (1965), ‘luisante’ (1998), ‘lutèce’ (1976), ‘luxor ‘(1977), ‘mireille’ (1971) bred by inra, cultivar ‘medalfa’ (1988) bred by inra and acvf, and cultivars ‘du puits’ (1950), ‘gamma’ (1952), ‘glacier’ (1975), ‘elga’ (1964), ‘emeraude’ (1952), ‘etoile du nord’ (1965), ‘hybride de crécy’ (1974), ‘polder’ (1972), ‘prima’ (1963), ‘oméga’ (1952), ‘orchésienne’ (1952), ‘verneuil’ (1968) bred by private breeders. other genetic materials in collections the seed-collecting campaign carried out in 2014 to collect natural populations of grass and legume species in southern france (see previous paragraphs) also enabled the collection of 11 populations of meadow fescue (festuca pratensis), two populations of red clover (trifolium pratense l.) and six populations of white clover (trifolium repens). in 2017, another collecting trip enabled the collection of 27 phalaris natural populations (phalaris arundinacea l. and phalaris aquatica l.) in southern france and corsica, which are currently in the process of seed regeneration. finally, the genebank maintains a small number of cultivars of red and white clovers, thimothy (phleum pratense l.), vetch (vicia sativa l.) and sainfoin (onybrychis viciifolia scop.), all from inra breeding. practical organization of the prairies genebank in 2008, the inrae research unit ur p3f formally set up the prairies genebank on its operational site in lusignan in order to gather genetic resources of forage and turf species previously maintained in several inrae sites across france. during the following years, substantial investments were achieved to equip the genebank with renewed facilities such as cold rooms for seed storage and a partitioned greenhouse to grow accessions in pollen isolation. accessions were inventoried and a database was created to record all necessary information (passport data of accessions, quality and quantity of seed lots, characterization and evaluation data) and to trace the activity (seed quality control, seed regenerations, seed distributions, etc.). 114 sampoux et al genetic resources (2025), (s2), 106–118 for each accession, seeds harvested after a regeneration event are split into three seed lots: an active lot weighing from 200 to 800g which is stored in a cold room (+5◦c and 30% hygrometry), a 20 to 30g safety lot stored at -20◦c, and a 1 to 2g long-term conservation lot stored at -20◦c in a different freezer than the safety lots. the genebank maintains active and safety seed lots only from the most recent regeneration event and longterm seed lots from all successive regeneration events and the initial seed batch introduced in the genebank. germination tests are performed every three years on active lots, if they are not replaced by lots from a new regeneration event, as well as on safety seed lots if the germination percentage of active seed lots is below the established thresholds. active and safety seed lots are regenerated when the germination percentage of active seed lots falls below 80%, i.e. every 10 to 15 years. species maintained in the genebank are allogamous and more or less self-incompatible. because of this sexual reproduction biology, natural populations, landraces and cultivars (synthetic varieties) from these species are polygenotypic populations in principle at panmictic equilibrium. therefore, the regeneration of accessions from these different kinds of genetic materials is performed by intercrossing a number of plants expected as sufficient to avoid genetic drift (i.e. 100 to 250 plants) in pollen isolation from any plant from the same species not belonging to the accession. different means are implemented to ensure pollen isolation. in field conditions, accessions from grass species are grown in spaced spots within a triticale–rye crop which acts as a pollen barrier. another way to achieve pollen isolation is the use of pollen-proof (for anemophilous grasses) or insectproof (for entomophilous legumes) tents and a partitioned greenhouse with 12 confined compartments. to date, 731 accessions made publicly available for sample delivery (table 1) can be browsed online on the siregal website of the biology and plant breeding department of inrae (https://urgi.versailles.inra.fr/s iregal), on the florilege website (https://florilege.a rcad-project.org/fr/collections) set up by the plant pillar (brc4plants) of the french national research infrastructure rare (bergheaud et al, 2025) and on the website of the european search catalogue for plant genetic resources eurisco (https://eurisco.ecpgr.org ) with the holding institute field filled in as fra001. these databases will be soon updated with the recently introduced accessions still missing in their records. all the species maintained in the prairies genebank are listed in annex i of the international treaty on plant genetic resources for food and agriculture (itpgrfa). therefore, 531 of the publicly available accessions were notified in 2014 as a contribution of france to the multilateral system of the itpgrfa. an additional set of 321 accessions has been proposed in 2024 for further contribution to the multilateral system (table 1). furthermore, passport data of 537 accessions from the natural diversity of grass species have been uploaded to the global biodiversity information facility (gbif) database (https://www.gbif.org/publisher/0b5846f7-2 0b5-410a-93c5-5de83b522deb). the prairies genebank was labelled as a biological resource centre by the network ibisa (french network of biology, health and agronomy infrastructures) in 2011 and approved as a genebank of the plant pillar of the french national research infrastructure rare in 2021. it was furthermore officially recognized as a curator of plant genetic resources for food and agriculture by the french ministry of agriculture in 2020. the prairies genebank manages the french network for conservation of forage and turf species genetic resources, which includes inrae and private companies actively breeding these species in france. in 2023, the prairies genebank as well as the biotechnology and chemistry facilities of ur p3f were acknowledged for iso9001:2015 certification. over the five last years, the prairies genebank has distributed around 1,300 seed samples to various recipients, which were, by decreasing order of magnitude, french and foreign academic research institutions, french and foreign plant breeding companies, farmers and hobby-growers. uniqueness of the collections the collections of natural populations maintained by the prairies genebank originate from areas not or little represented in other genebanks. notably, most of the grass and lucerne populations from northern africa kept by the genebank are not maintained in their country of origin, and sometimes do not grow anymore in their site of origin, because of changes in climate and land use, excessive grazing or replacement by cultivars from modern breeding. recent collections of natural populations undertaken by the inrae research unit ur p3f in france and other european countries have confirmed that the in situ conservation of these populations is often threatened by the continuous regression of natural and permanent grasslands, climate change, and the corruption of their genetic integrity by overseeding with unrelated cultivars. the ex situ collections of grass and legume natural populations maintained by the prairies genebank thus make a unique, although non-exhaustive, contribution to the preservation of the genetic diversity of these species. furthermore, the prairies genebank is the only repository of inrae cultivars removed from national lists. however, the polygenotypic nature and the allogamous sexual reproduction of forage and turf species make the seed regeneration of accessions costly, even though the seed market for these species, and consequently the means for genetic resources conservation, are relatively limited. international networks of genebanks, like the forages working group of the european cooperative programme for plant genetic resources (ecpgr), are thus essential to share conservation efforts in these species. in the coming years, the prairies genebank will aim to achieve a better sampling of the french heritage for genetic resources of forage and turf species in its collecgenetic resources (2025), (s2), 106–118 inrae prairies genebank for forage and turf 115 tions. present collections of orchard grass and tall fescue natural populations only sample a limited part of the french territory, and it will be aimed to complete the collections through new sampling campaigns in regions not visited so far. it will also be considered to sample the french natural diversity of legume species, such as white and red clover. another important goal will be to work towards a more representative collection of cultivars, bred by inrae and also other breeders, that were grown in france since the 1950s for all forage and turf species that had significant use. valorization of the collections initially set up to have genetic resources available to start breeding programmes, collections of natural populations may provide relevant materials to study the environmental distribution of interand intra-specific plant diversity. in this respect, 167 scientific papers from various international institutions have to date used the occurrence data of grass species forwarded by the prairies genebank to the gbif database. the main asset of these collections is their capacity to supplement occurrence data with phenotypic and genomic information already existing or newly generated. earlier in this paper, we reported the use of phenotypic data to document adaptive differentiation in fine-leaved fescue taxa (sampoux and huyghe, 2009) and adaptive variability within perennial ryegrass (balfourier and charmet, 1991). more recently, the facce-jpi eranet+ (https://www.faccejpi.net/en/faccejpi/actions/c ore-theme-1/facce-era-net-plus-on-climate-smart-agricu lture.htm) project grasslandscape gathered inrae, ipk (leibniz institut für pflanzengenetik und kulturpflanzenforschung institute of plant genetics and crop plant research, germany), ibers (institute of biological, environmental and rural sciences, wales, united kingdom) and ilvo (instituut voor landbouw-, visserijen voedingsonderzoek flanders research institute for agriculture, fisheries and food, belgium) to implement phylogeographic and landscape genomics analyses on a set of 470 accessions from the natural diversity of perennial ryegrass provided by 15 genebanks from european countries and the usda. high throughput genotyping of these accessions allowed for the reconstruction of the expansion history of perennial ryegrass across europe (blanco-pastor et al, 2019). then, a multivariate landscape genomics analysis retrieved 633 potentially adaptive loci associated with either winter cold or summer drought and heat stresses and pointed phenotypic traits putatively conferring adaptation to these stresses (blanco-pastor et al, 2020). the adaptive relevance of these traits was further confirmed by a functional ecology approach (keep et al, 2021). results of the project grasslandscape were then used to set up a european core collection of perennial ryegrass natural populations within the frame of an ecpgr activity (keep et al, 2020; sampoux and willner, 2022). adaptive phenotypic and genomic variations revealed by the project grasslandscape have furthermore been used to assess the future of perennial ryegrass local populations exposed to climate change by implementing genomic offset analyses (pégard et al, 2023b). for other forage species than perennial ryegrass, collections maintained in european genebanks, and beyond, could likely provide sufficiently wide sets of natural populations to set up environmental niche models of species (sampoux and badeau, 2009) and reveal adaptive phenotypic and genomic diversities within species (see for example boller et al (2010) for genetic resources and diversity in forage species). this information could be used to implement predictive analyses, such as genomic offset analyses, to foresee spatial shifts of adaptive diversity required for adaptation to ongoing changes in climate and other environmental factors (fitzpatrick et al, 2021; gougherty et al, 2021; hung et al, 2023; aitken et al, 2024; zou et al, 2024). on this basis, strategies for in situ conservation could be developed, taking into account ongoing environmental changes on a european scale. for most forage and turf species that can spontaneously persist and evolve in long-duration grasslands, in situ conservation planned in this way would be the cheapest and most sustainable means to maintain valuable functional and adaptive genetic diversity in the long term. incorporating adaption to future environmental changes across territories in in situ conservation programmes would contribute to maintaining an economically viable forage production on involved grasslands that would thus help the sustainability of these programmes. results of the project grasslandscape also showed that the diversity of perennial ryegrass forage cultivars currently grown worldwide represents only a small part of the natural diversity of the species (blanco-pastor et al, 2019). moreover, the h2020 european project eucleg (www.eucleg.eu) led by ur p3f studied a worldwide set of lucerne landraces and cultivars, which revealed clear phenotypic and genomic differentiation related to geographic origin and evidence that the cultivar diversity grown in europe represents a limited part of the whole genetic diversity of the species (pégard et al, 2023a). these results suggest that natural diversity, landraces and old cultivars of forage and turf species may still provide potentially useful genetic variability to adapt grown cultivars to upcoming challenges. such genetic resources may be useful sources to adapt species to new combinations of climate constraints (blancopastor et al, 2020) and to improve their tolerance or resistance to biotic stresses (sampoux and badeau, 2009; annicchiarico et al, 2015). they could also be valuable sources to develop cultivars adapted to cultivation in species mixtures. during the last five decades, forage and turf species have intensively been bred for performances in pure stands and it may be assumed that phenotypes more adapted to interactions prevailing in species mixtures (litrico and violle, 2015; sampoux et al, 2020) could be recovered from natural diversity or genetic material that experienced only limited breeding (e.g. old cultivars). furthermore, with the transition to agroecological practices, some forage 116 sampoux et al genetic resources (2025), (s2), 106–118 species have emerged as service crops. they could be used as intermediary cover crops, e.g. italian ryegrass, or as perennial living mulches, e.g. lucerne, red and white clovers, sainfoin (onobrychis viciifolia scop.) and birdsfoot trefoil (lotus corniculatus). cultivars bred for forage usage are probably not the best-adapted materials for these new uses (el-ghazzal et al, 2024) and genetic resources may help to breed more suitable phenotypes. however, introgressing allele diversity from raw genetic resources to elite cultivars may have become more and more challenging as continuously improved cultivars have been released. this may especially be the case for the allogamous and partially auto-incompatible forage and turf species, for which it can be assumed that the alleviation of the genetic load has been a substantial part of the genetic improvement (kimbeng and bingham, 1998; annicchiarico et al, 2015). genotyping based on genome sequencing should be expected to help the efficient introgression of desirable multigenic features from genetic resources to elite germplasm within a reasonable timespan. while investigations such as the ones of the project grasslandscape have proven that collections of natural populations can be used to reveal the signature of natural selection (loci whose allele frequency variation departs from neutral expectations), it could be expected that collections of cultivars punctuating the history of modern breeding may reveal the signature of artificial breeding (see for example welcker et al (2022) for such approach in maize). marker-assisted selection methods could then possibly be designed to optimize favourable allele frequencies at both loci involved in desirable natural adaptations and loci that determine agronomic performances in the cultivars of forage and turf species, essentially bred as synthetics. efforts to broaden, phenotype and genotype the collections of the prairies genebank are to be conceived in order to contribute in these directions. author contributions the manuscript was written by jps with contributions from bj and mg. all authors reviewed and commented the manuscript and approved the final version. conflict of interest statement the authors have no conflicts of interest to report. 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(2024). signatures of local adaptation and maladaptation to future climate in wild zizania latifolia. communications biology 7(1313). doi: https://doi.org/10.1038/s42003024-07036-1 https://doi.org/10.5507/vup.23.24463414 https://doi.org/10.5507/vup.23.24463414 https://doi.org/10.1534/g3.119.400809 https://doi.org/10.1534/g3.119.400809 https://www.ecpgr.org/resources/ecpgr-publications/publication/improvloliumcol-activity-report-2022 https://www.ecpgr.org/resources/ecpgr-publications/publication/improvloliumcol-activity-report-2022 https://www.ecpgr.org/resources/ecpgr-publications/publication/improvloliumcol-activity-report-2022 https://doi.org/10.1016/j.fcr.2024.109308 https://doi.org/10.1016/j.fcr.2024.109308 https://doi.org/10.1038/s41467-022-30872-w https://doi.org/10.1038/s42003-024-07036-1 https://doi.org/10.1038/s42003-024-07036-1 introduction genetic materials maintained by the prairies genebank collection of orchard grass natural populations other genetic materials collection of tall fescue natural populations other genetic materials collection of ryegrasses natural populations other genetic materials collection of fine-leaved fescues natural populations other genetic materials collection of lucerne history of the collection genetic resources currently available other genetic materials in collections practical organization of the prairies genebank uniqueness of the collections valorization of the collections author contributions conflict of interest statement genebank report genetic resources (2025), (s2), 41–48 doi: 10.46265/genresj.houu8356 https://www.genresj.org issn: 2708-3764 the inrae biological resource center ‘bracysol’: a french centre of valuable brassica, allium and solanum genetic resources for breeding florence esnault *,a, marie-pierre cann a, jean-eric chauvin a, anne-marie chèvre b, marie-ange dantec a, stéphane doré a, pascal glory b, marie-claire kerlan a, dominique kermarrec c, anne laperche b, lise-anna le ven a, maria manzanares-dauleux b, roland pellé a, jocelyne porhel a, julien quéran c, vincent richer a, catherine souchet a, sylvain théréné a, mathieu tiret b and nathalie nési a a igepp, inrae, institut agro, univ rennes, ploudaniel, france b igepp, inrae, institut agro, univ rennes, le rheu, france c rgco, inrae, ploudaniel, france abstract: the inrae biological resource center ‘bracysol’ belongs to brc4plants, the plant network of the french research infrastructure of agronomic biological resource centers (agrobrc-rare). it preserves more than 15,000 accessions belonging to different cultivated genera: brassica (cabbage, turnip, rape and mustard), allium (shallot and garlic) and solanum (potato and crop wild relatives). the brassica genetic resources are conserved as seeds in freezers or liquid nitrogen. the allium resources are maintained by vegetative propagation in fields or greenhouses and the solanum resources are maintained by vegetative propagation in fields, greenhouses, in vitro or in liquid nitrogen. these collections include old landraces, widespread cultivars, crop wild relatives and original scientific material. the accessions are described with passport, morphological or agronomic descriptors or traits. they have been included in various research programmes, at the national or international level, aiming at characterizing the diversity of these collections, studying the genetics of agronomic traits, developing molecular tools and creating pre-breeding lines helpful for breeding programmes. keywords: vegetative propagation, seeds, conservation, diversity, characterization, breeding, genebank citation: esnault, f., cann, m., chauvin, j., chèvre, a., dantec, m., doré, s., glory, p., kerlan, m., kermarrec, d., laperche, a., le ven, l., manzanares-dauleux, m., pellé, r., porhel, j., quéran, j., richer, v., souchet, c., théréné, s., tiret, m., nési, n. (2025). the inrae biological resource center ‘bracysol’: a french centre of valuable brassica, allium and solanum genetic resources for breeding. genetic resources (s2), 41–48. doi: 10.46265/genresj.houu8356. © copyright 2025 the authors. this is an open access article distributed under the terms of the creative commons attribution license (cc by 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. introduction the french national research institute for agriculture, food and environment (inrae) manages the biological resource center (brc) ‘bracysol’ which was set up in 2012 with the aim of establishing a collective management system of the different genetic resources collections maintained by inrae in ploudaniel (brittany, western part of france). the brc bracysol is managed ∗corresponding author: florence esnault (florence.esnault@inrae.fr) by two inrae units: the joint research unit institute of genetics, environment and plant protection (igepp, fra010) and the experimental unit genetic resources in oceanic conditions (rgco, fra179). currently, 18 permanent staff members are involved in the activities of the brc, representing about nine full-time equivalents. its operations are financed mainly by national or european research projects or by partnerships with private companies. it belongs to brc4plants (bergheaud et al, 2025), the plant network of the national research infrastructure of agronomic biological resource centres received: 25.10.2024 accepted: 18.12.2024 published online: 27.01.2025 https://www.genresj.org https://www.doi.org/10.46265/genresj.houu8356 https://www.genresj.org https://www.doi.org/10.46265/genresj.houu8356 mailto:florence.esnault@inrae.fr 42 esnault et al genetic resources (2025), (s2), 41–48 (brcs) named rare, for agronomic resources for research (agrobrc-rare). description of the collections the brc bracysol maintains collections of genetic resources of different genera: brassica (cabbage, turnip, oilseed rape and mustard), allium (shallot and garlic) and solanum (potato and related species). these collections were set up by researchers over the course of their research programmes. brassica collection the brassica collection is composed of: • 1,094 accessions of vegetable crucifers, including mainly landraces that were cultivated before the development of hybrid varieties. they were collected in france on farms in the 1980s (table 1). this material is not present in any other genebank. • 1,749 accessions of oilseed crucifers including lineage varieties representing the world variability (table 1). it also includes original scientific material like prebreeding lines presenting traits of agronomic importance such as resistance to different pests (leptosphaeria maculans (desmazières) cesati & de notaris or plasmodiophora brassicae (voronin)) or seed quality, but also mapping populations, core collections, doubled haploid plants or rlm (resistance to leptosphaeria maculans) genes differential set (balesdent et al, 2005). the accessions of this collection are long-term stored as seed samples in freezers (-18◦c). for each accession, three seed lots are formed according to an internal protocol: the first one is used for distribution or germination tests, the second one is a reserve lot which is used to make new distribution batches when the first seed lot is empty, and the third one is a safety lot which is conserved in another place than the first two seed lots. for the accessions that are landraces collected on farms, a fourth seed lot was formed and is conserved in a cryotank (-196◦c). the seed lots conserved in the freezers are regenerated every 10 to 15 years, depending on their germination performance which is tested the year of their obtention, after 4 and 8 years of conservation, and then every 2 years, using a germination method in petri dishes developed in house (100 seeds are placed in a petri dish on a paper soaked with 2ml of sterile water; the number of germinated seeds is counted after incubation at 20◦c for 7 days). until now, the seed lots that are in liquid nitrogen have never been regenerated. the regeneration protocol depends on the biological status of the accessions. below is a summary of the regeneration protocols for landraces and lines. landraces: the regeneration protocol for the landraces was determined to avoid genetic drift during successive multiplications (divaret, 1999). for each accession, 120 individuals are planted in insect-proof cages (figure 1). pollination is carried out by bumblebees and the seed lot is accepted if at least 75 plants have flowered and produced seeds. nevertheless, if morphological observations reveal genetic drift after several generations, a new cycle of multiplications can be performed, starting from the cryopreserved seeds which constitute a safety long-term conservation stock. lines: the seeds are produced by bagging a few inflorescences of each line and pollination is performed using flies. a final validation of the new seed lot is performed by observing the plants in the field obtained from this new seed lot and plants obtained from the previous seed lot. the collection is maintained in facilities including 3,000m2 of greenhouses, 1,500m2 of field space, a seed drying chamber, 16 freezers for a total capacity of 3,700l and a 170l cryotank. solanum collection the solanum collection includes about 11,000 accessions of potato and its wild relatives. the collection has been formed from research programmes since 1949. it is composed of: • 737 genotypes belonging to 29 wild potato species collected in south and central america (table 1). these accessions were introduced into the brc in the form of seeds provided mainly by the us potato genebank (sturgeon bay, usa). they are maintained by vegetative propagation as clones (figure 2). this way of maintaining these potato wild species constitutes a specificity of our brc. each clone was evaluated for resistance to different pathogens (mainly phytophthora infestans (montagne) de bary and cyst nematodes). these characterization data are therefore available for inrae researchers and project partners. • about 1,400 varieties representing world variability, including old varieties not maintained in any other european genebank (table 1). • original scientific material like interspecific hybrids, mapping populations, a core collection or dihaploid plants. this collection is maintained by vegetative propagation in the form of tubers (produced each year in the fields or in greenhouses for the wild species), in vitro plantlets (subcultured every 12 to 15 months) or cryopreserved shoot tips. the cryopreservation of the shoot tips is performed using a droplet vitrification method (kim et al, 2006). the in vitro collection is a safety duplicate of part of the field and greenhouse collections. some accessions are present only in vitro. so far, a small number of accessions is cryopreserved (123 clones). this long-term conservation method is used for the most valuable genetic resources (core collection, wild relatives clones, national collection). the facilities used to maintain this collection consist of 1,500m2 of greenhouses, 2ha of field space, in vitro genetic resources (2025), (s2), 41–48 43 culture facilities, cold storage rooms (950m3) and a 170l cryotank. allium collection the allium collection includes 108 garlic accessions and 246 shallot accessions (table 1). the collection has grown through research programmes since the 1970s. it is composed of landraces collected in france before the creation of the national catalogue in 1991, old and new french varieties and original scientific material for the selection of agronomic characteristics such as disease resistance (resistance to botrytis squamosa (walker) or peronospora destructor (berkeley) caspary) or dry matter content of bulbs. the accessions are maintained by vegetative multiplication in the form of bulbs produced every year in the field (for shallot) or in a greenhouse (for garlic). the facilities used to maintain this collection consist of 250m2 of greenhouses, 500m2 of field space and cold storage rooms (20m2). associated data the accessions of these collections are described with passport data using the multi-crop passport descriptors (mcpd) (alercia et al, 2015), with morphological descriptors defined by international experts according to the union for the protection of new varieties of plants (upov) guidelines and/or international plant genetic resources institute (ipgri) format (ibpgr, 1990; ipgri, ecpgr, avrdc, 2001; bioversity international, international potato center (cip), 2009) and with agronomic traits evaluated according to specific protocols of the research projects. the characterization data are recorded in ms excel files and stored on a local server. we are currently working on the development of a local database in which all these data will be gathered, facilitating the management of the available information and its subsequent transfer to the french portal florilège (htt p://florilege.arcad-project.org). the brc bracysol contributes to the french national collection of genetic resources (duval et al, 2023) that is made available as part of france’s international commitments in contributing to the implementation of the international treaty on plant genetic resources for food and agriculture (itpgrfa) (fao, 2001). so far, 132 potato varieties and 60 rapeseed lines are included in this national collection. part of these collections can be viewed on the french portal florilège (the garlic collection, the traditional cultivars and landraces of the shallot collection, the cabbage landraces of the brassica collection and the potato national collection). we are currently working on formatting the data for other parts of the collections in order to increase the number of accessions that can be viewed on this portal (starting with the potato variety collection and the rapeseed national collection). similarly, data on part of these collections can be viewed in the eurisco database (http://eurisco.ecp gr.org). we are currently working with the french national coordination for conservation of plant genetic resources (duval et al, 2023) to update these data and upload further data in eurisco. the management system of the brc bracysol has been certified under the iso 9001-2015 quality standard (iso, 2015) since 2021. distribution service the accessions maintained within the brc bracysol can be ordered via the french portal florilège. the distribution is performed according to the access conditions mentioned on the portal. the brassica genetic resources are distributed as seeds, the potato genetic resources as tubers or in vitro plantlets and the allium genetic resources as bulbs. the signature of a material transfer agreement is required (smta for the accessions included in the itpgrfa or inrae mta for the others). from 2019 to 2023, the brc bracysol distributed more than 6,000 accessions to users: 36% to inrae teams, 6% to french public institutes other than inrae, 13% to international public institutes, 28% to french private companies, 6% to international private companies and 11% to farmers, associations or private individuals. these distributions are performed by the brc bracysol in compliance with international regulations concerning sanitary issues (regulation (eu) 2016/2031, eu (2016)). in order to ensure the healthy status of the collections, various measures are taken and health diagnostics carried out. regarding the potato and allium collections, detection tests are carried out each year by an external service provider for the following pathogens: ralstonia solanacearum (smith), clavibacter michiganensis spp. sepedonicus (spieckermann & kotthoff), globodera pallida (stone) and g. rostochiensis (wollenweber), melöıdogyne fallax (karssen) and m. chitwoodi (golden, o’bannon, santo & finley) (for potato); ditylenchus dipsaci (kuehn) (for allium accessions). furthermore, enzyme-linked immunosorbent assays (elisa) (gan and patel, 2013) are also regularly performed to detect the main viruses that can infect these crops (potato virus y, potato virus x, potato virus a, potato virus s, potato virus m and potato leafroll virus for potato accessions, onion yellow dwarf virus and leek yellow stripe virus for allium accessions). if one of these viruses is detected in one plant, this plant is eliminated or isolated. recently, we showed that virus elimination in potato can be obtained with the routine cryopreservation method (souchet et al, 2024). however, for now, we have not implemented a cleaning programme for virus-infected accessions. finally, all the accessions imported from non-eu countries are subjected to quarantine. the distributions are also performed in compliance with international regulations concerning access and benefit sharing. we are supported for these legal issues by lawyers from inrae and use a dedicated decision support system developed in a project managed by agrobrc-rare (http://golo.cirad.fr/abs4brc web). bracysol: the french centre for brassica, allium & solanum genetic resources http://golo.cirad.fr/abs4brc_web 44 esnault et al genetic resources (2025), (s2), 41–48 table 1. composition of the collections.a, solanum taxonomy according to hawkes (1990) genera species no. of accessions biological status (%) wild traditional cultivar/ landrace breeding/ research material advanced/ improved cultivar brassica brassica carinata a. braun 2 100 brassica juncea (l.) czern. 111 100 brassica napus l. 1,706 5 76 19 brassica oleracea l. 972 4 92 1 3 brassica rapa l. 50 95 5 total 2,841 solanuma solanum tuberosum l. 10,000 1 84 15 solanum tuberosum subsp andigena 123 100 solanum andreanum baker 2 100 solanum albicans ochoa 1 100 solanum alandiae card. 4 100 solanum berthaultii hawkes 13 100 solanum bulbocastanum dun. 6 100 solanum brevidens phil. 46 100 solanum brachistotrichum (bitt.) rydb. 6 100 solanum chacoense bitt. 89 100 solanum commersonii dun. 2 100 solanum cardiophyllum lindl. 3 100 solanum demissum lindl. 115 100 solanum etuberosum lindl. 10 100 solanum fendleri asa gray 3 100 solanum gourlayi hawkes 21 100 solanum hougasii corr. 5 100 solanum kurtzianum bitt. et wittm. 2 100 solanum oplocense hawkes 4 100 solanum phureja juz. et buk. 42 100 solanum polyadenium greenm. 6 100 solanum polytrichon rydb. 14 100 solanum schenckii bitt. 33 100 solanum spegazzinii bitt. 40 100 solanum sparsipilum (bitt.) juz. et buk. 14 100 solanum stenotomum juz. et buk. 25 100 solanum stoloniferum schlechtd. et bché. 37 100 solanum tarijense hawkes 17 100 solanum trifidum corr. 8 100 solanum vernei bitt. et wittm. 46 100 total 10,737 allium allium cepa var. aggregatum g. don 246 9 81 10 allium sativum l. 108 28 64 8 allium oschaninii b. fedtsch 4 100 allium roylei stearn 1 100 total 420 genetic resources (2025), (s2), 41–48 45 figure 1. regeneration of brassica landraces under insect-proof cages figure 2. culture of potato-related wild species in the greenhouse partnership activities the brc bracysol is involved in various eu-funded projects related to the conservation and sustainable use of plant genetic resources: h2020 g2p-sol (ht tp://www.g2p-sol.eu); prima brasexplor (https://bra sexplor.hub.inrae.fr); horizon europe nememerge ( https://nem-emerge.eu) and prowild (https://www.p ro-wild.eu/). it takes part also in various projects financed by national funds or private partners (including promosol, gie colza, association des créateurs de variétés nouvelles de pomme de terre (acvnpt), fédération nationale des producteurs de plants de pomme de terre (fn3pt/inov3pt)). the objectives of these projects are 1) to explore, describe and analyze the genetic diversity of the collections (esnault et al, 2014; missinou et al, 2022; spanoghe et al, 2022), 2) to develop core collections (esnault et al, 2016), 3) to carry out genetic association analyses to identify the regions of the genome involved in resistance traits to different pests or abiotic stresses (kumar et al, 2018), 4) to introduce this diversity into pre-breeding material by exploiting recombination (boideau et al, 2021), 5) to develop markers that can be used in marker-assisted selection. recent results showed that the genetic resources maintained in the brc bracysol proved to be of great value to identifying sources of stable late blight resistance in potatoes and to introduce efficiently new variability in oilseed rape using its diploid progenitors (esnault et al, 2023). the most noteworthy current research activities are: exploitation of a genomic dataset (leuenberger et al, 2024b) developed on a panel of potato pre-breeding bracysol: the french centre for brassica, allium & solanum genetic resources 46 esnault et al genetic resources (2025), (s2), 41–48 clones maintained in the brc bracysol in genomewide association studies to identify genes involved in resistance to cyst nematodes (leuenberger et al, 2024a) or to late blight disease (phd work of leuenberger j.). in the framework of the european project brasexplor, collect, genotyping and phenotyping of brassica oleracea and b. rapa wild populations and cultivated landraces extending from the north atlantic coast to the southern algerian desert. the taxonomy of these collected accessions was checked by combining cytogenetic and molecular methods (falentin et al, 2024). this plant material is used to investigate the genomic regions involved in adaptation to climate change (wagner et al, 2023). as mentioned, brc bracysol has established longstanding collaborations with private partners, contributing in particular to breeding programmes. one of these collaborations consists of a convention signed in 1995 between inrae and the four french potato breeders gathered within acvnpt. acvnpt provides financial support to inrae for the conservation and characterization of the potato genetic resources maintained within the brc bracysol and in return gets free access, with a 5-year exclusivity period, to the pre-breeding material generated by inrae within the framework of its research activities using these genetic resources (kerlan et al, 2017). since 1995, inrae has selected 994 pre-breeding clones, improved mainly for resistance to different pathogens (including phytophthora infestans, globodera pallida, pectobacterium sp., melöıdogyne incognita (kofold & white) or potato virus y). so far, 41 potato varieties have been registered by the french breeders who used these pre-breeding clones, maintained by the brc, in their crossing programmes. promosol is another important partner, who funded several projects including probiodiv. in this project, it was demonstrated that it is possible to introduce efficiently new variability in oilseed rape using its diploid progenitors conserved in the brc bracysol. pre-breeding oilseed rape populations were created and seeds of 1,600 introgressed lines were provided to the breeders belonging to promosol (esnault et al, 2023). network and working group participation the brc bracysol coordinates two national networks for the conservation of plant genetic resources: the ‘oilseed crucifers’ network and the ‘potato’ network. these networks involve private and public partners and have defined the lists of accessions to be included in the national collection. in addition, the brc bracysol is part of the european cooperative programme for plant genetic resources (ecpgr) and is a member of the brassica, allium and potato working groups. it takes part in the following ecpgr activities that are currently being funded: ‘garliccs’ (genotyping-by-sequencing of the european garlic collection to develop a sustainable ex situ conservation strategy) and ‘euro-potatoes’ (collaboration action for updating the virtual european potato collection). conclusion the brassica, allium and solanum genetic resources maintained in the brc bracysol proved to be of great value in tackling agronomic issues currently faced by these crops. the brc aims at conserving the diversity and the good quality of these genetic resources and distributing them to researchers and breeders to further contribute to the development of more agroecological agriculture in a context of climate change. to achieve these goals, the brc bracysol works currently to improve the management of the characterization data associated with the accessions and enhance the visibility of these genetic resources. acknowledgements we acknowledge the experimental unit rgco for providing the fields needed for the maintenance of the genetic resources. author contributions fe managed and contributed to the overall writing of the manuscript. mpc, mad, lalv, rp, jp, jq and cs contributed to the writing of the solanum collection description, its associated data and distribution service. sd, pg, vr, and st contributed to the writing of the brassica collection description, its associated data and distribution service. dk and jq contributed to the writing of the allium collection description, its associated data and distribution service. fe, jec, amc, mck, al, mmd, mt and nn contributed to the writing of partnership activities and network participation. conflict of interest statement the authors have no conflicts of interest to report. references alercia, a., diulgheroff, s., and mackay, m. 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(2025) “a fast and effective method to distinguish cultivated fonio species: conservation and evaluation perspectives”, genetic resources, 6(12), pp. 83–94. doi: 10.46265/genresj.jdut8893. © copyright 2025 the authors. this is an open access article distributed under the terms of the creative commons attribution license (cc by 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. sandrine causse*,a,b, thomas kaczmareka,b,c, cécile duboisa,b, enoch g. achigan-dakod, joseph adjebeng-danquahe, richard y. agyaree, louise akanvouf, yacoubou bakassog,n, mamadou b. barryh, baye m. diopi, mame c. gueyei, abdou r. ibrahim bio yerimad,j, happiness o. oselebek, sani saidou idil,n, edak a. uyohm, sylvie vancoppenollea,b, adeline barnaudc, claire billota,b, jean-françois ramia,b, christian leclerca,b a cirad, umr agap institut, montpellier, france b agap institut, university of montpellier, cirad, inrae, institut agro, montpellier, france c diade, university of montpellier, ird, cirad, montpellier, france d unit of genetics, biotechnology and seed sciences, gbios, faculty of agronomic sciences, university of abomey-calavi, cotonou, republic of benin e council for scientific and industrial research—savanna agricultural research institute (csir-sari), ghana f département ressources génétiques, cnra, abidjan, côte d’ivoire g department of biology, faculty of science and technic, abdou moumouni university, niamey, niger h irag, conakry, guinea i isra, ceraas, thiès, senegal j department of rainfed crop production (dcp), national institute of agronomic research of niger (inran), niamey, niger k center for crop improvement, nutrition & climate change (ccincc), ebonyi state university, abakaliki, nigeria l department of plant production and biodiversity, faculty of agronomic and ecologic sciences, university of diffa, diffa, niger m department of genetics and biotechnology, university of calabar, calabar, nigeria n laboratory for the management and valorization of biodiversity in the sahel (gevabios), abdou moumouni university, niamey, niger * corresponding author: sandrine causse (sandrine.causse@cirad.fr) http://10.46265/genresj.jdut8893 mailto:https://doi.org/10.46265/genresj.apnr6909%0d?subject= https://www.genresj.org http://10.46265/genresj.jdut8893 mailto:sandrine.causse%40cirad.fr?subject= genetic resources (2025), 6(12), 83–9484 causse et al introduction crop genetic resources refer to the diversity of traditional landraces and modern cultivated varieties, including their crop wild relatives. the number of cultivated crops has been drastically reduced by the intensification of agriculture since the 20th century. today, around 30 species are used to satisfy 90% of humanity's needs, whereas 100 species were used at the beginning of the 20th century (gepts, 2006). in this context of crop genetic erosion, in situ and ex situ conservation of a wide range of crop diversity is essential to ensure food security and to face global changes (fao, 2010; khoury et al, 2014; fao, 2020). the ex situ approach involves safeguarding crop diversity outside of its native environment, typically within conservatories, or specialized infrastructures such as seedbanks. the primary goal is to conserve and propagate crop genetic resources and make them available for research, breeding and cultivation. this is particularly crucial for neglected and underutilized species (nus) that received limited scientific attention (stamp et al, 2012; hunter et al, 2019; ulian et al, 2020), whereas they could be used to face global changes and improve the quality and sustainability of food production (ulian et al, 2020). describing and characterizing nus accessions that are preserved in ex situ collections is essential for their management and sustainable use. accurate documentation of accessions enables informed decisions to be made on conservation, research, breeding and potential use (weise et al, 2020). however, this conservation approach requires the availability of accurate passport data, notably to avoid any taxonomic misidentification (guzzon et al, 2018) or geographic location errors, which can introduce spatial bias into databases and distort large-scale biodiversity analyses (beck et al, 2014). among nus, fonio is a key cereal, native to west africa, with valuable nutritional and agronomic qualities. fonio is highly adapted to harsh environmental conditions and plays a crucial role in food security within developing economies. the potential of fonio has earned it recognition by the value addition in cereal systems (vacs) initiative as a top cereal for west africa (karl et al, 2024). the accuracy of passport data is particularly critical in the case of fonio. fonio comprises two similar species with tiny seeds, both grown in west africa, sometimes in the same localities; identifying the two species is not obvious. the most common, digitaria exilis stapf, is known as white fonio and is cultivated in an area stretching from senegal to nigeria. the second, digitaria iburua stapf, is named black fonio and its distribution is limited to northern nigeria, togo and benin (animasaun et al, 2018). the black and white denomination for fonio refers to the colour of the seed husks, which are likely to be more intensely dark brown for d. iburua than d. exilis (adoukonousagbadja a-h, 2010) (figure 1). however, this criterion can vary within the two species, leading to confusion. the variability of this trait, plus the fact that fonio species are sometimes not distinguished by their common name (blench, 2016), can lead to misidentification in collections. figure 1. pictures of: a, black fonio (digitaria iburua) and b, white fonio seeds (digitaria exilis). genetic resources (2025), 6(12), 83–94 rapid identification of fonio species 85 improving the accuracy of genebank passport data concerning the identification of d. exilis and d. iburua is a key issue that needs to be overcome to preserve fonio genetic resources, and make their adaptive potential available to farmers. fonio identification could be based on vegetative, floral or spikelet characteristics, or molecular markers. for example, the growth habits of white and black fonio differ (figure 2), but using this trait as an identification criterion requires seed growing, as well as access to largescale cultivation areas or costly infrastructures. in addition, genebank collections often contain only limited seed samples for some fonio accessions. on the other hand, genetic identification methods, based on microsatellite genotyping or genome sequencing, require small samples (mondini et al, 2009). all these methods are expensive, destructive and time-consuming. the aim of this work was to develop a low-cost, nondestructive and rapid method, based on seed morphology, in order to assign fonio accessions to either white or black fonio species. to date, the seed morphometrics approach has never been applied to fonio crops. such an approach is highly relevant for fonio genebank collections to improve the quality of associated passport data and enhance the value of these collections. materials and methods plant material and sampling fonio accessions are maintained in the seed collection in montpellier, france, at the gamét resource centre (arcad) and the french national research institute for sustainable development (ird) and in national genebank collections across west africa. these collections of seeds (paddy grains), sometimes in small amounts (less than one gram), have been figure 2. pictures of several plants of: a, black fonio (digitaria iburua) and b, white fonio (digitaria exilis). built up since 1977 thanks to collection missions to farmers in the areas of origin and thanks to partnerships between french and african research institutions involved in various research projects. a sample of 118 accessions of d. exilis and d. iburua (98 and 20 respectively, supplemental table 1) previously sequenced in abrouk et al (2020) and kaczmarek et al (2025) was selected to maximize geographical coverage (figure 3) and thus climatic diversity. only accessions whose species had been genetically validated were selected. for each accession, one seed sample was prepared in a 2ml microtube, weighing between 110mg and 151.4mg. the variability in sample mass was linked to the quantity of seeds available and to the difficulty of handling very small seeds (in the millimetre range, figure 1). seed image analysis for each accession, the seed sample was poured and carefully laid on a 13.5 × 10.5cm surface of a flatbed scanner (epson expression 10000xl) to be scanned on a green background (canson-c200040066). an average of 232 seeds per accession was scanned. in total, 27,345 seeds were analyzed. the images were saved in .tif format (800dpi resolution). the images were analyzed with the rigatoni v.0.9.3 r package (rami, 2022), based on the ebimage r package. the rigatoni package was designed to analyze seed images acquired by scanning. in contrast to the colour of background pixels, the algorithm detects objects in an image and characterizes their size, shape and colour, with a total of 27 descriptors. each seed was individually cropped in the image using the krnel function. preliminary tests were carried out to calibrate the seed genetic resources (2025), 6(12), 83–9486 causse et al detection algorithm and define size and colour thresholds to avoid the detection of artefacts. it was observed that fonio seeds cannot be smaller than 500px (minimum size threshold for an object), nor larger than 2,000px (maximum size threshold). to find the seeds in the image, the hue range of the green background was set between 68° and 92° and the brightness threshold was set at 0.001. dimensions in pixels were converted to tenths-of-amillimeter (tmm), and the area was converted from pixels to square tenths-of-a-millimetre (tmm2). the hexadecimal colour code was also determined from each seed’s cropped image. for data analysis, this colour code in rgb components was then converted into h, s and v components with the colorspace v2.0-3 r package (zeileis et al, 2020). the advantage of the hsv colour system is that it is based on components perceived by humans to describe colours: hue (tint or predominant colour), saturation (colour intensity) and value (brightness), allowing intuitive interpretation of colour variations (hema et al, 2019). preliminary exploratory data analysis data validation a graphical exploratory analysis was performed on the values measured per seed, and revealed distributions that were sometimes highly asymmetrical with very extreme and therefore suspicious values. an automatic extreme values filter was applied with the boxplot.stats() function, using the interquartile range (iqr) and a whiskers coefficient of 3 times this length to eliminate only highly improbable values. for each descriptor and accession, any value below q1 3iqr, or above q3 + 3iqr was considered an extreme outlier, with q1 as the lower quartile and q3 as the upper quartile. seeds with figure 3. geographical map of west africa showing the spatial distribution of the 118 accessions of d. exilis and d. iburua used for seed measurements. at least one descriptor presenting an extreme outlier value were excluded from the data. for further data analysis, the morphometric values measured per seed were summarized, for each accession, by their median value. morphometric and colour descriptors an exploratory analysis of the 27 descriptors was carried out to assess their variation and reduce their number in the event of strong correlations. a graphical method was used to explore the relationship between descriptors in pairs (matrix of graphs, not shown), initially considering separately each category: size, shape, pixel intensity, colour, contour (see table 1 for descriptor details). thousand-grain weight was added as a usual descriptor for cereals. descriptors were selected in such a way as to retain only those descriptors that made sense, i.e. those that provided specific and easily understandable information on the sample variability. in the case of highly correlated variables, the selected variable was the one that made more sense. for example, s.area (seed area) and s.radius.mean (mean seed radius, table 1) were highly correlated (pearson correlation value 0.99), hence seed area, which made more sense than s.radius.mean, was selected. variables representing statistical dispersion parameters such as standard deviation, median absolute deviation, or quantile, were not selected. all variables that were complementary to each other, such as the h, s and v colour parameters, were selected. statistical analysis of morphometric data descriptive statistics were carried out using the stat.desc() function of the pastecs v.1.3.21 r package (grosjean et al, genetic resources (2025), 6(12), 83–94 rapid identification of fonio species 87 descriptor characteristics seed size (provided by rigatoni package) bbox.width* object bounding box width (in pixels, converted to tmm) measuring seed width bbox.height* object bounding box height (in pixels, converted to tmm) measuring seed length s.area* area, number of pixels in the shape (converted to tmm2) s.perimeter perimeter, number of pixels in the boundary of the object (converted to tmm) s.radius.mean mean radius (in pixels), average radius value from the centre of shape to boundary (converted to tmm) s.radius.sd standard deviation of the radius values (in pixels) s.radius.max max radius (in pixels), largest radius value from the centre of shape to boundary (converted to tmm) s.radius.min min radius (in pixels), shortest radius value from the centre of shape to boundary (converted to tmm) seed shape (provided by rigatoni package) m.eccentricity* elliptical eccentricity, values ranging from 0 (perfect circle) to 1 (straight-line). calculated with the longest axis (majoraxis) and the shortest axis (minoraxis) of the best-fitting ellipse: sqrt(1minoraxis2/majoraxis2). m.majoraxis largest axis of the best-fitting ellipse (in pixels, converted to tmm) m.cx, m.cy centre of the best-fitting ellipse coordinates (in pixels) m.theta object angle (in radians) pixels intensity (provided by rigatoni package) b.mean average of pixel intensity in the shape b.sd standard deviation of pixel intensity in the shape b.mad median absolute deviation of pixel intensity in the shape b.q (b.q001, b.q005, b.q05, b.q095, b.q099) quantile intensity of pixel intensity in the shape seed contour (provided by rigatoni package) poi.x, poi.y pole of inaccessibility coordinates, coordinates of the point farthest away from the boundary of the object poi.dist longest distance to the boundary of the object (in pixels, converted to tmm) seed colour (obtained by converting the rgb code provided by rigatoni package) h* hue (in degrees), predominant colour or tint, (values ranging from 0 to 360°) s* saturation, intensity of colour pigmentation (values ranging from 0 to 1) v* value, brightness of the colour (values ranging from 0 to 1) thousand-grain weight (calculated by accession) tgw* thousand seed weight (in grams) 2018) in order to characterize the two species. histograms of the median values per accession for each species (supplemental figure 1) showed non-normal (skewed and/or over-spread) distributions for most morphometric variables. these data were not suitable for the application of a t-test, and the two fonio species were compared by the mann-whitneywilcoxon rank test (wilcox.test() function). morphometric diversity of the 118 accessions was explored using principal component analysis (pca). pca was performed on the seven seed descriptors (table 1), using the pca() function in the factominer v.2.6 r package (lê et al, 2008). table 1. characteristics of the morphometric and colour descriptors. *, descriptors selected for the study; tmm, tenths-of-a-millimeter; tmm2, square tenths-of-a-millimeter; sqrt, square root. genetic resources (2025), 6(12), 83–9488 causse et al results the objective of this study was to develop an affordable, non-destructive and rapid method based on seed morphology to categorize fonio accessions into white or black fonio species. outliers detection the 3iqr method discarded 1.1% of the 27,345 seeds analyzed. nine accessions had no outliers. for the remaining 109 accessions, the percentage of outliers varied from 0.4% to 8.8% (supplemental table 2). no link could be established between the percentage of outliers and the parameters structuring the sampling design (species, country of origin and collection date). figure 4 shows an example of how outliers were detected for seeds with attached pedicels and seeds with open glumes. morphometrics description of black and white fonio d. iburua and d. exilis differed significantly for all variables (wilcoxon test at the p-value threshold < 0.05). it can be noted that seed area (s.area) and seed width (bbox.width) figure 4. picture showing the two main types of outliers (*). the two columns on the left show seeds with open glumes and, on the right, seeds with attached pedicels. the two rows represent: a, original seeds picture; b, bounding boxes plotted by the image analysis. showed no overlap at all between the two species for our sample of accessions (supplemental figure 1). the results presented below are based on the descriptors' median values. size and shape analysis d. iburua seeds were significantly wider (+19%), longer (+13%) and heavier (31%) than d. exilis seeds (wilcoxon test, p < 0.001, supplemental figure 1). d. iburua seeds were 19.1tmm long (bbox.height) and 11.1tmm wide (bbox. width), while d. exilis seeds were 16.9tmm long and 9.4tmm wide. the thousand-grain weight was equal to 0.71g for d. iburua and 0.54g for d. exilis (table 2). for seed width (bbox.width), the range of variation between the minimum and the maximum values showed a clear demarcation between the two species (supplemental figure 1). indeed, seed width varied for d. exilis from 8.2 tmm to 10.1tmm as compared to 10.5tmm to 12.0tmm for d. iburua accessions (table 2). seed area (s.area) values also highly differed (wilcoxon test, p < 0.001), revealing a distinct separation between the two species. for d. exilis, seed area varied from 90.6tmm2 to 133.7tmm2 whereas for d. iburua, seed area varied from 139.8tmm2 to 162.7tmm2 (table 2). these results confirmed that d. iburua seeds were significantly bigger than those of d. exilis. furthermore, m.eccentricity values were significantly (wilcoxon test, p < 0.05) higher for d. exilis (0.82) than for d. iburua (0.80). the distribution of values for each species (supplemental figure 1) showed a second peak at high values of m.eccentricity (around m.eccentricity = 0.83), indicating that some d. exilis accessions had, on average, more elongated seeds. colour analysis hue values were significantly different (wilcoxon test, p < 0.001) between d. iburua (h = 51.9°) and d. exilis (h= 56.4°). both values were in the yellow range, but d. iburua seeds had a warmer yellow-red hue than d. exilis seeds whose hue was closer to pure yellow (supplemental figure 2). moreover, colour brightness (v) values were significantly (wilcoxon test, p < 0.001) lower for d. iburua (v = 0.21) than for d. exilis (v = 0.32); reversely, colour saturation (s) values were significantly (wilcoxon test, p < 0.001) higher for d. iburua (s = 0.89) than for d. exilis (s= 0.77) (table 2). those contrasted v and s values, associated with h values correspond to the contrasted colour of the seed husks, which are dark brown for d. iburua and light brown for d. exilis (figure 1). morphometric diversity the pca analysis revealed a clear distinction between white and black fonio (figure 5). the first four principal components covered 99% of the total variance (supplemental figure 3). figure 5. principal component analysis carried out on the seed morphometric measurements of the 118 fonio accessions (n = 98 for digitaria exilis and n = 20 for digitaria iburua). a, correlation circle for the first two principal components, where bbox.width correspond to width, bbox.height to length, s to saturation of colour, h to hue of colour, v to brightness of colour, m.eccentricity to eccentricity, s.area to seed area, and tgw to thousand-grain weight. the supplementary variable tgw was coloured in blue. b, scatterplot of the 118 fonio accessions projected on the first two principal components plane. genetic resources (2025), 6(12), 83–94 rapid identification of fonio species 89 table 2. descriptive statistics for size and shape analysis of each fonio species. n, number of accessions. species variables minimum value (min) maximum value (max) median mean standard deviation (sd) coefficient of variation (cv, %) digitaria exilis (n = 98) s.area (tmm2) 90.63 133.72 113.28 112.80 71.91 7.5 bbox.width (tmm) 8.24 10.09 9.37 9.34 0.10 3.4 bbox.height (tmm) 14.34 19.05 16.86 16.85 0.95 5.8 m.eccentricity 0.74 0.86 0.82 0.82 0.00 2.9 h (°) 53.81 62.56 56.40 56.41 1.89 2.4 s 0.65 0.87 0.77 0.76 0.00 5.5 v 0.22 0.48 0.32 0.32 0.00 11.6 tgw (g) 0.31 0.70 0.54 0.53 0.00 12.2 digitaria iburua (n = 20) s.area (tmm2) 139.82 162.70 153.23 152.86 46.02 4.4 bbox.width (tmm) 10.54 11.96 11.11 11.14 0.14 3.3 bbox.height (tmm) 17.89 20.32 19.11 19.17 0.61 4.1 m.eccentricity 0.74 0.84 0.80 0.80 0.00 3.4 h (°) 48.24 71.71 51.86 53.63 35.54 11.1 s 0.80 0.94 0.89 0.89 0.00 3.7 v 0.11 0.37 0.21 0.23 0.01 35.3 tgw (g) 0.59 0.88 0.71 0.73 0.01 9.8 showed no overlap at all between the two species for our sample of accessions (supplemental figure 1). the results presented below are based on the descriptors' median values. size and shape analysis d. iburua seeds were significantly wider (+19%), longer (+13%) and heavier (31%) than d. exilis seeds (wilcoxon test, p < 0.001, supplemental figure 1). d. iburua seeds were 19.1tmm long (bbox.height) and 11.1tmm wide (bbox. width), while d. exilis seeds were 16.9tmm long and 9.4tmm wide. the thousand-grain weight was equal to 0.71g for d. iburua and 0.54g for d. exilis (table 2). for seed width (bbox.width), the range of variation between the minimum and the maximum values showed a clear demarcation between the two species (supplemental figure 1). indeed, seed width varied for d. exilis from 8.2 tmm to 10.1tmm as compared to 10.5tmm to 12.0tmm for d. iburua accessions (table 2). seed area (s.area) values also highly differed (wilcoxon test, p < 0.001), revealing a distinct separation between the two species. for d. exilis, seed area varied from 90.6tmm2 to 133.7tmm2 whereas for d. iburua, seed area varied from 139.8tmm2 to 162.7tmm2 (table 2). these results confirmed that d. iburua seeds were significantly bigger than those of d. exilis. furthermore, m.eccentricity values were significantly (wilcoxon test, p < 0.05) higher for d. exilis (0.82) than for d. iburua (0.80). the distribution of values for each species (supplemental figure 1) showed a second peak at high values of m.eccentricity (around m.eccentricity = 0.83), indicating that some d. exilis accessions had, on average, more elongated seeds. colour analysis hue values were significantly different (wilcoxon test, p < 0.001) between d. iburua (h = 51.9°) and d. exilis (h= 56.4°). both values were in the yellow range, but d. iburua seeds had a warmer yellow-red hue than d. exilis seeds whose hue was closer to pure yellow (supplemental figure 2). moreover, colour brightness (v) values were significantly (wilcoxon test, p < 0.001) lower for d. iburua (v = 0.21) than for d. exilis (v = 0.32); reversely, colour saturation (s) values were significantly (wilcoxon test, p < 0.001) higher for d. iburua (s = 0.89) than for d. exilis (s= 0.77) (table 2). those contrasted v and s values, associated with h values correspond to the contrasted colour of the seed husks, which are dark brown for d. iburua and light brown for d. exilis (figure 1). morphometric diversity the pca analysis revealed a clear distinction between white and black fonio (figure 5). the first four principal components covered 99% of the total variance (supplemental figure 3). figure 5. principal component analysis carried out on the seed morphometric measurements of the 118 fonio accessions (n = 98 for digitaria exilis and n = 20 for digitaria iburua). a, correlation circle for the first two principal components, where bbox.width correspond to width, bbox.height to length, s to saturation of colour, h to hue of colour, v to brightness of colour, m.eccentricity to eccentricity, s.area to seed area, and tgw to thousand-grain weight. the supplementary variable tgw was coloured in blue. b, scatterplot of the 118 fonio accessions projected on the first two principal components plane. genetic resources (2025), 6(12), 83–9490 causse et al the first principal component (pc1, 55.2% of the total variance) opposed seed size parameters and saturation (s) of the seed colour (positive values), with brightness (v) of the seed colour (negative values, figure 5a). seed size refers to seed area (s.area), seed width (bbox.width) and seed length (bbox.height), which were among the most influential characters on pc1 (supplemental table 3). variability of s.area and bbox.width variables was almost entirely represented by pc1 (cos2: resp. 90% and 85%, supplemental table 3). the pc2 (20.2% of the total variance) was mainly related to the m.eccentricity variable (contribution: 60.7%), which is a component of the seed shape, and to a lesser extent to seed length (bbox.height, contribution: 22.5%). the variability of the m.eccentricity variable is almost entirely represented by pc2 (cos2: 86%). pc3 (13.9% of the total variance) was mainly related to the tint (h, contribution: 80%). for pc4, which accounted for less than 10% of total variability, the brightness (v) of the seed colour was the most influential character (contribution: 37%) (supplemental table 3). the first axis (figure 5b) completely differentiated the two fonio species, with black fonio (right) seeds characterized by higher values of area, width, colour saturation (s) and height, and lower values of brightness (v), compared to white fonio (left) with the opposite characteristics. independent of this clear structure separating the species on the first axis, the second axis showed, within each species, a gradient of variation in seed shape (m.eccentricity), from the least to the most elongated. illustration of accessions by their country of origin (figure 6) revealed that the roundest seeds (bottom of axis 2) originated from nigeria only. moreover, the nigerian accessions were projected over the same range along this axis for both species. on the opposite side and on the top of axis 2, the white fonio accessions with the most tapered seeds mainly originated from mali and senegal. the first factorial plane concentrated 75% of the total variability and made it possible to characterize the morphometric differences between the seeds of the two species. the next pcs, calculated on the remaining variability, did not appear to be informative. they showed either particularities for some accessions (pc3), or more continuous variations which could not be linked to explanatory factors and therefore could not be interpreted (pc4). differentiation of the two species with only one morphometric descriptor figure 7 clearly showed that the seed width alone perfectly separated the two fonio species. descriptive statistics (table 2) specified the limiting values observed for these data: up to a value of seed width equal to 10.09tmm for d. exilis (n = 98) and from 10.54tmm for d. iburua (n = 20). in addition, the seed area parameter, which is strongly dependent on seed width and height, was also a differentiating trait between the two species (supplemental figure 4). discussion this work sought to distinguish the two species of cultivated fonio based on the characteristics of their seeds in a context of genebank conservation. seed morphometrics was used as a rapid, low-cost and non-destructive method to identify the two fonio species. morphometrics has largely figure 6. principal component analysis carried out on the seven selected morphometric variables and the 118 fonio accessions. the geographic origin of individuals is represented by symbols of different shapes and colours.  figure 7. scatterplot with marginal distribution of seed width (x) and seed length (y) of the 118 fonio accessions. genetic resources (2025), 6(12), 83–94 rapid identification of fonio species 91 been used to describe and compare organism shapes, allowing reliable species identification. this was confirmed on organisms as varied as orchids (chemisquy et al, 2009), mosquitoes (chaiphongpachara et al, 2022), indigo plants (soladoye et al, 2010), wheat species (goriewa-duba et al, 2018) and olive (terral et al, 2004; newton et al, 2014). the tiny size of the seeds of both species is a source of practical difficulties, particularly for handling and visual identification. we showed that the rigatoni r package (rami, 2022) could be adapted to detect very small objects in images. while this package has been initially developed for the analysis of irregular shapes like peanut pods, we used here a smaller number of rigatoni variables since fonio seeds have an oblong ellipsoid shape (idu et al, 2008). one consequence of small seed size is that seed lots can be heterogeneous due to the residual presence of undesirable biological material (pedicels, open glumes, foreign seeds), sand or stones in seed samples despite careful cleaning of the sample before measurement, as suggested by koreissidembélé et al (2013). a large number of seeds (over 200) per accession were scanned to ensure reliable results by limiting the influence of outliers and being able to detect them using robust quantitative methods. we implemented a fast method to remove extreme outliers from the analysis to make the identification more robust. depending on the accession, between 0.4% and 8.8% of seeds were identified as extreme outliers. white and black fonio are very similar as they share many agromorphological characteristics. according to adoukonousagbadja et al (2007), a clear-cut separation of both species was not possible using agromorphological traits such as plant for both species. on the opposite side and on the top of axis 2, the white fonio accessions with the most tapered seeds mainly originated from mali and senegal. the first factorial plane concentrated 75% of the total variability and made it possible to characterize the morphometric differences between the seeds of the two species. the next pcs, calculated on the remaining variability, did not appear to be informative. they showed either particularities for some accessions (pc3), or more continuous variations which could not be linked to explanatory factors and therefore could not be interpreted (pc4). differentiation of the two species with only one morphometric descriptor figure 7 clearly showed that the seed width alone perfectly separated the two fonio species. descriptive statistics (table 2) specified the limiting values observed for these data: up to a value of seed width equal to 10.09tmm for d. exilis (n = 98) and from 10.54tmm for d. iburua (n = 20). in addition, the seed area parameter, which is strongly dependent on seed width and height, was also a differentiating trait between the two species (supplemental figure 4). discussion this work sought to distinguish the two species of cultivated fonio based on the characteristics of their seeds in a context of genebank conservation. seed morphometrics was used as a rapid, low-cost and non-destructive method to identify the two fonio species. morphometrics has largely figure 6. principal component analysis carried out on the seven selected morphometric variables and the 118 fonio accessions. the geographic origin of individuals is represented by symbols of different shapes and colours.  figure 7. scatterplot with marginal distribution of seed width (x) and seed length (y) of the 118 fonio accessions. height, number of tillers, leaf length, fresh and dry biomass weight, panicle length, and yield. this study focused on seed morphology and seed weight. we showed that seeds of d. iburua were significantly larger, heavier and had a more intense and darker brown colour than those of d. exilis. seed width clearly distinguished d. exilis from d. iburua in our sample of accessions. our results thus confirmed a difference in seed size between the two species, as previously noted by echendu et al (2009) and jideani (2012). the distinctiveness of d. exilis and d. iburua was also confirmed with respect to seed weight (aliero et al, 2002; nyam et al, 2017). we showed that seed area was a descriptor also separating the two species; however, we focused on seed width, a onedimensional parameter whose variations are straightforward to interpret. it is worth noting that the differences between species, in seed size, weight and colour, were revealed despite the different conditions under which the accessions were collected and conserved. the species effect on these morphometric characteristics, therefore, appears stable, as it is greater than eventual environmental effects. inversely, the more or less tapered shape of the seeds did not show a clear difference between species, but varied in an apparently structured way according to country of harvest (pca, axis 2, figure 6). the projection on a map (supplemental figure 5) of seed shape values (m.eccentricity descriptor cut into classes), at harvesting sites, visually confirmed that the most tapered d. exilis seeds came mainly from the northern edge of the sampling zone (senegal, mali, northern burkina faso, niger), in drier climatic regions. this trend requires more in-depth studies in relation to climatic data. the number of accessions used in our study differed genetic resources (2025), 6(12), 83–9492 causse et al markedly between the two species. this is partly because black fonio is predominantly grown in central nigeria today, from where samples are available in genebanks. moreover, some geographical origins could not be used for d. exilis because of incomplete passport data. we can't rule out the possibility that a more balanced sampling design, both in terms of species frequency and geographic distribution, might nuance our results, and that the species classification might be less categorical in a context of greater morphometric variability. however, our sampling design was based on genetic studies that already maximized the diversity and species geographical range in their sampling. despite sampling constraints, pca provided some confidence in the results. the first principal component (figure 6), which differentiates the two species by seed size and colour saturation, did not appear to show any structure related to geographical origin. further studies focusing on the effect of geographic origin on seed morphology should investigate this issue more precisely, both within and between species. our morphometric results also open up new avenues for research in archaeobotany. the morphometric approach proposed in this paper could be used to identify fonio in archaeological records, contributing to the reconstruction of the evolutionary history of both cultivated fonio and its wild relatives. morphometrics of ancient seeds would provide a better understanding of the domestication and diffusion histories of d. exilis and d. iburua in west africa. indeed, morphometrics on crops allowed to confidently distinguish between domesticated versus wild forms and trace the evolution of cultivated forms through space and time, as in the case of grapevine (bouby et al, 2013; rôs et al, 2014; bonhomme et al, 2021; ucchesu et al, 2024) or date palm (terral et al, 2012; gros-balthazard et al, 2016). additional research with carbonization experiments is however needed to see the impact of charring on seed morphology in both species (ivorra et al, 2024). in order to preserve the existing fonio diversity from genetic erosion, germplasm collection and ex situ conservation is a necessity (dansi et al, 2010). correct taxonomic identification is all the more crucial for the plant genetic resources that are currently underrepresented in ex situ conservation facilities worldwide, as is the case for fonio, and therefore have a high priority for future collecting missions and urgent conservation measures (guzzon et al, 2018). labelling of accessions is essential to enhance the value of these collections and to make accessions usable. by enhancing passport data and combining it with additional information from other fields, new knowledge about plant genetic resources can be generated, which is crucial for the sustainable management of genebank collections. in the case of fonio species, the seed width could be used as the sole criterion for a simple and inexpensive method to make their taxonomic identification and genebank passport data more reliable. this approach is particularly useful when genetic data are not available. as a new genebank conservation procedure, we suggest that the method proposed in this paper be applied to fonio accessions already conserved in genebank collections to ensure the reliability of fonio identification in passport data. it could also be systematically applied to any new fonio accession before its integration in genebank collections, especially for fonio originating from regions where both species are grown. supplemental data supplemental table 1. sampling details: number of accessions by countries and species. supplemental table 2. information on the 118 accessions analyzed. supplemental table 3. results for the principal component analysis carried out on the seven selected morphometric variables and the 118 fonio accessions. supplemental figure 1. histogram of median morphometric values by accession and tgw (thousand grain weight) for the two species. supplemental figure 2. position of the two fonio species, according to their median hue values, on the diagram representing a part of the sequence of hue. supplemental figure 3. principal component analysis carried out on the seven selected morphometric variables and the 118 fonio accessions. scree plot: percentage of total variation explained by each principal component. supplemental figure 4. scatterplot with marginal distribution of seed area (x) and seed length (y) of the 118 fonio accessions. supplemental figure 5. projection on a map of seed shape values (m.eccentricity descriptor cut into classes). data and code availability statement dataset supporting the results of this article are available via dataverse: https://dataverse.cirad.fr/dataset. xhtml?persistentid=doi:10.18167/dvn1/zfztwp r script for the different analyses carried out throughout the paper are available on the cirad gitlab platform: https:// gitlab.cirad.fr/agap/fonio/seedfonio author contributions sc, tk, ab, cb, and cl designed the research. egad, ariby, ss, yb, bmd, mcg, rya, jad, la, mmb, eau, hoo, ssi, sv, tk, ab and cb contributed to the sampling of the biological material or the curation of collections. sc generated the data. sc analyzed the data with inputs from cd, jfr, tk, ab, cb, and cl. sc and cl wrote the paper with substantial inputs from tk, ab, cd, cb. conflict of interest statement the authors have no conflicts of interest to report. acknowledgments the exploration of fonio diversity over the past decade has been facilitated by various research initiatives. these projects received funding from waapp/ppaao 2a (cera58id06 se), agropolis fondation (specifically the agropolis resource center for crop adaptation and diversity https://www.genresj.org/index.php/grj/article/view/genresj.jdut8893/suppdata290 https://www.genresj.org/index.php/grj/article/view/genresj.jdut8893/suppdata290 https://www.genresj.org/index.php/grj/article/view/genresj.jdut8893/suppdata290 https://www.genresj.org/index.php/grj/article/view/genresj.jdut8893/suppdata290 https://www.genresj.org/index.php/grj/article/view/genresj.jdut8893/suppdata290 https://www.genresj.org/index.php/grj/article/view/genresj.jdut8893/suppdata290 https://www.genresj.org/index.php/grj/article/view/genresj.jdut8893/suppdata290 https://www.genresj.org/index.php/grj/article/view/genresj.jdut8893/suppdata290 https://dataverse.cirad.fr/dataset.xhtml?persistentid=doi:10.18167/dvn1/zfztwp https://dataverse.cirad.fr/dataset.xhtml?persistentid=doi:10.18167/dvn1/zfztwp https://gitlab.cirad.fr/agap/fonio/seedfonio https://gitlab.cirad.fr/agap/fonio/seedfonio genetic resources (2025), 6(12), 83–94 rapid identification of fonio species 93 [arcad] project and the cultivar project—id 1504-007) under the investissements d'avenir programme (labex agro: anr-10-labx-0001-01) within the i-site muse framework (anr-16-idex-0006). additionally, support came from the french government anr project (africrop project, anr-13bsv7-0017) and the european union horizon 2020 research and innovation programme (ewa-belt, 862848, ‘linking east and west african farming systems experience into a belt of sustainable intensification’). we would also like to thank crb gamét for providing us with seeds for our study (centre de ressource biologique gamét, arcad – umr agap institut de montpellier. https:// doi.org/10.18167/infrastructure/00007) references abrouk m., ahmed h.i., cubry p., šimoníková d., cauet s., pailles y., bettgenhaeuser j., gapa l., scarcelli n., couderc m., zekraoui l., kathiresan n., čížková j., hřibová e., doležel j., arribat s., bergès h., wieringa j.j., gueye m., kane n.a., leclerc c., causse s., vancoppenolle s., billot c., wicker t., vigouroux y., barnaud a., krattinger s.g. 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_heading=h.tcqbfmfp42vb _heading=h.r41e0oypmy9z _heading=h.n88480gngmh8 _heading=h.h9e1os3otme3 _heading=h.u29q4gi10zi6 _heading=h.a7qdcu3eo7c8 _heading=h.8tw4jwnxokzj _heading=h.85sgad5o6uf6 _heading=h.eb35vrbey5lf _heading=h.gwmvov3rqzh9 _heading=h.gcvgrni6qjuz _heading=h.c6vfmeyl98n3 _heading=h.j0umm8fys7k7 _heading=h.awsyhfqw7pad _hlk209424324 _hlk204761508 _hlk204867638 _heading=h.6w3o8sp7jw5j _heading=h.8kslk8txzsmk _heading=h.gd44fm55w8tu other articles genetic resources (2025), 6 (11), 57–70 doi: 10.46265/genresj.baiq2696 https://www.genresj.org issn: 2708-3764 across borders – the status and future opportunities for long-term conservation of nordic animal genetic resources ellen-louisa fagerheim white a, maria kjetså b, jaana peippo a, lucy morgan b, juha kantanen c, pierre comizzolid, lise lykke steffensen a, morten kargo e, tullis matson b, ian mayer f and mervi honkatukia *,a a the nordic genetic resource center (nordgen), alnarp, sweden b uk national livestock biobank, whitchurch, shropshire, united kingdom c natural resources institute finland (luke), jokioinen, finland d smithsonian’s national zoo and conservation biology institute, washington dc, united states of america e center for quantitative genetics and genomics, aarhus university, aarhus, denmark f faculty of veterinary medicine, norwegian university of life sciences, ås, norway abstract: the genetic diversity of multiple animal species is now declining rapidly, highlighting the need for action to protect and preserve animal genetic resources for the long term. the nordic countries house a broad range of farm and companion animal breeds and subspecies that play a critical role in environmental sustainability, food safety and security, and human activities. unfortunately, close to 80% of these breeds and subspecies are either endangered or critically endangered, with population sizes too small to ensure their long-term survival. in addition, almost half of them have either a declining or unknown demographic trend, and many of them suffer from high inbreeding. emerging pressures such as climate change, infectious diseases and public unrest further threaten the status of the populations, and urgent action is necessary to ensure their future survival. consequently, efforts for safeguarding the genetic diversity of animal genetic resources (angr) with additional in vitro or cryoconservation efforts need further consideration. the nordic conservation strategies for angr have traditionally been based on in vivo or live conservation. although cryoconservation efforts are in place for some species, the number of donors and doses varies considerably between breeds and species. due to the increasing demand for additional measures for safeguarding angr, this document discusses the status of active angr conservation measures in the nordic countries and emphasize the central role of regional cooperation in ensuring angr sustainability and long-term viability. further, the contributions of cryoconservation in mitigating genetic losses are discussed. keywords: in vivo conservation, live conservation, in vitro conservation, genebank, cryoconservation, nordic food security citation: white, e. f., kjetså, m., peippo, j., morgan, l., kantanen, j., comizzoli, p., steffensen, l. l., kargo, m., matson, t., mayer, i., honkatukia, m. (2025). across borders – the status and future opportunities for long-term conservation of nordic animal genetic resources . genetic resources 6 (11), 57–70. doi: 10.46265/genresj.baiq2696. © copyright 2025 the authors. this is an open access article distributed under the terms of the creative commons attribution license (cc by 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. ∗corresponding author: mervi honkatukia (mervi.honkatukia@nordgen.org) background the global decline in biodiversity also includes our farm animal breeds there is a growing global concern about the continued loss of animal biodiversity. evidence suggests that we have entered the “sixth mass extinction”; the first in earth’s history to be driven primarily by human received: 18.02.2025 accepted: 08.04.2025 published online: 28.05.2025 https://www.genresj.org https://www.doi.org/10.46265/genresj.baiq2696 https://www.genresj.org https://www.doi.org/10.46265/genresj.baiq2696 mailto:mervi.honkatukia@nordgen.org 58 white et al genetic resources (2025), 6 (11), 57–70 activity (cowie et al, 2022; wwf, 2022). currently, species are disappearing 10 to 1,000 times faster than the normal ‘background’ rate of extinction, and the number of individuals in multiple species is declining rapidly (wwf, 2022; iucn, 2024). while most attention has focused on wildlife, the conservation of the world’s numerous native breeds of farm animals, and the necessity to maintain genetic diversity within these species also need attention. the food and agriculture organization of the un (fao) published the global plan of action for animal genetic resources in 2007 (fao, 2007b) and the first state of the world’s animal genetic resources for food and agriculture (fao, 2007a). this was followed up in 2015, when fao published the second report on the state of the world’s animal genetic resources for food and agriculture (fao, 2015), to further highlight the importance of the need for additional measures under the global plan of action. the world’s commitment to the global plan of action was reaffirmed in 2017 by the 16th regular session of the commission on genetic resources for food and agriculture and the 40th fao conference. a third edition of the global plan of action for animal genetic resources is currently in preparation. despite the various implemented conservation efforts and the continuously increased awareness of the many important roles of native animal genetic resources (e.g. genetically, environmentally, economically and culturally), 6.7% of the worlds native breeds have become extinct, and 26% are classified as being at risk of extinction (fao, 2019). the loss of heritage breeds in current production systems may have a detrimental impact on efforts to improve future animal production systems (fao, 2007a; kantanen et al, 2015). the nordic commitment to conservation of animal genetic resources the nordic countries – denmark (including greenland), finland (including åland), iceland, norway, sweden, and the faroe islands – have a rich and diverse assemblage of local and regional transboundary farm animal breeds and subspecies (kierkegaard et al, 2020; white et al, 2024c). these animals hold strong cultural and historical importance, and both livestock species and companion animals like sheep, goats, cattle, horses, dogs and cats have a long history of association with human societies (ovaska et al, 2021; bläuer, 2024; kroløkke et al, 2024). the ancestors of some of the breeds even go back as far as 3,000–2,000 bce, and several of them were important during the viking age (bläuer, 2015). thus, these animals represent an invaluable part of the socioeconomic history of the nordic countries. notably, the grazing animals also play an essential role in ecosystem biodiversity and sustainability (bele et al, 2018; hall, 2018; fraser et al, 2022). unfortunately, at present, many of the local breeds face the risk of extinction due to dimishing population sizes, highlighting the urgent need to implement additional conservation measures before it is too late (fao, 2007a; white et al, 2024c). importantly, the nordic countries are committed to the “conservation of biological diversity, the sustainable use of its components and the fair and equitable sharing of benefits arising from genetic resources” as stated in the 1992 un convention on biological diversity (un, 1992). this commitment encompasses both wild and domestic animals and is further outlined for domesticated animals in the fao global plan of action for animal genetic resources (fao, 2007a), highlighting their responsibility to safeguard the nordic native breeds. additional approaches are needed while the conservation of nordic animal genetic resources (angr) has primarily focused on maintaining live populations (in vivo conservation), national efforts for cryoconservation of e.g., sperm, embryos and somatic cell tissue (in vitro conservation) of angr have also been carried out in all the nordic countries. however, current and future threats such as emerging diseases and climate change, combined with small populations with high inbreeding levels, highlight the importance of additional safeguarding for the breeds. plant safeguarding initiatives, including backup repositories in the millennium seed bank1 for wild plants in the uk and the global seed vault in svalbard2 for cultivated crops have been successfully operating for some time. however, there have not yet been significant regional efforts for farm animal cryoconservation in the nordics. the nordic network project titled ‘nordic animal genebanks added value through nordic cooperation’ (nordfrost, 2021–2024; white et al (2024a)), aimed to strengthen the collaboration and competence for cryopreservation of angr in the nordic region. nordfrost gathered stakeholders in the nordic countries to discuss the possibilities and challenges of genebanking in the region (white et al, 2024a). concerns on the costs versus benefits of cryoconservation were some of the topics that were raised as obstacles influencing genebanking activities. however, new methods for collection and preservation of material have made cryoconservation and utilization of genetic resources more cost-effective, allowing a wider range of stakeholders to participate in cryoconservation (blackburn et al, 2023; fao, 2023). many nordic countries are now recognizing the need to improve cryoconservation activities in combination with continued efforts for live conservation (for example, the national strategies of the nordic countries, supplemental material 1). 1 https://www.kew.org/wakehurst/whats-at-wakehurst/millennium-s eed-bank 2 https://www.croptrust.org/work/svalbard-global-seed-vault/ genetic resources (2025), 6 (11), 57–70 future opportunities for nordic angr conservation 59 aims and objectives this paper is a result of the work from the nordfrost network on finding new possibilities for optimized cryoconservation of nordic angr. it aims to advocate for active conservation measures that emphasize the central role of regional cooperation and adaptation of new techniques in ensuring the sustainability and long-term viability of angr in the nordic countries. thus, the status, challenges, opportunities and future possibilities of cryoconservation in the nordic countries are presented. animal genetic resources in the nordic countries and conservation strategies external threats to genetic diversity in animal populations from nordic countries the nordic native breeds face a range of external challenges such as economic and political unrest (e.g. economic fluctuations, warfare influencing prices and availability of feed), modern farming practices (e.g. intensification and industrialization), societal acceptance, climate change, and emerging diseases (fao, 2007a; clasen et al, 2020, 2021; white et al, 2024c,b). the effect of climate change, including global warming and more extreme weather patterns, continues to be of concern as temperatures continues to rise (simmons, 2022). overall, the nordic region is predicted to become both warmer and wetter, and these climatic changes will likely increase the spread and transmission of new disease epidemics threatening both farm animals and wildlife throughout the nordic region (gray et al, 2009; wang et al, 2011; yoo et al, 2016; sommer and cowie, 2020; ramos et al, 2021). some diseases are already threatening the nordic farm animal populations. for example, african swine fever (asf) and highly pathogenic avian influenza (hpai), infect several populations globally every year (cdc, 2023, 2024). further, hpai was reported on 71 fur farms in finland, causing the euthanasia of nearly half a million animals in 2023 (efsa et al, 2023). the neurodegenerative prion disease classical scrapie has significantly influenced the production of small ruminants in iceland (jónmundsson et al, 2016; hauksdóttir, 2021; thorgeirsdottir, 2022) and recent salmonella outbreaks on poultry farms in sweden have influenced the national egg supply (sva, 2024b). more recently, a new strain of vector-borne blue tongue virus has quickly spread through europe, and has now been detected in denmark, sweden and norway (defra, 2024; jordbruksverket, 2024; ndcc, 2024; sva, 2024b,a; veterinærinstituttet, 2024). such disease outbreaks can lead to euthanasia of the entire flock of animals on the farm and even on nearby farms to prevent expansion of the epidemic, thereby having critical consequences for the small populations of the local breeds (fao, 2007a). some small locally adopted breeds might only exist on one farm or in a small geographical area, which makes them particularly vulnerable. internal threats to genetic diversity in animal populations from nordic countries internal threats to animal populations are mainly those of inbreeding and low genetic diversity caused, for example, by a small population size or suboptimal breeding practices. this can lead to the accumulation of deleterious recessive genetic mutations in the population, which, in turn, leads to compromised fertility rates and increased disease rates (kadri et al, 2014). a small population size combined with inadequate breeding strategies can cause loss of genetic variation and lead the population further down in an extinction vortex, as illustrated in figure 1. effective management of genetic variation in small populations is therefore critical. strategies for managing small populations are comprehensively described in sustainable management of animal genetic resources (wooliams et al, 2005). advantages and limitations of live animal conservation and cryoconservation a population’s ability to adapt to its environment and achieve genetic gain is an important factor in both conservation and production populations. live conservation allows continuous adaptation to a changing environment, and breeding can lead to improved breed performance over time. cryoconservation is described as lacking the ability for continuous adaptation because the genetics are in a frozen state. nevertheless, cryoconservation can be seen as an important second layer of safeguarding the populations. further utilizing genetic variation stored in genebanks may help recover lost traits that are potentially valuable to adapt to new environments and improve performance. therefore, a combination of live conservation and cryoconservation is the optimal way to go. further, education regarding cultural heritage traditions, such as landscaping, is important for raising awareness surrounding unique breeds and their history as human companions and production animals. live conservation can directly promote both tradition and raise awareness of the breeds (fao, 2013), in a way that cannot be done by cryoconservation alone. however, it can be argued that by storing the genetic material, heritage is also conserved. key concerns surrounding the management and conservation of angr are related to different pressures, especially those relating to climate change, disease outbreaks and decline in genetic variation. live conservation in a geographically limited area is especially vulnerable to events such as political unrest, natural disasters and emerging diseases, as well as the decline in genetic variation due to suboptimal breeding practices or too small population sizes. while important tools for inbreeding control, such as optimum contribution selection (ocs) (grundy et al, 2000; woolliams et al, 2015) and biosecurity measures, mitigate genetic loss and the risk of disease, they do not remove the threat completely. cryoconservation protects against disease outbreaks and provides the possibility to improve the genetic 60 white et al genetic resources (2025), 6 (11), 57–70 figure 1. the extinction vortex. the figure above is a simple illustration of the consequences related to the continued loss of genetic variation in an already small population. loss of variation has a range of driving factors, both related to genetics and inbreeding and human decisions such as breeding strategies, economy and social acceptance. status of populations that may have suffered large losses. genetic materials from previous generations can be reintroduced into the populations by using cryopreserved material such as semen (blackburn et al, 2023; fao, 2023). implementing cryoconservation combined with live conservation can therefore increase the population carrying capacity, and possibly the effective population size, without influencing the living populations (eynard et al, 2018; blackburn et al, 2023; fao, 2023). further, evolving genomic methods and sampling techniques offer tremendous potential for effectively managing the nordic native breed populations. regarding costs, schematics demonstrating the different components needed for establishing a genebank are listed in figure 2. there is a lack of research evaluating the cost-effectiveness of cryoconservation compared with live conservation. some evidence suggests that genebanks can be cost-effective: for example, silversides et al (2012) determined that it could be approximately 90% cheaper to conserve chicken genetic resources by storing cryopreserved germplasm as opposed to living populations. however, it is important to consider that genetic resources (2025), 6 (11), 57–70 future opportunities for nordic angr conservation 61 this is an extreme example, only considering genebanking alone. importantly, it is possible to conserve populations cost-effectively in live populations by developing niche products or creating added value (e.g. animalassisted therapy, using animals in restoration and management of traditional ecosystems or the production of premium-quality food products). history, current conservation efforts and risk status globally, conservation efforts are still under development or insufficient to ensure the future security of angr (fao, 2015; erfp, 2021). meticulous recordkeeping and filling the observed gaps in knowledge, such as important genetic parameters and population development, are essential for the success of long-term conservation goals. however, the status of long-term conservation does not only rely on the conservation status of live populations but also on the status of cryoconservation. the nordic countries have 167 local and regional transboundary farm animal breeds and subspecies. the majority (71%) of the nordic breeds are endangered or critically endangered, while 5% are considered vulnerable. only 14% are not at risk, while the remaining 10% have an undetermined risk status (figure 3; white et al (2024c)). data for the nordic breeds collected from the domestic animal diversity information system (dadis) strongly indicate that the numbers of samples stored in national genebanks are not yet sufficient, or representative of the living populations’ genetic diversity. fao has recommended to cryoconserve breeds that are categorized as endangered or critically endangered; however, only 60 of the 119 nordic breeds with this risk status have cryoconserved materials, and the remaining 59 breeds have no additional safety measures in place (fao, 2024; white et al, 2024c). the nordic countries have so far mainly focused on maintaining living populations through live conservation measures. however, all nordic countries also have some cryoconserved material. the first nordic cryoconservation efforts included the establishment of semen banks for native cattle breeds in sweden, iceland, denmark and norway in 1967, 1969, 1971 and 1977, respectively (maijala, 2011). in finland, cryopreservation of semen and embryos from the finncattle breeds began during the 1980s (pehu et al, 2018). the vast majority of the collected material is semen from cattle and sheep breeds (table 1). the remaining species are highly under-represented in cryoconservation, and no material is stored from any avian species, pigs, cats or bees (table 1). cell types that include female genetic material including embryos, oocytes, dna and somatic cells are also lacking in nordic cryoconservation according to dad-is record of genebanking for conservation. number of donors presently used in cryoconservation in the nordic countries in addition to having at least some cryoconserved material, the breeds should ideally have sufficient samples preserved to be able to recreate healthy and sustainable future generations (white et al, 2024c). recent species-specific data from the centre for genetic resources, the netherlands (cgn) of wageningen university & research (wur) evaluates the sufficiency of stored samples according to different criteria. these include the number of donors and samples necessary for future reconstruction of a breed (van der sluis and schoon, 2024). the general consensus across all species is that a minimum of 50 male donor animals is required to reduce the risk of inbreeding depression. having 37 donors allow for some selection, but the estimated inbreeding rate per generation will be higher (0.67%), while 25 donor animals yield no possibility for selection because the inbreeding rate per generation will exceed 1%. it is also essential to consider the relationship between the donors for these evaluations (van der sluis and schoon, 2024). data from dad-is shows that 19 (24.36%) of the cryoconserved nordic breeds have sufficient semen doses from enough male donors for population sustainability (> 50 donors) (figure 4). furthermore, 7 (9%) of the breeds have cryopreserved embryos. so far, there have been no attempts to preserve somatic cells for genebanking purposes in any country (table 1, white et al (2024c)). notably, the largest proportion of material cryopreserved is from norwegian cattle and sheep breeds (table 1), which also constitute almost half of the breeds with sufficient male donors (figure 4; fao (2024); white et al (2024c)). further, the current collections lack female reproductive material and, in most cases, the genetic characterization of the donor living animals. the scarcity of stored material, combined with the number of breeds that are endangered and critically endangered, highlights the urgent need to improve the efforts to protect and conserve their genetic resources. in addition, the sanitary status of donors collected at artificial insemination (ai) stations is diagnosed according to eu regulations. however, information may be lacking for other genebank collections outside certified collection units (especially for female germplasm). furthermore, in some cases, current genebanks lack a link to animal pedigree information, genotypes and phenotypes. utilization of the samples without sufficient information about the sanitary status, pedigree or genotype risks introducing infectious diseases, unintended intensification of breeding, and unwanted genetic defects into the living population later (fao, 2023). importantly, utilization of breeding material from genebanks that are not eu-certified also conflicts with eu animal health regulations. challenges to conservation initiatives in the nordics challenges in collecting and obtaining samples one of the main reasons why so few species are represented in cryoconservation is that a lot of the stored 62 white et al genetic resources (2025), 6 (11), 57–70 figure 2. economic considerations for establishment of a cryoconservation genebank for animal genetic resources. genebank costs based on the dutch gene bank for farm animals. genetic resources (2025), 6 (11), 57–70 future opportunities for nordic angr conservation 63 table 1. distribution of cell types of cryopreserved samples between species in the nordic countries (fao, 2024). *, most of the collected dna and somatic cells until now have been stored to be used in scientific contexts, which is why they have likely not been registered as material that is stored for conservation purposes in dad-is. species number of breeds semen dna* embryos somatic cells * oocytes honeybee 0/1 0 0 0 0 0 cats 0/5 0 0 0 0 0 cattle 23/28 972,907 190 119 0 0 chicken 0/21 0 0 0 0 0 deer 1/1 200 100 1 550 0 dogs 12/29 1,634 0 0 0 0 ducks 0/6 0 0 0 0 0 goats 8/9 21,499 30 4 0 0 geese 0/6 0 0 0 0 0 horse 9/14 1,558 34 0 0 0 pig 2/5 140 0 0 0 0 pigeon 0/3 0 0 0 0 0 rabbit 0/5 0 0 0 0 0 sheep 26/34 75,291 215 108 0 0 figure 3. risk status per breed for the different species of nordic angr. the risk status is based on the classification system from fao (fao, 2013). the figure is adapted from white et al (2024c). samples come from species with commercial breeds and are collected in collaboration with commercial breeding partners (e.g. cattle). sheep, goats, horses and dogs are also species where ai is available, but not as widely used. for some groups of animals, collection of ejaculated semen is also not possible, either because the animals need to be trained beforehand, they are not tame enough, or there are no facilities close by. for avian species, cryopreservation protocols are not yet optimized, which limits the possibility for cryoconservation. consequently, for example, some poultry are conserved in live genebanks (ex situ, in vivo) in the nordics (brekke, 2017; sæther et al, 2018; berres et al, 2020). an important challenge is the cryopreservation of female tissue for genebanking. at present, female tissue (oocytes and embryos) is highly underrepresented in the current collections for cryoconservation in the nordic countries (table 1). collection of female reproductive tissues is more expensive than in males (table 2). however, the cost-effectiveness can be enhanced in species like cattle through hormonal stimulation to induce multiple ovulations. in contrast, for other species, such as horses, even hormonal intervention does not significantly improve efficiency, and collection of even two embryos at a time is rare. further, the successful use of ovarian tissue as an oocyte reservoir is very limited in domestic animals due to compromised developmental competence of young follicles. overall, many of the collection techniques require well-established laboratory facilities and staff with sufficient expertise. thus, establishing sufficient genebanking activities including both male and female tissues within each country can be difficult without enough funding and support. consequently, there are no collected oocytes at this time, and the cryoconservation of embryos is limited. challenges to the establishment and management of long-term genebanks the most important factors to consider relating to the establishment and management of long-term cryoconservation (genebanking) are long-term storage costs, number of facilities and management tools (e.g. database connecting samples to genotypes and phenotypes), sanitary issues, technical expertise and collection infrastructure, and prerequisites for collaboration (for cross-border cooperation). cryoconservation is a longterm commitment, and therefore, facility contracts and funding for long-term storage are essential for the management of genebanks (blackburn et al, 2023). in the nordic countries, nationally cryopreserved genetic material is often stored only in a single location for a given species or breed. such a form of storage is vulnerable, because it introduces the potential risk of loss of collected material due to unforeseen events such as system malfunction (fao, 2023). cryopreserved material is highly valuable and must be safeguarded against poten64 white et al genetic resources (2025), 6 (11), 57–70 figure 4. classification of cryopreserved semen donors from nordic angr breeds based on the criteria developed by the centre for genetic resources, the netherlands, including the total number of breeds represented in the samples. total number of breeds with samples: 78 (white et al, 2024c). table 2. comparison of cell types used in cryoconservation, and advantages and limitations of chosen sample types sample type advantages limitations semen cost-effective (large volumes per individual) optimized protocols (high survival rate) can be very useful in managing small population (large quantities can be used for sex-sorted semen well-known sanitary status can be used to re-create breeds by back-crossing expensive collection infrastructure (fewer males used for collection) collections done by small numbers of centralized systems of commercial companies (due to legislation) variation in the semen’s freezing ability between individuals. epididymal sperm can be collected from dead and living donors supports conservation of genetic diversity (i.e., can be collected from more males) can be used to re-create breeds by back-crossing cheap infrastructure lower quantity per dose sanitary status must be defined oocytes easy to collect can be collected from both live and dead donors (ovum pick-up, sterilization) collections can be repeated cheap infrastructure to collect samples from dead individuals decision upon mating can be postponed to the future developmental competence after cryopreservation is very low collection infrastructure from living donors is expensive number of oocyte per donor is variable and unpredictable early embryos good survival rates (depending on origin – in animal or in lab) expensive unpredictable mating decisions need to be made prior to cryopreservation somatic cells (sc) (e.g. skin tissue) can be collected from all individuals 100% of dna cheap infrastructure for sampling continuously developing techniques for using sc technologies for using sc can be expensive and not always successful ovarian and testicular tissues contain thousands of gametes at an early stage that can be developed in vitro. genetic value of prepubertal individuals can also be preserved collections can be either after sterilization or post-mortem genetic resources (2025), 6 (11), 57–70 future opportunities for nordic angr conservation 65 tial unexpected risks; therefore, it is recommended to set up and maintain at least two separate storage facilities in different geographical locations (fao, 2023), similar to recommended safety duplication strategies in plant genebanking. in cases where multi-country collaboration for cryoconservation is contemplated, there are other important factors to consider, such as legislation for cross-border transportation, sanitary requirements (i.e. biosecurity), and the vulnerability to alterations in national laws or disease outbreaks that could influence the retrieval of samples (blackburn et al, 2023). in europe, eu regulation 807/2014 (ec, 2014) introduced transitional provisions allowing the transport of samples across borders according to certain sanitary regulations. legislation and requirements for sanitary status can vary according to national legislation and regulations. in cases of collaboration, it has been recommended to implement the strictest regulations for sanitary issues (blackburn et al, 2023). while multi-country collaboration introduces some concerns, it also provides a range of benefits, such as reduced costs and improved expertise. the initial costs of establishing a facility for cryoconservation are often high, which is why many countries only have one national facility where collections are stored. collaboration between countries could mitigate the risk of malfunction while reducing the cost. obtaining the technical expertise necessary for processes such as collecting, freezing and thawing of samples is a key factor for the success of long-term cryoconservation and the possible use of the stored samples in the future. however, knowing the best practices for the collection and cryopreservation steps for each of the different species requires broad expertise. cross-border collaboration could promote transparency in knowledge and expertise between the countries involved, positively contributing to conservation. successful management of both longand shortterm cryoconservation facilities requires the necessary and relevant knowledge for the collected genetic diversity. blackburn et al (2023) argues that establishing a database with this information is not only important for contemporary management, but also for decisionmaking processes and the evaluation of the genebanking success. continuous evaluation of genebanks is essential for the future status of overall conservation, because it will highlight their possible strengths and weaknesses. future perspectives on angr conservation in the nordic countries collaboration across borders collaboration and shared knowledge of strategies and infrastructure offer future collective opportunities for the growth and security of nordic native angr. for plants, the global seed vault in svalbarddemonstrates that collaboration can be highly successful. further strengthening the collaboration for conservation of angr across country borders could be favourable in several ways. for example, exchanging duplicate material on a regional level could reduce the costs of cryoconservation while mitigating the risk posed by system malfunction. in europe, an important stakeholder for regional cooperation in cryoconservation for animals is the european gene bank network for animal genetic resources (eugena). this network serves as a coordinated digital platform that facilitates the exchange, management and conservation of genetic data from various farm animal species in europe. one of the main goals of eugena is to improve cryoconservation in europe by promoting transparency of both information and technology among the network’s member countries. in this manner, eugena promotes genebanking efforts both at a national level as well as at a regional level by supporting countries in fulfilling their individual roles and objectives, and by facilitating cooperation between their european member countries. all european countries, including the nordics can join the network if their genebanks meet certain criteria and are certified3. this network offers immense possibilities for cross-border collaboration. further, in the united kingdom, a proactive group to recognize the need for comprehensive genebanking of farm animals is the uk national livestock biobank (uknlb, https://www.livestockbiobank.com/), which could act as a valuable networking partner providing peer support for the nordic countries in their initiative and exploitation of the latest cryo-methodologies. comparable to the nordic region, up to 80% of the united kingdom’s native farm animal breeds are at risk of extinction, with the loss of such breeds highlighted as a significant threat to national food security (defra, 2021b,a). the uknlb is a farm animal biobank based on gamete preservation (sperm, egg and embryo), alongside collection and cryopreservation of skin samples for fibroblast cell line generation. the collection of skin samples alongside gametes is unique within the uk farming cryopreservation sector, and has numerous applications and opportunities, including preservation of the whole genetic profile of farm species and the regenerative genetic capture of female lines (see supplemental material 2 for more info). biobanking permits the indefinite storage of genetics beyond the lifespan of the original animal, ensuring the availability of resources for future applications (erfp, 2023). implementing state-of-the-art technologies for future possibilities of conservation the rapid progress in biotechnology paves the way to a future-oriented approach for genebanks. anticipated future advancements, including precision gene editing, synthetic biology and advanced reproductive 3 memorandum of understanding (mou) related to the european genebank network for animal genetic resources (eugena), accessible from https://www.eugena-erfp.net/en/about/how-to-become-amember 66 white et al genetic resources (2025), 6 (11), 57–70 technologies (blackburn et al, 2023), offer promising avenues (supplemental material 2). for example, currently explored methods such as dry-preservation of germinal vesicles offer a simpler and less expensive way for long-term conservation because the preserved material can be stored at room temperature (graves-herring et al, 2013; lee et al, 2019; lee and comizzoli, 2024). further, advances in sequencing and genomics allow improved characterization of traits as well as better representation of the genetic diversity within genebanks and effective evaluation of populations and inbreeding control. integrating these technologies into genebank models will enhance the ability to revive and restore endangered populations, ensuring the long-term viability of angr. the nordfrost network has introduced state-of-theart technologies that have previously been underutilized in traditional genebanking approaches to the nordic stakeholders, such as epididymal sperm collection and the cryopreservation of skin tissue (table 2; white et al (2024a)). utilizing epididymal sperm in other disciplines is not a new phenomenon, yet it has not been widely used as a cryoconservation method for angr. the advantage of using epididymal sperm is that it can be collected from both deceased and living donors as well as during castration, which means that sperm can be collected from more individuals. this supports the conservation of genetic diversity. further, like traditionally collected semen samples, it can be used to re-create breeds by back-crossing. for this method, it is important to consider that only one collection event is possible, and subsequently, the number of doses is lower than that of ejaculated semen. nevertheless, this method is highly valuable for species where routine semen collection poses challenges – e.g. for animals that are difficult to train for semen collection, or in situations where the necessary facilities are unavailable, or where animals die due to unforeseen events and the genetic diversity would otherwise be lost. this method has been successfully implemented in countries like finland, where epididymal sperm has been frozen from a variety of species, including cattle, horses, sheep, reindeer and roosters. the cryopreservation of skin tissue also holds significant potential, extending the range of future possibilities for long-term conservation and could act as an additional security measure alongside the traditional sampling of reproductive tissues. utilizing skin tissue offers an effective and less complicated way of capturing the genetic variation of individuals. for example, where gametes only capture half of the genetic information of the donor animal, skin samples and fibroblast cell lines effectively capture the entire dna (gorji et al, 2021). furthermore, while effective infrastructures for cryopreservation and thawing of sperm have been established for most breeds, equally effective procedures for female reproductive tissue have yet to be established (table 2; li et al (2009)). consequently, storing skin tissue samples in a genebank overcomes challenges that follow cryopreservation and thawing of egg cells (li et al, 2009). furthermore, there is also significant progress being made in the development of induced pluripotent stem cell technology, which would facilitate the development of sperm and egg cells from reprogrammed fibroblast cell lines (bhartiya et al, 2014; horer et al, 2023). the prospect of this technology is substantial as it would allow for the creation of individuals with a fresh genetic set, from a readily taken and cryopreserved skin sample. most development so far has been in lab-based mice, with pups already born from reprogrammed skin samples (mahabadi et al, 2018; moradi et al, 2019). more detailed descriptions of the increased recognition and utilization of cryopreserved skin tissue in conservation can be explored in supplemental material 2. final remarks to date, the nordic countries have maintained high sanitary standards for animal health. however, maintaining these standards is expected to become increasingly challenging. recent outbreaks of blue tongue, hpai and african swine fever spreading to the nordics highlight the importance of additional safeguarding measures for the conservation of animal genetic resources. cryoconservation is a possibility to add a second layer of conservation measures and safeguard animal genetic resources, as it protects against disease outbreaks and provides the possibility to improve the genetic status of populations that may have suffered large losses due, for example, to disease epidemics. one of the main goals for conservation in the nordic countries is to maintain healthy and stable populations, which implies maintenance of genetic variation within the populations. implementing both live conservation and cryoconservation allows for continuous adaptation and promotion of cultural heritage, while maintaining and/or improving genetic variation, and protecting against disease outbreaks that are difficult to contain. therefore, the combination of both live conservation and cryoconservation enhances the safeguarding of both current and future angr (fao, 2007a; blackburn et al, 2023). the nordic countries share the responsibility to secure their angr to the best of their knowledge (un, 1992). despite the increased efforts for utilizing cryoconservation as an additional safeguard, most of the nordic breeds are not secured and, in some instances, important information regarding both the samples and donors is missing. the nordfrost project highlighted the benefits of increased nordic collaboration on cryoconservation of angr. nordic cooperation in the field of live animal conservation and cryoconservation benefits from the growing recognition of the value of genetic diversity, the identification of shared threats and opportunities, and the development of new costeffective genebanking methods. stronger nordic collaboration on cryoconservation of angr could improve both the risk status and genetic genetic resources (2025), 6 (11), 57–70 future opportunities for nordic angr conservation 67 diversity of the nordic angr. collaborative conservation could play an important role in strengthening the future of angr by allowing shared responsibility, crossborder cooperation and knowledge surrounding both infrastructure and data collection. supplemental data supplemental material 1. national strategies of the nordic countries supplemental material 2. fibroblast cell lines and advancing technologies acknowledgements we sincerely acknowledge the contributions of: the nordic national coordinators & the members of nordgen’s animal genetic resource council: denmark: vivi hunnicke nielsen, aarhus university & clara nyegaard signori, ministry of food, agriculture and fisheries of denmark. the faroe islands: jens ivan ı́ gerkinum, the agricultural agency. finland: juha kantanen, natural resources institute finland & johanna rautiainen, lammasmaailma. iceland: birna krist́ın baldursdóttir, the agricultural university of iceland & thorvaldur kristjánsson, the agricultural advisory center (rml). norway: nina svartedal, the norwegian genetic resource centre. sweden: anna m. johansson, swedish university of agricultural sciences & karin olsson, the swedish board of agriculture. we gratefully acknowledge the financial support from the nordic joint committee for agricultural and food research (nkj). further, we would like to thank everyone who has participated in the nordfrost activities. lastly, we would like to thank annemieke rattnik and harvey blackburn for offering their knowledge about cryoconservation practices outside of the nordics. author contributions elfw – concept development, writing: original draft preparation, review and editing, data collection and analysis, visualization; mk – concept development, writing: review and editing, data curating, visualization; jp – concept development, writing: original draft preparation, 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(2005). sustainable management of animal genetic resources. nordic gene bank farm animals. woolliams, j. a., berg, p., dagnachew, b. s., and meuwissen, t. h. e. (2015). genetic contributions and their optimization. journal of animal breeding and genetics 132(2), 89–99. wwf (2022). living planet report 2022 building a nature-positive society, ed. almonds, r. e. a., grooten, m., bignoli, d. j., and petersen, t. (gland, switzerland: wwf). yoo, e. h., chen, d., diao, c., and russell, c. (2016). the effects of weather and environmental factors on west nile virus mosquito abundance in greater toronto area. ams journals . doi: https://doi.org/ 10.1175/ei-d-15-0003.1 https://www.sva.se/media/1dmhvulw/240725-l\%c3\%a4gesbild-salmonella-enteritidis.pdf https://www.sva.se/media/1dmhvulw/240725-l\%c3\%a4gesbild-salmonella-enteritidis.pdf https://www.cbd.int/convention/text/default.shtml https://www.vetinst.no/dyr/sau/status-blatunge-i-norge https://www.vetinst.no/dyr/sau/status-blatunge-i-norge https://doi.org/10.1603/me10117 https://doi.org/10.53780/lfnw7075 https://doi.org/10.53780/lfnw7075 https://doi.org/10.1175/ei-d-15-0003.1 https://doi.org/10.1175/ei-d-15-0003.1 background the global decline in biodiversity also includes our farm animal breeds the nordic commitment to conservation of animal genetic resources additional approaches are needed aims and objectives animal genetic resources in the nordic countries and conservation strategies external threats to genetic diversity in animal populations from nordic countries internal threats to genetic diversity in animal populations from nordic countries advantages and limitations of live animal conservation and cryoconservation history, current conservation efforts and risk status number of donors presently used in cryoconservation in the nordic countries challenges to conservation initiatives in the nordics challenges in collecting and obtaining samples challenges to the establishment and management of long-term genebanks future perspectives on angr conservation in the nordic countries collaboration across borders implementing state-of-the-art technologies for future possibilities of conservation final remarks supplemental data acknowledgements author contributions conflict of interest statement correction doi: 10.46265/genresj.jzqn2033 https://www.genresj.org issn: 2708-3764 received: 05.11.2025 | accepted: 05.11.2025 | published online: 06.11.2025 cristophe jenny (correct: christophe jenny) françoise nuissier (correct: franciane nuissier) error in table 1 in the published article, there was an error in table 1: [perennial plants in guyana (ppg)]: instead of “guyana”, it should have been “french guiana”. [coffea spp., theobroma spp., hevea spp., dalbergia spp.]: instead of dalbergia, it should have been aniba rosodora. the authors apologize for these errors and state that they do not affect the scientific conclusions of the article in any way. the pdf and html versions of the original article have been updated. published online: 06 november 2025 correction: organization of plant biological resource centers for research in france: history, evolution and current status valérie bergheaud *,a, jean-marc audergonb, arnaud bellecc, anne delaunay1, jérôme duminild, stéphane dussertd, florence esnaulte, emmanuel geoffriauf, brigitte gouesnardg, christophe jennyg,h, alain labeli, philippe lashermesd, najate maghnaouig,h, cécile marchalb, franciane nuissierj, nilda paulo-de-la-réberdiereg,h, aurélia prietk, valérie rieucau7,8, paule térèsg,h, anne-françoise adam-blondonl auniversité paris-saclay, inrae, bap, france binrae, paca center, ugafl, france cinrae, cnrgv french plant genomic resource center, france ddiade, ird, université montpellier, cirad, france einrae, institut agro, université de rennes, igepp, france finstitut agro, université d’angers, inrae, irhs, sfr quasav, france gumr agap institut, université montpellier, cirad, inrae, institut agro, france hcirad, umr agap institut, cirad, montpellier, france idiagonal, inrae, france jinrae, ur astro, guadeloupe, france kinrae, ur4 (ur p3f), nouvelle-aquitaine-poitiers center, france luniversité paris-saclay, inrae, urgi, france *corresponding author: valérie bergheaud (valerie.bergheaud@inrae.fr) correction to: genetic resources https://doi.org/10.46265/genresj.aszo2413; published online 04 march 2025 incorrect affiliation in the published article, there was an error in the affiliations of nilda paulo-de-la-réberdiere. instead of “[h,g]”, it should have been “[g,h]”. incorrect author names in the published article, two author names were incorrectly written: https://doi.org/10.46265/genresj.jzqn2033 https://www.genresj.org https://doi.org/10.46265/genresj.aszo2413 original article genetic resources (2026), 6 (11), 99–114 doi: 10.46265/genresj.mdso5816 https://www.genresj.org issn: 2708-3764 phenotypic traits in natural perennial ryegrass populations and relations to climate conditions at sites of origins across europe silvia bachmann-pfabe *,a,b,‡, mareike kavka a,‡, anna roschanski a,c, klaus j dehmer a, jean-paul sampouxd and evelin willner a a satellite collections north, oil and fodder crop collections, genebank department, leibniz institute of plant genetics and crop plant research (ipk), inselstraße 9, 23999 malchow (poel), germany b present address: faculty of agriculture and food sciences, plant nutrition and soil science, university of applied sciences neubrandenburg, brodaer strasse 2, 17033 neubrandenburg, germany c present address: institute of botany and landscape ecology, university of greifswald, soldmannstraße 15, 17487 greifswald, germany d national research institute for agriculture, food and the environment (inrae), centre nouvelle-aquitaine-poitiers, rd150, 8660, lusignan, france abstract: perennial ryegrass (lolium perenne l.) is one of the most important forage grass species in temperate climates. however, natural perennial ryegrass populations have been exploited to only a limited extent by breeders. therefore, 41 ecotypic lolium perenne populations collected across europe were studied for their agronomic performance in a 3-year common garden experiment located in north-eastern germany (poel island, mecklenburg western pomerania). agronomic performances were evaluated on 30 plants per population for 11 traits related to forage value and environmental adaptation. population means for studied traits were correlated to the values of climate variables at their collection sites. populations clearly differed in their phenotypic performance, and eight populations originating from belgium, france and germany outperformed the other populations by showing the lowest winter damage, strongest spring growth and regrowth capacity after cuts and low disease susceptibility. specifically, in the first experimental year, trait performances, in particular winter damage, spring growth and heading date, were related to the local climate at the site of origin of populations. acclimation to the climate conditions at the experimental site might explain why these correlations were less pronounced in the second and third experimental years. the characterized populations might now be considered to improve specific traits in breeding. keywords: ecotypes, phenotypic diversity, climate norms, lolium perenne citation: bachmann-pfabe, s., kavka, m., roschanski, a., dehmer, k. j., sampoux, j.-p., willner, e. (2026). phenotypic traits in natural perennial ryegrass populations and relations to climate conditions at sites of origins across europe. genetic resources 6 (11), 99–114. doi: 10.46265/genresj.mdso5816. © copyright 2025 the authors. this is an open access article distributed under the terms of the creative commons attribution license (cc by 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. introduction perennial ryegrass (lolium perenne l.) is one of the most important grass species for forage and turf uses in temperate climates worldwide and has undergone intensive breeding efforts (humphreys et al, 2010; ∗corresponding author: silvia bachmann-pfabe (pfabe@hs-nb.de) sampoux et al, 2011). natural perennial ryegrass populations are an important source of diversity for forage and turf breeding goals, but their wide genomic variability has been only partially exploited by breeding activities (blanco-pastor et al, 2019). the adaptation to local environments results in the differentiation of local ecotypes of perennial ryegrass with potentially valuable variability to improve growth seasonality, disease resistance or winter hardiness (humphreys, received: 14.02.2025 accepted: 13.05.2025 published online: 19.06.2025 ‡both authors contributed equally to the manuscript https://www.genresj.org https://www.doi.org/10.46265/genresj.mdso5816 https://www.genresj.org https://www.doi.org/10.46265/genresj.mdso5816 mailto:pfabe@hs-nb.de 100 bachmann-pfabe et al genetic resources (2026), 6 (11), 99–114 1989; charmet and balfourier, 1991; oliveira-prendes et al, 1997; hulke et al, 2007, 2008; kemesyte et al, 2010; willner et al, 2010; bachmann-pfabe et al, 2018). breeding increased the cumulative dry matter yield of forage-type perennial ryegrass cultivars between 0.3 and 0.9% annually during the past 40 to 50 years (wilkins and humphrey, 2003; mcdonagh et al, 2016). more specifically, summer and autumn yields increased, while spring dry matter yield remained almost unchanged (sampoux et al, 2011). however, early spring growth is an important breeding target in order to extend the growing season of perennial ryegrass, to provide enough fodder in temperate grasslands early in the year (goslee et al, 2017) and to adapt the growing period to a longer heat and drought summer season caused by ongoing climate change. for forage and grazing usage, plant breeders developed forage-type ryegrass cultivars with different maturity groups, from early heading types with high early-season yields to late heading types. the latter are best adapted to a long grazing season and permanent pasture use due to their increased summer and autumn growth (humphreys et al, 2010; laidlaw, 2005). disease resistance, especially to crown rust, improved in registered ryegrass cultivars for forage as well as for turf usage (sampoux et al, 2011, 2013). other current breeding goals are increased winter hardiness and persistence (kemesyte et al, 2010; goslee et al, 2017), reduced aftermath heading (hurley et al, 2007; mcgrath et al, 2010), tolerance to heat and drought stresses as well as increased nutrient and water uptake efficiency (crush et al, 2010; bothe et al, 2018). because intensive breeding in perennial ryegrass is a rather recent effort (20th century), useful traits of wild populations can still be easily integrated into breeding programmes (blackmore et al, 2016; blanco-pastor et al, 2019, 2021). documenting the relationship between the phenotype, including agronomic performance, of natural populations and the climatic conditions at their sites of origin, contributes to understanding climate adaptation in the natural diversity of perennial ryegrass. this is essential for future breeding progress, as it helps to identify desirable breeding goals and to point out suitable genetic resources to choose in genebanks or to collect in situ. the aim of this study was to combine phenotypic and environmental information in order to explore the trait variation between natural l. perenne populations along a geographical and climatic range. therefore, we cultivated 41 different natural l. perenne populations represented by 30 individuals per population in a spaced plant nursery and evaluated their agronomic performances over three years. material and methods collection sites and site-specific characteristics in 2015, 41 natural populations of perennial ryegrass (lolium perenne l.) were sampled across europe. in each population, a small number of living tillers were collected from 30–35 individual plants. the collection sites spanned areas of 100 to 290,000m2, with most sites larger than 1,000m2 (supplemental table 1). the 41 populations were collected in belgium (bel), germany (deu), estonia (est), france (fra), great britain (gbr), lithuania (ltu), the netherlands (nld), poland (pol), portugal (prt) and serbia (srb) (figure 1). the majority of the collection sites had an elevation ranging from 0 to 800m above sea level (masl). populations from sites above 800masl originated from germany (deu1, 1,040m), serbia (srb1, 1,076m; srb2, 1,159m) and france (fra5, 1,200m). the altitude of the sampling sites as well as their climatic conditions (blanco-pastor et al, 2021) are displayed in table 1. the annual mean temperature (bio1) of the collection sites ranged from 5.3◦c (population gbr8) to 14.8◦c (population fra3). the annual precipitation (bio12) averaged 902mm over collection sites and ranged from less than 580mm for populations deu7, deu9, deu10, est1 and fra3, to more than 1,300mm for populations deu1, fra1, fra5 and gbr6. the average daily maximum temperature of the warmest 14-day period of the year (bio5) was the highest for populations fra3, prt1, prt2 and srb3 (more than 28◦c). the average daily minimum temperature of the coldest 14-day period of the year (bio6) was lowest for ltu1, ltu2, srb1 and srb2 (less than -5◦c). furthermore, populations est1, fra3 and prt2 originated from the driest sites (precipitation <85mm in the driest quarter (bio17), table 1). as reported in the biological status of accessions (fao/bioversity, 2015) and the accession source (collsrc), the populations were predominantly collected from wild and semi-natural habitats (sampstat = 100, 110, 120; see supplemental table 1). these comprised natural grasslands, semi-natural pastures where no sowing or ploughing took place during the previous 15 to 70 years or roadsides. after collection, living tillers of the populations were transferred to the german federal ex situ genebank of the leibniz institute of plant genetics and crop plant research (ipk), satellite collections north at malchow/poel, germany for conservation. the passport data of the accessions are available via the ipk genebank information system gbis (https://gbis.ip k-gatersleben.de) and are also provided in supplemental table 1. individual plants within populations were pre-cultured in seedling trays using a turf-based planting substrate (einheitserde uetersen, pikiererde, uetersen, germany) and then transferred to the field. the ploidy level on all plants grown afterwards in the experimental garden was confirmed as diploid (2x = 14) using flow cytometry at inrae (ur p3f). genetic resources (2026), 6 (11), 99–114 trait-climate relations in perennial ryegrass germplasm 101 table 1. country of origin, leibniz institute of plant genetics and crop plant research (ipk) accession number, elevation and bioclimatic norms (1989–2010) at the collection sites of 41 l. perenne populations sampled across europe. bioclimatic norms over the period 1989–2010 were computed as per blanco-pastor et al (2021) (see also supplemental table 2). bio1, annual mean temperature; bio5, average daily maximum temperature over the warmest 14-day period of the year; bio6, average daily minimum temperature over the coldest 14-day period of the year; bio12, cumulated annual precipitations; bio17, cumulated precipitations of the driest quarter of the year. country of origin accession no. altitude (masl) bio1 (◦c) bio5 (◦c) bio6 (◦c) bio12 (mm) bio17 (mm) bel1 gr 13048 216 9.9 24.5 0.04 871 178 bel2 gr 13047 19 10.9 24.3 0.94 838 144 deu1 gr 13042 1,040 5.9 21.8 -4.50 1,584 333 deu2 gr 13041 748 8.0 23.1 -4.18 1,050 164 deu3 gr 13043 338 9.3 24.5 -1.30 860 169 deu4 gr 13030 33 10.1 25.4 -0.63 696 135 deu5 gr 13031 0 9.7 23.7 -0.75 823 145 deu6 gr 13034 0 9.0 22.5 -0.58 817 124 deu7 gr 13025 41 9.6 25.6 -1.90 576 104 deu8 gr 13023 0 9.3 23.4 -0.97 618 108 deu9 gr 13026 30 9.7 25.5 -1.87 532 96 deu10 gr 13027 8 9.7 25.7 -2.35 511 95 est1 gr 13052 3.5 7.2 22.1 -4.00 565 83.7 fra1 gr 13068 13 13.8 27.8 2.65 1,311 198 fra2 gr 13060 525 12.2 27.7 -0.10 1,097 190 fra3 gr 13072 1 14.8 30.2 3.05 573 51 fra4 gr 13062 223 12.0 26.8 2.38 979 182 fra5 gr 13067 1,200 6.6 21.1 -2.36 1,467 300 fra6 gr 13065 911 9.5 27.2 -3.65 1,160 167 fra7 gr 13063 800 8.9 26.1 -2.93 1,136 201 fra8 gr 13071 50 12.0 26.3 2.20 895 128 fra9 gr 13073 250 11.4 27.0 1.50 898 174 fra10 gr 13061 215 11.0 27.4 -0.41 837 165 fra11 gr 13070 297 10.0 26.3 -1.17 982 198 gbr1 gr 13078 142 9.6 22.5 0.51 663 118 gbr2 gr 13079 84 10.0 22.8 0.74 684 128 gbr3 gr 13080 134 10.7 22.4 1.38 774 148 gbr4 gr 13081 134 10.3 21.3 1.85 802 145 gbr5 gr 13082 161 10.0 19.1 2.07 968 171 gbr6 gr 13083 229 9.9 19.7 1.32 1456 229 gbr7 gr 13084 402 6.3 16.9 -1.60 1014 190 gbr8 gr 13085 402 5.3 15.4 -2.44 1047 195 ltu1 gr 13045 160 7.2 23.8 -5.59 624 105 ltu2 gr 13044 24 7.6 24.0 -5.11 637 107 nld1 gr 13033 0 9.8 22.4 -0.10 843 136 pol1 gr 13036 2 8.9 23.4 -1.94 656 119 prt1 gr 13075 675 11.3 28.7 -0.33 1129 92 prt2 gr 13076 875 11.6 29.3 -0.16 1030 73 srb1 gr 13050 1,076 6.2 22.9 -7.74 908 149 srb2 gr 13049 1,159 6.1 22.5 -8.12 836 135 srb3 gr 13051 149 11.7 29.8 -2.92 614 112 102 bachmann-pfabe et al genetic resources (2026), 6 (11), 99–114 figure 1. collection sites of the 41 lolium perenne natural populations collected across europe in 2015. blue sites are those with an annual cumulated precipitation norm (bio12) above 1,300mm, red sites are those with an annual mean temperature norm (bio1) above 12◦c and white sites are those with average minimum mean daily temperature norm below -5◦c during the coldest 14 days slice in the year (bio6). background map colours display variation of isothermality, i.e. annual mean of temperature diurnal range (bio2)/annual range of daily mean temperature (bio7). climate norms were computed for the period 1989–2010 (supplemental table 2). experimental design and traits evaluated the perennial ryegrass populations, originating from various ecogeographical conditions across europe, were cultivated in a field trial at the satellite collections north station in malchow/poel in northern germany (longitude 11◦28’26”e, latitude 53◦59’40”n, 10masl). the site is characterized by a mean annual precipitation of 544mm and a mean annual air temperature of 10.0◦c over the past 10 years. the predominant soil type is a sandy loam. the trial was set on a 38m × 18m field plot with homogeneous soil conditions. plants were transplanted to the field and arranged in a completely randomized design on 24 september 2015 (except for plants from populations collected in great britain, which were transplanted on 23 october 2015 and for populations deu8 and deu10, which were too weakly developed and were transplanted in march 2016). one clone of each originally sampled individual plant was randomly distributed in the field trial area without replication, except three plants per population, which were set up in two replicates (clones). a number of clonal replicates were planted in april 2016, as indicated in figure 2, because they were too weakly developed in september 2015. spacing between the individual plants was 45 × 50cm. an equalisation cut was made at the beginning of the growing season in 2016 after recording the winter damage score. three (2016) and four (2017) harvest cuts were completed, and fertilizers were applied after each cut, amounting to 240kg n/ha per year as calcium-ammonium-nitrate (table 2). a weather station located at the satellite collections north recorded the local weather conditions at the experimental site (supplemental table 3). the first winter after planting was characterized by mild temperatures in november and december (7.7 and 7.1◦c average daily temperatures, respectively). january was cold, with an average monthly temperature of -0.6◦c (long-term average (1991–2020): 1.5◦c in january in kirchdorf/poel (dwd, 2023a)) and 11 days with minimal temperatures below -5.0◦c, followed by a very dry spring with only 9.1mm rainfall in april 2016 (long-term average (1991–2020): 32.0mm in april in kirchdorf/poel (dwd, 2023b)). the experimental year 2017 was characterized by average air temperatures but high rainfall in june and july (supplemental table 2). the following traits were visually scored on a scale of 1 to 9 by an experienced genebank worker during the three experimental years of the trial: winter damage (wid), growing in spring (gsp), summer growth (gsu), genetic resources (2026), 6 (11), 99–114 trait-climate relations in perennial ryegrass germplasm 103 growing before winter (gwi) and general disease susceptibility (dis) (table 2). growth parameters were assessed from a visual impression of the aboveground biomass of the plant three times in each year: in april (gsp), in august (gsu) and before winter in october (gwi) expressed on a scale from 1 (low biomass) to 9 (strong biomass). wid describes the extent of the visually detectable damage after winter (proportion of dead tissues) on a scale from 1 (low damage) to 9 (strong damage). dis rates the severity of the disease occurrence on the basis of visually detectable leaf damage from 1 (low susceptibility) to 9 (high susceptibility). this score gives an overall visual impression of disease infection and does not differentiate between pathogens. heading date (hae) of plants was recorded in 2016 and 2017 as the number of days from the first of april to the day when the tips of five ears were visible. natural plant height was measured in spring (sph) before the first cut using a herbometer (herbometre electronique, framstore, france). plant height was measured again about 10 days after the first cut and is referred to as regrowth (reg 1), i.e. the capability to recover quickly and to develop new biomass after cutting. plant height was measured again before and after the second cut in july. the plant height 10 days after the second cut (reg 2) now gives information about the regrowth capability after the summer cut. height increment (hgtin) was calculated by subtracting reg1 from the height measured before the second cut (hsc) and serves as a measure of the strength of biomass growth between the cuts. finally, tussock size was measured several times throughout 2016 and 2017 using a ruler. tussock size was calculated as the mean of two perpendicular measurements of plant width; the highest value over recorded dates in 2016 and 2017 was used for data analysis (table 2). the number of living plants per population was counted 50 days after first planting date and at four later time points: (1) in spring 2016 (date of record of gsp in 2016) in order to determine survival after the first winter, (2) after heading and first cut (date of record of reg1 in 2016), (3) in spring 2017 (date of record of gsp in 2017) to determine survival after the second winter and (4) in spring 2018 (date of record of gsp in 2018) as an indicator of persistency. the number of living plants at the different record dates was expressed as a percentage of the number of plants counted 50 days after the first planting date to exclude weak plants that died right after the planting, plus later planted individuals. statistical analysis the software r (r core team, 2020) was used for statistical analyses. data processing only included plants surviving the second experimental year and populations with more than 15 surviving plants. this number is based on our considerations of finding a compromise between the minimum number of plants that must have survived to calculate a reliable population mean, and ensuring that the variance model does not become too unbalanced. at the same time, we wanted to avoid excluding too many populations from further analysis, as this would result in the loss of information. therefore, we set a threshold of at least 15 surviving plants per population, corresponding to at least 50% of the total plants/genotypes. to test for outperforming populations, a one-way analysis of variance (anova) with population as fixed effect was applied for each trait in each year. data across all populations were visually checked for normal distribution using q-q-plot and the lillie test (gross and ligges, 2015) and for variance homogeneity using the levene test of the package ‘car’ (fox and weisberg, 2011). population means were estimated with the ‘lsmean’ function and compared to the grand mean using the command ‘eff’ (lenth, 2016). populations were considered significantly different from the grand mean if p ≤ 0.05. if variance homogeneity was violated, the vcovhc function (heteroscedasticityconsistent covariance matrix estimation) of the package ‘sandwich’ (zeileis, 2004) was applied for post hoc comparison. for multivariate analysis, for each trait, population means were standardized (i.e. centred and reduced). euclidian distances between populations and between traits were calculated using the standardized population means and the ‘dist’ function of base r. clustering of the populations based on the recorded phenotypic traits was visualized in a heatmap generated with the ‘heatmap.2’ command of the ‘gplots’ package (warnes et al, 2020). values of populations for agronomic traits were correlated to values of bioclimatic variables and geocoordinates at their sites of origin and with each other (pearson’s correlation coefficient of the r package ‘hmisc’ (harrell, 2021)). correlations were considered significant if p ≤ 0.05. climate norms (period 1989–2010) of bioclimatic variables similar to the bioclim variables usually derived from the worldclim – global climate database, were used. they were already presented in blanco-pastor et al (2021) and are also described in supplemental table 2. fifteen bioclim-like variables were included in the correlation analysis. results survival rate in all populations, the plants survived the first winter without major losses as indicated by the high survival rates in spring 2016 (date of record of gsp 2016), which ranged from 96 to 100% (figure 2). of the additional, later planted individuals of deu5, grb1-4, grb6 and srb1-3, all but one each of srb1, srb2 and srb3 survived until the end of the trial. no survival rate is available for the first winter for populations deu8 and deu10, because the respective genotypes could not be planted before march 2016. the highest losses of plants were recorded after the first cut of 2016. the survival rate at the date of record (reg1 2016) clearly declined 104 bachmann-pfabe et al genetic resources (2026), 6 (11), 99–114 table 2. overview of main technical operations and trait records during the field experiment conducted from 2015 to 2018 to assess perennial ryegrass populations collected across europe. traita/managementb date of realization scale/comment2016 2017 2018 awinter damage (wid) 02 mar 02 mar 19 mar low (1) to strong (9) damage bequalisation cut 04 mar 09 mar bfertilizer application 04 mar 09 mar 80kg n/ha (can) agrowth in spring (gsp) 22 apr 20 apr 23 apr low (1) to strong (9) biomass aplant height spring (sph) 25 apr 26 apr mm aheading date (hae) april–may april–may days from 1st of april bfirst cut 06 jun 06 jun mm bfertilizer application 06 jun 09 jun 60kg n/ha aregrowth (reg1) 13 jun 19 jun mm aheight increment (hgtin) 18 jul 10 jul mm, hgtin = hsc-reg1 bheight + second cut (hsc) 18 jul 17 jul mm bfertilizer application 18 jul 17 jul 60kg/ha aregrowth (reg2) 25 jul 25 jul mm agrowth in summer (gsu) 23 aug 15 aug low (1) to strong (9) biomass adiseases (dis) 18 oct 04 sept low (1) to high (9) susceptibility bthird cut 25 aug 04 sept bfertilizer application 25 aug 04 sept 40kg/ha agrowth before winter (gwi) 17 oct 16 oct low (1) to strong (9) biomass bfourth cut 16 oct atussock size (tush) max 2016 max 2017 mm, mean of perpendicular width measurements for some populations to less than 70%, but these populations then stabilized, with no further dramatic losses in the following years. the populations est1 and ltu1 showed the highest losses at this timepoint, followed by fra1, fra2, prt2, fra3, deu1, fra7 and srb1. a high survival rate at the end of the 3-year experiment was recorded for populations fra8, deu2, deu5, deu7, deu9 and nld1, of which > 95 % of the cultivated plants survived (figure 2). phenotypic characterization of the populations the phenotypic diversity between populations was analyzed by a doubled cluster analysis on recorded traits and on populations. eleven phenotypic traits were measured in two or three consecutive years, resulting in 24 combinations of traits and year of record (table 2, supplemental tables 4 and 5). the cluster analysis on standardized population means grouped these 24 trait × year combinations into five major groups (figure 3). the most outstanding phenotypic cluster e contained the ordinal trait winter damage recorded in all three years. heading date and height increment between the first and second cut for both years grouped together in cluster d, while the two disease susceptibility ratings formed a separate cluster c. plant height in spring and growth in spring in 2016 were in cluster b together with growth in summer and winter and tussock size (gsu, gwi, tush). the plant height in spring in 2017 and growth in spring in 2017 and 2018 formed cluster a together with the two measured regrowth heights in 2016 and 2017. two populations with low survival rates (est1 and ltu1) were not included in the cluster analysis. the 39 remaining populations were assigned to five main clusters (figure 3). cluster 1 contained eight outstanding populations predominantly originating from germany but also from france and belgium. these were characterized by exceptionally low winter damage and strong and vigorous growth as indicated by high values (dark red colour) for these variables (sph, gsp, reg, tush, gsu, and gwi, figure 3). population deu2 was close to this cluster but had a later heading date (hae) and a lower height increment (hgtin). all the other populations were grouped along another branch of the clustering tree. an outstanding population was fra3 with high winter damage and low values for the other variables. cluster 2 (six populations from the north-eastern and central regions of the sampling area across europe) and cluster 3 (nine populations including five populations from great britain) differed for growing in spring and plant height in spring in 2016, but revealed no clear pattern for the other traits. srb2 and srb3 were separated from them by less vigorous growth but higher disease susceptibility and higher winter damage. cluster 4 contained one british and six french populations and showed high winter damage (cluster e) which was compensated for by a high spring and summer growth (gsp, gsu, cluster a). all five populations belonging to cluster 5 showed relatively high winter damage and low performance in all the other traits (figure 3). genetic resources (2026), 6 (11), 99–114 trait-climate relations in perennial ryegrass germplasm 105 figure 2. survival rate (in %) of populations calculated from the number of living genotypes 50 days after planting plus later planted individuals (number of clonal replicates given at the top) and from the number of living plants at the dates when growing in spring (gsp 2016, gsp 2017, gsp 2018) and regrowth after first cut in 2016 (reg1 2016) were scored. winter damage, growth in spring, growth before winter and heading date presented almost similar value ranges in the three trial years, while growth in summer and disease susceptibility fairly differed between the years (table 3). with a year-dependent average score of 5.4 to 6.1, winter damage was on an intermediate level, with the strongest damage after winter 2017. data from the three consecutive years indicated that populations from france generally had the highest winter damage in 2016 and 2017, in particular populations originating from southern france (fra1-fra4) (figure 3, table 3, supplemental table 4). populations from great britain (gbr3, gbr7), serbia (srb2, srb3) and portugal (prt2) also showed high winter damage. populations with the lowest winter damage originated from germany, but also from belgium (bel2), france (fra11) and lithuania (ltu2). the mean value of growth in spring ranged from 4.2 to 4.8 according to years. populations with strong growth in spring were bel2, fra8 and several german populations, in particular deu9. in contrast, the material from serbia exhibited low spring growth, particularly in 2017 and 2018. related to their late planting in 2015, also the british populations showed low spring growth. population fra3 differed from all the other populations by revealing the highest winter damage and the lowest growth in spring in all three experimental years. 106 bachmann-pfabe et al genetic resources (2026), 6 (11), 99–114 figure 3. clustering and heatmap based on the mean values of 11 different traits recorded in 39 natural l. perenne populations in two to three consecutive years under field conditions. traits were standardized prior to multivariate analysis and are displayed for each population ranging from low (= yellow) to high (= dark red) compared with other populations. wid, winter damage; gsp, growth in spring; gsu, growth in summer; gwi, growth before winter; dis, disease susceptibility; hae, heading date; sph, spring height; reg, regrowth height; hgtin, height increment; tush, tussock size (highest). clusters a-e grouped traits according to column means and clusters 1-5 grouped populations according to row means, both using euclidean distances. growth in summer, i.e. the biomass development of the plants in august after the two cuttings, was stronger in 2016 than in 2017. outstanding populations with very strong growth in summer were bel2, fra8, deu2, deu3 and deu9. growth before winter reached rather similar values in both years. well-developed plants with a high growth performance before winter were observed in populations bel2, fra8, deu2, deu3, deu7 and deu9. the average heading date was 47.0 and 48.5 days in the two years. late types (hae > 57 days) originated from belgium, in particular, bel2 with 67 days in 2017, northern germany (deu5-10) and northern britain (gbr8). although collected close to the site of gbr8, heading date of gbr7 was much earlier. next to several french populations, deu2 revealed the earliest heading date in the whole set with only ~25 days after 1 april in both years. general disease susceptibility, scored in september or october, reflected the general leaf health under the infection with several diseases. on average, the disease infestation was higher across populations in 2016 than in 2017. in particular fra6, deu2 and two out of three populations from serbia (srb2 and srb3) showed a higher disease susceptibility than the other populations, even in 2017 when disease pressure was lower. generally, based on all scored traits, bel2, fra8, deu3 and deu9 exceeded the performance of all other populations. additionally, deu2 revealed good biomass scorings throughout the experimental time, but tended to a higher disease susceptibility. deu2 and deu3 were both among the best populations for all scored traits, but strongly differentiated in heading date, with deu2 as an early heading type and deu3 as a late type genetic resources (2026), 6 (11), 99–114 trait-climate relations in perennial ryegrass germplasm 107 with an average of 60 days to heading. the weakest population was fra3, characterized by the strongest winter damage, the lowest spring and summer growths as well as the least developed plants before winter (supplemental table 4). plant height and diameter were measured at several time points during the experiment. on average across populations, plant height in spring, regrowth height after first cut and tussock size were higher in 2017 than in 2016, while regrowth height after second cut had similar average values in the two years. in contrast, height increment, as a measure of plant height development between first and second cuts, was only about half as high in 2017 as in 2016 (table 3). the spring height measurements confirmed the findings from growth in spring scorings. the average plant height in spring was 125mm in 2016 and increased to 205mm in 2017. populations with large spring heights were mostly from france (fra4, fra6, fra8, fra11) and germany (deu2, deu4, deu8, deu9). similar to growth in spring scorings, spring height was exceptionally low for all the material from great britain, particularly in 2016, and for material from serbia (figure 3, supplemental table 5). regrowth height 10 days after the first cut reached 137mm on average across populations in 2016 and 196mm in 2017. populations with strong regrowth after the first cut were fra7, fra8, fra9 and deu1, deu2, deu3, deu8 and ltu2. populations from prt and srb generally revealed a slow regrowth after the first cut. the regrowth height 10 days after the second cut was as on average 183mm and rather stable between the two years. the population with regrowth height after second cut stronger than most of the other populations, considering both years, originated from germany (deu3, deu5, deu7, deu8, deu9) and france (fra8). in contrast, regrowth capability below that of most of the other populations was measured for populations from portugal (prt1), serbia (srb2) and france (fra3). the increment of plant height, expressed as the height difference from regrowth height after first cut to the height at the second cut, declined on average across populations from 147mm in 2016 to 92mm in 2017. outstanding populations with a high height increment in both years and a late spring growth were deu9 and fra8. tussock size of the individual plants averaged 206mm at the end of 2016 and increased on average by 50mm by the end of 2017. highest tussock sizes (> 290mm) were generally recorded for the strong growing populations such as bel2, fra8, deu3, deu4, deu5 and deu8, while most populations from france, portugal and serbia had tussock sizes below 230mm. relation between performances of populations and bioclimatic variables at their sites of origin correlations between the phenotypic traits of the populations and the climatic variables at their sites of origin are displayed in figure 4. among all phenotypic traits, winter damage in 2016 highly correlated with almost all climatic variables. winter damage in 2016 showed the highest correlations with the mean annual temperature (bio1, r = 0.59, p < 0.001) and mean temperature in the driest quarter (bio9, r = 0.67, p < 0.001). the temperature daily range correlated with the susceptibility of the populations to cold winters as indicated by the correlation between winter damage in 2016 and temperature diurnal range (bio2, r = 0.45, p < 0.001). however, correlations were weaker and not significant in the following years. growth in spring correlated negatively with minimum temperatures during cold seasons (bio11, r = -0.40, p < 0.05), but only for 2016. hence, the lower the temperature during the winter period at the site of origin, the stronger was the plant growth in spring after transplanting. furthermore, temperature seasonality (bio4) was positively correlated with growth in spring 2016 with r = 0.42 (p < 0.01). days to heading correlated little with bioclimatic variables, except for mean diurnal range in both experimental years (bio2, r = -0.46, p < 0.01/-0.43, p < 0.01, figure 4). tussock size in both years correlated negatively with bio2 (r = -0.40, p < 0.05/-0.55, p < 0.001). disease susceptibility in both years correlated negatively with several temperature variables but not with precipitation indices. the lower the average daily minimum temperature (bio6, r = -0.53, p < 0.001/0.57, p < 0.001) and the lower the mean temperature of the coldest quarter (bio11, r=-0.51, p<0.001/-0.48, p < 0.01), the higher was the damage caused by diseases in the field experiment. accordingly, the higher the altitude of the site of origin, the higher the disease susceptibility in both years (r = 0.48, p < 0.01/0.53, p < 0.001). the latitude of the site of origin correlated positively with heading date, growth before winter and tussock size and negatively with winter damage in 2016 and 2017. other phenotypic traits showed only low correlations with the basic climate norms (figure 4). discussion we studied the agronomic performances of perennial ryegrass populations collected across europe and found large differences between the populations at the study site in northern germany. winter damage was high for accessions from warm and sunny origins with dry summers, as seen by positive correlations between winter damage and temperature (bio1, bio9, bio10) and negative correlations between winter damage and precipitation in the warmest quarter (bio18). especially populations from southern european regions with a warm climate at the site of origin, such as fra1-fra4 and prt2, showed high winter damage, followed by low spring growth and, finally, a high loss of plants after the first cut in 2016. this is consistent with previous results. keep et al (2021) and blanco-pastor et al (2021) reported that perennial ryegrass populations collected from areas with low winter temperatures were more resistant to winter damage in cold winters, while populations adapted to long and dry summers 108 bachmann-pfabe et al genetic resources (2026), 6 (11), 99–114 table 3. phenotypic traits measured in 39 natural l. perenne populations in two or three consecutive years under field conditions. given are average, minimum and maximum values of populations and populations significantly different from the grand mean. *, populations significantly higher than the grand mean for the traits gsp, gsu, gwi, sph, reg1, reg2, hgtin, tush; populations significantly lower than the grand mean for wid and dis; hae: the five significantly earliest populations and the five significantly latest populations; significance level p ≤ 0.05; see more detailed results in supplemental tables 4 and 5. trait year mean min max important populations* winter damage (wid) [score 1-9] 2016 5.8 4.8 8.3 low: deu9, fra11, gbr4, ltu2 2017 6.1 4.7 7.4 low: bel2, deu3, deu4, deu5, deu7, deu8, deu9, fra8 2018 5.4 4.2 6.9 low: bel2, deu5, deu7 growth in spring (gsp) [score 1-9] 2016 4.5 2.6 6.2 high: bel2, deu1, deu3, deu4, deu5, deu9, fra8, fra11 2017 4.8 2.9 6.6 high: bel2, deu4, deu7, deu9, fra8 2018 4.2 2.7 5.5 high: bel2, deu2, deu9, fra8 growth in summer (gsu) [score 1-9] 2016 5.9 3.2 8.2 high: bel2, deu2, deu3, deu9, fra8 2017 4.3 2.9 5.8 high: bel2, deu2, deu3, deu5, fra8 growth before winter (gwi) [score 1-9] 2016 4.7 2.9 6.4 high: bel2, deu3, deu7, deu9, fra8 2017 5.0 2.3 6.9 high: bel2, deu2, deu3, deu4, deu5, deu7, deu9, fra8 disease susceptibility (dis) [score 1-9] 2016 5.7 4.7 7.0 low: 2017 3.9 2.9 5.6 low: bel2 heading time (hae) [days from 1april] 2016 47.0 25.8 63.3 early: deu2, fra6, fra7, fra1, fra4, late: bel2, gbr8, deu10, deu5, deu6 2017 48.5 25.5 61.1 early: deu2, fra6, fra7, fra1, fra4, late: bel2, gbr8, deu10, deu5, deu6 spring height (sph) [mm] 2016 125 82.5 164 high: deu4, deu9, fra8, fra11, nld1, pol1, srb1 2017 207 159 264 high: deu2, deu4, deu8, deu9, fra4, fra6, fra8 regrowth height 1 (reg1) [mm] 2016 137 105 157 high: deu1, deu2, deu3, deu8, fra7, fra8, fra9, gbr2, ltu2 2017 196 165 222 high: deu2, fra6 regrowth height 2 (reg2) [mm] 2016 183 133 112 high: deu8, fra8 2017 182 156 205 high: deu3, deu5, deu7, deu8, deu9 height increment (hgtin) [mm] 2016 147 103 204 high: deu3, deu9, fra8, fra10, ltu2, pol1, srb1 2017 92.0 57.3 129 high: deu7, deu8, deu9, fra8 tussock size (highest, tush) [mm] 2016 206 122 253 high: bel2, deu3, deu4, deu5, deu9, fra5, fra8, fra11, gbr8, ltu2, nld1, pol1 2017 258 204 363 high: bel2, deu3, deu4, deu5, deu9, fra5, fra8, fra11, gbr3, gbr8, pol1 (and probably heat stress) revealed high aftermath heading and spike density but low persistency after cold winters. similarly, hulke et al (2007) found a higher tiller survival after cold winters in perennial ryegrass populations originating from northern, continental or alpine areas of europe than in populations from mediterranean areas with warmer climates. bachmannpfabe et al (2018) also found that perennial ryegrass populations from north-western spain were sensitive to low temperatures in winter. reports from bachmannpfabe et al (2018), blanco-pastor et al (2021) and keep et al (2021) confirmed that populations from southern europe with mild winter temperatures can grow in early spring and late autumn and even to some extent in winter. however, this ability makes them maladapted to the winter conditions of our northern germany experimental field, during which their living tissues were exposed to cold and frost. an adaptation to low winter temperatures is to stop growth during winter and start growing late in spring in order to escape cold stress (keep et al, 2021), which is not present in these southern european populations. in accordance with blanco-pastor et al (2021), who found late spike emergence in cold-adapted populations, winter damage correlated negatively with heading date in our study (supplemental figure 1). the correlation between winter damage and climate at sites of origin was only prominent in the first winter 2015–2016, but was negligible for the other years. that might be attributed to remarkably low temperatures in january 2016 (average -0.6◦c, minimal daily average temperature of -9.2◦c), which differentiated the populations originating from cold regions and the cold-sensitive populations from warmer regions. in the following years, acclimatization to the environmental conditions at the study site, i.e. by sufficient hardening, genetic resources (2026), 6 (11), 99–114 trait-climate relations in perennial ryegrass germplasm 109 figure 4. correlations between origin-specific variables (latitude, longitude, altitude and bioclimatic variables) and mean values of phenotypic traits of the 39 l. perenne populations. colour codes are pearson’s correlation coefficients (see sidebar on the top right). significant correlations are labelled with asterisks (*, p < 0.05; **, p < 0.01; ***, p < 0.001). dec lat, latitude; dec lon, longitude; bio1, annual mean temperature; bio5, average maximum temperature of the warmest 14/15-day period; bio6, average minimum temperature of the coldest 14/15-day period; bio8, bio9, bio10, bio11, mean temperature of the wettest, driest, warmest and coldest quarter, respectively; bio12, annual cumulated precipitations; bio16, bio17, bio18, bio19, cumulated precipitations in the wettest, driest, warmest and coldest quarter, respectively; bio2, mean diurnal range; bio4, temperature seasonality (standard deviation of average daily mean temperature per year); bio7, temperature annual range. wid, winter damage, gsp, growth in spring, gwi, growth before winter, dis, disease susceptibility, hae, heading date, sph, spring height, tush, tussock size (highest). only phenotypic traits with at least one correlation with r > 0.4 or r < -0.4 in at least one year are shown. especially since only plants that survived two years were considered in the analysis, could have contributed to the lack of climate-winter damage correlations. past climatic experience affects plant performance, as indicated by a study with 18 arrhenatherum elatius ecotypes, where tolerance to (late spring) frosts increased when plants were exposed to late frost in the preceding growing season (kreyling et al, 2012). for european perennial ryegrass accessions, the minimum lethal temperature, i.e. the temperature at which 50% of the plants would die, was determined to be between -3.0 and -13.9◦c (humphreys and eagles, 1988; hulke et al, 2008). hence, cold-sensitive genotypes in our experiment were not killed but probably damaged by the low temperatures in the first winter. these plants might survive freezing, but their growth is limited in subsequent spring (humphreys and eagles, 1988). accordingly, populations with strong winter damage, such as fra1-4, fra9 and prt2, revealed low growth in spring in our experiment. the low precipitation in march and april 2016 at the experimental site might have further contributed to a low spring growth of already winter-damaged plants and the subsequent loss of many genotypes after the first cut. unexpectedly, also populations srb1, srb3, ltu1 and est1 showed high losses (low survival rate) after the first cut in 2016, although they originated from regions with average temperatures in the coldest month reaching -7◦c. freezing tolerance is influenced by additional factors such as hardening, wind desiccation, snow cover or disease infestation (hulke et al, 2007). hardening prior to freezing is also critical to reduce cell damage caused by frost (humphreys and eagles, 1988). according to fuller and eagles (1980), the threshold for hardening in perennial ryegrass is between 5 and 7◦c for european germplasm, and between 2 110 bachmann-pfabe et al genetic resources (2026), 6 (11), 99–114 and 5◦c for less tolerant new zealand cultivars, while higher temperatures may even cause de-hardening. hence, the average temperatures of 7.7 and 7.1◦c in november and december 2015 might have hampered cold acclimatization and made the plants prone to other stresses. nevertheless, population ltu2 from lithuania is well suited to breed for winter hardiness, showing high survival rates and persistency as well as fast growth in spring and after cut (high sph, reg1 and reg2) including large tussock size. also, disease infestation might reduce frost tolerance and could explain the losses in populations from serbia. although disease infestation was not recorded prior to the first winter (in 2015), ratings from autumn 2016 and 2017 indicate a higher disease susceptibility in serbian populations, in particular in srb3. fungal diseases such as crown rust reduce the overall vigour and competitiveness of ryegrass (kimbeng, 1999) and thus may also negatively affect winter hardiness or the tolerance to drought in early spring. disease susceptibility in 2016 and 2017 was negatively correlated to temperature variables (bio1, bio6, bio9, bio11), indicating that populations from cold regions were more susceptible to disease infestation. geographical variation in the disease susceptibility of natural populations of grass species was already reported as associated with climatic variation. germplasm with higher tolerance to rust infection was, for example, identified in perennial ryegrass material collected in romania, crisana region (willner et al, 2010), and bulgaria (bachmann-pfabe et al, 2018). in a study with fine-leaved fescues across france, sampoux and badeau (2010) found that populations from warm and wet sites were the most resistant to diseases. climate conditions likely determine the presence/absence of pathogens, and the co-evolution of plant species with the pathogens to which they are exposed may lead to the development of resistance in plant genotypes. scores of biomass growth in spring, summer and before winter, are indicators of the potential fodder production of the studied populations in the different seasons. the cluster and heatmap analysis identified populations of cluster 1 (figure 3), namely bel2, fra8, deu2, deu3, deu4, deu5, deu7 and deu9, as best combining strong growth in spring, summer and autumn, large tussock size, late heading (except deu2) and low winter damage. these populations also showed a high survival rate throughout the experiment. these results might not be very surprising, since these populations originate from habitats with climate conditions very similar to the experimental site. they are more unexpected for population fra8, which comes from a site with milder winter temperatures and possible occurrence of drought events in summer. population fra3 clearly contrasted with populations of cluster 1 with the weakest vegetative growth, the smallest plant size and highest winter damage observed in the whole set of populations. the environment of this population is characterized by the highest winter temperature (bio6, 3.5◦c), the warmest summer temperature (bio5, 30.2◦c) and the lowest precipitation in the driest quarter (bio17, 50mm). reduced growth potential (especially in summer) and plant size (small tussock size) in this population are consistent with the growth-survival trade-off under the risk of summer heat and drought, a concept published by blanco-pastor et al (2021) and keep et al (2021). we also found that tussock size was positively correlated to precipitation in the wettest quarter (bio18) and negatively to maximum temperatures (bio5), mean diurnal temperature range (bio2) and annual temperature range (bio7). thus, we observed large tussocks (and high vegetative growth) in populations from the sites where the summer drought stress is the smallest (or without summer drought stress). wet climates with high rainfall, low summer drought stress and small diurnal and annual temperature ranges are found in oceanic regions. heading date is an important feature for perennial ryegrass as a forage crop. after heading, the proportion of reproductive tillers increases rapidly in the sward biomass, while the subsequent biomass growth slows down and the feeding quality decreases (hurley et al, 2007). plant breeders developed forage-type perennial ryegrass cultivars with different maturity groups, from early-heading types convenient for early-season cut to late-heading types best adapted to a long grazing season and used for permanent pastures (laidlaw, 2005; humphreys et al, 2010). heading date is a highly heritable trait with a broad sense heritability usually larger than 80% (keep et al, 2020); it was found to strongly correlate between two record years with pearson’s product-moment correlation of 0.82 in perennial ryegrass full-sib families by arojju et al (2016). our study is consistent with this well-known trend, as indicated by a correlation of 0.96 (p < 0.001) between heading date in 2016 and in 2017 (supplemental figure 1). populations bel2, deu5 and gbr8 were identified as the latest heading ones, while populations deu2, fra6 and fra7 were scored as the earliest heading ones. heading date of perennial ryegrass populations was previously reported as related to the climate and the solar radiation at sites of origin. barre et al (2018), blanco-pastor et al (2021) and keep et al (2021) typified perennial ryegrass populations from sites with cold winter and spring by slow spring growth and late heading. that corresponds with an escape strategy in which the peak of vegetative spring growth is scheduled to escape the risk of late cold stress (blanco-pastor et al, 2021). on the other hand, early heading and fast spring growth were reported in perennial ryegrass populations from sites with favourable conditions for growth, i.e. mild winters, cool summers and abundant rainfall (keep et al, 2021). accordingly, populations such as fra1-4, fra9 and prt2 with early heading date and thus early start of spring growth, were exposed to cold injuries and had high winter damage. ltu2 had few winter damages, although it is quite early heading, like fra11 and deu4. these populations could have acquired good winter genetic resources (2026), 6 (11), 99–114 trait-climate relations in perennial ryegrass germplasm 111 hardiness or may have other physiological patterns that enable them to cope with cold in winter and spring. heading date in our experiment showed no direct relationship to temperature norms at the sites of origin. but heading date was notably negatively correlated with mean diurnal temperature range across the year and positively with latitude. regions with large diurnal temperature ranges are usually continental regions with dry and cold climates with unfavourable conditions for growth and therefore later heading. similarly, higher northern latitudes have lower solar radiation and are often colder. since the germplasm used for this study was not compared to cultivars, we cannot assess the performance level, neither can we assess the performance in swards or mixtures. however, our study confirms the importance of breeding efforts for regionally adapted plant material. in general, the adaptation to the climate conditions at the site of origin in our newly collected l. perenne panel followed the same trends as observed by studies evaluating l. perenne genebank accessions collected from 1980 to 2000 (blanco-pastor et al, 2021; keep et al, 2021). therefore, we can assume that the natural distribution of perennial ryegrass phenotypic diversity across europe is still related to the average climate conditions that prevailed during the past decades, even if climate change has likely accelerated from the time of collection of genebank accessions to that of the newly collected ones in this study. conclusions the phenotypic characterization of l. perenne populations originating from habitats across europe in a common garden experiment showed that climate may be a major driver for phenotypic variation between populations. our results report on climate adaptation of a newly collected set of ecotypic populations of perennial ryegrass. this set of natural populations collected in contrasting environments is available as genetic resources for breeding and research at the collections for oil and fodder crops of the ipk genebank, and offers opportunities to widen the genetic base for selection. populations from environments similar to the experimental site had a better growth performance and may be integrated directly into breeding programmes for the north of germany, whereas others might be considered to improve specific traits. supplemental data supplemental table 1: collection material. passport data of the collected perennial ryegrass (lolium perenne l.) populations including information about the sample area. supplemental table 2: bioclimatic variables used to describe the climatic conditions at the sites of origin of the perennial ryegrass populations (bioclim like variables). supplemental table 3: weather conditions over the experiment duration at the field trial site. supplemental table 4: population means for the traits evaluated. supplemental table 5: adjusted means of the different populations in the field experiment from march 2016 to 2018 for the measured traits including statistics from a one-way anova (fand p-values). supplemental table 6: pearson correlation coefficients between the phenotypic traits (population means) and the climate norms at the site of origin of populations. supplemental figure 1: pearson correlation coefficients r between the phenotypic traits (population means). data availability the phenotypic data recorded in this experiment and presented in this manuscript are available via the genebank information systems of ipk https://gbis.ipkgatersleben.de/gbis2i/ by typing the accession number given in table 1 or the accession doi given in the supplemental table 1. furthermore, the data can be accessed via the eurisco search catalogue http://e urisco.ecpgr.org. the description of the bioclim-like climate variables and of the seasonal climate variables at sites of origin of populations was published by blancopastor et al (2021). acknowledgements the collection of ecotypic lolium perenne populations was organized and coordinated by the project partners from ibers, inrae and ipk. we thank all the partners for their help and support during the collection: centre of estonian rural research and knowledge, külli annamaa; institute of agriculture (lithuania), vilma kemesyte; west pomeranian university of technology (poland), marek bury; cgn, wageningen (netherlands), chris kik; ilvo, melle (belgium), an ghesquiere; forage grass breeding institute for forage crops kruševac (serbia), dejan sokolović; iniav, braga (portugal), ana maria barata; ibers, ian thomas (great britain); ipk satellite collections north, karla ploen, christine luckmann. special thanks to the whole team of the satellite collections north, oil and fodder crop collections, for the strong support and engagement during the maintenance of the field trial and during data collection. furthermore, we thank inrae (ur p3f) for the ploidy check of the populations. funding this research was carried out in the frame of the project grasslandscape funded by the 2014 faccejpi era-net+ call climate smart agriculture. funding for the project partner ipk satellite collection north was granted by the european commission (ec grant agreement n◦ 618105) via the german federal ministry of food and agriculture. https://www.genresj.org/index.php/grj/article/view/genresj.mdso5816/suppdata272 https://www.genresj.org/index.php/grj/article/view/genresj.mdso5816/suppdata272 112 bachmann-pfabe et al genetic resources (2026), 6 (11), 99–114 conflict of interest the authors declare no financial or personal conflict of interest. the funders had no role in the design of the study, in the collection, analyses, or interpretation of the data, in the writing of the manuscript or in the decision to publish the results. author contributions sbp analyzed the data, sbp and mk wrote the manuscript, ar and ew were responsible for the experimental design, trial management and collected the data, ew and kjd reviewed the manuscript, jps provided data on climate norms and climate variability and reviewed the manuscript. all authors read and approved the manuscript. references arojju, s. k., barth, s., milbourne, d., conaghan, p., velmurugan, j., hodkinson, t. r., and byrne, s. l. 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(2004). econometric computing with hc and hac covariance matrix estimators. journal of statistical software 11(10), 1–17. introduction material and methods collection sites and site-specific characteristics experimental design and traits evaluated statistical analysis results survival rate phenotypic characterization of the populations relation between performances of populations and bioclimatic variables at their sites of origin discussion conclusions supplemental data data availability acknowledgements funding conflict of interest author contributions review and position paper genetic resources (2024), s2, 6–12 doi: 10.46265/genresj.rfxb3570 https://www.genresj.org issn: 2708-3764 quality management in a genebank environment: principles and experiences at the centre for genetic resources, the netherlands (cgn) theo van hintum * and erik wijnker centre for genetic resources, the netherlands (cgn), wageningen university and research (wur), p.o. box 16, 6700 aa, wageningen, the netherlands abstract: to enhance the management of plant genetic resources by genebanks, implementing a quality management system is essential. such a system ensures the consistent quality of genebank operations through the establishment of a quality policy, the integration of quality planning and assurance, and the execution of continuous quality control and improvement measures. this structured approach also supports alignment with globally recognized standards, such as those established by the un food and agriculture organization (fao). the centre for genetic resources, the netherlands (cgn), with its significant e xperience i n q uality m anagement, i s p resented a s a c ase s tudy t o i llustrate t he m ethodology a nd i ts impact on genebank operations. by detailing operating procedures, a quality management system provides transparency, fostering trust and facilitating collaboration between genebanks. additionally, the potential for developing a certification system for genebanks – wherein an authorized body formally certifies that a genebank adheres to specific standards – is examined. keywords: ex situ genebank, genebank collaboration, quality management, certification system citation: van hintum, t., wijnker, e. (2024). quality management in a genebank environment: principles and experiences at the centre for genetic resources, the netherlands (cgn). genetic resources s2, 6–12. doi: 10.46265/genresj.rfxb3570. © copyright 2024 the authors. this is an open access article distributed under the terms of the creative commons attribution license (cc by 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. introduction genebanks play a vital role in safeguarding plant genetic resources (pgr) for future generations, ensuring that these resources remain accessible for developing crops essential to feeding the global population (fao, 2010). in addition to their long-term conservation efforts, genebanks also provide crucial materials to crop scientists and plant breeders, supporting their research and breeding programmes. these responsibilities are significant and demand a high level of commitment. consequently, it is essential that genebank operations maintain a high standard of quality, which must be consistently assured. effective quality management is therefore critical to ensure that genebanks fulfil their responsibilities and operate at the appropriate level to meet global food security needs. ∗corresponding author: theo van hintum (theo.vanhintum@wur.nl) the centre for genetic resources, the netherlands (cgn) holds the statutory responsibility for managing pgr on behalf of the dutch government. when this mandate was assigned by the dutch ministry of agriculture in 2004, the ministry also required cgn to implement a quality management system to ensure that public funds were being used effectively and that the dutch public could have confidence in cgn’s ability to perform its duties at a high standard. as a result, cgn became the first genebank in the world to achieve iso 9001 certification. as stated on the website of the international organization for standardization (iso, 2015), “iso 9001 is a globally recognized standard for quality management. it helps organizations of all sizes and sectors to improve their performance, meet customer expectations and demonstrate their commitment to quality. its requirements define how to establish, implement, maintain, and continually improve a quality management system (qms). implementing iso 9001 means your organization has put in place effective received: 29.10.2024 accepted: 13.11.2024 published online: 28.11.2024 https://www.genresj.org https://www.doi.org/10.46265/genresj.rfxb3570 https://www.genresj.org https://www.doi.org/10.46265/genresj.rfxb3570 mailto:theo.vanhintum@wur.nl genetic resources (2024), s2, 6–12 quality management in genebanks 7 processes and trained staff to deliver flawless products or services time after time.” each genebank holds unique collections that often reflect regional crops and wild relatives adapted to specific climates and conditions. preserving pgr is a shared responsibility that transcends borders. to fulfil this mission, genebanks must collaborate, sharing resources, data and expertise to prevent unnecessary duplication and ensure no vital genetic material is overlooked. working together also enables coordinated efforts to store, regenerate and monitor seed viability over time. successful collaboration relies on mutual trust, which in the context of genebanks means adhering to agreed-upon operating procedures and standards for managing pgr effectively. it is therefore not surprising that when the global crop diversity trust (gcdt) took on the coordination of the cgiar genebank platform and began contributing to the funding of cgiar genebanks, it recognized the need for a mechanism to monitor the performance and quality of these institutions. transparency in the operations of the genebanks was essential to assess their effectiveness and to identify areas for investment. consequently, the gcdt developed the ‘genebank quality management system’ and provided support to the cgiar genebanks in its implementation (lusty et al, 2021). the various elements of quality management within a genebank context are described and illustrated through the experiences of cgn. additionally, this discussion will introduce and explore certain aspects of the potential establishment of a genebank certification system. quality management in a genebank environment quality management quality can be managed, although it is difficult to define. according to the iso 9001 standard for quality management systems (iso, 2024), quality is defined as the “degree to which a set of inherent characteristics [or distinguishing features] of an object fulfils requirements”. an object is defined as “anything perceivable or conceivable, such as a product, service, process, person, organization, system or resource”. based on this definition the quality of a genebank can be defined as ‘the degree to which the genebank fulfils its objectives’. the objectives of a genebank are relatively welldefined and encompass two primary elements: the conservation of pgr for future generations and providing access to these pgr for the current generation of users (fao, 2014). while these elements are broadly agreed upon, interpretations of what constitutes ‘conservation’ and ‘access’ can vary among genebanks. discussions surrounding ‘conservation’ often focus on technical aspects, such as the required frequency of seed viability testing or the appropriate number of plants in a regeneration plot. in contrast, the concept of ‘access’ is more heavily influenced by policy considerations, addressing questions like who should have access to the material and under what conditions. regardless of differing perspectives on these definitions, it is essential to ensure that the objectives established for a genebank are met in the most effective manner possible. a quality management system serves as a valuable tool to assist genebanks in achieving these goals optimally. quality management, and this will appear obvious, involves overseeing all activities and tasks necessary to sustain a desired level of excellence, specifically to achieve established objectives. this process typically encompasses several key components, including quality planning, quality control and quality improvement. quality planning in the context of a genebank, quality planning involves establishing methods to measure or assess the achievement of objectives using key performance indicators (kpis), defining and updating standard operating procedures (sops), and ensuring that the desired quality level is maintained through an annual quality improvement plan (qip). these elements will be detailed below. after clearly defining objectives, methods need to be established to quantify or otherwise assess the achievement of these goals. this is typically accomplished through the identification of kpis, that can differ from genebank to genebank as they need to be tuned to the genebank’s organization and operations. the kpis may include metrics such as the number of accessions, the quantity of samples distributed, the number of regenerations conducted, the percentage of successful regenerations, and the number of viability tests performed. it is important to view these kpis as monitoring tools rather than strict performance assessments, as an overemphasis on these indicators could lead to unintended consequences. for example, a curator might feel compelled to distribute accessions solely to increase the number of samples sent out, potentially undermining the integrity of the genebank’s operations. the indiscriminate distribution of seed samples – such as fulfilling requests like “please send the entire lettuce collection” – can deplete inventory, necessitating earlier regeneration of the accession, which incurs additional costs and may impact the genetic integrity of the collection. therefore, in cases of large requests, a genebank should work collaboratively with the requester to identify an optimal selection of accessions that meets their needs while preserving the collection’s resources. the quality planning phase also encompasses the formulation of sops, which detail how essential operations are to be conducted. this is a critical component of effective genebank management and includes various elements such as protocols for acquisition, regeneration, seed processing (cleaning, drying, seed moisture content determination, viability testing, etc.), seed storage, distribution, information management and other 8 van hintum and wijnker genetic resources (2024), s2, 6–12 operational procedures. by clearly defining these sops, genebanks can ensure consistency and quality in their practices, thereby enhancing their overall effectiveness in achieving their objectives. the fao genebank standards for seed conservation (fao, 2014) categorize genebank operations into ten key areas: 1) acquisition of germplasm, 2) drying and storage, 3) seed viability monitoring, 4) regeneration, 5) characterization, 6) evaluation, 7) documentation, 8) distribution and exchange, 9) safety duplication, and 10) security and personnel. each of these essential elements can be associated with its own sop. however, depending on the scope of the quality management system, this framework can be expanded to encompass the promotion of use, research activities and other genebank-related functions. the level of detail in sops can vary significantly. in some instances, sops can serve as comprehensive guides, offering precise instructions on which actions to take and controls to operate in specific situations. in contrast, other sops may outline the principles and objectives of an operation without delving into the details of the actions required to achieve those goals. for example, a sop for the acquisition of germplasm typically emphasizes the need to adhere to all legal and phytosanitary regulations and specifies certain criteria for selecting materials suitable for inclusion in the collection. however, it may not detail where and how the material should be obtained (although it could be beneficial to include guidelines on handling materials during collection missions). sops not only clarify the processes involved but also serve as valuable resources for new staff members, helping them understand important considerations when performing genebank tasks. it is essential that these sops are not created in isolation but rather reflect existing practices and the current operational reality. during the documentation of procedures, it may become evident that certain practices are not ‘fit for purpose’ and require improvement. the previously mentioned fao genebank standards for seed conservation (fao, 2014) can often serve as a valuable reference point, providing guidance on what constitutes a high standard and an appropriate level of operation for genebanks. this alignment ensures that the sops are not only functional as a reference and training material but also effective in enhancing the overall quality of operations. quality planning is an ongoing process rather than a one-time task. it is typically conducted annually, producing a qip that is implemented throughout the year to achieve the desired quality standards. the qip incorporates elements such as user feedback, non-conformities, assessment of evolving policies, application of new technologies, and potential risks. quality control once the kpis and sops have been established, the genebank can implement a quality control mechanism. this process involves generating evidence that demonstrates compliance with the defined protocols, staff competency and user satisfaction. documenting this evidence should be integrated into the sops and may, for example, include maintaining logbooks for regenerations, which could record instances where protocols could not be adhered to, accompanied by justifications and approvals from a supervisor. additionally, the quality control mechanism may involve maintaining an overview of all requests for material, documenting the dates of the requests, the actions taken, the shipment dates of seeds, and potentially including feedback from the requestors of seeds. this systematic approach to evidence production not only ensures accountability but also fosters continuous improvements in the operational efficiency of the genebank. in addition to the user feedback, a significant and regular form of quality control comes from staff observations of potential deviations from established sops. these observations should be documented, processed and, together with other quality-related information, reviewed during internal audits to ensure comprehensive quality evaluation and drive continuous improvement through qips. ultimately, it should be feasible for an independent observer to assess and verify that the genebank is adhering to its established protocols. more critically, this observer should be able to ascertain that the staff possesses the requisite knowledge and skills to perform their duties as outlined in the sops. this principle is central to the certification process for the iso 9001 standard for quality management systems. as part of this iso 9001 certification, an auditor, selected by the certifying agency, will conduct an annual evaluation of the genebank. during this assessment, the auditor will verify that the genebank is operating in accordance with its sops and that management effectively oversees organizational operations, including initiatives for quality improvement. this external validation not only reinforces accountability but also enhances confidence in the genebank’s quality management practices. quality improvement the final component of quality management to be addressed here is quality improvement, which focuses on identifying operational flaws and implementing corrective measures. staff observations and feedback from genebank users play a crucial role in this process. when activities deviate from established sops, these non-conformities necessitate a thorough analysis to identify their root causes and facilitate appropriate adjustments to improve the protocols. user reports that highlight issues such as not receiving requested materials, receiving incorrect materials, or experiencing difficulties in germination of the received material are critical indicators that something is amiss. these signals may suggest problems with the ordering system, genetic resources (2024), s2, 6–12 quality management in genebanks 9 documentation errors, or seed viability concerns – all of which require immediate attention and action. this type of feedback from users can be asked when handling seed requests, but can also be collected in targeted questions and interviews. it is vital that all forms of feedback are taken seriously and addressed promptly. additionally, maintaining records of feedback and subsequent actions provides valuable information for auditors assessing the performance of a genebank. also here, the presence of sops detailing procedures of how complaints and issues are identified, addressed and improvements implemented makes sure that the qms itself fosters improvement. together, these elements constitute the quality management system of a genebank. given that each genebank is unique and the implementation of quality management practices remains relatively uncommon in this sector, there is currently no standardized model for a genebank quality management system. a standard of potential interest was published in 2018 for the biobanking community (iso 20387:2018) that through its focus on handling and storage of biological material, technical competence, risk management and data integrity may be of relevance to genebanks, albeit, to the best of our knowledge, no genebank currently uses this standard (iso, 2018). genebanks that have adopted quality management are often hesitant to publish their sops and related documentation. to date, cgn is one of the few genebanks that has made its complete quality management system publicly available (ecpgr, 2024). in the introductory text accompanying the sops, cgn states: “with these documents, cgn gives complete transparency regarding the reality of its genebank. as you will see, it is far from perfect. we hope this material will help others in setting up their quality management systems, and in providing transparency regarding their genebank operations. we also hope that it will start discussions and generate constructive feedback regarding our methods helping us to improve. in the end, we all want to conserve plant genetic resources as efficiently as possible, for the generations to come, and provide access to these resources for the current generation of users.” quality management at the centre for genetic resources, the netherlands (cgn) an important reason for implementing quality management, already referred to in the introduction section above, is to ensure the effective use of funding provided by supporting agencies. in 2004, cgn became the first genebank to achieve iso 9001 certification when their funding body, the dutch ministry of agriculture, mandated the establishment of a formal quality management system. pgr management was recognized by the dutch government as a key responsibility arising from international commitments, such as the convention on biological diversity (cbd) and the international treaty on plant genetic resources for food and agriculture (itpgrfa). the important responsibility of managing genetic resources was delegated to cgn, a division of wageningen university and research, which had been responsible for operating the genebank for wageningen’s agricultural institutes already since 1986. to ensure that cgn was fulfilling this statutory role effectively, the ministry required the implementation of a robust quality management system. setting up cgn’s quality management system already in 1993, cgn had produced an internal report titled cgn genebank protocol, which compiled the protocols followed by various curators, the seed manager and the documentation manager (van hintum and hazekamp, 1993). this report garnered considerable attention within the genebank community, as many institutions were keen to learn how a colleague genebank, cgn, conducted its operations. however, when the time came to formalize the sops for the quality management system, it became evident that the published cgn genebank protocol had outlined the procedures for an idealized scenario. for instance, the protocol might specify regenerating on the basis of a minimum of 50 plants, but in reality, if 55 were sown and 8 died, how should the curator proceed? the sops had to account not only for the ideal procedures but also specify the decisionmaking required in less-than-perfect circumstances. the process of drafting the sops sparked significant internal debate, curators learning from each other, asking the questions they never asked themselves, and ultimately leading to substantial improvements in quality. the establishment of an iso 9001-compliant quality management system at cgn was facilitated by an external consultancy firm. this firm provided expertise in the methodology, offering guidance on how to logically segment genebank activities, describe processes through flowcharts, and formulate the sops. as cgn was the first genebank to adopt a formal iso 9001 quality management system, there was no pre-existing standard to follow. this allowed cgn to analyze its activities and make a system that was ‘fit to purpose’ to its circumstances and reality. however, when compared to the quality management systems now used by other genebanks, the terminology employed by cgn is somewhat unconventional, and the level of detail is occasionally either excessive or insufficient, as compared to other systems. additionally, after two decades of operation and considerable evolution, the system’s internal coherence has eroded, suggesting that a comprehensive revision may be necessary. nevertheless, the system has significantly contributed to cgn’s success as a genebank, and still does. it enabled cgn to maintain consistent quality, as reflected by user feedback. the iso 9001 standard mandates that the cgn conduct annual external audits, carried out by an iso accredited conformity assessment agency. these audits generate reports that highlight areas requiring attention, including opportunities for improvement and, when applicable, instances of nonconformity with the iso 10 van hintum and wijnker genetic resources (2024), s2, 6–12 standard. in cases of nonconformity, cgn must submit an improvement plan, complete with a timeline and supporting evidence. in addition to the external audit, an internal audit is conducted annually. while cgn outsources this process to a specialized company, it retains the option to perform it in-house. the report of the internal audit serves as input for the subsequent external audit. every three years, recertification is conducted through a more comprehensive audit performed by the accredited auditing agency. costs and benefits estimating the costs of iso 9001 certification is nearly impossible. a general rule of thumb exists, but has very limited value. it suggests that establishing the system typically requires approximately c1,000 to c3,000 per employee, and to maintain it, 10–20% of the initial costs annually (personal observation). in the case of cgn, the estimate for the initial costs probably is conservative. the actual costs were never calculated and strongly depended on the significant staff time invested in drafting, revising and editing the sops. conversely, now that the system has been in place for an extended period, the annual maintenance cost is likely on the low side of the rule of thumb estimate. in fact, when accounting for the cost savings achieved through more efficient operations, it could be argued that cgn is actually realizing financial savings as a result of its quality management system. another important aspect to consider is staff perception and acceptance. at cgn, initial resistance to the introduction of quality management was significant, as it was viewed as a constraint on creativity, reducing employees to mere components of a mechanized system. additionally, the use of kpis to monitor processes was perceived as akin to ‘big brother’ surveillance. however, over time, staff members came to recognize and appreciate the benefits of a structured organization, and the importance of well-documented procedures became particularly evident during instances of succession, such as when retiring employees were replaced by new hires. a genebank certification system rationale for a genebank certification system effective collaboration and division of responsibilities are vital for the conservation of pgr required by future generations. however, successful collaboration necessitates mutual trust among genebanks. by adopting standardized practices and achieving a shared quality level, genebanks can establish reliance on one another’s efforts, thereby facilitating efficient collaboration. this partnership not only enhances the secure conservation and accessibility of pgr for users but also improves management efficiency by minimizing unnecessary redundancy; many genebanks currently conserve overlapping collections. the establishment of mutual trust enables the principle that ‘if you undertake this task, i do not need to do so’, thereby reducing long-term conservation costs and reallocating resources to address gaps in collective pgr collections and investments in quality improvement. this, in turn, enhances pgr utilization through improved characterization, documentation and better user interfaces. in europe, the pgr community, organized under the european cooperative programme for plant genetic resources (ecpgr), recognizes the necessity of implementing a genebank certification system. in its plant genetic resources strategy for europe, launched on 30 november 2021, the european pgr community calls on the establishment of an economically sustainable certification system accessible to genebanks (ecpgr, 2021). also, the fao intergovernmental technical working group on plant genetic resources for food and agriculture has underscored the importance of a quality assurance system, preferring the term ”acknowledgment system” aligned with the fao genebank standards (fao, 2014). they have recommended that the fao investigate capacity-building and evaluation mechanisms to support genebanks in adhering to these standards (fao, 2023). beyond the benefits of quality management for individual genebanks, certification will provide a framework to ensure that these institutions meet community-agreed standards for conservation and access, and that continuity is guaranteed. the fao genebank standards (fao, 2014) are well accepted for the operating procedures concerning handling material and the standard material transfer agreement (smta) of the itpgrfa could provide the basis for the distribution of pgr (fao, 2024). in addition, procedures for guaranteeing continuity will need to be set up. should a genebank lose its certification, another certified genebank should be able to assume responsibility for the material from the ‘lost’ institution, thereby ensuring that pgr once integrated into the system remains preserved and accessible. consequently, a certification system is essential for enhancing efficiency, reliability, transparency and accountability, given that the conservation and accessibility of pgr represent a global responsibility that must be upheld by all credible stakeholders, including international, regional and national genebanks. components of a certification system implementing a certification system for genebanks necessitates several key elements. firstly, the genebanks seeking certification must establish a robust qms that enables an external auditor to assess both the activities undertaken and the methodologies employed. secondly, the sops utilized within the genebanks must align with community-agreed standards. lastly, a certification mechanism must be developed and administered by an organization endowed with adequate authority. an increasing number of genebanks are in the process of establishing qms; however, international support and coordination remain limited. the absence of coordinated genetic resources (2024), s2, 6–12 quality management in genebanks 11 international oversight for pgr activities has resulted in a lack of responsibility for guiding genebanks in these critical advancements. consequently, there is a risk that genebanks will repeatedly reinvent processes and protocols. the establishment of a central hub to provide training materials, templates and examples of effective qms tailored to various types of genebanks and operations could significantly expedite this process. the fao genebank standards (fao, 2014) serve as an excellent foundation for defining minimum operational quality levels. while some adaptations will be necessary to incorporate current technology and evolving insights, the fundamental objectives – namely, to conserve plant genetic resources for future generations while ensuring their availability to present users – are clearly articulated. moreover, details concerning access provision to pgr must be formulated, alongside procedures to ensure continuity. the groundwork has already been laid through the initial set of genebank standards (fao, 2014). a critical element still to be finalized in the development of a genebank certification system is the appointment of a certifying agency. this agency would have several key responsibilities. firstly, it would need to establish the general competency requirements for genebanks, reflecting the consensus of both the scientific and genebank communities. secondly, it would be tasked with creating a verification process to ensure these requirements are met. this process should outline how the requirements are to be fulfilled and how their fulfilment will be assessed. typically, this includes certification audits every three to five years, along with intermediate audits to monitor ongoing quality management within the genebank. several organizations are currently under consideration for the role of certifying agency, with the goal and expectation of arriving at a suitable solution. discussion the qms of cgn was developed independently, without following a predefined standard, leaving scope for further improvement. rather than positioning this qms as an exemplary model, it has been made publicly accessible to provide transparency and encourage constructive feedback (ecpgr, 2024). an open dialogue regarding quality management practices and procedures in genebanks would benefit all involved by providing exposure to actual qms approaches in genebanks and stimulating discussion about sops. furthermore, it will inspire the harmonization of these systems and enhance the quality of all genebanks involved. recently, cgn initiated an evaluation of the alignment of its procedures with the fao genebank standards (fao, 2014), revealing certain divergences in practice. for instance, cgn’s approach to seed viability testing, which employs fixed thresholds, contrasts with the fao’s recommendation to test for specific declines in viability (wijnker et al, 2024). while cgn’s alternative approach is obviously based on a considered rationale, feedback from the genebank community and possibly a certifying body could provide valuable input for further improvement. in its commitment to ensuring the accessibility of pgr, cgn currently lacks a formal contingency plan should it cease operations or be unable to provide access to the genetic resources in its collections, thus jeopardizing access to pgr. a network of certified genebanks could play a critical role in these circumstances, taking over and potentially keeping the pgr currently in cgns collection conserved and accessible. a genebank certification system would provide the credibility needed. moreover, in combination with the legal assurances provided by the smta, it could provide a robust foundation for continued access to these resources in an open network of certified genebanks. conclusions quality management serves as a crucial instrument for enhancing the effectiveness and efficiency of genebanks, establishing a foundation for collaboration. the experiences of various genebanks, particularly those of cgn, demonstrate the positive impacts of implementing quality management practices. by integrating quality management with community-agreed minimum standards for genebank operations, a foundation is established for genebank certification. this certification would represent a significant advancement toward ensuring the proper conservation of, and access to, pgr for both present and future generations of users, ultimately contributing to global food security. acknowledgements the work of cgn was and is supported by the dutch ministry of agriculture, fisheries, food security and nature as part of the statutory research task ‘wot-03 genetische bronnen’. an important driving force behind the establishment of a genebank certification system is the eu-funded project pro-grace (pro-grace, 2024). this project has received funding from the european union’s horizon europe research and innovation programme under grant agreement no 101094738. conflict of interest statement the authors declare that they have no conflicts of interest. references ecpgr (2021). plant genetic resources strategy for europe (rome, italy: european cooperative programme for plant genetic resources). url: https: //bit.ly/pgrstrategy. ecpgr (2024). cgn, the netherlands sets precedent with public access to standard operating procedures. url: https://www.ecpgr.org/resources/ latest-news/news-detail/cgn-the-netherlands-setshttps://bit.ly/pgrstrategy https://bit.ly/pgrstrategy 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(2021). a performance management system for longterm germplasm conservation in cgiar genebanks: aiming for quality, efficiency and improvement. plants 10(2627). doi: https://doi.org/10.3390/plants10122627 pro-grace (2024). promoting a plant genetic resources community for europe. url: https://www. grace-ri.eu/pro-grace. accessed date: 2024-10-22 van hintum, t. j. l. and hazekamp, t. (1993). cgn genebank protocol. march 1993. centre for plant breeding and reproduction research (cprodlo), centre for genetic resources, the netherlands, wageningen, the netherlands 15p. wijnker, e., bouchaut, d., van treuren, r., and van hintum, t. 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https://www.fao.org/plant-treaty/areas-of-work/the-multilateral-system/smta/en/ https://www.fao.org/plant-treaty/areas-of-work/the-multilateral-system/smta/en/ https://www.iso.org/standard/62085.html https://www.iso.org/standard/62085.html https://www.iso.org/standard/67888.html https://www.iso.org/standard/67888.html https://www.iso.org/quality-management#toc1 https://www.iso.org/quality-management#toc1 https://doi.org/10.3390/plants10122627 https://www.grace-ri.eu/pro-grace https://www.grace-ri.eu/pro-grace https://doi.org/10.1007/s10722-024-02019-x https://doi.org/10.1007/s10722-024-02019-x introduction quality management in a genebank environment quality management quality planning quality control quality improvement quality management at the centre for genetic resources, the netherlands (cgn) setting up cgn's quality management system costs and benefits a genebank certification system rationale for a genebank certification system components of a certification system discussion conclusions acknowledgements conflict of interest statement original article genetic resources (2025), 6 (11), 115–121 doi: 10.46265/genresj.oadz7911 https://www.genresj.org issn: 2708-3764 genebank peer reviews: a powerful tool to improve genebank quality and promote collaboration theo van hintum a, sharon balding b, gergana desheva c, john dickie b, maŕıa josé dı́ezd, luis guasch e, pavol hauptvogel f, vojtěch holubec g, dagmar janovská g, ulrike lohwasserh, isaura mart́ın e, ludmila papoušková g, beate schierscher-viret i, lise lykke steffensen j, katya uzundzhalieva c, patrizia vaccino k and josé vicente valcárceld a centre for genetic resources, the netherlands (cgn), wageningen university and research (wur), wageningen, the netherlands b millennium seed bank (msb), royal botanic gardens kew, wakehurst place, ardingly, uk c agricultural academy, institute of plant genetic resources ”konstantin malkov” (ipgr), sadovo, bulgaria d institute for the conservation and improvement of the valentian agrodiversity (comav), polytechnic university of valencia, spain e national institute for agricultural and food research and technology (inia-csic), spanish plant genetic resources centre (crf), alcalá de henares, madrid, spain f research institute of plant production (ripp), national agricultural and food centre (nppc), piěštany, slovakia g czech agrifood research center (carc), ruzyně, prague, czech republic h leibniz institute of plant genetics and crop plant research (ipk), gatersleben, germany i agroscope, changins, nyon, switzerland j nordic genetic resource center (nordgen), alnarp, sweden k research center for cereal and industrial crops (crea-ci), council for agricultural research and economics (crea), vercelli, italy abstract: the conservation of plant genetic resources (pgr) is critical to ensuring global food security and agricultural sustainability. genebanks play a vital role in ex situ conservation, complementing in situ strategies by preserving crop diversity (incl. their wild relatives) and providing access to biological materials for research, breeding and farming. however, maintaining high conservation standards and ensuring accessibility remains a global challenge. to address this, the ‘genebank peer review’ system was developed as a collaborative quality assessment and improvement mechanism. this system facilitates reciprocal evaluations among genebanks, promoting transparency, capacity building and continuous improvement in conservation practices. implemented in europe since 2019, the peer review process involves structured self-assessments, site visits and expert evaluations, culminating in publicly available reports that guide genebanks in enhancing their operations. feedback from participating institutions highlights the system’s effectiveness in fostering knowledge exchange, strengthening professional networks and improving genebank management practices. despite its success, challenges remain, particularly regarding expert availability and resource constraints. future efforts should focus on institutionalizing mentorship programmes to sustain and expand the impact of genebank peer reviews and monitor improvements. keywords: crop diversity, ex situ conservation, plant genetic resources, peer review, quality improvement citation: van hintum, t., balding, s., desheva, g., dickie, j., d́ıez, m. j., guasch, l., hauptvogel, p., holubec, v., janovská, d., lohwasser, u., mart́ın, i., papoušková, l., schierscher-viret, b., steffensen, l. l., uzundzhalieva, k., vaccino, p., valcárcel, j. v. (2025). genebank peer reviews: a powerful tool to improve genebank quality and promote collaboration. genetic resources 6 (11), 115–121. doi: 10.46265/genresj.oadz7911. © copyright 2025 the authors. this is an open access article distributed under the terms of the creative commons attribution license (cc by 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. received: 07.03.2025 accepted: 20.05.2025 published online: 24.06.2025 https://www.genresj.org https://www.doi.org/10.46265/genresj.oadz7911 https://www.genresj.org https://www.doi.org/10.46265/genresj.oadz7911 116 van hintum et al genetic resources (2025), 6 (11), 115–121 introduction to ensure a sufficient food supply for the growing global population, it is crucial to conserve plant genetic resources (pgr) and make them accessible for crop research, plant breeding and cultivation, both now and in the future. since the 1960s, genebanks have been established to complement traditional in situ conservation methods (engels and ebert, 2021). this shift from in situ to ex situ was necessary due to changes in agriculture, land use, and environmental conditions that threatened to erode the in situ biodiversity. genebanks offer several advantages over in situ conservation, including improved access to both information and biological material. however, the task of conserving pgr is huge, and no single genebank or country can manage it alone. it requires a global effort with contributions from various actors. many research institutions involved in plant breeding have established genebanks, and most countries maintain a national genebank or a network of genebanks, often linked to national breeding programmes. according to the food and agriculture organization (fao, 2025), in 2022 the global genebank network conserved approximately 5.9 million accessions across 871 genebanks composed of 13 international genebanks (such as those managed by cgiar), 6 regional and the remaining 852 national genebanks – more than half of them in europe. the global effort to conserve pgr is supported by international treaties and collaborations, such as the international treaty on plant genetic resources for food and agriculture (itpgrfa, (fao, 2009)), which aims to ensure the conservation and sustainable use of pgr and the fair sharing of benefits arising from their use. this collaborative approach is essential for maintaining the genetic diversity needed to adapt to future agricultural challenges and ensure food security. obviously, not every genebank intends to contribute to the ‘global effort’. included in the 871 genebanks are many local – what could be called ‘working collections’, not meeting any standards (fao, 2014) in terms of conservation practice or access. for example, only 123 out of the 852 genebank deposited seeds at the svalbard global seed vault (https://www.seedvault.no/). this could be due to well-considered decisions, but in most cases, it may be a lack of awareness, with resulting shortcomings in procedures. to be considered part of the ‘global effort’ a genebank should have adopted two principles: (1) the material should be properly conserved and (2) the material should be accessible for use. the first principle, proper conservation, is a technical issue. obviously one can disagree about the technical details, such as the frequency of germination tests, the population size of regenerations, and the need to triplicate at the svalbard global seed vault, but the objective is clear. technical disputes are about the balance between security (genetic integrity and seed quality) and costs: the higher the security, the higher the costs per unit and as a result the lower the number of units that can be conserved. the second principle, access, is not only technical, but also has a political dimension. on the technical side, one could ask what information should be provided about the material, what percentage of the material conserved should be readily available for distribution, or what a genebank should be willing to do, to get the material to the requestor (in terms of plant passports, import permits, non-gmo statements, etc.). on the political side, it can be more complex. considerations will have to take account of access and benefit-sharing policies: who is allowed to receive material and under what conditions? fortunately, there is a broad base of experience to draw on. regarding the conservation issues, there are the fao genebank standards (fao, 2014) and extensive experience from genebanks. regarding the access issues, there are the itpgrfa, the eu regulation on the nagoya protocol (eu, 2014) and organizations such as the european cooperative programme for plant genetic resources (ecpgr) setting standards or proposing best practices (https://www.ecpgr.org/). in the domain of genebank performance monitoring, a certain level of experience has been accumulated within the community. a number of genebanks have adopted the iso 9001 standard for quality management, thereby ensuring adherence to established protocols, effective risk management, and the implementation of continuous quality improvement mechanisms, including procedures for addressing user feedback and complaints. additionally, the global crop diversity trust has developed the genebank quality management system (gqms) and has provided support for its implementation across cgiar genebanks (lusty et al, 2021). nevertheless, the establishment and maintenance of such systems entail substantial investments, both from the genebanks themselves and from the entities responsible for review and auditing (see also van hintum and wijnker (2024)). in a situation where the genebank community has community standards, the challenge is to ensure that the members of this community meet those standards, especially where funds are scarce. some genebanks will already meet the standards to a certain extent, while others will need changes in protocols or even additional equipment or staff. in the attempt to improve the quality of operations in the community, the obvious first step is to determine the status quo. the members of the community, the genebanks, need to determine how they are doing, and identify where improvements can or should be made. for this reason, the ‘genebank peer review’ system was designed to create clarity and transparency about the status of genebanks and to build capacity to improve collections’ status where needed and possible. initially, this peer-review system is aimed at the european national genebanks wanting to be part of the global effort to conserve pgr for future generations and make it available to the current. genetic resources (2025), 6 (11), 115–121 genebank peer reviews 117 history of the genebank peer review system the pgr community in europe is very heterogeneous, consisting of hundreds of genebanks (fao, 2025), with a huge range of objectives, sizes and methodologies. ideally, europe could set up a system similar to the usda national plant germplasm system (https://ww w.ars-grin.gov/npgs/), i.e. including some central and some sub-regionally specialized facilities, with proper coordination, quality management and a clear policy regarding access. ecpgr, as the collaborative umbrella organization for european countries, has made efforts to address the heterogeneous and fragmented landscape but has yet to achieve significant improvements in coordination. initiatives such as aegis (www.ecpgr.o rg/aegis), a catalogue of pgr managed according to standards in various participating european genebanks, were promising but still need major steps forward to become effective (van hintum et al, 2021). during an ecpgr meeting about ’assessing current practices and procedures to strengthen aegis’ held in madrid in december 2018, a plan was presented to improve transparency and build capacity in the european genebank community based on mutual visits of genebank staff members (engels et al, 2019). this plan – called ‘improving pgr conservation and access in europe; a plan to create a voluntary genebank’s peer review system’ – was prepared and presented by the centre for genetic resources, the netherlands (cgn), based on its involvement in the reviewing process organized by the global crop diversity trust coordinating and supporting the operations of the cgiar genebanks (lusty et al, 2021). the plan was received positively and it was decided that: “peer review proof of concept will be tested (colleague experts visiting, discussing and learning from each other – not an auditing or policing exercise)” and that “under the coordination of cgn, first peer-review cycle will be completed in early 2019”. in the first months of 2019, cgn formulated protocols for peer reviews, including elements of aquas, the quality management system of aegis (ecpgr, 2025a). in the period from february to april 2019, a pilot cycle of peer reviews was organized, partly in the framework of the eu project genres bridge (https://www.genresbrid ge.eu/). this project aimed “to strengthen conservation and sustainable use of genetic resources by accelerating collaborative efforts and widening capacities in plant, forest and animal domains”. the pilot cycle involved the ihar-bip genebank in radzików, poland, the comav genebank in valencia, spain and cgn in wageningen, the netherlands. after each visit a short report was written with observations and recommendations, and after the complete cycle, a summary of experiences and conclusions was written and made public on the ecpgr website (van hintum et al, 2019). based on this pilot cycle of reviews, the protocols for the peer reviews were further improved and the concept was used in another eu-funded project, agent (activated genebank network, https://agent-project.e u/). this project aimed “to unlock the full potential of biological material stored in gene banks worldwide by utilizing fair international data standards and an open digital infrastructure for managing plant genetic resources”. in this context another four cycles of reviews were organized in the period 2022–2024, with 12 reviews involving eleven genebanks; cgn was involved in two cycles. the concept of genebank peer reviews the fundamental principle of genebank peer reviews is straightforward: within a cycle of three reciprocal visits, experienced staff members from two genebanks evaluate the facilities and operations of a third institution. these visits are preceded by a self-assessment conducted by the host genebank, the results of which are made available to the reviewers. during the review process, the visiting experts are granted full access to relevant information. following the evaluation, the reviewers compile a report, which is subsequently published online, contingent upon the consent of the reviewed genebank. further details, primarily based on the genres bridge report on this activity (genres bridge, 2021), are provided below. objectives the primary objective of the peer review process is to provide a comprehensive description of the management, facilities and procedures of a genebank and evaluate them. this serves two key purposes: (1) enabling expert colleagues to offer technical feedback on these aspects, and, when necessary, (2) facilitating the development of an expert-driven improvement plan. such an improvement plan, informed by the review report, may be utilized in fundraising efforts. the transparency fostered by these peer reviews is expected to benefit the pgr community by identifying both strengths and weaknesses within the pgr management infrastructure. this, in turn, will allow for the optimization of strong points and the implementation of targeted measures to address areas requiring improvement. the peer-review process aims to assess all pertinent aspects of the genebank under evaluation, including management, facilities and procedures, within a 2-to-3day visit. preparation the initial phase of the process entails identifying appropriate partners and systematically planning the reviews, as detailed below: • three participating genebanks are selected. this selection is based on the context of the reviews, i.e. the genebanks participating in the project that organizes the peer reviews, and obviously the enthusiasm of the genebanks to be included. • each genebank designates a qualified representative who will actively participate in all three 118 van hintum et al genetic resources (2025), 6 (11), 115–121 reviews. this individual should possess extensive knowledge of their respective genebank and expertise in pgr management. the representative will serve as the primary contact throughout the review process. in exceptional cases, a genebank may appoint two representatives if warranted. • tentative dates for the three reviews are determined, with each review expected to last between two and three days, depending on the complexity of the operations. for each review: • the reviewed genebank provides, when available, technical information relevant to its operations, including management structures, facilities and procedures. this is achieved through the completion of the operational genebank manual, which should be compiled using the template provided by ecpgr (2010), or a version slightly expanded version for the peer reviews (see supplemental material 1). • reviewers may request specific data to facilitate their preparatory work. • the reviewed genebank prepares a draft agenda, allowing reviewers to provide feedback and propose amendments beforehand. • the reviewed genebank arranges accommodation for the reviewers and covers the associated costs. • reviewers organize and finance their own travel to the genebank’s country. review process • the reviewed genebank assumes responsibility for organizing local transportation during the review and covering related expenses. • the reviewers are granted full access to the genebanks records, staff and facilities upon request. should any restrictions apply, these must be formally documented along with a rationale. • throughout the visit, the reviewers engage in comprehensive discussions and visits encompassing all relevant aspects of the genebank’s operations, including those outlined in the operational genebank manual. additionally, broader issues such as funding stability, management structures and organizational effectiveness are examined. a checklist of key discussion points is available (see supplemental material 2). • the review concludes with a final session dedicated to discussing the observations made by the review team. post-review process • the reviewers prepare a preliminary report summarizing their findings, structured according to the checklist of discussion points. • the reviewed genebank is given an opportunity to verify the factual accuracy of the report and propose necessary corrections. • the reviewers finalize the report and provide it to the reviewed genebank for internal use in improvement planning and fundraising efforts. • the reviewed genebank determines which sections of the report should remain confidential. the public version of the report will reference restricted sections, allowing interested parties to request additional information bilaterally. • the public report is made available to relevant stakeholders including colleague genebanks and funding agencies (ecpgr, 2025b). additional considerations the review process is designed to be collaborative and founded on mutual trust. the purpose of the review is not to critique individuals but rather to enhance the efficiency and management of the reviewed genebank and exchange knowledge. consequently, review teams should be kept small, and all activities beyond the core review tasks should be conducted collectively. furthermore, the proposed cost-sharing arrangement – whereby the host institution covers all local expenses while visiting team members are responsible only for their travel costs to the host country – aims to alleviate financial burdens, particularly for genebanks located in regions with lower cost levels. experiences and lessons learned the concept of genebank peer reviews was formulated in 2019, and since then five cycles of three visits each have been organized in europe in the frame of genres bridge and agent projects, involving 12 genebanks (cgn was involved in three cycles, ihar in two). an overview of the reviews is given in table 1. when asked to evaluate their experiences with the genebank peer review, the feedback from the genebanks involved was predominantly positive. the quotes in this paragraph are from this feedback and the evaluation of the first pilot cycle of reviews (van hintum et al, 2019). many participants initially needed to familiarize themselves with the concept of genebank peer reviews. as one participating genebank noted, “although the initial impression was merely that of undergoing an evaluation process to assess the work carried out at the bank, once the process began, the perception shifted to viewing it as an opportunity to receive guidance from personnel with extensive experience in managing germplasm collections.” the process of developing the operational genebank manual was widely regarded as a constructive exercise, offering new insights into the genebank’s operations – insights that some genebank managers had not previously considered. however, for genebanks that already had established quality management systems (van hintum and wijnker, 2024), the exercise was sometimes perceived as redundant, as it primarily involved restructuring existing quality manuals to fit the operational genebank manual format. genetic resources (2025), 6 (11), 115–121 genebank peer reviews 119 table 1. the genebank peer reviews performed in the period 2019–2024. for the reports and additional information see https:// www.ecpgr.org/aegis/aquas/peer-visits. insitute of reviewed genebank location visited date review visit reviewers centro de conservación y mejora de la agrodiversidad valenciana (comav) valencia, spain 7-8 february 2019 theo van hintum (cgn), wieslaw podyma (ihar-pib) centre for genetic resources, the netherlands (cgn) wageningen, the netherlands 6-8 march 2019 wieslaw podyma (ihar-pib), maŕıa josé d́ıez & josé vicente valcárcel (comav) national center for plant genetic resources (ihar-pib) radzików, poland 16-17 april 2019 maŕıa josé d́ıez & josé vicente valcárcel (comav), theo van hintum (cgn) crop research institute (cri) prague, czech republic 12-13 may 2022 pavol hauptvogel (ripp), ulrike lohwasser (ipk), theo van hintum (cgn) leibniz institute of plant genetics and crop plant research (ipk) gatersleben, germany 19-20 july 2022 dagmar janovská & ludmila papoušková (cri ), pavol hauptvogel & iveta čičová (ripp) research institute of plant production (ripp) pieš̌tany, slovakia 23-24 august 2022 dagmar janovská & ludmila papoušková & vojtěch holubec (cri), ulrike lohwasser (ipk) centro nacional de recursos fitogenéticos (crf) madrid, spain 7-8 july 2022 katya uzundzhalieva & gergana desheva (ipgr), theo van hintum (cgn) centre for genetic resources, the netherlands (cgn) wageningen, the netherlands 19-20 july 2022 isaura martin & luis guasch (crf), katya uzundzhalieva & gergana desheva (ipgr, bulgaria) institute of plant genetic resources ’konstantin malkov’ ipgr) sadovo, bulgaria 6-7 october 2022 luis guasch & isaura mart́ın (crf), theo van hintum (cgn) nordic genetic resource center (nordgen) alnarp, sweden 29-30 june 2023 john dickie (msb), theo van hintum (cgn) millennium seed bank (msb) ardingly, uk 6-7 july 2023 theo van hintum (cgn), lise lykke steffensen (nordgen) centre for genetic resources, the netherlands (cgn) wageningen, the netherlands 21-22 september 2023 lise lykke steffensen (nordgen), john dickie & sharon balding (msb) national center for plant genetic resources (ihar-pib) radzików, poland 21-23 october 2024 beate schierscher-viret (agroscope), patrizia vaccino (crea-ci) research center for cereal and industrial crops (crea-ci) vercelli, italy 23-24 september 2024 maja boczkowska (ihar), beate schierscher-viret (agroscope) agroscope changins (agroscope) nyon, switzerland 25-26 september 2024 patrizia vaccino (crea-ci), maja boczkowska (ihar) the cycle of mutual visits was unanimously regarded as a worthwhile investment of both effort and time. “as a host, although the initial sensation was that our genebank was under evaluation, this feeling disappeared as soon as the review started because it was carried out in a friendly atmosphere and we quickly realized that we could take profit of many of the reviewers’ suggestions.” the feedback provided by reviewing peers was generally perceived as constructive and encouraging. “the opinion of colleagues that are dealing with equivalent responsibilities and problems in another country has given us a better understanding of the strengths and weaknesses of our institution and has served as a starting point for identifying areas of improvement.” moreover, for the reviewing genebanks, the opportunity to closely examine a colleague’s genebank proved to be a valuable learning experience. observing different practices and methodologies served as a source of inspiration, stimulating improvements in approaches and protocols within their own institutions. furthermore, it strengthened the contacts between genebank managers, or as formulated by one of the participating genebanks: “the two days spent at each genebank not only offered us an opportunity for indepth discussions on plant genetic resources with a team of experts but also provided an opportunity to strengthen professional relationships with colleagues committed to the same field. we were truly aware of the advantages of being part of a genebank community.” 120 van hintum et al genetic resources (2025), 6 (11), 115–121 the reports contained between 2 and 21 recommendations by the reviewers. the recommendations addressed very practical issues such as “in order to have unique identifiers for the accessions digital object identifiers (dois) should be implemented.” or “consider establishing a lower ceiling to the amount of seeds to be stored of one accession to avoid unnecessary use of space in the -18°c storage room.” to policy-oriented recommendations such as “consider the possibility of introducing handling fees to reduce the requests of hobby growers as the genebank seeds are too expensive to distribute to that category.” the reports are available on the peer review website hosted by ecpgr (ecpgr, 2025b). assessing the impact of follow-up actions following the review cycles is challenging, as numerous additional factors have also influenced the development of the genebanks involved. most genebanks utilized the reports to set priorities for their activities and/or to advocate for funding to support specific infrastructural improvements. additionally, the publicly available operational genebank manuals and review reports (available on the ecpgr/aquas website (ecpgr, 2025a)) may have provided valuable insights to other genebanks, potentially inspiring improvements in their operations. at a broader level, these reviews and resulting resources can be expected to have also contributed to increased transparency in genebank practices and methodologies. however, the extent of this influence remains difficult to quantify, although the overall impression is very positive. when asked, one of the genebanks concluded “after several years, participating in the peer review process has led to a substantial improvement in the germplasm bank”, a conclusion that is shared by most participating genebanks. discussion and conclusions the conclusions that can be drawn after 15 genebank peer reviews, are largely consistent with those derived from the initial cycle of reviews (van hintum et al, 2019). firstly, these peer reviews have demonstrated their cost-effectiveness as a means of enhancing the quality of genebank operations. beyond offering a comprehensive evaluation of various operational aspects, the reviews also play a vital role in motivating and inspiring staff members, thereby promoting continuous improvement. the reviewers, in general, expressed appreciation for the process, particularly valuing the opportunity to observe and discuss the operations of their colleague genebanks. the social aspect of briefly working alongside international colleagues was also highly regarded. the functioning of the review teams was generally effective. it became evident that, ideally, one of the genebanks involved should be a well-established institution, capable of serving as an inspiration for others. the review cycle, which involved three genebanks and two or three reviewers for each review, was particularly successful as it allowed for a sufficient level of intimacy, ensuring both confidentiality and transparency. the reviewers were typically sufficiently senior and experienced, enabling them to critically assess their colleagues’ work. the duration of most reviews ranged between one and a half to two days, a timeframe that was deemed short but appropriate. shorter reviews risked remaining superficial, while longer reviews would have allowed for more in-depth feedback but would also consume more time from the experts involved and thus resources. the self-assessment process, particularly the preparation of the operational genebank manual prior to the review, emerged as a crucial component in fostering transparency and expanding the hosting experts’ own understanding of the procedures within the genebank. in some instances, this process revealed issues that had previously gone unnoticed by the genebank manager. since the manual does not refer to genebank standards explicitly, the evaluation of the procedures in the context of e.g. the fao genebank standards (fao, 2014) was one of the aspects of the review. the genebank peer review approach holds potential for further development into a comprehensive tool not only for enhancing but also for sustaining the quality of genebank operations. to this end, it could be formally integrated into the quality management systems of genebanks, contingent upon its institutionalization within the framework of ecpgr or a comparable overarching body, potentially extending beyond the european context. such institutionalization would necessitate stable financial support and the establishment of a semipermanent pool of expert reviewers. moreover, the systematic follow-up on review recommendations could be embedded as a structural component of genebank quality management, including formalized reporting mechanisms. however, these developments may affect the currently appreciated informal character of the peer review process. at a broader level, the outcomes of genebank reviews could serve as a valuable resource for informing the prioritization of funding initiatives aimed at strengthening the global system for the conservation of plant genetic resources. overall, the genebank peer reviews have had a significant positive impact on the quality of the genebanks involved and have strengthened the connections between genebank experts. they were considered by the reviewers as a cost-effective tool for quality improvement. however, a key challenge remains the reliance on the availability of senior experts, particularly those from well-established institutions. setting up a pool of experienced genebank experts to ‘mentor’ the reviews could be a solution. supplemental data supplemental material 1: template for operational genebank manual supplemental material 2: checklist of key discussion points https://www.genresj.org/index.php/grj/article/view/genresj.oadz7911/suppdata280 https://www.genresj.org/index.php/grj/article/view/genresj.oadz7911/suppdata280 genetic resources (2025), 6 (11), 115–121 genebank peer reviews 121 acknowledgments the author(s) declare financial support was received for the research, authorship, and/or publication of this article. this work was supported by two projects that received funding from the european union’s horizon 2020 research and innovation programme: the genres bridge project under grant agreement no. 817580 and the agent project under grant agreement no. 862613. the authors wish to express their gratitude to their colleagues in the genebanks for their invaluable support in managing daily operations and ensuring the effective functioning of these institutions. their willingness to provide full transparency regarding protocols, activities, and experiences during the review process was instrumental in shaping the foundation of this study. author contributions tvh developed the concept of the paper, all other authors commented on the draft and contributed to various extents to the writing. conflict of interest statement the authors declare that they have no conflicts of interest. 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(2024). quality management in a genebank environment: principles and experiences at the centre for genetic resources, the netherlands (cgn). genetic resources (s2), 6–12. doi: https://doi.org/10.46265/genresj.rfxb3570 https://www.ecpgr.org/fileadmin/templates/ecpgr.org/upload/aegis/documents/procedures_examples/template_for_the_preparation_of_operational_genebank_manuals.doc https://www.ecpgr.org/fileadmin/templates/ecpgr.org/upload/aegis/documents/procedures_examples/template_for_the_preparation_of_operational_genebank_manuals.doc https://www.ecpgr.org/fileadmin/templates/ecpgr.org/upload/aegis/documents/procedures_examples/template_for_the_preparation_of_operational_genebank_manuals.doc https://www.ecpgr.org/fileadmin/templates/ecpgr.org/upload/aegis/documents/procedures_examples/template_for_the_preparation_of_operational_genebank_manuals.doc https://www.ecpgr.org/aegis/aquas/overview https://www.ecpgr.org/aegis/aquas/peer-visits https://doi.org/10.3390/plants10081557 https://www.ecpgr.org/fileadmin/bioversity/publications/pdfs/aegis_workshop_report_-_final_01_03_2019.pdf https://www.ecpgr.org/fileadmin/bioversity/publications/pdfs/aegis_workshop_report_-_final_01_03_2019.pdf https://www.ecpgr.org/fileadmin/bioversity/publications/pdfs/aegis_workshop_report_-_final_01_03_2019.pdf https://eur-lex.europa.eu/legal-content/en/txt/pdf/?uri=celex:32014r0511 https://eur-lex.europa.eu/legal-content/en/txt/pdf/?uri=celex:32014r0511 https://www.fao.org/3/i0510e/i0510e.pdf https://openknowledge.fao.org/server/api/core/bitstreams/612be5af-72cc-4017-afc2-936e9be6c6ed/content https://openknowledge.fao.org/server/api/core/bitstreams/612be5af-72cc-4017-afc2-936e9be6c6ed/content https://openknowledge.fao.org/server/api/core/bitstreams/612be5af-72cc-4017-afc2-936e9be6c6ed/content https://www.fao.org/wiews https://www.genresbridge.eu/fileadmin/templates/genres/uploads/documents/reports/d.3.5.pdf https://www.genresbridge.eu/fileadmin/templates/genres/uploads/documents/reports/d.3.5.pdf https://doi.org/10.3390/plants10122627 https://doi.org/10.3390/plants10122627 https://www.ecpgr.org/fileadmin/templates/ecpgr.org/upload/aegis/peer_reviews/pilot_genebank_peer_reviews_-_observations_and_conclusions_rev.pdf https://www.ecpgr.org/fileadmin/templates/ecpgr.org/upload/aegis/peer_reviews/pilot_genebank_peer_reviews_-_observations_and_conclusions_rev.pdf https://www.ecpgr.org/fileadmin/templates/ecpgr.org/upload/aegis/peer_reviews/pilot_genebank_peer_reviews_-_observations_and_conclusions_rev.pdf https://doi.org/10.3390/plants10102165 https://doi.org/10.3390/plants10102165 https://doi.org/10.46265/genresj.rfxb3570 introduction history of the genebank peer review system the concept of genebank peer reviews objectives preparation review process post-review process additional considerations experiences and lessons learned discussion and conclusions supplemental data acknowledgments author contributions conflict of interest statement original article genetic resources (2025), 6 (12), 26–38 doi: 10.46265/genresj.zdew3901 https://www.genresj.org issn: 2708-3764 received: 14.03.2025 | accepted: 11.06.2025 | published online: 18.08.2025 phenotypic variability of smallanthus sonchifolius germplasm of peru abstract: smallanthus sonchifolius (yacon) is a functional food native to the south american andes. its tuberous root and leaves are the main parts consumed; however, few studies have been carried out on its phenotypic variability. this study aimed to characterize 214 yacon accessions from the germplasm bank of the instituto nacional de innovación agraria (inia), peru. twelve qualitative and seven quantitative variables were used. accession y-74 showed the largest leaf dimensions, while y-28 showed the highest productivity per plant. multiple correspondence analysis and principal component analysis revealed that the variables propagule color, leaf shape, root pulp color, leaf length and width, root weight per plant, and yield contributed significantly to the discrimination and identification of promising accessions. the geographical grouping of the accessions showed differences between accessions from the north and south of peru. the qualitative phylogenetic tree showed 12 morphological groups discriminated mainly by leaf morphology and root characteristics, while the dendrogram analysis identified four clusters, with cluster ii standing out with an average yield of 73.5t/ha of tuberous roots. these results are important, as they allowed the identification of promising accessions and useful traits that can contribute to improving productivity and promoting the expansion of yacon cultivation at national and international levels. keywords: germplasm, phenotypic, functional food, yacon, andes citation: santa cruz-padilla, a. e., vásquez-orrillo, j. l., rodríguez lópez, s. y., eugenio leiva, a., bardales-lozano, r. m., seminario, j. f. and murga-orrillo, h. (2025) “phenotypic variability of smallanthus sonchifolius germplasm of peru”, genetic resources, 6(12), pp. 26–38. doi: 10.46265/genresj.zdew3901 © copyright 2025 the authors. this is an open access article distributed under the terms of the creative commons attribution license (cc by 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. angel esteban santa cruz-padilla*, a, jorge luis vásquez-orrillo*, a, silvia yanina rodríguez lópeza, araceli eugenio leivaa, ricardo manuel bardales-lozanob, juan f. seminarioc and hipolito murga-orrillod a instituto nacional de innovación agraria (inia), estación experimental agraria baños del inca, subdirección de recursos genéticos, jirón. wiracocha s/n, 06004, los baños del inca, perú b universidad nacional de la amazonía peruana (unap), departamento académico de suelos y cultivos, facultad de agronomía, jirón nauta, 16002, iquitos, perú c universidad nacional de cajamarca. facultad de ciencias agrarias, programa de raíces y tubérculos andinos. av. atahualpa 1050. c.p. 06003. cajamarca, cajamarca, perú d universidad nacional autónoma del alto amazonas (unaaa). escuela profesional de ingeniería agrónoma, prol. libertad 1220 1228 yurimaguas, alto amazonas, loreto, perú * corresponding authors: angel esteban santa cruzpadilla (asantacruz@inia.gob.pe), jorge luis vásquezorrillo (jorge.vasquez.orrillo@gmail.com) introduction smallanthus sonchifolius, known as yacon, is a perennial species native to the andes of south america (caballero and colonia, 2018; de sales et al, 2021). it is cultivated from venezuela to northern argentina, between 900 and 3,500masl (huaycho et al, 2016). however, its remarkable plasticity has facilitated its adaptation to climates outside the andes (seminario et al, 2003; mansilla et al, 2006; wagner et al, 2019), in countries such as the czech republic, the united states, brazil (quaresma et al, 2020), new zealand and germany (lachman et al, 2007). in the peruvian andes, tuberous root yields vary from 8 to 96t/ha depending on the genotype (santa cruz and vásquez-orrillo, 2023). yacon maintains a historical and cultural value, as it has been an important functional food for andean populations since pre-columbian times (huaycho et al, 2016; loperamarín, 2020). the roots and leaves have benefits for human health. the roots are usually eaten fresh, and the leaves in infusions (lebeda et al, 2004; moreira et al, 2020). the health benefits of yacon are due to its antioxidant, antimicrobial, hypolipidemic, antidiabetic and even anticancer properties https://www.genresj.org mailto:https://doi.org/genresj.zdew3901?subject= mailto:asantacruz%40inia.gob.pe?subject= mailto:jorge.vasquez.orrillo%40gmail.com?subject= https://doi.org/10.46265/genresj.zdew3901 genetic resources (2025), 6(12), 26–38 yacon characterization in peru 27 (baek et al, 2018; myint et al, 2019; adriano et al, 2019; minchola-castañeda et al, 2022). the consumption of extracts from the leaves and tuberous roots of yacon regulates the glycemic state and increases the concentration of insulin in the blood (santos et al, 2017; ferraz et al, 2020); it also contributes to the reduction of body weight (honoré et al, 2018). in this context, the therapeutic benefits of yacon highlight the need to conserve and document the genetic heritage of this species (garcía et al, 2022; wagner et al, 2019). yacon germplasm exhibits phenotypic variability among different accessions. variations in shape, weight and oligofructan content have been revealed in tuberous roots (valentová et al, 2006), and differences in isoenzymes and phenolic content in leaves (valentová et al, 2006; mansilla et al, 2006). morphological characterization has identified multiple morphotypes and ecotypes of both cultivated and wild yacon (polanco and garcía, 2013; ignacio et al, 2017). genetic diversity analysis using molecular markers has shown distinct groupings among accessions, with the highest diversity observed in central peru (mansilla et al, 2006). variations in reproductive biology, including flowering time and pollen viability, have also been reported among accessions (mansilla et al, 2010). furthermore, studies have found differences in total phenolic content, antioxidant activity and chemical composition among local yacon phenotypes (lachman et al, 2007; russo et al, 2015). this phenotypic variability of yacon makes it a valuable resource for breeding programmes and agroindustrial applications. in peru, the instituto nacional de innovación agraria (inia) conserves yacon germplasm from 11 regions distributed throughout the andes, currently counting 214 accessions. this diversity has highlighted the need to update phenotypic characterization studies. in this context, it is hypothesized that this germplasm has significant phenotypic variability, which will allow the identification of accessions with superior agronomic characteristics, suitable for use in future genetic improvement programmes. this study aimed to characterize the phenotypic variability of 214 yacon accessions from the inia germplasm bank, conserved at the estación experimental agraria baños del inca, cajamarca, peru. materials and methods plant material the study was carried out between june 2021 and march 2022. a total of 214 yacon accessions were used, originating from the regions of piura, cajamarca, amazonas, la libertad, ancash, pasco, junín, cusco, ayacucho, apurímac and puno (figure 1 and supplemental table 1) and conserved ex situ since 1986. these accessions are part of the andean roots germplasm bank of inia, estación experimental agraria baños del inca, cajamarca, peru. figure 1. map of collecting locations of 214 accessions of smallanthus sonchifolius collected in 11 regions of peru and conserved in the inia germplasm bank. genetic resources (2025), 6(12), 26–3828 santa cruz-padilla et al experimental area in the inia cochamarca experimental annex, located at 7.28137 s, 78.21987 w and 2,835masl, in the province of san marcos, cajamarca region (figure 1). the experiment was installed in an area of 1,796m2, prepared to a depth of 30cm, where island guano (1,200kg/ha), diammonium phosphate (164kg/ha) and potassium chloride (73kg/ha) were applied before sowing. each experimental unit had an area of 6.3m2 (0.9m x 7m). planting was done in a systematic way (each accession in a row), at distances of 0.90m between rows and 0.50m between plants (22,220 plants per hectare). each experimental unit consisted of 15 plants, 10 plants being evaluated and 5 plants as borders. soil analysis of the experimental plot indicated the following: ph of 5.7, organic matter 2.62%, 33.39ppm of phosphorus and 165ppm of potassium, and sandy loam texture (laboratorio de suelos, aguas, abonos y foliares de la estación experimental agraria baños del inca inia). during this period of study execution, a mean temperature of 14.3ºc, a minimum of 7.7ºc and a maximum of 20.8ºc were recorded; and the mean monthly rainfall was 117.9mm (senamhi, 2024). qualitative and quantitative descriptors used in characterization twelve qualitative morphological descriptors and seven quantitative ones were used (table 1), which were assessed over several agricultural campaigns. colors were defined using the royal horticultural society colour chart (rhs, 2001). table 1. qualitative and quantitative descriptors used in the characterization of yacon accessions descriptor type descriptor acronym period of assessment qualitative secondary stem color and its distribution sscd preflowering stem branching sbra preflowering pigmentation of the vein on the underside of the leaf pvul preflowering leaf blade shape lbsh at 50% flowering shape of the leaf base stlb at 50% flowering leaf blade edge lbed at 50% flowering ray flower shape rfsh at 50% flowering petal tooth slit depth ptsd at 50% flowering storage root surface color srsc at harvest flesh color of the storage root fcsr at harvest clefts in the storage roots csro at harvest color of the propagules copr at harvest quantitative number of stems per plant nspl at the end of flowering plant height (cm) plhe at the end of flowering leaf length (cm) lele at 50% flowering leaf width (cm) lewi at the end of flowering weight of storage roots per plant (kg) wsrp at harvest number of storage roots per plant nsrp at harvest yield of storage roots (t/ha) yosr at harvest data analysis the characterization data were subjected to multivariate statistical analyses. initially, the overall structure of the dataset was explored using a factor analysis of mixed data (famd), which simultaneously integrated both qualitative and quantitative traits. to further investigate specific patterns, a multiple correspondence analysis (mca) was applied to the qualitative variables, and a principal component analysis (pca) to the quantitative ones. in addition, associations among the quantitative traits were assessed using pearson’s correlation coefficient. to define phenotypically differentiated groups among the accessions, a hierarchical cluster analysis was performed, employing euclidean distance as the dissimilarity measure and the complete linkage method for clustering. the optimal number of clusters was determined by inspecting the resulting dendrograms, selecting the cut-off point that maximized within-group homogeneity. the biological validity of the clusters was confirmed by evaluating their phenotypic coherence based on the descriptors analyzed. to assess quantitative differences among the defined clusters, mean comparisons of the traits were conducted using tukey’s hsd test (p < 0.05). the analyses were conducted using the factoextra (kassambara & mundt, 2020) and factominer (lê et al, 2008) packages for mca and pca, respectively. dendrograms were generated using the cluster (maechler et al, 2021) and circlize (gu et al, 2014) packages, while visualization of results was carried out with ggplot2 genetic resources (2025), 6(12), 26–38 yacon characterization in peru 29 (wickham, 2016) and itol: interactive tree of life (letunic & bork, 2024). mean comparisons were performed using the agror package (shimizu et al, 2023). all analyses were conducted in the rstudio statistical software (r core team, 2024). results the phenotypic characterization data recorded for the 214 yacon accessions conserved in the inia germplasm bank are presented in supplemental table 2. these include 12 qualitative and 7 quantitative traits, which were used to assess phenotypic variability. factor analysis of qualitative and quantitative traits figure 2 shows a joint analysis using the ‘scree plot’ and ‘variable contribution’ on the first two principal components, based on a mixed data set including 19 traits (qualitative and quantitative) analyzed for the 214 accessions. the scree plot (figure 2a) shows the values of the first ten components, with the first and second components having the highest values adding up to 8.55, out of a total value of 22.53. in the contribution of variables (figure 2b), those above the mean contribution line stand out: copr, fcsr, lele, lewi, ptsd, srsc, lbed and sscd (for explanation of acronyms see table 1). of these, copr and fcsr are the most relevant qualitative variables, while lele and lewi are the most prominent among the quantitative variables. due to these findings, and considering that the traits wsrp and yosr are relevant for breeding programmes, further analysis was conducted to explore in detail the specific contribution of each type of variable to obtain a more complete understanding of their influence on the dataset. figure 2. factor analysis of a mixed data set in 214 yacon (smallanthus sonchifolius) accessions from inia, peru. a, scree plot of ten principal components; b, contribution of qualitative and quantitative variables. acronyms are the same as those in table 1. qualitative characteristics of yacon the characters showed a cumulative variability between dim1 and dim2 of 19.9%. the copr and fcsr traits showed the highest similarity in the mca (figure 3a), emphasizing their high contribution to the observed differentiation. in addition, a pattern of grouping of the accessions according to their geographical origin was evident, showing that accessions from piura and la libertad were associated with those from cajamarca; similarly, accessions from puno and cusco showed proximity. individual associations were also identified, such as those from junín and pasco (figure 3b). hierarchical analysis of qualitative yacon characters the hierarchical analysis presented in figure 4 shows the formation of 12 groups based on qualitative morphological traits, which are structured according to vegetative and reproductive characteristics of the accessions, as well as geographical and possibly environmental factors. the groups are mainly distinguished by leaf morphology (shape, base and margin), branching pattern, as well as surface and pulp coloration of the reservoir root and propagules. the analysis of table 2 and the information in supplemental table 1 shows that the regions with the lowest representation are junín (9 accessions), puno (14 accessions), la libertad (9 accessions) and pasco (1 accession), with only two clusters for junín and puno, three for la libertad, and one for pasco. this low frequency is possibly due to the fact that the accessions from these regions exhibited lower morphological diversity, were restricted to specific characteristics, or there was limited availability of accessions for analysis. the presence of accessions from junín and puno in only a few clusters suggests reduced variability, which could indicate local adaptations that have occurred over a shorter evolutionary period or under more homogeneous environmental conditions. genetic resources (2025), 6(12), 26–3830 santa cruz-padilla et al on the other hand, regions such as cajamarca and apurímac, with a high frequency of accessions (presence in seven clusters), showed greater morphological diversity. this is reflected in a wide range of characteristics such as leaf shape, flesh color and stem structure, indicating a wide genetic variability and adaptive capacity. figure 3. multiple correspondence analysis (mca) of twelve qualitative traits in 214 yacon (smallanthus sonchifolius) accessions from inia, peru. a, contribution of qualitative variables to the mca; b, geographical clustering based on collection locations. acronyms correspond to those listed in table 1. small dots represent individual accessions; larger dots indicate the centroid of each geographic group. quantitative characteristics plants had between 1 and 5 stems (nspl), with a mean of 2.71 (supplemental table 2). fourteen accessions reached the minimum value, while three accessions reached the maximum. in the case of plant height (plhe), a minimum value of 41.6cm and a maximum value of 164cm were observed, corresponding to accessions y-206 and y-154, respectively. on the other hand, accession y-74 had the longest leaves (lele) with 25.4cm, while y-174 recorded the lowest value with 9.6cm. the latter also had the lowest leaf width (lewi) value with 9.2cm, while the highest value was measured in accession y-65 with 29cm. accessions y-111, y-114, y-137, and y-182 coincided in the lowest storage root weight (wsrp) with 0.5kg, while accession y-28 reached the maximum value with 4.7kg, positioning itself as a promising accession due to its high genetic resources (2025), 6(12), 26–38 yacon characterization in peru 31 table 2. qualitative morphological group distribution in relation to the main traits of the yacon accessions, as identified through hierarchical cluster analysis. cluster n° of accessions regions of provenance characteristics i 74 cajamarca, amazonas, la libertad, piura stem with purple secondary color at nodes and internodes; triangular leaves, with predominant basal branching; surface of storage roots light yellow; yellow-orange pulp; dark greyish purple propagules. ii 40 apurímac, ayacucho, cajamarca, cusco, la libertad, piura triangular leaves; elliptic to oblong ligulate flower; variability in flesh color: yellowish white, yellow-orange and light orange. iii 9 junín stem with purple secondary color in internodes; triangular leaves with sagittate base; absence of pigmentation on the veins on the underside of the leaf; light greyish purple storage root surface ; light orange and orange-yellow flesh. iv 7 amazonas, apurímac, cajamarca, cusco, puno, junín secondary color absent on stem; triangular leaves with subhastate base; dark greyish purple storage root surface; white flesh; purplish red and white propagules. v 15 amazonas, ancash, apurímac, ayacucho, cajamarca, piura secondary color absent on stem; triangular leaves; light greyish purple storage root surface; yellowish white or orange-yellow flesh color of the storage roots; purplish red and dark greyish purple propagules. vi 40 ancash, apurímac, ayacucho, cajamarca, cusco, puno stem with purple secondary color at nodes and internodes; triangular leaves with hastate base; light yellow storage root surface, with white flesh mottled with greyish purple or reddish purple tones; white and purplish red propagules. vii 7 apurímac, cajamarca, cusco no secondary color on stem; triangular leaves with subhastate base; light greyish purple storage root surface; yellowish white or orange-yellow flesh color of the storage roots; purplish red and dark greyish purple propagules. viii 10 amazonas, ancash, apurímac, ayacucho, cajamarca, la libertad green stem with purple tinges at nodes and internodes; triangular leaves with subhastate base; light yellow storage root surface; purplish red to dark greyish purple propagules. ix 6 cajamarca, la libertad branching along the entire length of the stem; triangular leaves with truncate or subhastate base; light yellow storage root surface; yellow-orange flesh color of the storage roots; white and dark greyish purple propagules. x 3 ayacucho, cusco branching along the entire length of the stem; deltoid leaves with truncate base; oblong ligulate flower; light yellow storage root surface; white flesh color of the storage roots; violet-blue propagules. xi 1 cajamarca stem with purple secondary color at nodes and internodes; basal branching; leaves cordate with lobed base; light yellow storage root surface; light orange flesh color of the storage roots; purplish red with white propagules . xii 2 pasco, ayacucho stem with purple secondary color at nodes and internodes; branching along stem; triangular leaves with sagittate base; light greyish purple storage root surface; light orange or yellowish white with reddish purple pits flesh color of the storage roots; purplish red propagules. productivity. regarding nsrp, accessions y-42, y-103, y-126, y-181 presented the lowest number of storage roots (nsrp) with six units, while accession y-144 reached the maximum production with 32 units. the accessions ranged from 10t/ha to 94t/ha of storage root yield (yosr). finally, the coefficients of variation ranged between 18.9% and 45.7%; nspl, wsrp, nsrp and yosr were the characters with a variability higher than 30%. principal component analysis and correlation the pca results (figure 5) show that the first two principal components together account for 63.80% of the total variance (pc1 = 41.60%; pc2 = 22.20%). using this analysis, the accessions were grouped into six categories based on the flesh color of the storage root. accessions with white and genetic resources (2025), 6(12), 26–3832 santa cruz-padilla et al light orange flesh are mainly distributed in quadrants i and iv; accessions with yellowish-white flesh are distributed in the four quadrants; accessions with yellowish-white flesh with irregular reddish-purple speckles are mainly located in quadrant i; accessions with yellowish-white flesh with irregular dark greyish-purple speckles are found in quadrant iv; and accessions with yellowish-orange flesh are distributed in quadrants ii and iii. in this case, wsrp and yosr vectors point towards the upper left quadrant suggesting a strong association of accessions with negative pc1 and positive pc2 values, mainly related to yellow-orange pulp color. on the other hand, plhe and nsrp show a higher correlation with positive pc1 and pc2 values, being associated with pulp color accessions a, b, c and f. the variables lele, lewi and nspl correlate with negative values in both pc1 and pc2, suggesting their relationship with accessions in the lower left quadrant. correlation between quantitative traits the correlation matrix for quantitative traits is presented in table 3. a strong positive correlation was observed between leaf length and leaf width. both traits also showed moderate positive correlations with the weight of storage roots per plant and with total root yield. the weight of storage roots per plant exhibited a high correlation with total yield. in contrast, the number of storage roots per plant did not show a significant correlation with either root weight or yield. plant height was negatively correlated with leaf length, leaf width, and the number of stems per plant. additionally, the number of storage roots per plant showed negative correlations with leaf dimensions. figure 4. phylogenetic hierarchical tree of 214 accessions of yacon (smallanthus sonchifolius) based on 12 qualitative characters of the germplasm of inia, estación experimental agraria baños del inca, cajamarca. clusters are colour-coded and numbered as in table 2. figure 5. principal component analysis on 214 accessions of yacon (smallanthus sonchifolius) from inia, peru, grouped according to the flesh color of the storage root (with 95% confidence ellipses): a, white; b, yellowish-white; c, yellowish-white with irregular reddish-purple speckles; d, yellowish-white with irregular dark greyish-purple speckles; e, yellow-orange; f, light orange. acronyms are the same as those in table 1. genetic resources (2025), 6(12), 26–38 yacon characterization in peru 33 light orange flesh are mainly distributed in quadrants i and iv; accessions with yellowish-white flesh are distributed in the four quadrants; accessions with yellowish-white flesh with irregular reddish-purple speckles are mainly located in quadrant i; accessions with yellowish-white flesh with irregular dark greyish-purple speckles are found in quadrant iv; and accessions with yellowish-orange flesh are distributed in quadrants ii and iii. in this case, wsrp and yosr vectors point towards the upper left quadrant suggesting a strong association of accessions with negative pc1 and positive pc2 values, mainly related to yellow-orange pulp color. on the other hand, plhe and nsrp show a higher correlation with positive pc1 and pc2 values, being associated with pulp color accessions a, b, c and f. the variables lele, lewi and nspl correlate with negative values in both pc1 and pc2, suggesting their relationship with accessions in the lower left quadrant. correlation between quantitative traits the correlation matrix for quantitative traits is presented in table 3. a strong positive correlation was observed between leaf length and leaf width. both traits also showed moderate positive correlations with the weight of storage roots per plant and with total root yield. the weight of storage roots per plant exhibited a high correlation with total yield. in contrast, the number of storage roots per plant did not show a significant correlation with either root weight or yield. plant height was negatively correlated with leaf length, leaf width, and the number of stems per plant. additionally, the number of storage roots per plant showed negative correlations with leaf dimensions. figure 4. phylogenetic hierarchical tree of 214 accessions of yacon (smallanthus sonchifolius) based on 12 qualitative characters of the germplasm of inia, estación experimental agraria baños del inca, cajamarca. clusters are colour-coded and numbered as in table 2. figure 5. principal component analysis on 214 accessions of yacon (smallanthus sonchifolius) from inia, peru, grouped according to the flesh color of the storage root (with 95% confidence ellipses): a, white; b, yellowish-white; c, yellowish-white with irregular reddish-purple speckles; d, yellowish-white with irregular dark greyish-purple speckles; e, yellow-orange; f, light orange. acronyms are the same as those in table 1. table 3. correlation matrix among quantitative characters. significant correlations at *p < 0.05, **p < 0.01, ***p < 0.001; ns: not significant. the character acronyms are the same as those used in table 1. character nspl plhe lele lewi wsrp nsrp yosr nspl 1.00 plhe -0.23 *** 1.00 lele 0.34 *** -0.23 *** 1.00 lewi 0.30 *** -0.18 ** 0.90 *** 1.00 wsrp 0.21 ** -0.10 ns 0.39 *** 0.35 *** 1.00 nsrp -0.09 ns 0.22 ** -0.23 *** -0.24 *** 0.10 ns 1.00 yosr 0.20 ** -0.11 ns 0.37 *** 0.35 *** 0.98 *** 0.11 ns 1.00 hierarchical analysis of quantitative yacon characters the circular dendrogram in figure 6, along with the corresponding information in table 4, shows a grouping of the accessions in four clusters. cluster i, with 120 accessions, has a mean of three stems per plant and 116.3cm plant height. this group exhibits a mean leaf length of 20.1cm and leaf width of 20.9cm. the weight of storage roots per plant is 1.8kg, with 13.1 storage roots per plant and a mean yield of 36.91t/ha. this cluster is characterized by mean values for vegetative development and root production compared to the other clusters. cluster ii is composed of 19 accessions that together have the most outstanding agronomic characteristics of all the groups. with a plant height of 104.3cm and a mean of three stems per plant, the accessions in this group have the largest leaf dimensions, with 21.1 and 21.9cm length and width, respectively. this cluster is particularly distinguished by its high root productivity, with 3.6kg of storage root weight per plant, 15.2 storage roots per plant, and a mean yield of 73.5t/ha. cluster iii groups a total of 55 accessions with a plant height of 115.4cm and a mean of 2.3 stems per plant. the leaf dimensions of this group are 16.0 cm leaf length and 15.9 cm leaf width, indicating moderate leaf development compared to clusters i and ii. furthermore, in comparison with clusters i, ii and iv, it presents the lowest values in storage root weight per plant (1.3kg), number of storage roots per plant (11.6) and yield (26.5t/ha). cluster iv includes 20 accessions, which represent 9.3% of the total. these accessions are distinguished by a plant height of 131.5cm, the highest among the clusters. in addition, it has a mean of 1.7 stems per plant; together with a reduced leaf development in length and leaf width, with 14.9 and 14.7cm, respectively. in terms of production, the storage root weight per plant is 1.46kg, with 21 storage roots per plant and a yield of 37.37t/ha. this cluster stands out for its high plant size and a higher number of roots per plant. descriptive analysis and comparison of means of quantitative traits of yacon the analysis of the mean values of the quantitative traits (table 4) revealed significant differences (p < 0.05) among the clusters, indicating a clear structuring of the yacon accessions into four groups with distinct agronomic profiles. these groups enable the identification of materials with potential for different objectives: selection aimed at high yields (cluster ii), balance between growth and productivity (cluster i), or evaluation of accessions with agronomic limitations that may require specific improvements (clusters iii and iv). genetic resources (2025), 6(12), 26–3834 santa cruz-padilla et al figure 6. circular dendrogram of yacon (smallanthus sonchifolius) germplasm of inia, peru table 4. descriptive analysis and comparison of means between quantitative traits of the clusters. the character acronyms are the same as those used in table 1. sd, standard deviation; cv, coefficient of variation. *, means followed by the same letter in the rows do not differ statistically from each other, by tukey’s test (p < 0.05). character mean character values* and sd cluster i cluster ii cluster iii cluster iv cv(%) nspl 3.00±0.77 a 3.05±0.52 a 2.29±0.71 b 1.75±0.55 c 26.51 plhe (cm) 116.33±20.86 b 104.31±25.5 b 115.45±28.05 b 131.5±16.97 a 19.78 lele (cm) 20.15±2.27 a 21.42±3.21 a 16.03±3.65 b 14.93±2.09 b 14.77 lewi (cm) 20.96±3.34 a 21.99±4.01 a 15.92±4.25 b 14.76±2.72 b 18.82 wsrp (kg) 1.87±0.59 b 3.68±0.56 a 1.34±0.50 c 1.46±0.66 c 30.94 nsrp 13.18±3.52 bc 15.26±3.33 b 11.64±3.27 c 21.05±3.95 a 25.43 yosr (t) 36.91±11.93 b 73.58±11.11 a 26.58±9.98 c 29.2±13.18 c 31.29 genetic resources (2025), 6(12), 26–38 yacon characterization in peru 35 discussion qualitative characteristics the qualitative traits contributed heterogeneously to the phenotypic variability of 214 yacon accessions (figure 3a), explaining 19.9 % of the total variability in the first two dimensions of the analysis. although the contribution was moderate, it was observed that the color of the propagules and flesh color of the storage root traits stand out for their high discriminatory capacity between accessions, indicating their relevance in group differentiation. in particular, the relevance of fcsr is supported by previous studies that identified it as one of the three most important traits for evaluating yacon hybrids (vegas et al, 2015). the geographical analysis in figure 3b revealed that phenotypic variability is influenced by the adaptation of the accessions to specific environmental conditions, observing clustering patterns according to their geographical proximity. accessions from piura, la libertad and cajamarca formed particular and related groups, probably due to ecological, anthropogenic and genetic conditions, suggesting a strong relationship of the accessions with the environment where they thrive (da silva et al, 2019). this finding is consistent with those obtained by polanco and garcía (2013) who noted that yacon genotypes are adapted and specialized to specific agro-ecological conditions. complementarily, the hierarchical analysis presented in figure 4 and detailed in table 2 provides a more detailed view of the morphological diversity of the accessions, classifying them into 12 qualitative morphological groups according to their vegetative and reproductive characteristics. this grouping reflects the genetic complexity and adaptation of the plants to different ecological conditions. the differences observed in branching patterns, leaf morphology (including shape, base and margin), as well as the coloration of the storage root and propagules, support a grouping based on their phenotypic characteristics. the junín and pasco accessions showed phenotypic characteristics differentiated from the rest, probably due to the influence of unique microenvironments and genetic factors, which would have driven the evolution and differentiation of these accessions. this finding is supported by molecular studies conducted by mansilla et al (2006) and soto (2012), who identified accessions specific to central and southern peru, while in the north, they showed greater homogeneity. the results suggest the existence of important centres of diversity for the conservation, genetic improvement and sustainable use of yacon. quantitative characteristics the descriptive analysis of quantitative traits revealed variability among the accessions with evident differences in plant height and yield traits. pca (figure 5) showed the greatest contribution of leaf size and yield traits in the phenotypic differentiation of the yacon accessions. a significant proportion of accessions with yellow-orange flesh color were associated with the wsrp and yosr vectors, showing a phenotypic differentiation centred on the storage root, suggesting that these accessions were adapted to optimize the accumulation of reserves. this result is congruent with polanco and garcía (2013) who determined that yacon has been subjected to anthropogenic selection aimed at obtaining highly productive storage root genotypes. accessions with white and light orange flesh were grouped with those exhibiting yellowish-white flesh marked by irregular reddish-purple speckles or irregular dark greyishpurple speckles. this grouping, as observed in figure 5, shared morphological traits related to plhe and, to a lesser extent, to nsrp. plhe was inversely correlated with lele and lewi (table 3). this indicates that accessions with higher plant height had smaller leaf dimensions, while those with lower plant height had larger leaf dimensions (table 4). in contrast, accessions with yellowish-white flesh exhibited greater dispersion in the four quadrants, indicating greater variability, probably associated with their phenotypic plasticity. given the relationship of the traits assessed in the pca, we can select lele, lewi, wsrp and yosr as valuable traits to discriminate accessions within the species. according to the quantitative traits, the accessions were distributed into four clusters (figure 6). the analysis of the distribution of accessions suggested that accessions with larger leaf dimensions were associated with higher yields, since a larger leaf area implies greater light uptake, a larger surface area for gas exchange, and greater accumulation of water and nutrients. consequently, photosynthate production increased, leading to a greater biomass in the storage roots. leaf dimensions and their relationship with yield have been correlated in other crops such as potato (solanum tuberosum l.) and tomato (solanum lycopersicum l.) (león-burgos et al, 2021), as well as in common bean (phaseolus vulgaris l.) (warnock et al, 2006). these studies suggest that increased photosynthate accumulation in sink organs is related to optimal development of the source organs. however, further studies are required to determine the direct impact of leaf size on yield. on the other hand, clusters grouping lower-yielding accessions showed limitations in biomass mobilization to storage roots, possibly attributable to vegetative or environmental factors influencing the phenotype. this finding is consistent with douglas et al (2007) who established a significant positive relationship between yield and both planting time and climatic conditions. this observation suggests the need to investigate genotype–environment interaction to identify accessions that maximize the translocation of assimilates to storage roots under different environmental conditions. descriptive analysis and comparison of means of yacon quantitative traits (table 4) provided valuable information on variability and performance of the accessions. these results showed significant relationships between wsrp, yosr and leaf dimensions. this indicates that selection of promising individuals should focus on clusters with significant and outstanding traits in yield and associated traits (foliage) to maximize productivity in future breeding programmes. this finding coincides with the study by rodríguez lópez et al (2022), who identified promising genotypes based on their yield and morphological characteristics such as leaf area and number of stems, among others. genetic resources (2025), 6(12), 26–3836 santa cruz-padilla et al conclusions the qualitative traits copr and fcsr, together with the quantitative traits lele and lewi, were key determinants in the phenotypic differentiation of the 214 yacon accessions. morphological variability exhibited a clear geographical structuring. accessions from northern peru (piura, la libertad and cajamarca), the south (cusco and puno) and the central region (junín and pasco) formed well-defined groupings based on phenotypic similarity. the hierarchical analysis based on quantitative traits identified cluster ii, comprising 19 accessions, as having the greatest agronomic potential, with an average yield of 73.5t/ ha, a storage root weight of 3.6kg per plant, and an average of 15.2 storage roots per plant. positive correlations were observed between yosr and both wsrp, lele and lewi, suggesting that foliar development may serve as a reliable predictor of yield performance. supplemental data supplemental table 1. geographical origin and coding of 214 accessions of yacon from the inia germplasm bank, cajamarca, peru. supplemental table 2. agromorphological characterization data of 214 yacon accessions from the inia germplasm bank, peru. acknowledgements this work was supported by the subdirección de recursos genéticos de la estación experimental agraria baños del inca of the instituto nacional de innovación agraria (inia). the authors would like to thank armando linares estrada and sebastián llico sánchez for their invaluable support in the field. author contributions angel esteban santa cruz-padilla and jorge luis vásquezorrillo: conceptualization, formal analysis, writing – original, research, data curation, resources, methodology, proofreading and editing; silvia yanina rodríguez lópez and araceli eugenio leiva: research, data curation, resources, methodology, proofreading and editing; ricardo manuel bardales-lozano and hipolito murga-orrillo: formal analysis, writing – original, research, methodology, proofreading and editing; juan f. seminario: research, resources, methodology, proofreading and editing. conflict of interest statement the authors have declared that no competing interests exist. references adriano, l., dionísio, a., de abreu, f. carioca, a., zocolo, g., wurlitzer, n., de oliveira pinto, c., de oliveira, a., and de carvalho sampaio, h. 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(2016). ggplot2: elegant graphics for data analysis. url: https://ggplot2.tidyverse.org https://www.senamhi.gob.pe/servicios/?p= estaciones https://agronomiar.github.io/agrorpackage/index.html https://agronomiar.github.io/agrorpackage/index.html https://hdl.handle.net/10568/126005 https://doi.org/10.1021/jf052645u https://doi.org/10.1021/jf052645u https://doi.org/10.15381/rpb.v22i2.11352 https://doi.org/10.15381/rpb.v22i2.11352 https://doi.org/10.3390/su11174581 https://ve.scielo.org/scielo.php?script=sci_arttext&pid=s0002-192x2006000100002&lng=es&tlng=es https://ve.scielo.org/scielo.php?script=sci_arttext&pid=s0002-192x2006000100002&lng=es&tlng=es https://ve.scielo.org/scielo.php?script=sci_arttext&pid=s0002-192x2006000100002&lng=es&tlng=es https://ggplot2.tidyverse.org editorial genetic resources (2025), (s2), 1–5 doi: 10.46265/genresj.blds6319 https://www.genresj.org issn: 2708-3764 ex situ conservation of plant genetic resources in europe – a journey through history, mission, challenges and future opportunities filippo guzzon *, sandra goritschnig, nora capozio and lorenzo maggioni european cooperative programme for plant genetic resources (ecpgr), c/o alliance of bioversity international and ciat, via di san domenico 1, rome, 00153, italy abstract: the special issue entitled: ‘ex situ conservation of plant genetic resources in europe – a journey through history, mission, challenges and future opportunities’ presents 16 original articles, including 11 genebank reports from 7 european countries (belgium, france, germany, hungary, italy, norway and russia) and 5 review and position papers presenting relevant concepts to improve plant genetic resources (pgr) conservation and access. they provide an overview of pgr ex situ conservation in europe, and reflect on the history and future directions of the collections. important topics in pgr conservation and use are explored, including quality management systems for genebanks, the role of community seedbanks, the importance of collaborative research projects and national and international research infrastructures for pgr, and the integration of in situ and ex situ pgr conservation. from this article collection, the key importance of genebanks clearly emerges – not only in the long-term conservation of plant biodiversity but also in supporting and enabling plant breeding, research in plant biology and in situ conservation initiatives, highlighting important topics that should be prioritized for the efficiency and continuous improvement of pgr conservation activities. this article collection sparks discussions on future directions of ex situ plant conservation to further increase the impact of genebanks and their contributions to sustainable development. keywords: agricultural biodiversity, crop diversity, genebanks, germplasm banks, plant conservation citation: guzzon, f., goritschnig, s., capozio, n., maggioni, l. (2025). ex situ conservation of plant genetic resources in europe – a journey through history, mission, challenges and future opportunities. genetic resources (s2), 1–5. doi: 10.46265/genresj.blds6319. © copyright 2025 the authors. this is an open access article distributed under the terms of the creative commons attribution license (cc by 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. introduction human societies have created, organized and conserved living collections of plant species since ancient times, in all areas of the world and for multiple reasons (granziera, 2001). since the end of the 19th century, researchers have conceptualized and highlighted the importance of the conservation and availability of plant genetic resources (pgr) for crop breeding and research, especially of landraces and crop wild relatives (cwr) (plucknett et al., 1987). the modern concept of a genebank – a facility for the long-term ex situ conservation of reproductive samples of pgr acces∗corresponding author: filippo guzzon (f.guzzon90@gmail.com) sible for breeding and research purposes – was first realized at the beginning of the 20th century at what is now the n. i. vavilov all-russian institute of plant genetic resources (vir) in saint petersburg, which pioneered the collection, identification and description of the diversity of cultivated plants (loskutov, 1999; loskutov et al., 2025). genebanks have since been established in many countries to preserve and keep available pgr and prevent the loss of wild plant populations and landraces due to substitution with modern high-yielding varieties (lehmann, 1981). in europe, one of the earliest genebanks was established in gatersleben, then east germany, after world war ii, and subsequently became the german genebank after reunification in 1990. other collections were established in several countries of eastern europe in the 1950s, including bulgaria, czechosloreceived: 10.05.2025 accepted: 12.05.2025 published online: 23.05.2025 https://www.genresj.org https://www.doi.org/10.46265/genresj.blds6319 https://www.genresj.org https://www.doi.org/10.46265/genresj.blds6319 mailto:f.guzzon90@gmail.com 2 guzzon et al genetic resources (2025), (s2), 1–5 vakia, hungary and poland. investments in national collections in western europe came later, after awareness was raised by the food and agriculture organization of the un (fao) and technical conferences held in the 1960s, alerting about the risks of genetic erosion (i.e. “the loss of genetic diversity and variation in a crop”, van de wouw et al. (2010)) due to displacements of old varieties by modern ones (pistorius, 1997). the genebank of the federal republic of germany in braunschweig started operations in 1971, the italian genebank in bari in 1974, the nordic genebank of the five nordic countries in lund, sweden, in 1979 and the dutch genebank in wageningen in 1985. many countries did not establish centralized genebanks, but their collections were created by universities as well as public and private research institutes. the splitting and sprouting of nations due to political changes after 1989, and the concept of sovereignty over genetic resources introduced by the convention on biological diversity in 1992 (unep, 1992), opened the way to the expansion of conservation institutions. a large diversity of historical backgrounds and foundational motivations has given rise to a wide number (around 400) of institutes conserving pgr listed in the european search catalogue for plant genetic resources (eurisco, http://eurisco.ecpg r.org), summing up to more than 2 million accessions of pgr currently conserved ex situ in europe. these accessions account for half of the total pgr accessions listed in the global genesys database (genesys pgr, www.gen esys-pgr.org) and about one-third of all pgr accessions actively conserved in genebanks globally (fao, 2025). these ex situ collections vary in their missions, expertise, financial sustainability, legal arrangements, size of collections, conservation and distribution methods, data information and quality management systems. the ex situ conservation of pgr in genebanks is currently considered the most effective strategy to avoid losses in plant diversity and enhance the availability of these resources (davies and allender, 2017). this is particularly relevant considering that pgr represent a fundamental asset to widen and diversify the genetic basis of modern crop cultivars and provide useful traits for breeding in the current scenario of climate change and with the need to minimize negative impacts of agricultural production on natural ecosystems (mccouch et al., 2013; pixley et al., 2023). moreover, genebanks conserve and keep available landraces, old cultivars and neglected crops that are being rediscovered, after decades of genetic erosion in several european areas (see e.g. hammer et al. (1996)), often linked with traditional foods and products and offering new opportunities for farmers and food industries (see e.g. helicke (2024)). fostering collaboration among genebanks in different countries and involving different stakeholders can be an important strategy to strengthen the conservation and use of pgr (engels et al., 2024). in the european scenario, the european cooperative programme for plant genetic resources (ecpgr) is a collaborative programme, active since 1980, among most european countries working together on multiple initiatives aimed at ensuring the long-term conservation and utilization of pgr in europe. ecpgr recently published the plant genetic resources strategy for europe (ecpgr, 2021). this document highlights gaps and necessary actions that should be addressed in the coming decade to ensure long-term pgr availability through their sustainable use and conservation. consolidating and sustaining ex situ conservation is a priority action within the strategy, with several important targets identified that should be achieved by 2030. in this context, this genetic resources special issue aimed at providing the opportunity to disclose, at a so far unpublished level of detail, a comprehensive overview of the history, mode of operation, strengths and weaknesses of some exemplary european institutions conserving plant germplasm as well as related mechanisms influencing their operation. most of these data and observations are of high relevance for the pgr community, yet they are often difficult to publish in regular research articles or remain scattered across various publications, often in national languages. this collection not only showcases examples of successful initiatives but also serves as a valuable resource for policymakers, helping them to understand the state of the art in view of identifying opportunities for better cooperation and sharing of responsibilities. content of the special issue this special issue is composed of 16 original articles in addition to this editorial, grouped into two main categories: (1) reports from genebanks describing their history, composition of the collections, key activities and future perspectives, and (2) review and position papers on emerging topics aimed at enhancing the conservation and sustainable use of plant diversity in europe. eleven genebank reports from seven european countries are presented in this issue, namely: belgium (dumont et al., 2025), france (esnault et al., 2025; feugey et al., 2025; ricou et al., 2025; sampoux et al., 2025), germany (weise et al., 2025), hungary (ay et al., 2025), italy (alberti et al., 2025; palombi et al., 2025), norway (asdal, 2025) and russia (loskutov et al., 2025). these reports are just a snapshot of the more than 400 ex situ collections registered in eurisco (figure 1). however, they cover all the main ex situ conservation techniques for plant germplasm (longand medium-term orthodox seed storage, in vitro conservation, cryopreservation and field conservation, see fao (2014)) and vary significantly in terms of number of accessions and plant species conserved as well as management practices (covering multi-species as well as cropand species-specific collections). while most of the genebank reports in this issue deal with the conservation of pgr for food and agriculture, reports on other collections are also included (i.e. the versailles arabidopsis stock centre, collections of ornamental species and nonfood industrial crops, the pannonian seed bank as a congenetic resources (2025), (s2), 1–5 ex situ conservation of pgr in europe 3 figure 1. locations of european genebanks contributing reports to this special issue. in red dots, the location of all institutions providing data to eurisco (extracted from fao wiews). blue diamonds indicate the locations of the eleven institutions that published genebank reports in the special issue (some collections are conserved in different sites). the seven countries where these contributing genebanks are located are highlighted in green. servation seedbank for wild species), and the svalbard seed vault (norway), the largest global seed collection of safety duplicates of crop genebank accessions. review and position papers offer a view on important topics for pgr conservation and use: quality management systems for genebanks (van hintum and wijnker, 2024), an overview of community seedbanks in europe (bocci et al., 2025), the importance of collaborative research projects and national and international research infrastructures to promote pgr conservation and use (bergheaud et al., 2025; goritschnig et al., 2025), and the integration between in situ and ex situ conservation of pgr (maxted et al., 2025). key messages this special issue provides an overview of the diversity and complexity of ex situ conservation activities of plant diversity across europe and on emerging topics to enhance the long-term conservation and use of pgr. the presented genebanks started assembling their pgr collections during the 20th century, particularly after the 1950s. collecting activities and the acquisition of new accessions are still ongoing, focusing especially on cwr, landraces and crop species that have often been neglected and are now being re-evaluated by research and breeding, promising adaptation to changing environmental conditions. most genebanks today focus on improving the documentation, characterization, evaluation, access and use of conserved accessions and their associated data. the genebank reports highlight the diverse functions that these institutions have within their national pgr conservation programmes and seed systems. the distribution activities of conserved samples across the years highlighted the importance of these genebank collections for research and breeding, for both the private and the public sectors. genebank collections are fundamental sources of useful agronomic and stress tolerance traits for plant breeding and also provide plant germplasm material for hobby growers, repatriation and rematriation activities (ocampo-giraldo et al., 2020). genebanks are also important to support the registration of newly selected or conservation varieties and to conserve and keep available old cultivars formerly registered in national variety lists. they serve as central nodes in networks and collaborative programmes, including private and public institutions and on-farm networks, aiming at enhancing pgr conservation and use. genebanks are often active in outreach activities, raising awareness about the importance of agricultural biodiversity and its long-term conservation, while also promoting the use of pgr. most genebanks are open to, and actively engage in, international collaborations for research and exchange of genetic resources. however, the opportunity to evolve towards a more integrated system for the conservation and management of genetic resources at the regional level is rarely acknowledged as a shared goal. evidently, the benefits that could result from shared management and use of resources – such as economy of scale, reduction of redundancies and gaps, and integration of expertise – are not immediately recognized at the local level. this is in line with the challenges faced by initiatives like aegis (european genebank integration system) in gaining traction (van hintum et al., 2021). in the framework of this special issue, important topics emerged that should be considered priority actions for the continuous improvement of pgr conservation activities in europe: • fully implement quality management systems for genebanks, including the creation of a certification agency specialized in genebank activities to continuously improve the efficiency, reliability and transparency of all genebank operations. • establish safety duplicates, not only for orthodox seed accessions but also in vitro, cryo and field collections. this is an important step to reduce the risk of losing these priceless resources. • coordinate and integrate in situ and ex situ conservation strategies, acknowledging the role 4 guzzon et al genetic resources (2025), (s2), 1–5 of community seedbanks and on-farm conservation programmes. this integration will foster a dynamic management of pgr to ensure that the highest degree of plant genetic diversity is conserved and accessible to users at all times. • support ongoing multi-omics characterization and evaluation of conserved accessions. this will help refocus conservation efforts, identify collection gaps and allow the mining of collections for useful traits. • improve data management and accessibility of accession data, integrating passport data with novel multi-omics characterization and evaluation data collected during routine regenerations and as part of collaborative research projects. better documentation of pgr including cwr and landraces will increase their value and therefore their use for research and breeding. • test and employ new technologies to enhance automation and digitization of routine processes in the management of collections to reduce errors and increase data quality. • establish national and pan-european research infrastructures that can coordinate conservation activities and streamline scientific services and research on pgr conservation and use. improved pan-european coordination will help align the diverse and often heterogeneous pgr conservation activities, improving financial and operational efficiency and access to services. achieving these ambitious targets will improve the longterm conservation and accessibility of our priceless natural resources, which are pivotal to face present and future challenges related to food security, environmental sustainability and the implementation of nature-based solutions. overall, the tangible and invaluable contribution of genebanks to the long-term conservation of, and access to, plant diversity clearly emerged from this article collection. furthermore, as the importance of pgr in breeding continues to grow and with it the increasing volume of pgr-related data, the scope of genebanks is widening to becoming bio-digital genetic resources centres (maxted et al., 2025; mascher et al., 2019). the genebank reports underline the value of documenting and sharing the history of genebanks with the broadest community to inform collections’ management and establish future priorities. we encourage more genetic resources centres to share the fascinating history of why and how their collections were assembled, how their conservation and research methodologies have evolved and reflect on challenges encountered over the years and their corrective actions. this special issue can also be a useful source for young professionals interested in pgr to obtain an overview of genebanking in europe and its future goals. finally, we hope that initiatives such as this article collection can spark discussions on the future directions of ex situ plant conservation to 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(2025). agro-morphological and molecular characterization of argentine maize (zea mays l.) landraces of ‘cristalino colorado’ race. genetic resources 6 (12), 14–25. doi: 10.46265/genresj.tsjg3884. © copyright 2025 the authors. this is an open access article distributed under the terms of the creative commons attribution license (cc by 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. raquel alicia defacioa,*, natalia mercedes paza, ana rosa schlatera, marcelo edmundo ferrera, sergio jorge bramardib,c a instituto nacional de tecnología agropecuaria, estación experimental agropecuaria pergamino, cc31 pergamino 2700, buenos aires, argentina. b universidad nacional del comahue, departamento de estadística, neuquén, argentina c centro de investigaciones en toxicología ambiental y agrobiotecnología del comahue (conicet – unco), neuquén, argentina introduction maize (zea mays l.) landraces originated from longterm cultivation under natural and artificial selection in different environments and under different cultural management schemes (xiang et al, 2010). these landraces maintain high genetic variation and good adaptation to the natural and anthropological environment where they have evolved (lucchin et al, 2003; di pasquale et al, 2024). the mechanization of agriculture, the increase of urban areas, changes in consumption patterns and production systems has led to the replacement of landraces by improved varieties or hybrids (pilling et al, 2020). mechanization of agriculture and new market demands forced breeders to generate more uniform and productive crops with stable yield (esquinas alcázar, 2005). this homogeneity resulted in an irreversible loss of genetic variability, known as genetic erosion, with the consequent increase of the vulnerability of agricultural crops to future attack by biotic and abiotic stresses (salhuana et al, 1998; troyer et al, 1988; esquinas alcázar, 2005). genetic resources have long been an important source of new alleles for commercial plant breeding. however, high variability conserved in germplasm banks worldwide is poorly used because breeders prefer crosses among elite inbred lines for their improvement programmes (vigouroux et al, 2008). extensive exploitation of landraces is hampered by their high heterogeneity, low performance, seed underproduction and negative genetic load (gorjanc et al, 2016).* corresponding author: raquel alicia defacio (defacio.raquel@inta.gob.ar) http://doi.org/10.46265/genresj.tsjg3884 https://www.genresj.org http://10.46265/genresj.tsjg3884 mailto:defacio.raquel@inta.gob.ar genetic resources (2025), 6(12), 14–25 characterization of argentine maize landraces 15 using conserved germplasm in future plant breeding requires systematic evaluation of desired traits (xiang et al, 2010; balconi et al, 2024). for this purpose, there are many descriptors, such as agro-morphological traits and molecular markers, which contribute different and equally important information. germplasm characterization and evaluation must be complemented with appropriate statistical analyses to obtain a more complete description of the landraces and establish relations among them. properly studied and catalogued plant genetic resources can be available for plant breeding programmes (bramardi, 2023). in argentina, as in other countries of the americas, there is a large diversity of maize types. argentine maize landraces are classified into 44 races mainly based on specific traits related to ear and grain descriptors, such as shape, colour and texture, and use (cámara hernández and miante alzogaray, 2003; solari, 2007). argentina has a leading role in the production of export maize ‘plata’, typical of the ‘cristalino colorado’ race, being currently the only producer of this type of maize worldwide. the kernel of ‘cristalino colorado’ maize is of intense orange colour; the endosperm is mostly hard and glassy in the periphery and floury in the centre, lacking indentation (secretaría de agricultura, ganadería, pesca y alimentación, 1997). this kind of grain is widely used in dry milling processes for human consumption and poultry feeding. this grain contains higher carotenoid concentrations than dent corns (chandler et al, 2013); when included in the diet of chicken, it gives a desirable colour to the skin and egg yolks, without the addition of synthetic pigments. besides, true metabolizable energy values of ‘cristalino colorado’ maize are higher than those of the dent maize due to the higher concentration of oil in the grain. ‘cristalino colorado’ maize can supply the calories required by cattle and pigs, with no need for additional oil in their diet. a ‘cristalino colorado’ hybrid is available in the argentine market; however, it is of lower quality than traditional genotypes (paz, 2009). previous studies of maize landraces conserved in the active germplasm bank at ‘instituto nacional de tecnología agropecuaria’ (inta) pergamino (bap) revealed a high degree of molecular and agro-morphological variability (salhuana et al, 1998; defacio et al, 2005; paz et al, 2005; defacio, 2009; paz, 2009; defacio, 2017; heck et al, 2020; rivas et al, 2022), as well as in disease resistance (presello et al, 1996; presello et al, 2006; iglesias, 2008; defacio et al, 2018) and grain quality traits (lópez et al, 2005; heck et al, 2019). the aim of the present study was to characterize the variability of 36 maize landraces of ‘cristalino colorado’ race collected from buenos aires province, argentina, based on agro-morphological traits and ssr markers, and the relationship among them. materials and methods plant material thirty-six maize landraces of ‘cristalino colorado’ race conserved at bap were evaluated. these landraces were collected in buenos aires province between 1951 and 1963 (luna and safont lis, 1978). landrace passport descriptors and races are presented in table 1. four synthetic openpollinated (op) varieties developed by the inta pergamino corn breeding programme, payagua inta, candelaria inta, sp1234 and bs13p, were included as checks. payagua inta and candelaria inta have semi-dent endosperm and sp1234 belongs to the ‘cristalino colorado’ race. bs13p has dent endosperm and was developed through recurrent selection applied to bs13. agro-morphological characterization evaluations were performed in two environments, pergamino (33°53′01″ s, 60°34′01″ w) and ferré (34°07’30” s, 61°08’27” w), buenos aires province, argentina, during the 2004/2005 growing season. both are characterized by typical argiudol soil, (inta, 1972). the climate is classified as humid temperate, characterized by annual average rainfall of 1,000mm and average temperature of 16–18 ºc. figure 1 presents meteorological data recorded as long-term averages (1982–2005) and during the 2004/2005 growing season for the two environments. minimum and maximum temperatures and long-term precipitation data were obtained from the nasa power package in r (sparks, 2018), while precipitation during the 2004/2005 growing season was manually recorded in the field. both trials were conducted using a randomized complete block design with two replications. each plot was planted in two 5m rows with a spacing of 70cm and 30 plant hills. standard agronomic practices were followed for successful crop growth. fifteen quantitative traits, based on maize descriptor (cimmyt/ibpgr, 1991) were evaluated: days to anthesis (gdu, growing degree units), days to silk (gdu), anthesissilking interval (gdu), ear length (cm), ear diameter (mm), number of kernel rows (number), kernels per row (number), kernel width (mm), kernel length (mm), plant height (cm), ear height (cm), plant height/ear height ratio (index), 1,000-kernel weight (g), yield (kg/ha), and prolificacy (index). phenological traits, 1000-kernel weight, yield and prolificacy were measured on the complete plot. morphological traits were collected on ten plants per plot, randomly selected in each plot, using the average of the 10 units for performing the analyses. a principal component analysis (pca) was performed using the standardized data matrix obtained from the arithmetic means of the agromorphological quantitative variables corresponding to both environments and replications, in order to obtain an average characterization throughout the environments (zuliani et al, 2018). pearson correlation was computed to assess the relationship among traits prior to pca. molecular characterization landraces were evaluated using a set of five public ssr markers (phi080, phi072, phi034, bnlg439 and phi96100) located on different chromosomes, with a high degree of polymorphism. oligonucleotide sequences are publicly available at the maize data bank (www.agron.missouri. edu/coop/ssr-probes/ssr1.html). dna was extracted from young leaves of 25 plants per landrace, according to kleinhofs et al (1993). pcr reactions were carried out in a mj research ptc-100 thermocycler (usa). the amplification products were visualized in 6% polyacrylamide gels and were http://www.agron.missouri.edu/coop/ssr-probes/ssr1.html http://www.agron.missouri.edu/coop/ssr-probes/ssr1.html genetic resources (2025), 6(12), 14–2516 defacio et al table 1. landrace passport descriptors and races. identifier location department altitude (masl) latitude longitude race arzm01001 acevedo pergamino 70 33°46’ s 60°27’ w cristalino colorado arzm01002 rancagua pergamino 69 34°02’ s 60°30’ w c. colorado – c. amarillo arzm01003 rancagua pergamino 69 34°02’ s 60°30’ w cristalino colorado arzm01005 arroyo dulce salto 75 34°06’ s 60°24’ w cristalino colorado arzm01006 tacuarí salto 69 34°13’ s 60°19’ w c. colorado – c. amarillo arzm01007 salto salto 51 34°18’ s 60°15’ w cristalino colorado arzm01008 salto salto 51 34°18’ s 60°15’ w cristalino colorado arzm01012 arenales general arenales 84 34°19’ s 61°18’ w c. colorado – c. amarillo arzm01013 rojas rojas 69 34°12’ s 60°44’ w c. colorado – c. amarillo arzm01014 chacabuco chacabuco 69 34°38’ s 60°29’ w cristalino colorado arzm01015 salto salto 51 34°18’ s 60°15’ w cristalino colorado arzm01016 arroyo burgos bartolomé mitre   34°04’ s 60°07’ w cristalino colorado arzm01017 san pedro san pedro 27 33°42’ s 59°41’ w c. colorado – c. amarillo arzm01022 ortiz basualdo pergamino 64 34°03’ s 60°39’ w c. colorado arzm01025 hunter rojas 50 34°15’ s 60°32’ w c. colorado – c. amarillo arzm01026 ferré general arenales 88 34°08’ s 61°08’ w cristalino colorado arzm01027 carabelas rojas 83 34°03’ s 60°52’ w cristalino colorado arzm01028 colón colón 90 33°59’ s 61°06’ w cristalino colorado arzm01030 conesa san nicolás 58 33°36’ s 60°22’ w cristalino colorado arzm01033 el paraíso ramallo 33 33°34’ s 59°59’ w c. colorado – c. amarillo arzm01036 conesa san nicolás 58 33°36’ s 60°22’ w cristalino colorado arzm01039 rancagua pergamino 69 34°02’ s 60°30’ w cristalino colorado arzm01044 chivilcoy chivilcoy 55 34°54’ s 60°01’ w cristalino colorado arzm01048 la violeta pergamino 55 33°44’ s 60°11’ w cristalino colorado arzm01058 chivilcoy chivilcoy 55 34°54’ s 60°01’ w c. colorado – c. amarillo arzm01062 chacabuco chacabuco 69 34°38’ s 60°29’ w c. colorado – amarillo ocho hileras arzm01066 rojas rojas 69 34°12’ s 60°44’ w cristalino colorado arzm01082 nueva roma torquinst 285 38°06’ s 62°14’ w cristalino colorado arzm01086 nueva roma torquinst 285 38°06’ s 62°14’ w cristalino colorado arzm01087 nueva roma torquinst 285 38°06’ s 62°14’ w cristalino colorado arzm01092 pigüé saavedra 298 37°41’ s 62°24’ w cristalino colorado arzm01096 coronel suárez coronel suárez 234 37°28’ s 61°56’ w cristalino colorado arzm01102 carhue adolfo alsina 112 37°11’ s 62°45’ w cristalino colorado arzm01124 trenque lauquen trenque lauquen 96 35°58’ s 62°44’ w cristalino colorado arzm01151 mones cazón pehuajó 88 35°48’ s 61°53’ w cristalino colorado arzm01152 carlos tejedor carlos tejedor 96 35°23’ s 62°25’ w cristalino colorado detected by silver nitrate staining (benbouza et al, 2006). the relative allelic frequencies were calculated using the direct counting method from the individual genotypes found in each landrace. the prevosti distance (prevosti, 1974) was calculated from the relative allelic frequencies of the molecular markers for each landrace to infer the relationship between landraces using a principal coordinate analysis (pcoa). the allele number was determined for each group. subsequently, expected heterozygosity (he) was calculated for each group identified in the pcoa, which was calculated using the following equation: where, pi is the frequency of the i-th allele, and k is the number of alleles. joint analysis for joint analysis, generalized procrustes analysis (gpa) (gower, 1975) was performed. this method gets a better adjustment for the information provided by both pca and pcoa. a consensus configuration was performed and represented in a two-dimensional space. a minimum spanning tree (mst) from the euclidean matrix obtained from the first k he = 1 ∑ p i 2 i=1 genetic resources (2025), 6(12), 14–25 characterization of argentine maize landraces 17 figure 1. meteorological data recorded as long-term averages and during the 2004/2005 growing season in ferré (a) and pergamino (b), buenos aires province, argentina. prec, long-term precipitations; prec_04/05, precipitations in 2004/2005 growing season; tmax, long-term maximum temperatures; tmax_04/05, maximum temperatures in 2004/2005 growing season; tmin, long-term minimum temperatures; tmin_04/05, minimum temperatures in 2004/2005 growing season. two gpa coordinates was added in the principal plane. a mantel test was performed to quantify the relationship between the molecular, agro-morphological and consensus distance matrix. to determine concordance of molecular and agromorphological characterizations at the landrace level, we calculated the euclidean distance between two analogous points, i.e. points corresponding to a single landrace in the agro-morphological and molecular configurations. all statistical analyses were carried out using the ntsys programme (numerical taxonomic system ver. 2.11) (rohlf, 2002). results and discussion agro-morphological characterization table 2 presents the mean values, standard errors, and ranges for each trait, calculated across landraces in each environment under evaluation. in the ferré 2004/2005 growing season, plants exhibited shorter height, a shorter anthesis-silking interval, and higher grain yields. these differences may be attributed to the higher precipitation levels recorded in ferré during january, which match with the critical period for determining grain yield (30 days centred around flowering (fischer and palmer, 1984)). maize is particularly sensitive during this stage, and any stress can increase the anthesis-silking interval, potentially leading to pollination failure and grain yield loss (tao et al, 2023). the other traits did not show differences between the two environments. the correlation matrix (table 3) revealed strong and significant (p < 0.01) correlations between several traits: days to silking and days to anthesis (r = 0.88), plant height and ear height (r = 0.87), yield and ear diameter (r = 0.79), ear height and plant height/ear height (r = -0.78), yield and ear length (r = 0.74), and kernel length and ear diameter (r = 0.73). similar correlations among these traits have been reported by other authors during the evaluation of maize landraces (defacio, 2009; javed et al, 2021; de faria et al, 2022). genetic resources (2025), 6(12), 14–2518 defacio et al table 2. mean values, standard errors (s.e.), and ranges for each trait evaluated in two environments. table 3. correlation matrix between evaluated traits. el, ear length; ed, ear diameter; nkr, number of kernel rows; kw, kernel width; kl, kernel length; kr, kernels per row; hkw, 1,000-kernel weight; ph, plant height; eh, ear height; ph/eh, plant height/ear height ratio; prol, prolificacy; da, days to anthesis; ds, days to silking; asi, anthesis–silking interval; ns, non-significant (p > 0.05); *, significant at p < 0.05; **, significant at p < 0.01. environment pergamino 2004/2005 ferré 2004/2005 trait mean s.e. range mean s.e. range ear length (cm) 15.99 0.14 13.10–19.50 16.56 0.11 14.00–18.70 ear diameter (mm) 41.46 0.31 33.40–48.70 42.04 0.30 34.50–48.10 number of kernel rows 13.16 0.17 10 .00–16.60 13.09 0.15 9.80–16.40 kernel width (mm) 8.48 0.09 6.00–9.80 8.77 0.07 7.20–10.20 kernel length (mm) 8.19 0.12 5.40–12.00 10.11 0.10 8.00–12.00 kernels per row 31.25 0.40 21.60–38.40 34.90 0.28 29.90–40.00 prolificacy (index) 0.94 0.02 0–1.36 1.01 0.01 0.66–1.33 1,000-kernel weight (g) 281.30 3.78 174.00–355.00 285.67 3.31 201.00–346.00 yield (kg/ ha) 5576.90 176.80 1341.10–9440.80 6838.30 172.90 2623.80–11561.30 plant height (cm) 163.98 1.56 122.50–193.00 143.42 1.18 118.50–165.50 ear height (cm) 96.88 1.22 68.00–117.50 84.20 1.10 65.00–106.50 plant height/ear height ratio (index) 1.70 0.01 1.52–2.03 1.71 0.01 1.43–2.09 days to anthesis (gdu) 1032.30 3.86 944.70–1100.15 903.23 3.49 837.60–1017.55 days to silking (gdu) 1069.75 3.09 991.10–1143.75 974.92 5.10 895.90–1089.80 anthesis-silking interval (gdu) 37.45 1.79 12.80–108.12 71.69 2.91 15.00–155.30 el ed nkr kw kl kr hkw yield ph eh ph/ eh prol da ds asi el 1 ** ns ns ** ** ** ** ** ns * ns ns ns ns ed 0.56 1 ** ns ** ns ** ** ns ns ns ns ns ns ns nkr 0.12 0.49 1 ** * ns ns * * ns ns ns ns ns ns kw 0.16 -0.08 -0.46 1 ns ns * ns ** * ns * ns ns ns kl 0.51 0.73 0.36 0.10 1 ns ** ** ns ns ns ns ns ns ns kr 0.61 0.10 -0.17 0.18 0.26 1 ns ns * ns ns ns ns ns ns hkw 0.62 0.64 0.09 0.38 0.66 0.11 1 ** * ns ns * ns ns ns yield 0.74 0.79 0.34 0.05 0.70 0.27 0.75 1 ns ns ns ** ns ns ns ph 0.46 0.15 -0.32 0.50 0.20 0.40 0.43 0.28 1 ** * ns ns ns ns eh 0.23 0.02 -0.28 0.41 0.11 0.27 0.26 0.11 0.87 1 ** ns * * ns ph/eh 0.14 0.11 0.10 -0.13 0.03 -0.02 0.04 0.11 -0.37 -0.78 1 ns ** ** ns prol 0.27 0.24 -0.16 0.33 0.27 0.10 0.35 0.44 0.22 0.07 0.14 1 ns ns ns da -0.04 -0.03 -0.07 0.06 0.18 0.09 -0.11 -0.11 0.25 0.43 -0.50 -0.01 1 ** ns ds -0.02 0.04 -0.09 0.11 0.10 -0.02 -0.04 -0.09 0.29 0.43 -0.45 0.10 0.88 1 ** asi 0.04 0.15 -0.06 0.12 -0.13 -0.21 0.13 0.03 0.14 0.10 -0.01 0.22 -0.03 0.45 1 genetic resources (2025), 6(12), 14–25 characterization of argentine maize landraces 19 el ed nkr kw kl kr hkw yield ph eh ph/ eh prol da ds asi el 1 ** ns ns ** ** ** ** ** ns * ns ns ns ns ed 0.56 1 ** ns ** ns ** ** ns ns ns ns ns ns ns nkr 0.12 0.49 1 ** * ns ns * * ns ns ns ns ns ns kw 0.16 -0.08 -0.46 1 ns ns * ns ** * ns * ns ns ns kl 0.51 0.73 0.36 0.10 1 ns ** ** ns ns ns ns ns ns ns kr 0.61 0.10 -0.17 0.18 0.26 1 ns ns * ns ns ns ns ns ns hkw 0.62 0.64 0.09 0.38 0.66 0.11 1 ** * ns ns * ns ns ns yield 0.74 0.79 0.34 0.05 0.70 0.27 0.75 1 ns ns ns ** ns ns ns ph 0.46 0.15 -0.32 0.50 0.20 0.40 0.43 0.28 1 ** * ns ns ns ns eh 0.23 0.02 -0.28 0.41 0.11 0.27 0.26 0.11 0.87 1 ** ns * * ns ph/eh 0.14 0.11 0.10 -0.13 0.03 -0.02 0.04 0.11 -0.37 -0.78 1 ns ** ** ns prol 0.27 0.24 -0.16 0.33 0.27 0.10 0.35 0.44 0.22 0.07 0.14 1 ns ns ns da -0.04 -0.03 -0.07 0.06 0.18 0.09 -0.11 -0.11 0.25 0.43 -0.50 -0.01 1 ** ns ds -0.02 0.04 -0.09 0.11 0.10 -0.02 -0.04 -0.09 0.29 0.43 -0.45 0.10 0.88 1 ** asi 0.04 0.15 -0.06 0.12 -0.13 -0.21 0.13 0.03 0.14 0.10 -0.01 0.22 -0.03 0.45 1 trait pc1 pc2 ear lenght 0.8324 0.0588 ear diameter 0.7851 0.3346 number of kernel rows 0.2442 0.6234 kernel width 0.2345 -0.5806 kernel lenght 0.7884 0.1752 kernels per row 0.4068 -0.2193 prolificacy 0.3596 -0.1912 1,000-kernel weight 0.8402 0.0398 yield 0.8861 0.2641 plant height 0.5499 -0.6681 ear height (cm) 0.3948 -0.7835 plant height/ear height ratio -0.0741 0.6170 days to antesis 0.1024 -0.5616 days to silking 0.1209 -0.6186 anthesis-silking interval 0.0623 -0.2467 table 4. axis loadings corresponding to pc1 and pc2 figure 2. principal component analysis of agro-morphological traits. landraces are identified with the last numbers in their identifier (e.g. 13 corresponds to arzm1013). genetic resources (2025), 6(12), 14–2520 defacio et al results from the pca (figure 2) show that the first and second principal components (pc1 and pc2, respectively) accounted for 27.42% and 20.86% of the total variation, respectively. the axis loadings corresponding to pc1 and pc2 are shown in table 4. pc1 was positively and strongly associated with yield, 1000-kernel weight, kernel length, ear diameter and ear length. pc2 was negatively and moderately associated with plant architecture traits (plant height and ear height), days to anthesis, and days to silking. landraces were classified in four groups by pca, based on the distances observed between individuals in the direction of both established gradients. g1. this group included bs13p, candelaria inta and payagua inta. these op varieties were associated with the highest values of yield and its components, as well as shorter plant and ear height and fewer days to anthesis and silking than the rest of the evaluated landraces. this result agrees with the fact that these genotypes were selected for yield purposes. g2. represented by only one accession (arzm01044) that showed the lowest yield and the smallest ear, grain size and 1,000-kernel weight of all landraces. this accession presented medium to low plant height and intermediate to fewer days to anthesis and silking. g3. this group included landraces with average yield, 1,000-kernel weight, kernel length, ear diameter, and ear length, displaying shorter plants, lower ear height and fewer days to anthesis and silking than the g4 cluster. g4. represented by landraces with average yield, 1,000-kernel weight, kernel length, ear diameter and ear length, and highest values for days to anthesis and silking, high plant and ear height. molecular characterization in this study, a set of five ssr markers was employed for the preliminary molecular characterization of maize landraces. other authors (di pasquale et al, 2024; joshi et al, 2020; ignjatović micić et al. 2013) have also used a low number of ssrs, ranging from 5 to 10, to evaluate maize landraces. a total of 48 alleles were detected. the overall number of alleles per locus varied from 6 (phi034 and phi072) to 21 (bnlg439), with an average of 9.6 (table 5). six alleles were unique to landraces (unique or private alleles) while five other different alleles were present in two landraces (rare alleles). the average number of alleles per locus obtained from landraces (9.6) was higher than the values reported by reif et al (2003) (5.9), warburton et al (2002) (6.3), labate et al (2003) (6.5) and di pasquale et al (2024) (7.4), but lower than those reported by barcaccia et al (2003) (20.75), rivas et al (2022) (19.05) and torres-morales et al (2023) (25.39). in the op varieties, the assayed loci scored a mean number of alleles equal to 7.6, lower than landraces (9.6). this result is consistent with that obtained by barcaccia et al (2003) of 10.25 vs. 19.75, showing that even though op varieties have genetic variability, they originated from a narrow genetic base. first and second principal axes of pcoa (figure 3) accounted for 10.79 and 8.96% of the total variation, respectively. landraces were distributed in four groups based on their relative distances on the principal plane, which differed from those obtained using pca. g1. represented by arzm01082 and arzm01086 landraces. g2. this group included arzm01124, arzm01102, arzm01152, bs13p and candelaria inta. g3 and g4. these groups included most of the evaluated landraces and were separated by the dispersion of the second principal coordinate (pco2). this clustering was not associated with the presence of rare or private alleles. some landraces exhibited rare or private alleles (arzm01003, arzm01049, arzm01102 and sp1234) but were clustered with other landraces. allele numbers for five ssr markers and expected heterozygosity were calculated for each group identified by the pcoa (figure 4). the number of alleles observed varied among groups, increasing from g1 to g4 in parallel with the number of landraces included in each group. this pattern suggests greater genetic variability in g4, in concordance with the broader dispersion of landraces observed for this group in the pcoa (figure 3). the expected heterozygosity (he) also varied among groups, but no clear relationship was observed with the total number of alleles. joint analysis the correlation between agro-morphological and molecular data matrices was very low and not significant (r = 0.07, p-value = 0.77). the different configurations obtained with both types of variables indicate that individual characterization offers additional information that can be used complementarily to know the genetic variability among landraces. low correlation values between agronomic and molecular traits were found in 41 varieties of cucumber (cucumis sativus l.) (bramardi et al, 2005), in 37 patagonian isolates of yeast (saccharomyces cerevisiae) (lopes et al, 2006), in 57 red clover landraces (trifolium pratense l) (dias et al, 2008), and in a set of banana (musa sp.) clones (ermini et al, 2016). for this reason, it is necessary to use a technique that gathers molecular and agro-morphological information. according to the gpa results (figure 5), seven groups of landraces were identified. some landraces denoted a high correspondence between molecular and agro-morphological characterizations. however, most landraces denoted a great discordance between agro-morphological and molecular data. distance between both types of data presents a range between 0.02 table 5. numbers of alleles per locus across landraces ssr markers no. of alleles phi080 8 phi072 6 phi034 6 phi96100 7 bnlg439 21 average 9.6 genetic resources (2025), 6(12), 14–25 characterization of argentine maize landraces 21 figure 4. allele numbers for five ssr markers and expected heterozygosity (he) for each group identified by the pcoa figure 3. principal coordinate analysis of molecular traits. landraces are identified with the last numbers in their identifier (e.g. 13 corresponds to arzm1013). figure 5. generalized procrustes analysis of agro-morphological and molecular traits in the first two axis with minimum spanning trees. landraces are identified with the last numbers in their identifier (e.g. 13 corresponds to arzm1013). genetic resources (2025), 6(12), 14–2522 defacio et al (arzm01017) and 0.45 (arzm01082), with an average of 0.24 (table 6). the discrepancies observed between agromorphological and molecular markers may be explained by the fact that the ssr used in this work are neutral and not linked to the agro-morphological traits under evaluation. expression of these phenotypic traits is strongly influenced by environmental conditions and the selection performed table 6. distance between two analogous points, i.e. points corresponding to a single landrace in the agro-morphological and molecular configurations identifier distance between molecular and agro-morphological traits arzm01001 0.26 arzm01002 0.27 arzm01003 0.21 arzm01005 0.27 arzm01006 0.23 arzm01007 0.37 arzm01008 0.24 arzm01012 0.32 arzm01013 0.42 arzm01014 0.10 arzm01015 0.19 arzm01016 0.06 arzm01017 0.02 arzm01022 0.17 arzm01025 0.14 arzm01026 0.30 arzm01027 0.37 arzm01028 0.23 arzm01030 0.15 arzm01033 0.40 arzm01036 0.06 arzm01039 0.31 arzm01044 0.08 arzm01048 0.24 arzm01058 0.11 arzm01062 0.24 arzm01066 0.24 arzm01082 0.45 arzm01086 0.42 arzm01087 0.25 arzm01092 0.21 arzm01096 0.43 arzm01102 0.26 arzm01124 0.37 arzm01151 0.08 arzm01152 0.23 bs13p 0.16 candelaria inta 0.16 payagua inta 0.17 sp 1234 0.26 by farmers according to local preferences. this selection contributed to phenotypic differentiation, which may not be reflected in neutral genomic regions, such as those assessed by ssr markers (javed et al, 2021). consensus configuration grouped the four op varieties and the six landraces. interestingly, arzm01044 formed a group by itself when evaluated by agro-morphological traits and was included in a group with a larger number of landraces when evaluated by molecular markers and in consensus analysis. arzm01082 and arzm01086 landraces were assigned to a separate group, according to molecular markers. however, according to agro-morphological traits, these landraces were grouped with other landraces. in gpa, these landraces were grouped with op varieties. a similar situation was observed with checks candelaria inta, payagua inta and bs13p, which formed a distinct group according to agro-morphological analysis but grouped together with other landraces according to the molecular markers and gpa. the correlations among the three distance matrices (molecular, agro-morphological and consensus) were estimated using a mantel test. a greater correlation was found between the consensus and the molecular and agro-morphological characterization (0.20 and 0.47, respectively) than between molecular and agro-morphological characterization (0.07). this result indicates that gpa allows the simultaneous characterization of a set of accessions with agro-morphological traits and ssr markers. there is no unique pattern of association among landraces, which emphasizes the importance of studying the different descriptors jointly to obtain the best description and interpretation of genetic diversity. in conclusion, both agro-morphological and molecular variation were detected among the studied landraces, highlighting the importance of integrating both types of characterization to evaluate genetic diversity. gpa is a powerful statistical technique to align genetic and agromorphological descriptors. increasing the knowledge of the available genetic diversity in maize germplasm will facilitate the establishment of core collections. furthermore, integrating agronomic performance with genetic data is critical to developing and optimizing future breeding strategies. currently, maize breeding relies on a narrow genetic base. incorporating landraces into crosses with elite varieties offers a promising approach to introduce novel alleles and broaden the genetic base of maize breeding. moreover, the local adaptation exhibited by landraces represents a valuable source of germplasm for future needs in sustainable agriculture, particularly in the context of climate change. to enhance the understanding of the genetic diversity of the conserved landraces, it is recommended to incorporate more molecular markers as well as landraces from other races and origins. author contributions all authors contributed to the study conception and design. material preparation, data collection and analysis were performed by raquel defacio, natalia paz and sergio bramardi. the first draft of the manuscript was written by raquel defacio and all authors commented on previous versions of the manuscript. all authors read and approved the final manuscript. genetic resources (2025), 6(12), 14–25 characterization of argentine maize landraces 23 acknowledgments we are grateful to the staff of germplasm bank and molecular markers laboratory at inta pergamino for their assistance in field trials and laboratory experiments, respectively. we also thank dr. juliana iglesias for her precious collaboration and valuable suggestions. this work was financed by the instituto nacional de tecnologia agropecuaria (inta) and the agencia pictr2002-00109 ‘conservación, valoración y desarrollo de recursos genéticos vegetales mediante el uso de nuevas tecnologías.’ data availability statement accession-level data are available from the corresponding author upon reasonable request. conflict of interest statement the authors have no competing interests to declare that are relevant to the content of this article. references balconi, c., galaretto, a., malvar, r. a., nicolas, s. d., redaelli, r., andjelkovic, v., revilla, p., bauland, c., gouesnard, 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(2018). comparación de técnicas de análisis multivariado mediante simulación para caracterización de recursos fitogenéticos en función de caracteres susceptibles a interacción genotipo-ambiente. revista fave sección ciencias agrarias, universidad nacional del litoral. 17(1): 75-86. https://doi.org/10.14409/fa.v17i1 https://doi:10.4081/ija.2023.2206 https://doi:10.4081/ija.2023.2206 https://doi.org/10.2135/cropsci1988.0011183x002800030010x https://doi.org/10.2135/cropsci1988.0011183x002800030010x https://doi.org/10.3732/ajb.0800097 https://doi.org/10.3732/ajb.0800097 https://doi.org/10.2135/cropsci2002.1832 https://doi.org/10.14409/fa.v17i1 _hlk188542971 _hlk199684286 _hlk203027396 _hlk199063987 _hlk188543607 _hlk188543854 _hlk188544429 original article genetic resources (2025), 6 (12), 39–56 doi: 10.46265/genresj.apnr6909 https://www.genresj.org issn: 2708-3764 received: 28.02.2025 | accepted: 07.07.2025 | published online: 19.08.2025 the promise of access and benefit-sharing is met through holistic policy reform: insights from colombia’s genetic diversity and innovation landscape during cop16 abstract: to tackle the global biotechnological innovation divide, parties to the convention on biological diversity (cbd) are negotiating policies to fairly share the benefits from the use of digital sequence information (dsi) on genetic resources. the policies aim to transfer money, knowledge and technologies from technology-rich developed to biodiversity-rich developing countries in order to bolster the latter’s capacities to achieve the cbd’s objectives. however, by focusing predominantly on scientific capacities, these policies overlook the complex interactions between various actors, conditions and infrastructures that collectively constitute a country’s innovation capacity. in the first-time application of the national innovation system approach in this policy context, we identify many factors contributing to an innovation gap in colombia, the host country of cop16, resulting in barriers to study and valorize biodiversity and in lost opportunities for the country to benefit from new technologies. this analysis calls for consideration of broader policy reforms in access and benefit-sharing (abs) negotiations, and illustrates how holistic policy interventions are needed in countries that benefit from abs instruments to effectively use financial, scientific and technological resources. without such an approach, efforts to enhance benefit-sharing from genetic resources and dsi risk reinforcing inequalities in innovation capacity. finally, we discuss actions countries could take to use their current resources better, as well as how scientists and companies as users of genetic resources and dsi can pursue mutual interests by tackling innovation bottlenecks. [para una versión en español del resumen, por favor consulte los datos suplementarios – for a spanish version of the abstract, please see supplemental data] keywords: bioprospecting, access and benefit-sharing, digital sequence information, capacity building, distributive justice, cali fund, innovation divide, national innovation system citation: kreiken, b. and asveld, l. (2025) “the promise of access and benefit-sharing is met through holistic policy reform: insights from colombia’s genetic diversity and innovation landscape during cop16”, genetic resources, 6(12), pp. 39–56. doi: 10.46265/genresj.apnr6909. © copyright 2025 the authors. this is an open access article distributed under the terms of the creative commons attribution license (cc by 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. bob kreikena,b, lotte asvelda,c a section ethics and philosophy of technology, delft university of technology, jaffalaan 5, 2628 bx, delft, the netherlands b centre for genetic resources, wageningen university & research, droevendaalsesteeg 4, 6708 pb, wageningen, the netherlands c section biotechnology and society, delft university of technology, van der maasweg 9, 2629 hz, delft, the netherlands * corresponding author: bob kreiken (b.e.kreiken@tudelft.nl) introduction the completion of the human genome kick-started the 21st century for biotechnology and bioinformatics. with the rapid decrease in sequencing costs, large swathes of genetic sequence data from wild and domesticated species are being generated. these data help researchers and companies understand the threats species face and identify valuable genetic traits in them, such as drought resistance or the ability to break down plastic. globally, however, there is a growing divide between countries with and without this capacity to reap scientific and economic benefits. for many years, most benefits from the use of digital sequence information (dsi) on genetic resources have been accrued by high-income countries (hic), while most biodiversity, and therefore potential dsi, is found in lowand middle-income countries (lmic). this inequality has been the subject of access and benefit-sharing (abs) negotiations under the un convention on biological diversity (cbd) (rohden & scholz, 2022). at cop16, held in cali, colombia, in 2024, the cbd negotiated the functioning of a multilateral mechanism for benefit-sharing from the use of dsi, including the cali fund for the disbursement of monetary benefits, and https://doi.org/10.46265/genresj.apnr6909 https://www.genresj.org https://www.genresj.org/index.php/grj/article/view/genresj.apnr6909/suppdata277 mailto:https://doi.org/10.46265/genresj.apnr6909%0d?subject= mailto:b.e.kreiken%40tudelft.nl?subject= genetic resources (2025), 6(12), 39–5640 kreiken and asveld called upon large and medium-scale businesses that use dsi to contribute 1% of their profit or 0.1% of their revenue (cbd, 2024). this mechanism, which, according to some, could potentially generate usd billions per year (lse roundtable team, 2024), is expected to be used by recipient governments to fund conservation projects, meeting the self-identified needs of indigenous peoples and local communities (iplc), technology transfer and capacity-building. scientists and companies are expected to contribute to the latter activities under the banner of non-monetary benefit-sharing. literature on a multilateral benefit-sharing mechanism has focused on alignment with research needs, its underlying ethical principles and directions for allocation of the funds (bagley, 2021; deplazes-zemp, 2019; scholz et al, 2022). while the cali fund details are being further negotiated, it remains unclear why certain countries fare better in developing their innovation capacity to access, generate and utilize genetic resources and dsi than others. without that knowledge, benefit-sharing from the cali fund risks being ineffective and even unjust. cop host colombia, an upper-middle-income megadiverse country with advanced science but a small biotech sector, and a likely beneficiary, is an excellent case study to investigate this research question. initially, innovation scholarship assumed a linear relationship between government-funded basic and applied research, the development of products and their diffusion in society (godin, 2006). abs frameworks arguably mirror this view by regarding research on genetic resources as a stepping stone for commercial bioprospecting and benefit redistribution by governments (secretariat of the convention on biological diversity, 2011). biotechnological trajectories, however, are embedded in and formed by institutions and their interactions (chaturvedi, 2005; hall, 2005). from early on, the capacity to create and share benefits as an incentive to promote conservation has been part of the rationale behind abs policies (sirakaya, 2022). however, assumptions that benefit-sharing automatically translates to enhanced innovation capacities are far too simple. bilateral abs agreements have long been criticized for oversimplifying how genetic resources are used in research and development (r&d) (sherman et al, 2025). the factors that make r&d in a country possible in the first place are, however, still overlooked in the abs literature. it is important that abs policies also recognize this institutional complexity so that to-be-shared benefits strengthen these interactions. that gap in understanding is evident in the recurring tendency to attribute scientific capacity development challenges to resource deficiencies. for example, making more data, information and communication technologies, and training resources available may increase individual scientists’ capacity but obscures “insidious” patterns of inequality (bezuidenhout et al, 2017). precisely because knowledge production is sustained by institutional, economic, organizational and political factors (mormina, 2019), taking into account and strengthening the knowledge structures wherein monetary and non-monetary benefits are created and received is as important as facilitating benefit-sharing itself. in a nutshell, these insights call for holistic, countryand issuespecific capacity-building and investments by the cali fund and by users of genetic resources and dsi. the moral value at stake here is the fair and equitable sharing of benefits from genetic diversity. we reiterate two distributive justice claims here. distributive justice requires a fair distribution of both benefits and scientific capabilities to create benefits (mormina, 2019). that means that aside from an equal distribution of resources and opportunities in science, structural biases and barriers in the use of genetic resources and dsi are dismantled. furthermore, the (non-) monetary benefit transfers do not change the overall direction of r&d, but distributive justice demands from users of genetic resources and dsi an integration of the needs and priorities of beneficiaries upstream at the onset of the r&d cycle (kreiken & mccarthy, 2025; de jonge & korthals, 2006). so, instead of maintaining the status quo, hic as dominant valorizers of dsi and lmic as beneficiaries, we argue that the cbd and its stakeholders should target countries’ innovation capacity gaps to create and retain benefits. the reason for undertaking this study is to assess how a country’s creation and retention of benefits from genetic diversity is influenced by institutional, economic, historical, organizational and political factors. we now turn to the policy rationale behind this study in relation to abs policymaking and the ongoing development of the cali fund. while most capacity-building programmes of the abs initiative and global environment facility focus on legislative capacities to implement abs policies, there are so far fewer programmes focused on scientific and innovation capacity deficits to use dsi and genetic resources. recipients of the cali fund are expected to primarily direct funding towards activities that contribute to conservation and sustainable use of biodiversity, which can include scientific research and capacity-building to “generate, access, use, analyse and store [dsi]” (article 18 of annex decision 16/2 (cbd, 2024)). innovation and institutional capacities to valorize scientific research on dsi and genetic resources are overlooked, however. currently, there are indications that the vast majority of their economic value (‘the pie’) is captured at the end of the bioprospecting value chain in patents acquired in hic (dunshirn & zhivkoplias, 2024). while historically lmic have benefited greatly from conserving and developing their biodiversity, for example, by having a rich crop and animal breed variety with nutritional and medicinal value, not all of these benefits have translated to financial gains or technological development. this valorization gap should be considered significant in the context of the premise of abs to transfer money back to lmic for conservation and capacity-building purposes. charting out a path to economic self-sustenance in abs policy is important because the contributions to the cali fund so far remain voluntary, making expectations about it being a sustainable source of finance for lmic perhaps unrealistic. additionally, the covid-19 pandemic laid bare the vaccine dependency of lmic, leading to calls for greater biotechnological sovereignty (guzman et al, 2024). simply put, if the innovation divide is left unchanged, the monetary benefits that lmic will receive through abs (‘crumbs’) are marginal relative to the economic gains realized in hic in the long term. for context, in unequal exchanges in raw materials and labour with the global north, the losses the south incurs exceed the aid it receives thirtyfold (hickel et al, 2022). disregarding the innovation divide is a missed opportunity because countries with genetic diversity-based industries may direct innovations and tax revenue to nationally relevant goals, including conservation and scientific research, also because we assume that r&d activities in lmic are more easily matched to the needs of the country and its vulnerable groups than downstream r&d activities in hic. in addition, we expect that companies will be more willing to contribute to the genetic resources (2025), 6(12), 39–56 innovating with genetic diversity in colombia 41 fund if beneficiary countries have clearer ideas of issues that can be addressed through the fund and have long-term plans for greater economic self-sustenance. these assumptions do not disregard the need for fair and equitable benefit-sharing. therefore, this article’s insights into the factors that hamper or boost scientific research and innovation for conserving and sustainably using genetic diversity, contribute to informing investment priorities for cali fund recipients and broader business engagement. in the next sections, we first elaborate on the national innovation system model that guides our analysis and data collection. after an overview of colombia’s relevant laws, state of biodiversity and bioeconomy, the findings are categorized per aspect of the value chain and linked back to components of the analytic model. finally, we make a call to action to rethink domestic and abs policymaking and the cali fund’s investment priorities. materials and methods framework: national innovation model in this section, we explain how the recognition of institutions and interactions enhances our ability to answer the research question. figure 1 represents a simplified value chain of genetic resources and dsi in colombia, according to a linear innovation view. figure 1. linear representation of innovation in colombia. adapted imagery from icons8. to integrate institutional complexity, science policy analysts have used the national innovation system (nis) since the 1980s to analyze individual systems of innovation and their interactions, like the alignment of education with business priorities (godin, 2009). the commonly used framework for nis is shown in figure 2 and includes actors and processes that enable knowledgeand innovation-based economic development (kuhlmann & arnold, 2001). as a whole, the nis model reflects the underlying mechanics of a society’s innovation capacity, which is “the context-specific range of skills, actors, practices, routines, institutions and policies needed to put knowledge into productive use in response to an evolving set of challenges, opportunities, and technical and institutional contexts” (hall, 2005). following this definition, we include various users and providers of genetic resources, contextualize the work in relation to abs and science policy in colombia, and consider biodiversity loss as the main challenge and the bioeconomy as the main opportunity. for the purpose of the article, and not uncommonly, we enlarge the basic nis model with three additions to form a nature-based biotechnological innovation system (see figure 3). in line with the potential of niss for positive environmental impact (brás & robaina, 2024; fernandes et al, 2022), we hypothesize that increased benefitsharing contributes to the conservation and sustainable use of biodiversity. because genetic resources and dsi can be considered inputs to the innovation system (bruynseels, 2020), we include a ‘natural system’ and a detailed genetic resources (2025), 6(12), 39–5642 kreiken and asveld subcategory for dsi-related research infrastructure. secondly, traditional knowledge associated with genetic resources and dsi is included in the education and research system. thirdly, because supranational science, technology and innovation policies are gaining more influence on niss (weerasinghe et al, 2024), and because we want to know the (potential) impact of international abs policies, we include the ‘international policy and political system’. altogether, figure 3 shows that the value chain of genetic resources and dsi, as represented in figure 1, is sustained and influenced by various systems and interactions. although this is the first application of the nis model in this policy context, we are cognizant of the empirical gap and challenges with its application to developing countries. in a general sense, the developing context is characterized by weaker intellectual property rights (iprs), incremental technological development, unstructured business interactions, and low levels of knowledge, demand and investment (egbetokun et al, 2017). many developing countries also lack adequate data to allow for international comparison (weerasinghe et al, 2024). data collection at the start, we conducted a short scoping review of literature and policy documents on biodiversity research, biotechnology and the bioeconomy. with approval from a human research ethics committee, online and in-person semi-structured interviews with professionals throughout figure 2. the national innovation system model (adapted from kuhlmann & arnold (2001)). the nis shows all institutions and actors in various systems that play a role in driving a country’s innovation. well-performing linkages between systems, represented by the arrows, are equally important. for example, actors in the industrial system react to consumer demand and government demand for r&d by commercializing innovations that were developed or co-developed with actors in the education and research systems. activities in the industrial, education and research systems are influenced by a country’s infrastructure, which can range from (un)available venture capital to code of conduct and strong/weak protection of intellectual property rights. the enlarged nis were conducted during one month of fieldwork during and after cop16 in the fall of 2024 (table 1). cops are a good field site because host countries position themselves strongly with regard to the cbd’s objectives (lee et al, 2021), and because they have an unprecedented concentration of stakeholders. the research questions and conceptual framework were revised cyclically during and after the fieldwork (lew, 2010). beforehand, interviewees were identified and contacted via linkedin, based on their contributions to relevant research articles and webinars. further interviews were secured at cop16, which was separated into a blue zone for negotiations and associated events, and a green zone for more colombia-specific events. furthermore, two business conferences were attended, the expo bioingredientes in cali and the open innovation and investor summit in bogotá. visits to the biochemical laboratory of icesi university in cali, and the bioprospecting laboratory of invemar in santa marta, complemented findings on research infrastructure. finally, three ecotours to farallones, chingaza and tayrona park helped to understand the conservation context. the english and spanish transcripts from the 53 interviews were open-coded and thereafter clustered under one or more of the nis model’s components. preliminary findings were presented to bystanders in the hall of the cop16 blue zone’s venue, and later in a seminar on the cop outcome and dsi at universidad de los andes for several key stakeholders. interviewees were requested to validate the results section and give written permission to be cited anonymously or with their full name. genetic resources (2025), 6(12), 39–56 innovating with genetic diversity in colombia 43 figure 3. overview of a nature-based biotechnological innovation system. the different aspects of the value chain of genetic resources and dsi are positioned near the related systems, and new relationships are included compared to figure 2. the international policy and political system interacts with the political system (e.g. domestic implementation of un policies, tax system as framework condition), demand (e.g. accessibility to new markets), and infrastructure (e.g. technology transfer and monetary benefit-sharing). the political system influences access to genetic resources and the conservation of the natural system, and potentially the availability of intermediaries (e.g. funding of incubator programmes). results the results are represented in order of the key aspects of the value chain (figure 1) and with the relevant nis components with which they interact (figure 3). policies for biodiversity and bioeconomy nis components: framework conditions, natural, research and political systems colombia has clear policies to boost its nature-based biotechnological capacity that are grounded in its natural, political, historical and socio-economic context. in between the pacific and atlantic oceans and divided by the andes and amazon, colombia is the world’s second most biodiverse country. because of its proximity to key markets, biodiversity is described as an international “competitive advantage” (melgarejo, 2013) and regarded as an opportunity for nationwide cultural and economic transformation (aparicio, 2022). minister of the environment and cop16 chair susana muhamad (resigned in february 2025) aspired to increase the share of gdp from the bioeconomy from 0.8% to 3% by 2030 (the city paper bogota, 2024a). internationally, the government repeatedly associates the country with its natural wealth. in his cop16 opening speech, president gustavo petro referenced the words of indigenous peoples about creating the “idea [of colombia] as a world power of life as a national mission” (presidencia de la república de colombia, 2024). petro envisions development without neoliberalism, the use of fossil fuels, or the extraction from nature, and “harmony with nature” is included in the national development plan 2022–2026 (vallejo zamudio, 2023). the minority afro-colombian and indigenous populations have endured violence, land-grabbing and subjugation to western scientific ontologies and christianity, first under spanish colonization, later under colombian governments, and recently by corporations and armed groups (goyes & south, 2016; chaves-agudelo et al, 2015). in a “vicious cycle of biopiracy”, genetic resources and traditional knowledge from these marginalized and impoverished groups are at risk of misappropriation (goyes & south, 2016). biopiracy, particularly dsi-enabled ‘digital biopiracy’, also causes national concerns, leading susana muhamad to call for measures to ensure “sovereignty over genetic information” (the city paper bogota, 2024b). colombia has a broad legal basis for ipr to protect innovations abroad (procolombia, 2024), which is similar to that of other countries. biopiracy related to genetic resources genetic resources (2025), 6(12), 39–5644 kreiken and asveld through patent acquisition is inhibited by colombia’s ipr system. particularly important to genetic resources is andean decision 486 (andean community, 2000) on the common provisions on industrial property which contains various provisions to prevent biopiracy (salas, 2020), most importantly: (1) the requirement in article 3 that biological and genetic heritage and traditional knowledge underlying inventions was acquired in accordance with the law, so as not to breach provisions of decision 391 on abs, (2) the exclusion in article 15 sub b of patents on biological processes and material and genomes or germplasm, and (3) a requirement in article 26 sub h to disclose an access contract in the patent filing if traditional knowledge of iplcs was obtained. the state of biodiversity in colombia today is heavily influenced by the aftermath of the 2016 peace agreement between the government and the farc guerrilla group, which has enabled increased deforestation and illicit coca production, but also biodiversity exploration (huddart et al, 2022; irwin, 2023). without measures to curb the expansion of agriculture, a major employment sector in colombia, biodiversity loss could accelerate by 38 to 52% by 2033 (guerrero-pineda et al, 2022). faced with the need to conserve and simultaneously sustainably use biodiversity and the need table 1. overview of interviewees in colombia (details available in supplemental material 1) type of system and work no. of interviews education and research animal biology 4 botany and crop research 8 omics and bioinformatics specialists 4 (industrial) biotechnology and -chemistry 5 students biochemistry (group interview) 7 bioprospecting specialists 6 law, ethics and human rights 6 subtotal 40 biotrade, -tech and -economy biotrade companies 3 biotech start-ups and spin-offs 4 bioeconomy experts 1 innovation broker 1 subtotal 9 politics and policy policymaker and diplomat 1 politician 1 embassy worker 1 subtotal 3 other non-governmental organization 1 total 53 to integrate thousands of people from previous conflict zones back into society, then president manuel santos reinvigorated the 2015 colombia bio programme, a nationwide policy agenda focusing on biodiversity research, bioprospecting, product valorization, institutional strengthening of value chains and public awareness of biodiversity (irwin, 2023). according to both interviewees and expert institutes, colombia bio is internationally recognized as an exemplary bioeconomy programme. colombia defines the bioeconomy as an “economy that efficiently and sustainably manages biodiversity and biomass to generate new products and processes with added value, based on knowledge and innovation” (consejo nacional de política y economía social, citation, 2018), p. 26, as translated in johnson et al (2022)). the bioeconomy mission, a national policy launched in 2020, has five focus areas: biodiversity and ecosystem services, sustainable agricultural production, biomass and green chemistry, biointelligent colombia, and health and wellbeing. central to achieving each of these goals is boosting the use of biotechnology, omics and bioinformatics. access to in situ genetic resources nis components: framework conditions, research, industry, natural, political and international policy system, physical and ipr and information infrastructure considerable barriers to researchers’ access to and collection of colombian genetic resources are administrative, legislative burdens, customs and safety challenges. for fair access to genetic resources, a balance must be struck between user burdens and user rights (collins et al, 2020). but among biodiversity researchers, colombia’s access regulation is notoriously burdensome, sometimes leading to researchers giving up a study (fernández, 2011; wight, 2019). to date, colombia has not ratified the nagoya protocol . the legal bases for abs are article 81 of the constitution (senado de la república de colombia, 1991), andean decision 391 (andean community, 1996), and various subsequent decrees (reep, 2025). access permits are evaluated and granted by the genetic resources team in the ministry of environment and sustainable development. users usually have to report yearly to this team and negotiate another contract in case of commercial interest. in addition, users have to comply with the national parks service’s and other regulations for responsible and sustainable sampling. this patchwork of regulation means that to access just one sample, a foreign scientist may have to acquire seven different documents (collins, 2019). while most interviewees did not express concern with the objective and content of the abs legislation, they experienced high red tape and delays related to its implementation, which affected graduate and short research projects the most. this, in turn, affects international collaborations, as exemplified by the experience of a colombian university biologist: “one time, i had my application filed already three months before a research visit of five months in germany. after personally returning to colombia, it took another three months before the sample could be exported to germany, long after the visit ended.” interviewees report stories of researchers secretly shipping samples in their luggage to avoid delays. abs’s genetic resources (2025), 6(12), 39–56 innovating with genetic diversity in colombia 45 adverse impacts on research are not well-received. among interviewees, law enforcement gaps in extractive industries created the strong feeling that regulation “harms honest people while bad people continue to destroy biodiversity.” fortunately, scientific access has become easier over the past years with regulatory changes. another promising development is that colombian institutions are increasingly signing a memorandum of understanding with international collaborators to facilitate standardized access to samples. however, red tape still looms large for commercial research, which is key to kickstarting the bioeconomy (silvestri, 2016). back in 2013, less than a third (27%) of all bioprospecting permits were accepted and three-quarters of applications took longer than eight months to be processed (güiza & bernal camargo, 2013). the red tape and delays caused a high degree of informality, estimated at threequarters (77%) of bioprospecting activities (güiza & bernal camargo, 2013). apparently, some companies manage the business risks posed by red tape by delaying permit applications for genetic resources with unknown or prior obvious commercial potential until after completing r&d and reaching the final investment decision stage. according to an interviewed policymaker, the major cause of the permit delays is insufficient human resources in the team to handle the requests, which are only increasing. legal unclarity and “coordination failure between institutions” are to blame for the delays (güiza & bernal camargo, 2013). but delays are also caused frequently by users who submit insufficient and inaccessible documentation. interviewees shared that in-house legal counsel for scientists is essential to gaining permits fast and avoiding legal repercussions. this highlights how administrative burdens disproportionately affect small research institutions and companies. until recently, there were no specific procedures for access to genetic resources on indigenous and afro-colombian lands, complicating bilateral abs negotiations (silvestri, 2016). the state pursued an extractivist policy for genetic resources tailored to industrial interests, while indigenous peoples were hardly consulted (nemogá, 2014). the interior ministry has to verify whether a consultation with iplcs is necessary before the genetic resources team can grant an access permit. but history-related distrust and the self-protective attitude of iplcs, that an interviewee describes as “¿gano yo?” (”what do i win from this?”), in combination with legal unclarity, have made such negotiations very complicated. regulatory changes alone will not rebuild that trust. sampling also involves costs for already constrained research budgets. the government instituto agropecuario de colombia charges between 500 and 3,000usd for risk analysis services before seed of a species can be imported (agribrasilis, 2022). all subsequent importers receive a waiver, disincentivizing first-users to pay the fees for species without direct economic benefit. the prohibitive cost or lack of cargo services is another challenge. interviewees have experienced degradation or destruction of samples due to delays in customs, caused by personnel’s distrust of equipment like nitrogen containers, and because samples were not stored in the right conditions during transit at some airports in colombia. this has considerable negative effects because fieldwork in remote regions is often too costly to undertake twice. a last factor of limitation in sampling is violence. while the safety situation has improved considerably since 2016, narco-trafficking is rampant in remote regions. one research team was limited to conducting research directly in the surroundings of an army base and later had to abort the project due to a deteriorating security situation. we assume that such security precautions increase fieldwork costs and limit participatory processes with iplcs. conservation of ex situ genetic resources nis components: framework conditions, research, political and international policy system, financial, physical, and standards and norms infrastructure colombia’s biocollections mostly face financial and organizational challenges, while there are opportunities to be found in increased research, education and benefit-sharing activities. ex situ conservation is organized at various scales, including in botanical gardens, the future seeds genebank of the centro internacional de agricultura tropical (ciat), and in four public research institutes, namely agrosavia for crop research, sinchi for amazonian research, invemar for marine and coastal research and the humboldt institute for nationwide biodiversity research. universities also maintain their own, sometimes outsourced biocollections, and some iplcs store seeds of nutritious and culturally relevant plants in community seedbanks. generally, interviewees reported insufficient funding for biobanking, although agricultural research receives more support than biodiversity research. economic challenges are energy price swings and the salaries of permanent contract staff. meanwhile, research funding is decreasing and the government’s re-valuation of grants at the end of each year creates a lot of job insecurity, making it hard for institutes to retain their staff. interviewees indicated that the decoupling of biocollection funding from research project funding would be desirable. university biocollections experience unclear assignments of responsibilities and degrading infrastructure. some scientists have a curator’s responsibility on top of their day-to-day research tasks, leading to decreased vigilance for incidents. at one time, a researcher lost almost a complete tissue collection when a freezer thawed without raising an alarm. the lack of dedicated curators also means that access to others’ samples becomes more dependent on personal favours by the researcher who facilitates access, thereby slowing down overall research. an opportunity for cost reduction lies in the centralization of biocollections and service provision. an interviewee noted that here, again, violence is a risk. during the major unrest of the national strike in cali in 2021, protestors blocked off entire roads, including towards palmira, where the future seeds bank is located. only at the last moment, a truck carrying liquid nitrogen was exempted by the protestors, showing the external fragility of even the most secure collections. altogether, these threats to biocollections must be seen in the light of sequencing efforts, since when funding for sequencing finally becomes available, sample quality must be maintained, especially for long reads. additionally, the informational value of dsi that becomes available through sequencing builds on the characterization work and advanced regeneration practices at biocollections. enthusiasm for the use of next-generation technologies could bias capacitybuilding efforts toward sequencing and ignore current capacity deficits in ex situ conservation. however, both genetic resources (2025), 6(12), 39–5646 kreiken and asveld capacities need to be strengthened to realize mutual benefits. interviewees also considered it important that samples in biocollections “do not just sit there” and additional measures are taken for value creation. the humboldt institute’s seed bank in boyacá, which has species from the whole country, for example, hired an ethnobotanist to add more value to the collection for society. the marine research institute, invemar, is exploring the expansion of its natural history museum, which is currently limited to a small exposition in the wet collection, to teach the public about marine biodiversity. apart from creating value for the public, biocollections can address concerns specific to iplcs by helping them conserve seeds that are vulnerable to weathering in glass and mason jars with training, freezers, and seed repatriation. these ideas highlight that capacity-building activities aimed at biocollections should not only focus on conserving genetic diversity ex situ but also support biocollections’ aspirations to maximize value for the public and stakeholders. generation and storage of dsi nis components: research, industry and political system, physical infrastructure contrary to ‘each its own sequencer’ thinking, the capacity to generate dsi in colombia is primarily constrained by import costs of reagents, infrequent maintenance of sequencing equipment, and customs issues to import sequencing, laboratory equipment and reagents. this, among other factors, has caused significant biodiversity data gaps in colombia. dsi is only available for one in twenty species, with the vast majority of the available data describing bacteria or being related to just a few projects (noreña et al, 2018). to increase the availability of genome sequence data, in 2019, a new node of the earth biogenome project network was founded, ebp-colombia, which was also embedded in bioeconomy programmes like colombia bio (huddart et al, 2022). however, there have been no updates for some years now, raising the impression that the project has been discontinued due to dried-up funding. despite decreasing costs, sequencing is still a costly exercise in latin america (noreña et al, 2018; vilaça et al, 2024). in colombia, it is much cheaper to ship samples abroad for sequencing. although some institutions have sequencing capacity, others face limited or no access to these machines. additionally, to make a purchase of such equipment cost-effective it is necessary to process a high volume of samples. maintenance costs and delivery, as well as reagent costs, however, form the major bottleneck. there are long waiting times for maintenance workers to repair machines. furthermore, whereas researchers in the usa can order reagents and get them delivered almost instantly, colombian researchers have to wait for extremely lengthy periods, frequently more than a couple of months. the first cause for this delay is bureaucracy in academic institutions, which restricts purchase authorization to a small number of people. secondly, obligated by national import regulations, researchers have to submit orders to licensed intermediaries that can import the reagents. but because there are few such intermediaries in colombia, companies can charge higher prices, which is the main reason why reagents are two to five times as expensive as the original price in the exporting country. when the order is finally shipped, delays, damage and loss in customs are possible: “a reagent for a rt-lamp test took three months to arrive. the kit has a ph indicator, which usually is cherry red, which turns yellow with a positive test. but the kit arrived orange, meaning it can’t be used anymore. we discovered that the cold chain broke during the shipping process because customs did not put it in a fridge for four days.” [university biologist] with significant delays, technology software and service support by the sequencer vendor can become obsolete. one research team that faced more than two years of delay, therefore, renegotiated with the technology provider for a newer sequencer machine. conversations with interviewees suggest that to improve this part of the value chain, waste of research budget and time can be avoided by having the government shake up the intermediary market to enable researchers to access reagents more cheaply. existing sequencing capacity can be used more efficiently if institutions advertise and rent or centralize their sequencers to achieve cost reduction. greater sequencing capacity could come from investments in new businesses that produce reagents or provide maintenance in the latin american region. a recommendation to increase the availability of dsi from remote regions of colombia is for research institutions to set up collaborations with businesses that collect biodiversity data through environmental dna (edna) and other techniques for conducting environmental impact assessments. although large-scale storage of dsi is organized by genetic databases in other countries, institutions may need local data servers and portals for digital genebanking and pre-analyses. here, negotiations with software and cloud providers form an opportunity for cost reduction. a mentality shift in biological research is probably also needed. according to an interviewee, instead of the “catch them all” mindset in biodiversity sequencing, researchers could perhaps better focus on collaborations on the generation of fewer high-quality dsi. likewise, countries with overlapping biodiversity may avoid duplicate work and achieve scale benefits by forming regional collaborations wherein sampling, ex situ conservation, and the generation and storage of dsi are coordinated across borders. in biodiversity genomics europe, for instance, tasks, resources, lessons learned and capacities gained along the genomic pipeline are distributed over institutions in different countries. because diversity in abs regulations may pose an issue, colombia could best collaborate inside the andean community with bolivia, ecuador and peru, as each country’s abs legislation builds on decision 391 (ljungqvist et al, 2025). lastly, through international collaborations with producers of sequencing equipment, which are under the scope of the cali fund (cbd, 2024), researchers in lmic can gain access to grants for generating dsi (pacbio, 2020). publication of and access to dsi nis components: education, research and international policy system, financial infrastructure while challenges for lmic scientists are reported with regard to data access and compliance with fair data standards in the literature (shanahan & bezuidenhout, 2022; bezuidenhout et al, 2017), no particular issues were reported by the colombian interviewees. when new biodiversity data standards are adopted, scientists require both capacity genetic resources (2025), 6(12), 39–56 innovating with genetic diversity in colombia 47 building and efforts to demonstrate their benefits – an experience that is common across countries. there are, however, significant challenges with regard to publishing dsi that originated from iplc territories. their unfamiliarity with dsi and distrust make initial conversations between them and scientists difficult. there are no standards yet for respecting traditional knowledge and maintaining best practices for intellectual property. iplcs also reject unrestricted open data: “when we have spoken with them about data sharing, they usually say: “we need some specific rules and safeguards about publishing the dna data and how we will be reflected in these publications” they have their own needs that we, as scientists and data managers, need to meet.” [biodiversity data specialist] the alignment of data-driven research with community needs is recognized as an ethical priority by the c3biodiversidad consortium (c3biodiversidad, 2018a). lessons to navigate this engagement are found in a collaboration of the humboldt institute and the organization wise ancestors with a paisa community in antioquia to produce two reference genomes for two critically endangered birds (wise ancestors, 2024). five local collaborators received one year of salary and a broad training in sampling, bioinformatics and biomonitoring while wise ancestors guided them in genomics-based conservation management actions. because the generation of a genome sequence as a research outcome is not directly relevant to the community, the project also worked on developing ecotourism as an alternative livelihood, and helped the collaborators to cultivate the edible mortiño berries (vaccinium myrtillus), which benefit both the antioquia brushfinch (atlapetes blancae), also called the ‘montañerito paisa’, and the community. increasing the social value of genome sequencing required a new mindset: “i think in the long run, with these social benefits, resources are better spent because the project is filling a community need that helps them to conserve nature. there is a growing detachment between what people learn in universities and the needs of colombia. studying the gene for a bird to be blue or yellow has very important scientific value, but that kind of information may not be in colombia’s list of highest priorities.” [gustavo a. bravo, curator of ornithology at instituto alexander von humboldt] for this change of mindset, university curricula have to include more ethics and responsible business conduct. it also necessitates a reorganization of research funding because, in a project with a strong participatory component like this, a sequence can be five times as expensive to obtain. those costs illustrate that while scientists in the dsi discussions managed to be exempted from monetary benefit-sharing, the organization of research funding is indeed related to financial benefits for communities of interest. yet, it is so far not clear to the project leaders how these benefits can be sustained over time because a genome publication is a one-time event and scientific project funding will dry up at some point. that means that sequencing projects by themselves are inadequate sources of funding compared to standard conservation funding. dsi and genetic resources use in science nis components: framework conditions, education, research, industry, political and international policy systems, financial and physical infrastructure in colombia, advanced scientific capacity is held back by structural underfunding, brain drain, a lack of cyberinfrastructure, and inconsistent and increasingly short research grants. opportunities arise mainly in public– private research partnerships and international scientific collaboration. interviewees indicated that there already is advanced scientific knowledge, lab quality and technological development in colombia. however, relative to comparable countries in the region, it has notably fewer biotechnological and bioinformatics publications (benitez-paez, 2010; martinez et al, 2014). this may be explained by the following factors. the first reason is related to education. in higher education, less than 10% of students follow bioeconomy-related disciplines (alviar et al, 2021). for university graduates, there are few research positions, to such an extent that fewer are enrolling in phd programmes, and many emigrate. some researchers with a strong motivation to give back to the country eventually return, but their patience gets exhausted too. this poor economic perspective is also experienced by employed researchers, exemplified by a story from a public university scientist: “in just one year, all three of my phd students left because they were not being paid by the university as contractually promised. no research was done. many professors tell me that the only students who end up graduating are the ones who are independently wealthy or those with scholarships.” [university biotechnologist] this outflow of trained personnel, in combination with short-term research grants, places a burden on principal investigators who have to repeatedly train new researchers. another limiting factor is the cyberinfrastructure. colombia has sufficient developers, administrators and bioinformatics expertise to run such infrastructure, but the availability of high-performance computing, computational training resources and data storage is limited (de vega et al, 2020; c3biodiversidad, 2018b). in other words, the strong human resource component is suppressed by a weak physical resource component (figure 3). both interviewees and c3biodiversidad (2018b) indicated that insufficient and unstable investment in r&d is the major challenge to knowledge production. between 2000 and 2020, total r&d expenditure as a share of gdp in colombia was, on average, 0.22% (world bank, 2024). although not uncommon in latin america, that is very low when compared to regional leader brazil, with 1.11%, and the oecd average of 2.40% over that same period. meanwhile, under the current government of gustavo petro, the budget for higher education is declining by one quarter in real terms, even though the ambition was to increase r&d expenditure to 0.5% of gdp by 2026 (fernández, 2023). as research grants decrease in size, competition among researchers increases, and the scope of projects becomes more limited. structural underfunding and swings in government budgets particularly affect public universities, which receive less funding through tuition fees and private investment than private universities. genetic resources (2025), 6(12), 39–5648 kreiken and asveld interestingly, the tendency of new governments to tie science funding to political themes, thereby limiting research groups’ consistency, has had a knock-on effect on public universities’ collaborations with companies that prefer longterm and stable research relationships. additionally, funding often does not arrive in time. ten percent of the sistema general de regalías, a system for the distribution of royalties from industry, is invested in science. yet only half of the budget was delivered during its first year of operation, raising suspicions of corruption with interviewees (organisation for economic co-operation and development (2014), p. 118). irregularities and lost resources are recurrent to this day. opportunities mainly appear in public–private collaborations. the share of business expenditure in r&d (berd) in colombia in 2020 stood at 0.15% compared with the oecd median of 1.15%. four-fifths of businesses in colombia are reported to not invest in r&d (dane (2023), as cited in organisation for economic co-operation and development (2024a)). berd can complement university funding when basic research is co-financed by applied research. for example, in the icesi sustainable industries and applied science labs in cali, one of the most advanced in latin america, companies hire university researchers to design, validate or prototype bioprocess tests. eafit, a private university in medellín, secured high-performance computing, known as the apolo platform, in partnership with purdue university, which now enables them to conduct paid services for companies. hybrid positions wherein a researcher or student works part-time in a company are also smart from both a science funding and valorization standpoint. despite these synergies, public–private research collaborations can have trade-offs, including the skewing of the research agenda toward commercial applications over basic research that can benefit the conservation and sustainable use of biodiversity, and a possible misalignment between the private sector’s need to maintain a quick pace and patience-requiring participatory processes with stakeholders. there are also improvements to be made in international science policies. slightly less than half (43.5%) of colombian scientists experienced feelings of language-based discrimination in article revisions and rejections (ramírezcastañeda, 2020). the country has low english literacy, and translating takes up a lot of research time and budget. high fees for open-access publishing can also be a barrier to publishing in high-impact and high-visibility journals. bilateral science diplomacy can help to enhance international scientific collaborations without the need for extra funding. the united kingdom embassy in bogotá contributed to the creation of bridge colombia, a network of colombian and british scientists. academics in bridge colombia then secured funding from the global challenges research fund for a flagship project called grow colombia, which aims to boost the country’s bioeconomic innovation capacity. bilateral science diplomacy can also help to flag science issues directly to governments or agencies and businesses. feedback from scientists on the prohibitive cost of sequencing in colombia, for example, was communicated through the embassy to british technology providers. “science diplomacy helps to connect uk science with colombian priorities. there was a time when the uk came to ‘teach’ colombian scientists. that is over now as a result of their joint research work. the bilateral collaboration operates now under a logic of equitable research partnerships. that is possible because colombian science has been advancing to a point at which scientists from both countries work on a peerto-peer basis.” [luis calzadilla, head of science and innovation at uk embassy in bogota] this facilitative role in accessing funding and enhancing equity in research is very important. interviewees remarked that they increasingly have to find non-colombian collaborators to be able to apply for grants abroad, which feels awkward and exploitative. in the opposite direction, it was felt by an interviewee that some foreigners just seek collaboration with colombian scientists to access samples, without leaving meaningful work on the publication for their counterparts. this type of sample-focused “helicopter research” in collaborations with colombian institutions is already reported in human genomics (cock-rada & gomez, 2018). industrial use of genetic resources and dsi nis components: framework conditions, demand, education, research, industry, political and international policy systems, intermediaries, financial and ipr and information infrastructure there is a major valorization gap in the country that is caused by a lack of entrepreneurship support and culture, investment unavailability, administrative delays, export challenges and tax burdens. colombia has fewer large bioprospecting centres and biotechnology-based companies than similar countries in the region (bueno & ritoré, 2019). the country seems to suffer from “an absence of scientific governance” to direct r&d toward commercialization and toward knowledge gaps in genetic diversity conservation (bueno & ritoré, 2019). once university scientists find bioprospecting value, they struggle to valorize the findings. marine botanist enrique peña at universidad del valle in cali initiated studies for the application of two species of invasive red algae (sargassum fluitans and sargassum natans) that pollute the tourist beaches of san andrés island in the caribbean as a feedstock for fertilizer production. building a pilot plant on campus would cost approximately 1 million usd. although peña has already attracted interest from companies, domestically and abroad, funding or investment to initiate building the plant is still insufficient. peña noted that an entrepreneurial mindset is still uncommon in public universities. private universities, once founded by business leaders, offer more support for entrepreneurship and valorization, such as business and finance courses in life sciences programmes, incubators, and mentorship programmes. sciphage is a biotechnological start-up with its own r&d and some patents from research at universidad de los andes. it develops phage therapy for the livestock sector, an alternative method to antibiotics that has various health and environmental benefits (mishra et al, 2024). sciphage has already built a production plant outside bogotá and is seeking investment to genetic resources (2025), 6(12), 39–56 innovating with genetic diversity in colombia 49 scale up production. the pontificia universidad javeriana has another model where it creates spin-offs with its own patents. dreembio is one spin-off that develops phytomedicines for cancer treatment based on ethnobotanical plant knowledge. the company is working with campesino or farmer communities to develop the raw material value chain. these companies clearly show that innovation can have socio-environmental benefits. according to arturo luna, the former minister of science, technology and innovation, while the bioeconomy does require advanced biotechnology, it can include low-tech businesses too: “there are already low-tech businesses in colombia that add value to biodiversity and fit perfectly with the bioeconomy model. we have to invest in these businesses because this virtuous cycle will generate employment and financial resources and opportunities for biodiversity conservation. however, in order to add value to our biodiversity, more and sustained investments in biotechnology are required.” [arturo luna, freelance] take, for example, kahai s.a. it managed to produce the jungle cacay tree (caryodendron orinocense) on plantations. because the nut’s oil outperforms argan oil in some cosmetic applications, there is an enormous biotrade potential. to collaborate with international development agencies and have a better investment case, the company built a strong corporate social responsibility (csr) component, including a reforestation programme and inclusion of iplcs. these three home-grown companies represent textbook examples of successful bioprospecting and biotrade. but they face numerous challenges, which are illustrative of the kinds of issues that limit the development of genetic resources and dsi in the natural products industry. first, there is an investment gap in colombia. regular investors, like banks, lack advanced knowledge of biotrade and biotech. throughout the country, there is an over-demanding investment culture with sometimes “aggressive pushing for unrealistic targets” and requests for proof of traction and several letters of intent from buyers before pre-seed or seed funding can be acquired. in other countries, interviewees experienced more eager investors who understand that break-even is far away and that not all investees will succeed. the second major challenge relates to the issuing of sales permits by instituto nacional de vigilancia de medicamentos y alimentos (invima), the government agency that approves products for human and animal health. biotech companies experience the licensing process as a severe regulatory obstacle, which is attributed to the agency’s perceived unfamiliarity with novel technologies, insufficient capacity and personal hesitancy to take risks to avoid liability. biotrade companies also struggle, primarily with delays. both kahai s.a. and pangea natural products, a company that sells medicinal herbal supplements, waited roughly five years for approval of their product, though the hefty permit costs of over 3,000usd were already paid. for starting companies, these costs are high, and delays can mean ‘make it or break it.’ another hurdle is the lack of domestic demand for natural products based on scientific research: “demand creation is a cultural process where you teach about the environment and the values of the product, but also [the positive] impacts for communities.” [legal expert] colombia has the character of a ‘follower market’ and its consumer culture requires that companies first demonstrate product success abroad. while adapting to customer needs, companies struggle with strict import regulations such as those imposed by the eu. network brokers, like local chambers of commerce, have an important role in helping companies access markets. reducing global conference fees would help companies gain exposure to investors and clients. the fourth challenge is colombia’s tax and fiscal policy, which, according to interviewees, stifles the growth of their companies and makes them strongly consider commercializing abroad. compared with an average of 23.7%, colombia’s 35% corporate income tax rate is the highest of all oecd countries (organisation for economic co-operation and development (2024b), p. 20-26). although the 19% value-added tax is close to the oecd average, it is still among the highest in latin america. a policy proposal for a phased decrease in tax rates for small and large businesses is being debated in parliament. because the results show that universities strategically use patents for founding startups and spin-offs, it is also important to discuss the challenges and opportunities for the ipr system (described earlier in section ‘policies for biodiversity and bioeconomy’). in 2024, the colombian government issued a compulsory license to start producing generic and more affordable versions of the drug dolutegravir for tackling an emerging hiv crisis. this type of action, however, could have negative effects such as diplomatic repercussions, arbitration or decreased market entry by foreign drug companies (landis, 2024). such risks may be lower if colombia can source more products from domestic companies. but here again, the country’s funding landscape will need drastic adjustment to guide startups with acquired patents through the financially challenging valley of death phase between r&d and revenue generation. on the other hand, there are opportunities at a national and international level. to begin with, universities should continue investing in their technology transfer and innovation offices that support researchers during r&d and connect them to industry. on top of that, universities can cover the costs of researchers’ patent applications and create room in work schedules for entrepreneurship (calza et al, 2020). at a national level, the government can implement patent pilot programmes to review the efficacy of patent dispute resolution by courts (salas, 2020). bilateral diplomacy related to ip can also facilitate the protection and export of colombian products and technology. one tool is the patent prosecution highway, which countries use to fast-track patent applications by companies that acquired patents in another country. to stimulate bilateral technology transfer between colombia and countries with divergent ipr systems, it can develop mutual transfer agreements that contain colombia’s abs provisions but are flexible enough to incorporate the other country’s priorities (fajardo et al, 2025). there are also unresolved tensions in the ipr system with regard to the protection and empowerment of iplcs, genetic resources (2025), 6(12), 39–5650 kreiken and asveld caused by cbd decisions that reinforced national sovereignty over genetic resources to the detriment of indigenous selfgovernance (fredriksson, 2019). martha gomez lee, an abs scholar, argues that the self-governance of iplcs can be promoted by embedding article 31 of the un declaration on the rights of indigenous peoples in the abs system, which states: “… they also have the right to maintain, control, protect and develop their intellectual property over such cultural heritage, traditional knowledge, and traditional cultural expressions.” empowerment of iplcs during the development of intellectual property may carry implications for the governance of dsi: “in my opinion, the crux of the matter is that even before intellectual property rights, any initial digitization of a genetic resource and its deposit in any database must have explicit prior informed consent.” [martha gomez lee, teacher-researcher in abs and traditional knowledge at universidad externado de colombia] finally, it is worth noting here that companies think that working with campesinos is much easier than with iplcs because the former are generally better organized (so engagement has a faster pace) and the latter have complex cosmovisions to engage with. also, companies can use publicly available scientific knowledge on the function of species that may have been discovered by iplcs in the pre-abs era. this perhaps explains why there are, to date (and as confirmed by martha gomez lee), zero signed annexes by providers of traditional knowledge as an intangible component to genetic resources, although the latest figures show that around 20 commercial abs contracts and 400 abs contracts overall have been concluded with the ministry (ministerio de ambiente y desarrollo sostenible, 2021). discussion despite colombia’s large bioeconomic potential, many structural challenges remain. in figure 4, we present the current issues (in text boxes) and provide recommendations (in italics). the majority of issues relate to underfunding, investment and regulatory capacity, and red tape. existing research capacity is not used efficiently because of siloed r&d, high equipment and maintenance costs, and outflow of trained personnel. moreover, the companies with bioeconomic potential that do emerge struggle to grow inside colombia. without considering these domestic policy issues, monetary payments, scientific capacity building and technology transfer as typical abs tools, will have little effect on achieving that potential. here, we reflect on what could be accomplished by colombia itself, how specific needs could be addressed by abs policies, the cali fund and non-monetary benefitsharing from dsi, and lastly, the value of innovation system models for both holistic and targeted policy interventions. although subsequent governments have expressed strong bioeconomic intent, policies are being discontinued, and there is a coordination failure between government bodies. this aligns with aparicio's (2022) finding that colombia’s narrative is focused on future high-tech-driven biodiversity exploration as a precondition for the bioeconomy, while questions over the current performance of value chains are pushed aside. on the other hand, the findings showed that the fostering of cultural pride in home-grown science and companies may be the next step for growing national interest in biodiversity and bioprospecting. colombia’s combination of underinvestment and high taxation further lowers the creation of and erodes benefits from genetic diversity. this shows that bioeconomy policy should expand its scope from technology and biodiversity to bureaucracy, government capacity, science funding, factors for business growth and retention of companies. immediate priorities to tackle are the delays in various permits and the degradation of biocollections. in our opinion, the bureaucracy, particularly, is a ‘talent grinder’ for people who intentionally do research or business for the benefit of the country. greater engagement of scientists and entrepreneurs in policy-making that concerns them is recommended in order to help address their needs and decrease red tape. mobilizing intermediaries and network organizations would be useful for this purpose and for enabling public–private partnerships that help scientists access new funding sources (figure 4). also, the export focus of companies due to a lack of domestic demand leads to missed opportunities to address local needs through valuable products, retain jobs and tax revenue. the government should therefore stimulate more demand for biotech and biotrade products and services (weerasinghe et al, 2024), and, although very complex, incentivize private sector alignment with green rather than fossil-fuel biotech pathways: “with some companies, there is a mentality that when you provide jobs, you already do enough. but business conduct is not per se sustainable and not per se benefit-sharing.” [ana maria castillo, director of competitiveness and internationalization at the cali chamber of commerce] the analysis has various implications that (non-)parties and observers to the cbd must consider. first, they should engage potential beneficiaries of the cali fund, like colombia, to critically evaluate the effects of various policies on the full value chain of genetic resources and dsi. to enhance synergies between the political system and international policy and political system (figure 4), (non-)parties to the cbd can link the biodiversity-focused negotiations to bilateral and multilateral diplomatic processes over scientific, technological and industrial cooperation, ip and technology transfer, taxation, development aid, and customs. furthermore, the nis model and similar approaches can help to diagnose issues and identify bottlenecks in countries, and steer a more effective and efficient use of monetary benefits from the fund. for instance, it is smarter to invest in lowhanging fruits and missing cogs, such as public–private partnerships and reagent cost reduction for sequencing than in new sequencers. additionally, investing in biodiversitypositive and iplc-inclusive companies may have a higher return on investment in the long term than grants for shortterm conservation projects. ultimately, (non-)parties and observers to the cbd have to look further than physical infrastructural (figure 4) capacity needs related to “generate, access, use, analyse and store” dsi (cbd, 2024). to embed this holistic perspective in abs policy, parties to the cbd are recommended to incorporate standard language related to innovation systems in the negotiation documents on dsi and fi gu re 4 . i de nt ifi ed c ha lle ng es in c ol om bi a’ s na tu re -b as ed b io te ch no lo gi ca l i nn ov at io n sy st em w ith a re as fo r im pr ov em en t p re se nt ed in it al ic s. a cr on ym s: s m al l a nd m ed iu m en te rp ri se s (s m es ); r es ea rc h an d de ve lo pm en t ( r & d ); s ci en ce , t ec hn ol og y an d in no va tio n (s ti ); in te lle ct ua l p ro pe rt y (i p) ; i nt el le ct ua l p ro pe rt y ri gh ts ( ip r s) . genetic resources (2025), 6(12), 39–56 innovating with genetic diversity in colombia 51 pushed aside. on the other hand, the findings showed that the fostering of cultural pride in home-grown science and companies may be the next step for growing national interest in biodiversity and bioprospecting. colombia’s combination of underinvestment and high taxation further lowers the creation of and erodes benefits from genetic diversity. this shows that bioeconomy policy should expand its scope from technology and biodiversity to bureaucracy, government capacity, science funding, factors for business growth and retention of companies. immediate priorities to tackle are the delays in various permits and the degradation of biocollections. in our opinion, the bureaucracy, particularly, is a ‘talent grinder’ for people who intentionally do research or business for the benefit of the country. greater engagement of scientists and entrepreneurs in policy-making that concerns them is recommended in order to help address their needs and decrease red tape. mobilizing intermediaries and network organizations would be useful for this purpose and for enabling public–private partnerships that help scientists access new funding sources (figure 4). also, the export focus of companies due to a lack of domestic demand leads to missed opportunities to address local needs through valuable products, retain jobs and tax revenue. the government should therefore stimulate more demand for biotech and biotrade products and services (weerasinghe et al, 2024), and, although very complex, incentivize private sector alignment with green rather than fossil-fuel biotech pathways: “with some companies, there is a mentality that when you provide jobs, you already do enough. but business conduct is not per se sustainable and not per se benefit-sharing.” [ana maria castillo, director of competitiveness and internationalization at the cali chamber of commerce] the analysis has various implications that (non-)parties and observers to the cbd must consider. first, they should engage potential beneficiaries of the cali fund, like colombia, to critically evaluate the effects of various policies on the full value chain of genetic resources and dsi. to enhance synergies between the political system and international policy and political system (figure 4), (non-)parties to the cbd can link the biodiversity-focused negotiations to bilateral and multilateral diplomatic processes over scientific, technological and industrial cooperation, ip and technology transfer, taxation, development aid, and customs. furthermore, the nis model and similar approaches can help to diagnose issues and identify bottlenecks in countries, and steer a more effective and efficient use of monetary benefits from the fund. for instance, it is smarter to invest in lowhanging fruits and missing cogs, such as public–private partnerships and reagent cost reduction for sequencing than in new sequencers. additionally, investing in biodiversitypositive and iplc-inclusive companies may have a higher return on investment in the long term than grants for shortterm conservation projects. ultimately, (non-)parties and observers to the cbd have to look further than physical infrastructural (figure 4) capacity needs related to “generate, access, use, analyse and store” dsi (cbd, 2024). to embed this holistic perspective in abs policy, parties to the cbd are recommended to incorporate standard language related to innovation systems in the negotiation documents on dsi and fi gu re 4 . i de nt ifi ed c ha lle ng es in c ol om bi a’ s na tu re -b as ed b io te ch no lo gi ca l i nn ov at io n sy st em w ith a re as fo r im pr ov em en t p re se nt ed in it al ic s. a cr on ym s: s m al l a nd m ed iu m en te rp ri se s (s m es ); r es ea rc h an d de ve lo pm en t ( r & d ); s ci en ce , t ec hn ol og y an d in no va tio n (s ti ); in te lle ct ua l p ro pe rt y (i p) ; i nt el le ct ua l p ro pe rt y ri gh ts ( ip r s) . genetic resources (2025), 6(12), 39–5652 kreiken and asveld abs, for example, ‘take into account and strengthen countries’ capacity to innovate with genetic resources and dsi’. the decision to distribute the fund’s money via national biodiversity funds instead of project-based applications deserves attention, too. some companies’ patent royalties that have been paid to the ministry of the environment and sustainable development through abs mechanisms have not been distributed yet. thus, governments’ capacity to distribute funds and iplcs’ capacity to receive them deserve immediate attention to maintain company engagement and minimize overhead costs. also, because many beneficiary countries are unequal (colombia’s gini coefficient is among the highest in the world (organisation for economic cooperation and development, 2024a), care is warranted so that benefits are directed to the most disadvantaged actors in beneficiary countries. apart from iplcs, that would be female researchers (paz & pardo-díaz, 2024), particularly in public universities in the underdeveloped regions of colombia. seeing these issues, new opportunities for benefit-sharing emerge. even though research and academia remain, rightly so, ‘off the hook’ from monetary benefit-sharing, their handling of resources certainly involves distributive questions. abs policy should coordinate more with international efforts to map and decrease costs for lmic scientists, such as open-access publishing and conference fees. and, as the wise ancestors and bridge projects illustrate, north-south scientific collaborations and capacity-building projects may consider the fair allocation of research funding and joint application for research grants in hic. although unconventional, there is also a big potential for non-monetary benefits from companies. to understand this, we draw on the concept of political corporate social responsibility (csr). it holds that in the era of globalization, countries, especially developing ones, cannot fully regulate business conduct and that csr activities have an increasingly political nature. this not only manifests itself in voluntary actions and initiatives for self-regulation but also in collaborations with governments to fill governance gaps or to provide public goods like science (azizi, 2020; frynas & stephens, 2015). the cop16 decision reflects a narrow sense of justice in exchange by stating that users who make a payment “are considered to have fairly and equitably shared monetary benefits” (cbd (2024), para 15). but do benefits have to necessarily take the form of tick-the-box payments? our findings show a clear need for companies in lmic to access investment and loans, and mentorship in r&d, company leadership, market access and regulatory affairs. eligible companies for payment to the fund could play a huge role here and, in some cases, pursue mutual interests. for this, the cbd could also undertake a networking function to match companies between hic and lmic as a form of development aid (an example is pum in the netherlands), thereby allowing for better alignment of r&d than general capacity-building projects. parties could consider legitimizing existing or new activities as monetary benefit-sharing. the risk is that the functioning of the fund for biodiversity finance is undermined, and that the cost figures of the activities could potentially be skewed to meet the 1% of profit or 0.1% of revenue mark. in any case, initiatives should build and not erode trust in the un and recipient governments. analysts using the nis approach may include an international industry system to guide these interactions, especially for analyzing cross-border industries. on the nature-based biotechnological nis (figure 3), we note that the wealth of findings and the wide scope of the study limit the discussion of specific interactions between systems or actors in detail. while the article adopts a nationwide lens, many interactions happen on a local or regional level, especially in innovation hubs such as medellín. a closer look may reveal regionally different interactions and systems, and possibly inequalities. scholars using the nis approach may reconsider the marginal position of traditional knowledge by including it under a separate system. it is recommended that their and civil society’s perspectives be included more strongly in further research, although an effort was made to secure a balanced selection of interviewees (table 1). we emphasize that because of the approach, we highlighted issues while the situation is not black and white. many issues are also found in other countries, including hic, and colombia, as a case study, is likely not unique in that respect. more comprehensive studies and texts would allow for more comparison and insights into strengths and weaknesses. it is important to tailor the nis to the unique context of the country of analysis, since the promotion of ‘one-size-fits-all’ ideas for development can disregard other ways of learning (casadella & tahi, 2023). conclusion facing accelerating technological progress and growing innovation divides, the cbd stands at a crossroads. will it finally consider the fair distribution of scientific and innovation capacities and thereby put shared benefits to use effectively? clearly, a business-as-usual continuation of capacity-building and benefit-sharing activities is unfit for current and emerging biotechnological innovation trajectories. the global political push to share benefits from dsi, therefore, has to be coupled with concerted efforts for policy reform in beneficiary countries, in abs, and unconventionally in other policy domains. holistic policy interventions, by principle, require governments to consult those who create benefits, educators, scientists, ceos, and, of course, iplc representatives. but perhaps not all can or should be solved by governments. governance gaps invite country-specific contributions by users of genetic diversity that match technological niches and satisfy mutual interests. we eagerly await new research on innovation systems in the context of bioprospecting, so that policymakers can learn from countries’ best practices and take advantage of regional opportunities. supplemental data supplemental material 1. list of interviewees supplemental material 2. resumen en español (spanish abstract) author contributions bob kreiken: conceptualization, formal analysis, investigation, methodology, project administration, validation, visualization, writing – original draft, writing – review & editing lotte asveld: conceptualization, funding acquisition, project administration, supervision, writing – review & editing https://www.genresj.org/index.php/grj/article/view/genresj.apnr6909/suppdata277 https://www.genresj.org/index.php/grj/article/view/genresj.apnr6909/suppdata277 genetic resources (2025), 6(12), 39–56 innovating with genetic diversity in colombia 53 conflict of interest statement the authors report no conflict of interest. ethics 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(2025) “genetic variation of burgo chicken from bengkulu, indonesia, based on the nd1-mitochondrial dna gene”, genetic resources, 6(12), pp. 153–159. doi: 10.46265/genresj. nzsl1498. © copyright 2025 the authors. this is an open access article distributed under the terms of the creative commons attribution license (cc by 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. jarulisa,*, aceng ruyanib, nurmeiliasaric, ahmat fakhri utamaa amagister program of biology, department of biology, faculty of mathematics and natural sciences, universitas bengkulu. jl. w.r. supratman, kandang limun, bengkulu 38371, bengkulu, indonesia bbachelor program of biology, department of biology, faculty of mathematics and natural sciences, universitas bengkulu, jl. w.r. supratman, kandang limun, bengkulu 38371, bengkulu, indonesia cdepartment of animal science, faculty of agriculture, universitas bengkulu, jalan w. r. supratman, kandang limun, bengkulu, indonesia * corresponding author: jarulis (jarulis@unib.ac.id) introduction the domestication of wild animals is part of the journey of human civilization. one of the most commonly domesticated animals is the chicken. chickens are bred for egg and meat production. the red partridge is the first chicken that was successfully domesticated in southeast asia and southwest china (fumihito et al, 1994; väisänen et al, (2005); liu et al, 2006; miao et al, 2013). studies indicate that the domestication process of red partridges in asia began around 3,000 years ago, leading to the species now known as the domestic chicken (gallus gallus domesticus). domestication of the red partridges (gallus spp) in east asia occurred in the mid-late holocene. (miao et al, 2013; larson et al, 2014). domestication has influenced changes in the behaviour, physiology and productivity of chickens; however, some similarities persist between domestic chickens and their ancestors, such as aggressive behaviour during mating and urinary protein excretion, which remain consistent with that of their wild counterparts. (al-nasser et al, 2007). meanwhile, local chickens found in indonesia have continued to develop since this successful domestication process. this situation has led to indonesian chickens forming a different genetic clade from other chickens in asia. therefore, indonesia is considered one of the centres of chicken domestication in asia (sulandari et al, 2007). in https://doi.org/10.46265/genresj.nzsl1498 https://www.genresj.org https://doi.org/10.46265/genresj.nzsl1498 https://doi.org/10.46265/genresj.nzsl1498 mailto:jarulis@unib.ac.id genetic resources (2025), 6(12), 153–159154 jarulis et al indonesia, there are red partridges (g. gallus bankiva and g. gallus spadiceus) and green jungle fowl (g. varius) with a total of 31 strains spread across the regions of sumatra, java, bali and nusa tenggara. (sibley and monroe, 1990; nataannjaya, 2000). one of the local chicken breeds found in indonesia is the burgo chicken. burgo chickens are fertile and can produce a high number of offspring, as well as five times more eggs than the red partridges, averaging 32 eggs per period (sutriyono, 2016). in addition, burgo chickens have a distinctive crowing sound and beautiful feather colours, which encourage people to raise them as ornamental animals and livestock. the burgo chicken population is found in all districts of bengkulu province, sumatra island (putranto et al, 2017). however, there has been no research into their genetic relationship and characteristics, so it remains unclear whether it is the result of inherited genetics or the impact of environmental factors. as a source of germplasm, burgo chickens are threatened by various anthropogenic factors, including habitat fragmentation, which causes isolation in these species, further threatening their populations. moreover, as one of the local chicken clades, the taxonomic position of burgo chickens remains unknown. taxonomic determination is generally based on morphological and genetic characteristics. studies related to the morphology of burgo chickens have been conducted previously (rafian et al, 2017; safitra et al, 2022). mitochondrial dna (mtdna) has been widely used to analyze genetic variation between populations and species due to the high number of dna copies, making it suitable for analysis with a limited amount of dna or easily degraded dna (ni'mah et al, 2016). one of the mtdna genes used for species identification is nadh dehydrogenase subunit 1 (nd1) (amin and mushlih, 2020). the nd1 gene is part of complex i, also known as nadh. ubiquinone oxidoreductase is the first and largest enzyme complex in the mitochondrial respiratory chain, playing a role in oxidizing nadh to release electrons that assist in the translocation of protons to the inner membrane, producing proton gradients (hirst, 2010). the genetic diversity of the gallus genus, based on the mitochondrial dna coi gene, shows a genetic similarity of 98% between red partridges from bengkulu and south sumatra (jarulis et al, 2022). several previous studies have utilized the nd1 gene. for instance, bowles and mcmanus (1993) revealed interand intraspecies variations in echinococcus from 59 isolates; raharjo et al (2018) detected rat meat contamination in meatballs using the nd1 gene; and widayanti et al (2022) successfully identified mutations at three sites within the 972-nucleotide sequence of the nd1 gene of indonesian catfish. therefore, we investigated the potential of the nd1 gene to determine the level of genetic similarity among burgo chicken populations, other chickens in indonesia, and other gallus species. no comparative genetic study of burgo chickens, particularly based on the mitochondrial dna nd1 gene, has ever been conducted. therefore, this research is essential to provide data on the genetic diversity and variation among burgo chicken populations and between species of the gallus genus in indonesia. the findings will support the bengkulu provincial government’s efforts to identify and designate the bengkulu burgo chicken cluster for submission to the central government, as part of future conservation initiatives aimed at preserving the population’s genetic diversity. materials and methods blood collection blood samples were collected from 28 burgo roosters owned by members of the bengkulu burgo chicken hobbyists. there three locations where the burgo chicken samples were taken are bengkulu city, kepahiang, and rejang lebong. blood samples were drawn through the carpal joints and pectoralis veins. preserved using edta tube according to seutin et al (1991) and stored in a freezer at -20°c, before use. all blood samples were analyzed in the molecular biology laboratory, department of biology, universitas bengkulu. dna extraction and purification the blood samples (10-20µl) were preserved in edta tubes. the dna was isolated using the dneasy® blood and tissue kit cat. no. 69504 (50), following the spincolumn protocol qiagen procedure with modification. in our research, the elution solution used was 50µl with three repetitions. the isolated dna was observed on 1.2% agarose gel using electrophoresis and stored in a freezer at -20°c, before the amplification process. polymerase chain reaction (pcr) dna the nd1 gene of burgo chickens was replicated using a pcr technique with a dna template derived from the total dna product. the nd1 gene sequence used to design the specific primer in this study was obtained from the complete genome of mitochondrial dna from g. gallus from kalimantan (genbank accession number ky039421). nd1the primers were brnd1f (5'cccaccctaacaaaccttctaatc-3') and brnd1r (5'tagggtgacttcgtat gagat tgt-3'), which amplified a 450bp fragment of the 974 bp nd1 sequence. all reaction mixtures followed the existing protocol gotaq green. the reaction mixture contained 25µl gotaq green, 1.5 µl forward primer, 1.5µl reverse primer, 3µl dna template, and 19µl nuclease-free water. pcr amplification was performed using a simpliamp thermal cycler with the following programme: denaturation at 94°c (1 minute), annealing at 55°c (45 seconds) and elongation at 72°c (1 minute) for 30 cycles. furthermore, the successful amplification samples were sent to pt. genetika sains for sequencing. data analysis the bioedit 7.0.9 software (hall, 1999) was applied to edit the nd1 gene sequence and visualize the electrograms and nucleotide base sequences.the nucleotide sequence (forward and reverse) products were aligned using clustal w of the mega 11.0 programme (tamura et al, 2013). each individual's gene sequence was compared with the nd1 reference to determine the similarity level of the samples. the genetic distance between individuals was calculated using the 2-parameter kimura (k2p) method (kimura, 1980). the phylogeny tree was constructed using the neighbourjoining (nj) method with 1,000 replications (tamura et al, 2013). additional nd1 g. gallus gene sequences found in genbank were downloaded and included in the phylogenetic tree reconstruction analysis (see table 1). genetic diversity parameters, namely haplotype (hd) and nucleotide (π) diversity were calculated using dnasp v6.12.03 software genetic resources (2025), 6(12), 153–159 genetic variation of burgo chicken, indonesia 155 (rozas et al, 2017). the haplotype analysis was presented in a sequence location distribution map/operational taxonomic unit (otu) and haplotype network images to depict the latest connectivity and genetic distribution between populations using model median-joining by network v10.2.0.0 software (bandelt et al, 1999). results single nucleotide polymorphism the nucleotide sequence of the nd1 gene observed in table 1. snp between individuals of burgo chickens from bengkulu based on the nd1 gene (450bp). sample code indicates accession numbers of sequences sourced from genbank. dots (.) indicate identical nucleotide to the reference sequence for nd1 (ky039420.1). a, adenine; c, cytosine; g, guanine. 450bp between burgo chicken species from bengkulu had two nucleotide polymorphisms (snp) that differed among individuals at positions 52 and 375 (table 1). site 52 showed a transversion substitution in burgo chicken individuals from rejang lebong regency and kepahiang regency, namely from cytosine (c) to adenine (a), while a transition substitution at site 375 was found among burgo chicken individuals from central bengkulu, namely from the nucleotide base adenine (a) to guanine (g). no. sample code location/source local name site number haplotype group 52 375 1 ky039420.1 genbank red junglefowl c a hap 2 2 ky039418.1 genbank red junglefowl . . hap 2 3 ky039422.1 genbank red junglefowl . . hap 4 4 ky039421.1 genbank red junglefowl . . hap 5 5 ap003323.1 genbank bankiva . . hap 2 6 nc007238.1 genbank green junglefowl . . hap 6 7 nc007240.1 genbank grey junglefowl . . hap 7 8 nc007239.1 genbank ceylon junglefowl . . hap 8 9 br1 central bengkulu burgo . g hap 1 10 br2 central bengkulu burgo . . hap 2 11 br3 central bengkulu burgo . . hap 2 12 br4 central bengkulu burgo . . hap 2 13 br5 central bengkulu burgo . g hap 1 14 br6 central bengkulu burgo . . hap 2 15 br7 central bengkulu burgo . . hap 2 16 br8 central bengkulu burgo . . hap 2 17 br9 central bengkulu burgo . . hap 2 18 br10 central bengkulu burgo . . hap 2 19 br11 central bengkulu burgo . . hap 2 20 br12 central bengkulu burgo . . hap 2 21 br13 central bengkulu burgo . . hap 2 22 br14 central bengkulu burgo . g hap 1 23 br15 central bengkulu burgo . . hap 2 24 c1f2 rejang lebong burgo . . hap 2 25 c2f3 rejang lebong burgo a . hap 3 26 c4f2 rejang lebong burgo . . hap 2 27 c5f2 rejang lebong burgo . . hap 2 28 k1f1 kepahiang burgo . . hap 2 29 k2f1 kepahiang burgo . . hap 2 30 k3f2 kepahiang burgo . . hap 2 31 k4f2 kepahiang burgo a . hap 3 32 k5f2 kepahiang burgo . . hap 2 33 k10f2 kepahiang burgo . . hap 2 34 k11f2 kepahiang burgo . . hap 2 35 k12f2 kepahiang burgo . . hap 2 36 k13f3 kepahiang burgo . . hap 2 genetic resources (2025), 6(12), 153–159156 jarulis et al haplotype network in this study, network reconstruction was performed using median-joining (bandelt et al, 1999). twenty-eight samples of burgo chickens were complemented with additional genetic data of eight samples of the gallus genus from genbank, including g. gallus from kendu (ky039420.1), g. gallus from garut (ky039418.1), g. gallus from nunukan (ky039422.1), g. gallus from tarakan (ky039421.1), g. gallus bankiva (ap003323.1), g. varius (nc007238.1), g. sonneratii (nc007240.1), and g. lafayetii (nc007239.1). we succeeded in identifying eight haplotypes with a sequence length of 450bp. in burgo chicken samples, three haplotypes were found: hap 1, hap 2 and hap 3 (figure 1). each haplotype is separated by a single nucleotide base, represented by a small horizontal line connecting the haplotypes. figure 1. the haplotype network of the gallus spp. population, based on the nd1 gene alignment. yellow, burgo chicken (this study); blue, g. gallus from kalimantan (ky039422.1; ky039421.1); green, g. gallus bankiva (ap003323.1); orange, g. gallus from java (ky039420.1; ky039418.1); red, g. varius (nc007238.1); grey, g. sonneratii (nc007240.1); army green g. lafeyetii (nc007239.1). genetic distance genetic distances were analyzed using pairwise distances with the mega 11 software (table 2). in general, genetic distance is divided into three groups, namely genetic distance between individuals (intraspecific), genetic distance between g. gallus species, and genetic distance between species of the gallus genus (interspecific). in this study, a slight change was observed in the interspecific genetic distance compared to all burgo chicken samples, incorporating genetic data from red partridges and subspecies from genbank. meanwhile, the outgroup comparison involved all red partridges and their offspring with all other partridges. the intraspecific genetic distance among three districts in bengkulu province, based on the nd1 gene, showed the lowest value of 0%, while the highest distance was 0.4%. table 2. intraand interspecific genetic distance in burgo chickens based on the nd1 gene (450bp) genetic distance maximum minimum average intrapopulation of burgo chicken 0.4% 0.0% 0.1% burgo chicken versus other gallus gallus 0.4% 0.0% 0.12% interspecies of gallus spp. 5.9% 0.2% 3.62% genetic resources (2025), 6(12), 153–159 genetic variation of burgo chicken, indonesia 157 phylogeny phylogenetic trees were reconstructed using the nd1 gene to determine the taxonomic position of burgo chickens in comparison with the available reference data. the phylogeny of the burgo chicken sample was grouped into one clade with red partridge from java, kalimantan, and the subspecies of g. gallus bankiva (figure 2). however, five samples formed a small group, namely br3, br14 and br1 from central bengkulu, and c2f3 and k4f2 from rejang lebong and kepahiang. this is due to the discovery of mutations in the sequence that caused a slight change; however, the shape of the phylogenetic tree remains stable. discussion human intervention drives the continuous domestication of chickens. domestication has led to the development of many chicken breeds worldwide. indonesia has 31 local chicken breeds that have adapted over tens to hundreds of years. each local chicken breed has characteristics influenced by its specific region (nataamijaya, 2010). these characteristics are intrinsically linked to their genetic foundation, such as snps. snps are often used to interpret variations and identify species or individuals (torres, 2016). in our study, there were two differentiation sites, namely sites 52 and 375. at site 52, a change was present in nucleotide bases from c to a (c2f3 and k4f2), while a change from a to g was found at site 375 (br1, br5, and br14). these changes are caused by mutations. according to warmadewi et al (2020), mutations can enhance adaptability by eliminating original traits. sometimes, the treatment such as maintaining high stocking density and implementing accelerated growth diets of domestic chickens has negative impacts such as health problems, brittle bones, and even sudden death (hirsch, 2003; meseret, 2016). however, it is not yet known for certain whether the changes that occur in burgo chickens have a positive or negative impact on their ability to adapt, so more in-depth research is needed regarding the morphometry and morphology of burgo chickens. the c2f3 and k4f2 samples were burgo chickens obtained from kepahiang and rejang lebong districts, while the br1, br5 and br14 samples were burgo chickens from central bengkulu district. the landscapes in each location differ: kepahiang and rejang lebong are highland areas, whereas central bengkulu is a lowland area, leading to different adaptation processes. based on the snp data, burgo chickens are grouped into three haplotypes according to their sequence similarity, namely hap 1, hap 2 and hap 3. the 450bp alignment of the nd1 gene yielded eight haplotypes of the entire sequence (figure 1). similar genetic data is present from several species, including burgo chicken, g. gallus from java, and g. gallus bankiva. this is interesting because several burgo chickens share the same genetic components as g. gallus (java) and g. gallus bankiva; however, the results of the haplotype analysis may be influenced by the number of samples and population. research by wang et al (2020) using 863 native and domestic chicken genomes showed that crossbreeding occurred among red partridge subspecies. therefore, it is possible that all three originated from the same ancestor. these data are strengthened by previous studies that revealed the origin of red partridge as the ancestors of local chickens worldwide. sulandari et al (2008) found 69 haplotypes in the  genetic characterization of local indonesian chickens and local chickens outside indonesia using d-loop, besides discovering the indonesian chicken genes in other countries. although our data only used three breed populations in bengkulu province, they revealed a direct relationship between burgo chickens, red partridge, and their descendants, as indicated by haplotype 2. genetic distance is one of the tools used for species identification, alongside morphological and morphometric data. lately, bird research has been relying on genetic data to facilitate the identification process. each species has a threshold value for genetic distance; if the genetic distance is equal to or greater than 3%, species separation occurs (fouquet et al, 2007). based on genetic distance identification, the distance between burgo chickens and red partridges (intersubspecific) is 0.1–0.4%. meanwhile, the distance among burgo chickens (intraspecific) ranges from 0 to 0.4%, indicating a close relationship at the species level. this suggests that they likely originate from a closely related or similar population. therefore, burgo chicken can be identified as a new breed of red partridge. however, further study on morphometry as well as sound identification is required to ensure this theory. similarly, utama et al, (2023) obtained the genetic distance between burgo chickens and red partridges as 0–0.8% using the coi gene. in addition, zein and sulandari (2008) reported that the genetic distance between native chicken populations in lombok, using the d-loop, ranged from 0.1–1.7%. the distance between red partridges from bengkulu and south sumatra based on the coi gene has also been confirmed to range from 0–1.4% figure 2. phylogenetic tree construction with neighbour-joining (nj) modelling of 28 burgo chickens from bengkulu using a k2p model and 1,000-time bootstrap, based on the nd1 gene (450bp). genetic resources (2025), 6(12), 153–159158 jarulis et al (jarulis et al, 2022). the genetic distance between red partridges and domesticated individuals, as measured by the d-loop and coi gene, exhibits a divergence range of 0–1.7%, which is higher than the divergence observed in the nd1 gene. therefore, the nd1 gene is more conserved. the results of the  phylogenetic tree reconstruction of 28 burgo chickens from bengkulu using the nj model and 1,000 bootstraps are presented in figure 2. nj is one of the phylogenetic analysis methods based on the difference in the evolution rate of each branch. the components in nj analysis are the operational taxonomic units and evolutionary distance. based on the phylogenetic tree, all burgo chickens form a large clade, joined by g. gallus bankiva and g. gallus from kalimantan and java. this suggests that, genetically, burgo chicken still have a direct relationship as descendants of the red partridge. however, there are two small groups among the individual burgo chickens, due to nucleotide base differences at sites 52 and 375. several factors can cause differences in nucleotide bases, including geographical and environmental factors, as well as the duration of isolation, all of which can trigger mutations. in general, the genes used for identification are the coi gene and the non-coding region (d-loop). many studies have focused on these two genes (zein and sulandari, 2012; bilgin et al, 2016). however, several previous studies have stated that the nd1 gene can also be used for identification because it contains conserved regions (bowles and mcmanus, 1993; raharjo et al, 2018; widayanti et al, 2022). therefore, the use of the nd1 gene in species identification can be applied as an alternative to the coi gene with more stable traits in the region. however, this method is not yet accurate in determining the taxonomic position or discovering species history because our study only used 450bp (± 50%) of the total length of the nd1 gene (974bp). conclusion the nd1 gene sequence of mitochondrial dna from the original burgo chicken in bengkulu has been successfully obtained. snps were identified at two sites of the nd1 gene, with a sequence length of 450bp. the average genetic distance within the burgo chicken population was 0.1%, while the distance between burgo chicken to other chicken populations was 0.12%. all burgo chickens formed the same clade in the phylogenetic tree, though two individuals (c2f3 and k4f2) showed slight differences, forming small groups based on variations in nucleotide bases. genetic differences among burgo chickens from bengkulu, other chicken species in indonesia, and several locations worldwide are present but non-significant. our data show that burgo chickens may be genetically distinct from other chickens found in indonesia and globally. however, further research on the morphology and morphometrics of burgo chickens is needed to confirm these findings. acknowledgements we would like to thank the ministry of education, culture, research and technology of the republic of indonesia for research funding assistance through the fundamental research scheme in 2024 with contract number no. 3930/ un30.15/pt/2024. we also appreciate the bengkulu burgo chicken community for their assistance and all the people who have helped with the research. author contributions jarulis and ahmat fakhri utama contributed to the study’s conception and design. data collection was done by jarulis. data analysis and writing of the first manuscript draft were performed by jarulis and ahmat fakhri utama. all authors commented on the various versions of the manuscript, and read and approved the final manuscript. ethics statement all experiments were approved by the local ethics committee of the university of bengkulu, indonesia. animal procedures were conducted in accordance with the ethical code guidelines no. 15/ker-lppm/ec/2023. conflict of interest statement the authors declare no conflicts of interest. references al-nasser, a. et al. 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(2025). community seedbanks in europe: their role between ex situ and on-farm conservation. genetic resources (s2), 147–161. doi: 10.46265/genresj.ohnk3179. © copyright 2025 the authors. this is an open access article distributed under the terms of the creative commons attribution license (cc by 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. introduction this article describes the role of community seedbanks (csbs) in europe in the plant genetic resources for food and agriculture (pgrfa) community and their contribution to the conservation and sustainable use of agrobiodiversity in complementarity with the ex situ management system. recently, two eu horizon projects (dynaversity: www.dynaversity.eu and farmers’ pride: https://more.bham.ac.uk/farmerspride/ ) have worked on this complementarity promoting the emergence of a european network of actors involved in pgrfa conservation and use. both projects have involved csbs in their activities and developed manuals and guidelines for the management of csbs in connection with public genebanks. but what are the main differences between csbs and genebanks? if the complementarity between ex situ and onfarm conservation of pgrfa has been accepted by the scientific community in the last 20 years, less attention has been given to investigating the role of csbs in relation to these two systems. only a few scholars have studied such collective endeavours (vernooy et al, 2015) while national seed policies hardly include csbs among the relevant institutions for conservation and sustainable use of pgrfa. a quantitative study on the impact of csbs and civil society organizations (csos) in the european conservation system of pgrfa is still lacking. many questions about their role can be addressed. how can they act as an intermediate between genebanks and farmers/gardeners? how can they increase the awareness of citizens on biodiversity conservation? how can they work with public research centres to support participatory plant breeding programmes? how can they diversify european seed systems and, subsequently, food systems? it becomes important to better understand the functions as well as the practical and collective actions of csbs, based on concrete experiences. this article aims to fill this gap, presenting three case studies from italy, switzerland and austria, based on the received: 03.12.2024 accepted: 11.03.2025 published online: 04.04.2025 https://www.genresj.org https://www.doi.org/10.46265/genresj.ohnk3179 https://www.genresj.org https://www.doi.org/10.46265/genresj.ohnk3179 148 bocci et al genetic resources (2025), (s2), 147–161 personal experiences of the authors, who work with the three organizations, and on the results of surveys and workshops organized within the framework of four eufunded research projects – diversifood (https://div ersifood.eu/), dynaversity, farmers’ pride and prograce (https://www.grace-ri.eu/pro-grace). genebanks and csbs should not be considered as competitors but as complementary actors, each having specific objectives, targets and rules as summarized in table 1 (bartha et al, 2021). perhaps the most relevant difference is that genebanks are committed to the long-term conservation of pgrfa and to granting facilitated access to a wide range of users, while csbs aim at short-term conservation and easy availability of seed for the aims of the community managing the csb. this complementarity means that together, they can contribute to creating inclusive and integrated conservation strategies at national and regional levels. community seedbanks in europe for more than 40 years, csbs around the world have emerged as part of the so-called informal seed system to counteract the loss of locally adapted varieties through the development of collective seed systems (vernooy et al, 2015). most of the studies and articles on the subject refer to experiences from the global south. however, a comprehensive analysis of the impact and role of csbs on seed systems in industrialized countries is still missing. in 2017, within the framework of the european project diversifood, a group of seed networks including arche noah, rete semi rurali (rsr), prospecierara (psr), réseau semences paysannes (rsp) and red de semillas (rds) organized a regional survey on csbs in europe, to start understanding their distinctive features. the results of the survey, briefly presented in this paper, showed the great diversity of initiatives in terms of age, size and internal structures. differences emerged also in their approaches to pgrfa management, with some leaning towards a more ‘dynamic management’ approach. since 2005, the number of csbs in europe seems to be rapidly growing, at least in some regions (koller and bocci, 2018). the first csbs in europe were established in northern and central european countries (austria, denmark, germany, the netherlands, sweden, switzerland, united kingdom), mainly by seed saver organizations and managed by gardeners. the us-based seed savers exchange inspired the birth of many of these organizations. the uk garden organic’s heritage seed library, known at the time as henry doubleday research association, was founded in 1975. prospecierara (switzerland) was founded in 1982 and arche noah (austria) in 1990. since then, other organizations have sprung up in european countries, with an accelerated increase occurring since the mid-2000s, especially in france and spain. the diversifood survey was able to map only a few experiences from south-eastern europe. the survey revealed that there is not one type of csb that fits all. this diversity is related to the fact that csbs in europe have followed two different pathways. in the older cbss in europe, mainly from central and northern europe, private gardeners had a key role, following the example of the seed savers in australia and the usa. on the contrary, in southern europe small farmers played a major role, adapting the experiences of social movements in the global south, where csbs emerged to provide seeds to farmers in marginal areas or after conflict. however, most experiences converge around the key ideas of diversity, conservation, exchange, community and sovereignty. regardless of the crop, most of the accessions they conserve are landraces, farmers’ varieties, old commercial varieties (open-pollinated varieties) or breeding populations. what is important to note is that many initiatives have moved from just conservation to more dynamic approaches, where participatory and decentralized plant breeding plays an increasing role. this change was achieved through dedicated training activities for all the members of the csbs, which raised awareness of breeding for diversity and local adaptation. many csbs have also been able to create strong links with citizens, often using public campaigns focused on the importance of plant and seed diversity, and protecting local varieties. through these activities, they have promoted more diversified, sustainable and resilient food systems that are better suited to face climate challenges. the main obstacles raised by the participants to the survey have been the lack of financial resources and an enabling legal environment. in fact, seed and food policies have often promoted uniform and formal seed systems, through regulations on seed marketing. for example, the interpretation of seed exchanged by farmers varies across countries in europe: some countries allow it, while others consider it as commercialization following the rules of seed marketing. only recently more diversity entered this picture with the concept of conservation varieties (eu commission directives 62/2008 (eu, 2008), 145/2009 (eu, 2009) and 60/2010 (eu, 2010)), even if its impact is still questioned (didonna et al, 2024). networking and cooperation as well as mutual support and social learning have been indicated as strategies to overcome these barriers. within this large and diversified movement of seed conservation, sharing and breeding, the idea of creating a european umbrella organization emerged as members of different csbs started connecting through european meetings. in 2005, european seed networks organized the first european meeting – ‘let’s liberate diversity’ – in poitiers, france. after that meeting, rsp, rsr and rds started the process of formalizing a regional-wide organization which would group the different associations involved in seed saving, on-farm conservation and agrobiodiversity management. after seven years of negotiations and meetings, the european coordination let’s liberate diversity (eclld) was formally registered in 2012 as a non-profit organization in belgium, and as of genetic resources (2025), (s2), 147–161 community seedbanks in europe 149 table 1. the main conceptual framework of community seedbanks (csbs) and genebanks (bartha et al, 2021). genebank community seedbank organizational structure public institution from single-person initiatives to community-based organizations (association, foundation, network without legal status, etc.). centralized structure partly decentralized (network structure) actors employees (scientists, practitioners), occasionally farmers and breeders (if project available) network member volunteers (gardeners, farmers, horticulturists, etc.), employees (scientists, practitioners). funding structure state, public–private partnerships (ppp), projects private (members, sponsors, foundations, etc.), public (european, state, region, municipality), commercial activities, non-profit organizations (npo) communication strategy; know-how transfer towards scientific and breeder community. case by case policymakers too. specific and science-focused communication. specific communication to farmers. broad public (sponsors, donors), practitioners (farmers, gardeners, horticulturists), governmental decision-makers and politicians. integrative and comprehensive communication. quality management for plant genetic resources aiming at a common and internationally agreed certification system based on protocols and standardized procedures. monitoring only internal genebank activities. aiming at quality systems that are best adapted to the needs and actual situation (financial and structural) of the csb. monitoring is based on the control of the whole network. choice of plant material based on national breeding programmes, genebank managers’ interest, national agrobiodiversity strategy (if existent). only recently international coordination and sharing of responsibilities (e.g. aegis, a european genebank integrated system). based on csb strategy developed by network members based on public or founder’s interests as well as financial and network capacity. often local, national or regional focus. breeding providing pgr for breeders for targeted breeding activities mostly for resistance. aiming at specific and homogenous varieties. varieties adapted to industrial agriculture. evolutionary breeding mostly aims for tolerance. varieties with a less homogenous calibration spectrum. aiming at varieties that keep their adaptation capacity to different agricultural systems. governance public mission based on national and international law/agreements/protocols based on common agreed values, shared visions and missions and agreed statutes and bylaws. social aspects are key. hierarchic, top-down from hierarchic to democratic structures, bottom-up bound to governmental obligations civil society organizations, representing the interests of the community access to materials mainly through the easy standard material transfer agreement (smta) and the rules of the international treaty on plant genetic resources for food and agriculture (itpgrfa) it could vary from one csb to another. there are csbs fully compliant with itpgra, others that have specific mutually agreed terms type of materials mainly old varieties and landraces in the public domain. some conserve also breeding lines or commercial varieties with dedicated access rules from old varieties and landraces in the public domain to new heterogeneous materials bred through participatory breeding programmes 2024, it brings together 22 organizations from 21 countries, encompassing over 170 national organizations. eclld is dedicated to promoting the dynamic management of cultivated biodiversity and farmer-led seed systems across europe and aims to bring diversity back into our food systems. by connecting csbs, researchers, civil society groups, seed savers, and farmers, eclld operates through three core focus areas: policy, community seedbanks, and communities. as a platform for policy engagement, eclld supports capacity-building initiatives and fosters exchanges among its members to enhance their advocacy efforts toward policies and regulations that promote and sustain agrobiodiversity. in the area of csbs, eclld empowers local groups by facilitating knowledge sharing, supporting exchanges on practices, and the integration of participatory plant breeding (see as reference the three technical manuals on csbs, galluzzi et al (2021c,b,a) https://liberate diversity.org/knowledge/readings/). finally, through its work on communities, eclld fosters peer-to-peer learning, enabling stakeholders to share practices, exchange experiences, innovate, build connections and collectively 150 bocci et al genetic resources (2025), (s2), 147–161 drive action on cultivated diversity. events like the let’s liberate diversity and let’s cultivate diversity forums, are central to these community-building efforts (for further details visit www.liberatediversity.org). three case studies our case studies describe the csb experience of three different european organizations which are eclld members: pro specie rara (psr, switzerland), arche noah (austria) and rete semi rurali (rsr, italy). they were chosen since they exemplify the diversity among european csbs and are well connected to their respective national systems for pgrfa conservation. moreover, the three organizations have all made efforts, although in different ways, to engage with the so-called formal seed systems, with activities such as seed marketing, registration of local varieties/populations, characterization of the accessions conserved and traceability of the work of the csbs through dedicated databases. for each organization, we will describe the history, turning points, pgrfa managing system, the networks they are involved in and their perspectives. a brief summary of the main characteristics of the three organizations is presented in table 2. they have different members (single persons in the case of psr and arche noah, and other organizations for rsr), activities (psr is working also on animal breeds), facilities and access rules. psr and rsr have integrated the easy standard material transfer agreement (smta) of the international treaty on plant genetic resources for food and agriculture (itpgrfa) for providing accessions for research and breeding, meanwhile arche noah has its own access rules with an obligatory compliance check: if companies or organizations work with gmos or patents, they are excluded from seed access. rete semi rurali history rete semi rurali (rsr) is the italian seed network, an umbrella non-profit association grouping organizations involved in the sustainable use of agrobiodiversity, within an agroecological framework. rsr was set up in 2007 by seven founders; in 2024 it consisted of 36 profit and non-profit members. rsr’s mission is to diversify our food systems, starting from seeds and varieties. its projects are directed primarily towards increasing diversity in agricultural systems, starting with the management of diversified seed systems in organic farming. rsr’s strategy aims to recognize the role of farmers and other actors in breeding and seed production and enable a legal framework for the dynamic management of agrobiodiversity (bocci and galluzzi, 2015). rsr activities cover four different work areas: 1. action research: this area supports the diversification of agricultural systems, by promoting a different model of agricultural research which brings research back to farmers’ fields (decentralization) and involves different food system actors (participation). 2. community seedbanking: this area supports diversified seed systems by promoting csb development at a local level (see figure 1). 3. communities: working on seeds means working with the communities that grow them, process and consume their products. rsr works to build and support vibrant communities in which food system actors interact with each other, being aware of their complexity and diversity. 4. policies: to promote changes in agricultural systems, it is necessary to build a political, legal, economic and social environment that makes these possible. rsr is active in fostering this enabling environment at local, regional, national and european levels. in 2013, after the eclld meeting let’s cultivate diversity in tuscany, rsr started to create its own csb dedicated to different cereal species. subsequently, the csb expanded its structures and functions, engaging new members, opening hubs in new regions, establishing field trials for different crops, and engaging the communities in the evaluation of varieties and sensory analysis of the products (petitti et al, 2022). great emphasis was placed on participatory approaches to bottomup seed system innovation, focused on the development and dissemination of dynamic crop populations and their management within organic farming systems (de santis et al, 2022). the concept of community biodiversity management (deboeuf et al, 2013) was adopted, believing in its great potential for change and adaptation, and for making agricultural systems the places where site-specific innovation takes place. each year, rsr’s csb organizes two seed distribution campaigns, one for winter and one for spring crops, through which a catalogue of landraces and populations is released and from which farmers or gardeners can ask for small seed samples. rsr has developed a dedicated material transfer agreement to trace the exchange of the materials and keep track of the flows from the csb. the samples distributed are small, but generally larger than the ones of formal genebanks. for more uniform varieties, rsr provides around 200 or 300gr and for populations up to 2kg, to avoid reducing the diversity by sampling a small amount of seeds. in 2019, rsr inaugurated its new headquarters, called the house of agrobiodiversity, a multifunctional space, which includes the first agrobiodiversity library in italy, a fully operational seedbank and a training centre. in 2022 rsr launched diversitas – the digital ecosystem of rsr. it collects and manages all the accessions in the csb and the data from the experimental fields. diversitas is designed to track the flow of seeds in and out of the csb. turning points from 2010–2019, thanks to eu projects solibam (w ww.solibam.eu) and diversifood, rsr moved from genetic resources (2025), (s2), 147–161 community seedbanks in europe 151 table 2. main features of rete semi rurali, prospecierara and arche noah rete semi rurali prospecierara arche noah date of founding 2007 1982 1990 website https://rsr.bio www.prospecierara.ch www.arche-noah.at legal structure non-profit organization; umbrella org. foundation association board, staff 5 board members, 2 employees, 15 consultants 7 board members, 35 employees 9 board members, 57 employees (approx. 39 full-time equivalents) members 35 entities (profit and non-profit) 13,000 donors and 4,400 active seed savers and rare-breed holders. farmers, breeders, gardeners, researchers, etc. 10,000 members + 7,000 extra donors mission diversification of farming and seed systems maintain and promote the genetic and cultural diversity of plants and animals. conservation and development of crop diversity in regional and europe-wide networks and advocacy for an enabling policy framework main collections 3,487 accessions. crops: soft and durum wheat, barley, rye, oat, other cereals, rice, maize, tomato, sunflower, soybean, chickpea, bean and lupine. 5,600 cultivated plants and 32 rare breeds 5,500 seed accessions and 550 fruit cultivars main activities central seed storage (climatic chamber and freezer). database: diversitas. central seed storage (climatic chamber and freezer), tuber storage facility (climatic chamber), nursery and greenhouse and tunnel. webpage for seed and breeds exchange. database for dynamic on-farm management central seed storage (climatic chamber and freezer), one visitor’s garden and one multiplication garden incl. tunnels. online shop and shop in the visitor’s garden. internal and external database. main network activities seed saving, knowledge exchange, courses, markets, collection holders, data collection, breeding seed saving, knowledge exchange, courses, markets, collection holders, data collection, seed saving and regular multiplication incl. data collection, educational programme with approx. 50 courses per year, political campaigning, participatory vegetable breeding networks, arche noah diversity farms main projects (2024 status) implementing the itpgrfa in italy, ecpgr eva network, 5 horizon europe projects 72 different projects; label for psr products; horizon 2020 projects; projects within the frame of the national action plan for pgrfa no patent on seeds-campaign, participatory vegetable breeding, fruit monitoring austria, supporting community biodiversity management in south-eastern europe via small-scale grants. online seed savers index the mere preservation of local varieties and landraces to actively breeding for diversity, developing evolutionary populations of soft and durum wheat (triticum aestivum l. and t. turgidum subsp. durum (desf.) husn.), and barley (hordeum vulgare l.). this work on participatory and decentralized plant breeding, and in particular on evolutionary populations, was done in collaboration with dr salvatore ceccarelli, a breeder who worked at the international center for agricultural research in the dry areas (icarda), one of the solibam partners, and then directly with rsr. icarda’s evolutionary populations of soft and durum wheat and barley were evaluated and tested in different and contrasting farming environments (bocci et al, 2020; ceccarelli and grando, 2020). this move from agrobiodiversity conservation to breeding for diversity was the first important turning point in the history of rsr. over time, the work on wheat and barley was expanded to other crops: rice (oryza sativa l.), tomato (solanum lycopersicum l.), oat (avena sativa l.), lupin (lupinus 152 bocci et al genetic resources (2025), (s2), 147–161 figure 1. community seedbank at rete semi rurali albus l.), sunflower (helianthus annuus l.) and recently other legumes, following the increasing interest among farmers in crop populations for organic farming systems. in the beginning, the populations remained within rsr’s network and were exchanged during the seed campaigns, since marketing this kind of seed was not legally possible. in 2014, a second relevant turning point occurred: thanks to the lobbying done by solibam partners, mainly the organic research centre (uk), fibl (switzerland), itab (france) and rsr, the european commission opened the space for marketing the seeds of these populations by an experimental derogation (eu commission implementing decision 150/2014 (eu, 2014)). using this derogation, rsr supported farmers to engage in the process of seed production, multiplication and marketing of the populations they were growing and adapting, by registering as small seed companies. in 2017, the first soft wheat population was officially certified by public authorities and two farmers (one in tuscany and one in sicily) started marketing its seeds. at the same time, rsr developed its label for the seed packages using and adapting the open-source pledge promoted by the open source seed initiative in the us (https://osseeds.org). the last relevant turning point was the approval of the new eu regulation 848/2018 (eu, 2018) on organic production and labelling of organic products that entered into application in january 2022. this regulation created a new varietal category, the organic heterogeneous material (ohm), which took up the concept of populations contained in the decision of 2014. since then, rsr has worked to support the implementation of ohm in italy, notifying one rice, one sunflower and one soft wheat ohm. collaboration with institutions since its foundation, rsr has collaborated with the ministry of agriculture, being one of three partners of the national programme for the implementation of the itpgrfa. this programme involves 29 research facilities of the council for agricultural research and agricultural economics analysis (crea), the institute of plant genetics of the national research council (cnr) in bari and rsr. thanks to the programme, rsr supports its csb as well as the italian delegation within the itpgrfa framework on negotiations related to the sustainable use of agricultural biodiversity and farmers’ rights. thanks to the above national programme and its involvement in horizon projects, rsr has developed dedicated agreements with a range of european organizations, including one with the cnr genebank in bari for the multiplication and regeneration of some of the accessions conserved there. other agreements on participatory and decentralized plant breeding have been signed with the universities of florence, bari, turin, milano bicocca, viterbo and the sant’anna genetic resources (2025), (s2), 147–161 community seedbanks in europe 153 school of advanced studies in pisa. the aim of these agreements is to place rsr as an intermediary organization between the public research system and farmers. networks rsr is a member of several italian alliances and networks including azione terrae, the coalition for agroecological transition (https://azioneterrae.com). this coalition is made up of 7 international cooperation associations (acra, cisv, cospe, deafal, lvia, mani tese, terra nuova) and two italian and european civil society networks (rsr and agroecology europe), engaged in experimentation, promotion, training and dissemination of different aspects of agroecology, involving both research and farmers’ organizations. azione terrae plays a crucial role in the promotion of agroecology in italy and west africa, while the role of rsr is to strengthen the link between good farming practices and seed systems, putting the development of diversified seed systems at the core of activities of the coalition. at the national level, rsr has also been involved in the campaign cambiamo agricoltura, which unites over 70 organizations actively engaged in the negotiations of the common agricultural policy (cap). at the european level, rsr is a full member of the european consortium for organic plant breeding (ecopb, https://www.eco-pb.org/), which aims at facilitating knowledge exchange and supporting breeding programmes for organic farming. as mentioned, rsr is one of the founding associations of the european coordination let’s liberate diversity. at the international level, rsr is a member of the global coalition of open source seed initiatives (gossi, https://www.opensourceseeds.org/en/go ssi), an international coalition of organizations, individuals (farmers, seed keepers, plant breeders, activists) working to ensure that seeds can be freely used and shared in perpetuity. rsr contributes actively to the debate and negotiations on the european regulatory framework and is involved within the itpgrfa in the development of policies on the sustainable use of pgrfa and farmers’ rights. perspectives rsr has become a complex, inclusive and dynamic network dealing with local field experimentations as well as international processes. it aims to maintain a fruitful dialogue among practitioners, researchers and policymakers. its work demonstrates that we must enlarge the vision of agrobiodiversity, focusing not only on mere conservation but on innovation and breeding for diversity, i.e. delivering new varieties that are sufficiently diverse (rather than narrowly responding to the standard criteria of distinctness, uniformity and stability (dus) of modern varieties) to be able to adapt to climate change and low-input farming systems. the belief in the importance of diversifying seed, farming and food systems is the reason why rsr has recently moved towards projects and research activities that involve not only seed diversification and breeding but also intercropping, rotations and soil microbiome. these will be the challenges for rsr in the coming years. regarding the csb and its database diversitas, the next steps will be the possibility of implementing the itpgrfa easy smta directly from its website for the accessions distributed from the csb and the digital object identifier (doi) for some of the conserved accessions. prospecierara history psr was founded in 1982 in st. gallen (switzerland) and its first activities were related to safeguarding rare breeds. in 1985, collecting activities for fruits, field crops and vegetables started. in 1988, the first employee was hired with a fixed salary. around the same time, the network of seed savers was established and the seedbank (called ‘seed library’) was founded. very successful tv broadcasts and some national exhibitions about rare breeds and fruit varieties organized by psr and its partners and volunteers helped to raise awareness among the broader public. in the 1990s, the first private foundations started funding the projects of psr and private donors supported the organization as well as many volunteers who helped to propagate seeds as seed savers. in addition, many breeders joined the different breeding associations created by psr to coordinate the conservation of the different endangered breeds. after 15 years of existence, psr encountered about 2,000 donors, 250 seed savers and over 2,000 breeders organized in 15 different breeding associations. turning points the first important turning point for psr’s activities was the ratification of the convention of biological diversity by switzerland in 1995, followed by the development of the national plan of action for the conservation of plant genetic resources for food and agriculture (nappgrfa) in 1998. the department of agriculture decided to create the swiss commission for the conservation of cultivated plants (skek/cpc) in which psr became a leading member of the governing body to implement the nap-pgrfa. this mandate was and still is accompanied by some funds (3.2 million chf per year). at the time, the commission developed a conservation strategy with conservation standards for various crops and a national database that relates to the european search catalogue for plant genetic resources (eurisco). psr’s own database has an interface with the national database to transfer and exchange passport and characterization data. today about 15% of the turnover of psr is covered by these public funds. a second important turning point was the collaboration with coop, the biggest supermarket chain in switzerland. together with this impactful partner, psr participated in one of the biggest national exhibitions called expo02, which attracted millions of people over https://www.opensourceseeds.org/en/gossi https://www.opensourceseeds.org/en/gossi 154 bocci et al genetic resources (2025), (s2), 147–161 six months. the interest of the visitors in the topic was so big that coop decided to fix the collaboration through a contract. this collaboration has lasted until today. accordingly, psr’s focus shifted more and more from pure conservation towards the sustainable use and development of pgrfa. psr developed a label for the promotion of traditional and endangered varieties to keep or reintroduce them in the value chain. aside from the coop outlet, farmers and horticulturists who are part of the psr conservation network use this label for their own marketing activities, helping them showcase the added value of their products to consumers. in 2023, psr’s database revealed that about 600 people use the psr label and surveys reported that around 30% of the swiss population know this label. another important turning point in the swiss pgrfa conservation activities occurred when the government implemented article 147a in the agriculture law (sr 910 1 art. 147a lwg): ”the confederation may promote the conservation and sustainable use of genetic resources. it may manage genebanks and conservation collections or have them managed and support measures such as in situ conservation, in particular with financial contributions.” under this provision, psr and other stakeholders in this field could apply for funding for activities going beyond pure conservation such as on-farm development and improvement of pgrfa. thanks to the development of a suitable legal framework (e.g. the national long-term strategy for pgrfa and its accompanying measures) as well as through adequate funding opportunities, psr was able to grow further, engaging new stakeholders for on-farm management of pgrfa. today governmental financial support constitutes only about 20% of their total turnover but is still crucial because it guarantees the financing of conservation activities that depend on a long-term financing source (e.g. on-farm fruit and berry collections). development and monitoring prospecierara went on to develop its network of seed savers (for vegetable species) and collection holders (for fruits and berries). today, 400 seed savers maintain 1,208 vegetable varieties, 270 people care for 1,012 ornamentals, 1,000 people host 2,436 fruit and 422 berries varieties all over the country. the psr staff is responsible for managing the network and monitoring conservation activities. for seeds, this work is facilitated by the central seed library, located in wildegg (ag) and consisting of a climatized room with 2,000 accessions stored as seeds. this repository functions as a backup of the in situ collection, with seed savers regularly sending back a reference seed lot of the variety/accession they maintain and regenerate. the reference lots are sown in psr’s different demonstration gardens, where quality and varietal identity are verified. all the exchanges between the seed savers and psr are registered in a specific database for on-farm conservation and can be traced back. every year, the 1,653 label holders (i.e. registered and validated seed savers) receive a request to fill in a checklist to describe the status of the genetic resources they are maintaining. this checklist is provided by psr on its portal (https://www.prospecier ara.ch/it.html). based on the results of this monitoring activity, psr delivers a report about the status of each of the 5,600 accessions maintained by the people of the network. this report also allows the seed library manager to establish how many seed lots are in the genebank and their storage time. the manager will also know how many seed savers are maintaining the accession, how many are marketing seeds, fresh and processed products and how many of them are offering seeds on the variety finder portal of psr. by compiling all this information, that is facilitated by the database, the manager can judge the conservation status of an accession and decide if an accession is endangered and must be propagated quickly or not. in addition, different training courses are organized for beginners and advanced seed savers to improve their knowledge and skills. sustainable use over time, psr’s activities shifted more and more from pure conservation to sustainable use of pgrfa and the development and improvement of varieties, e.g. for niche markets and with niche varieties. the shift of psr’s activities towards sustainable use was backed by a national law (sr 916.151.1 art. 2.4 2.7, 27, 29) for the marketing of seeds brought into force in 2010: besides registered and certified varieties, a new category called ‘niche varieties’ was created to include varieties that don’t fulfil the dus criteria. the government considered that certain varieties could be very interesting for niche markets or small-scale farmers or private gardeners (see figure 2). allowing to register these varieties and being able to place them on the market would enrich the diversity of vegetables and field crops in the fields and on consumers’ tables. this positive legal environment led to breeding activities to improve landraces, minor or ‘opportunity’ crops, or develop populations, with the support of government and private (e.g. coop) funds. some of the breeding activities included participatory methods and, in some cases, citizen participation when a broader range of information about plant growth and development in different agricultural and horticultural contexts had to be collected and compiled. while often breeding activities take the form of mass selection to advance a population variety, in some cases, crossbreeding is the only way to make a variety fit for on-farm utilization (see table 3). further development as a csb in the future, open-pollinated crops will become even more important for alternative, independent, locally adapted and innovative agricultural systems, a grassroots alternative to hybrids that are more and more taking over intensive and industrial agricultural production of food (ipes-food, 2016). on the other hand, citizens genetic resources (2025), (s2), 147–161 community seedbanks in europe 155 figure 2. carrot variety ‘gniff’ from ticino from pro specie rara collections being commercialized table 3. cross-breeding activities initiated by prospecierara (psr) for new niche varieties species varieties origin breeding daucus carota ‘gniffola’ landrace ‘gniff’ x ‘purple haze’ sativa rheinau, 2012–ongoing solanum lycopersicum ‘cuore di bue’ ‘cuore di bue’ x resistant varieties against cladosporium sp. sativa rheinau, 2012–ongoing brassica rapa subsp. rapa ‘albedo viola’ (rejected name) ‘guringa’ several old varieties of psr, the swiss genebank and from commerce landrace ‘bosco gurin’ x several varieties of the same type sativa rheinau, 2016–2024 allium cepa ‘piri’ old variety ‘birnenförmige’ x (‘yankee f1’ x (‘rijnsburger’ x ‘yankee f1’) ) sativa rheinau, 2012–2022 tragopogon porrifolius salsify populations 11 salsify lines from psr, the swiss genebank and from commerce psr & sativa rheinau together with psr network (participatory breeding), 2024–ongoing cucurbita pepo var. cylindrica striped zucchini old variety ‘de gênes striée vert-jaune’ x other striped zucchini sativa rheinau, 2024–ongoing are increasingly interested in agroecological food production systems that foster biodiversity, protect the environment and provide healthy food for them. this is one of the reasons why urban farming and gardening movements are popping up all over the country in and around cities. well-educated young people are applying new and better-adapted farming systems to local conditions. several community-supported agriculture systems around cities like geneva, basel or zurich have contacted psr to get access to bigger amounts of seeds to be able to start their trials on a bigger scale and select those varieties that fit best to their local and specific needs and establish production plots for the marketing of produce. for psr this development causes different challenges: first, quantities of seeds requested are greater than those normally distributed for purely experimental purposes; second, these new actors require training in order to acquire the ability to produce and regenerate their own seed each year. finally, these users demand more detailed information about the pgrfa stored in psr’s seed library. to face these challenges, psr is considering a series of new developments. to produce and distribute greater quantities of seed to growers, it could become a small seed company or start collaborating with local seed companies. in general, psr would like to act as a knowledge hub or be part of a knowledge platform that provides 156 bocci et al genetic resources (2025), (s2), 147–161 information to growers and helps them to make decisions tailored to the specific environmental and/or economic and social conditions they operate in – a kind of one-stop shop for farmers. networks since the early 2000s, psr has entered several partnerships with research institutions, whether within the framework of the nap-pgrfa for the inventory, description and conservation of pgrfa, or within the framework of production and distribution with coop. an important factor in the success of many of these collaborations was the clear definition of partners’ roles. the typical collaboration between psr and research institutions covers three steps along the continuum from conservation to the sustainable use of pgrfa: 1. conservation: ex situ, i.e. in vitro and genebank conservation by research institutions (e.g. agroscope) linked with the in situ and on-farm conservation by psr and its partners. the research institutions use their scientific expertise to carry out ex situ conservation measures. on the other hand, psr has an interest in testing pgrfa in on-farm conditions to possibly promote them among farmers, while network partners can use their pgrfa collections as sources of material for the multiplication and commercialization of seeds. 2. evaluation: genetic and morphological characterization by research institutions (e.g. agroscope) linked with the quality assessment, value description (e.g. cultural history, or market value), and determination of the utilization potential by psr. while research institutions provide the expertise and technical infrastructure for characterization, psr and its network can evaluate the pgrfa in terms of their socio-economic impact. 3. environmental adaptation for agricultural use: selection of pgrfa, breeding activities and marketing activities by psr and partners linked with agronomic evaluation and case studies by research institutions with a strong link to farmers (e.g. fibl). psr initiates breeding activities to recover the varieties and bring them to the market. the ‘prospecierara’ label provides a tool to enhance the product’s credibility with consumers. on the other hand, it is important for farmers to know the expected quality and quantity of the pgrfa available before accepting it for large-scale cultivation on their farms. research organizations advise farmers about the agronomic and quality features of the pgrfa available through psr enabling them to make informed choices depending on their context and needs. perspectives psr is dedicated to further developing the plant and animal genetic resources it maintains in its broad network. as this example shows, there are strong incentives for collaboration between csbs, research institutions and genebanks. there is a great need for research on the characterization and evaluation of pgrfa, and the role of research institutions and genebanks is undeniable. at the same time, psr experience shows that successful promotion of pgrfa is only possible with many dedicated actors, which come together in the csb network. this collaboration will not only enable better integration of pgrfa into society through innovative agroecological farming systems but will also return a wealth of real-world data and experiences about pgrfa to the research institutions. arche noah history the development of arche noah can be divided into four phases: 1. pioneer phase. emerging from predecessor organizations, arche noah was founded in 1990 by farmers and gardeners who had formed a network among crop collectors and seed savers. early on, the organization issued a seeds handbook (not published, for members only) to stimulate the exchange of seed and other plant reproductive materials and began compiling a collection of rare and endangered varieties. the 1990s were a time of rapid growth of the collection, stimulated by collecting missions (austria, croatia, romania) but also by research into commercial open-pollinated and heirloom varieties. eventually, in 1994, the organization opened a garden for crop multiplication that was also open to the public for educational purposes. seed production was organically certified which gave it a unique position among seed savers organizations as well as in the agricultural scene. arche noah was a pioneer in the then-emerging austrian organic farming scene. 2. differentiation phase. around 2000, the organization had become well-known and grew to 6,000 members. the annual plant market attracted many visitors and became a meeting point for the organic gardening community. arche noah took up the direct marketing of transplants. to manage risks from commercial activities and to clearly separate commercial from non-profit activities, a company with limited liability was founded. along with these developments, the number of employees at the organization rose and departments developed, specializing in plant collections (seed archive, fruit collection), gardening, seed savers activities, sales and event management. an early act of policy advocacy was to secure exemptions for unregistered pgrfa under the austrian national seed legislation. this was achieved through persistence, as well as a rare window of opportunity for alternative farming approaches at that time. since then, the legal exemptions have allowed for marketing small quantities of seeds, making austria a unique environment for heirgenetic resources (2025), (s2), 147–161 community seedbanks in europe 157 loom varieties in the eu. beyond that, arche noah advocated for lighter registration procedures for conservation varieties. the demand for seeds was growing and it became necessary to open the registration process for plant materials not conforming to industrial standards and feasible for small seed companies. in the educational sector, a series of books was launched, setting off with a handbook about seed gardening (heistinger, 2013). in 2008, the education programme on organic gardening and crop diversity was expanded, comprising nowadays up to 80 courses per year, training people in seed-saving techniques, crop diversity and gardening skills. 3. integration phase. in the late 2000s, a bundle of new strategic targets pushed the enlargement of the organization. with the slogan ‘eating up what we want to save’, arche noah built a wider network with home gardeners and farmers, incentivizing access and marketing of rare varieties in the form of seeds, plantlets and products through different channels (plant markets, arche noah shop, farmers markets). the focus shifted from ‘home gardeners’ to the urban consumer as a new target group. this was accompanied by systematic on-farm research – also within eu projects – to evaluate varieties and their potential uses. the cooperation with the organic seed company reinsaat led to an ever-increasing demand from consumers and retailers. since then, arche noah has been registering several heirloom varieties in the eu catalogue per year and offering seeds via the arche noah webshop. 4. association phase. as of 2010, the political and international cooperation expanded: arche noah hosted several international policy workshops and advocated for seed law issues, in some cases directly in brussels. in 2015, the cooperation with the campaign ‘no patents on seeds’ started. at the same time, arche noah started to coordinate the austrian participatory tomato breeding network. the financing structure was expanded thanks to austrian and european research funds and new fundraising models, first targeted sponsorships for on-farm breeding and multi-location fruit conservation. since then, the number of employees and supporters of arche noah has remained stable, while noticing a shift from association members to sole financial donors. multiplying accessions and making accessions available in comparison to public genebanks, the association focuses more on cooperation with farmers and gardeners in terms of conserving, describing, spreading and further developing pgrfa. the activities of arche noah centre on the seed archive and the gardens which are responsible for maintenance, variety descriptions and seed quality testing. documentation of each variety or accession is supported by a database of text descriptions and photos for each variety or accession, complemented by data about the origin, plant health status, quantity and germination performance of harvested or stored seed lots. recently, the database was enforced with crop-specific descriptors, mainly based on upov and ipgri standards, but supplemented by more user-related descriptors such as taste, utilization class, yield or lodging. distribution of plant material (mainly seeds) is done via various production lines (table 4). seed surplus of non-registered varieties from the multiplication garden are offered as ‘treasures from the seed archive’ in strictly limited quantities in accordance with the austrian seed regulation revised in 2006 (ris, 2025). two additional production lines pursue the aim to provide seeds of registered varieties and varieties of non-regulated species on a regular basis to a wider audience, having the arche noah webshop and local sale points as main channels, but also serving major retailers such as a supermarket chain. these marketing activities are carried out by the associated company vielfalt erleben, which is fully owned by the non-profit arche noah. most of the offered varieties are registered in the eu variety catalogue as “varieties with no intrinsic value for commercial crop production but developed for growing under particular conditions” and are multiplied by contracted farmers. the main target group for arche noah seeds are amateur gardeners in austria and neighbouring countries (see figure 3). accessibility of rare fruit varieties is limited to a set that is managed by certified organic nurseries cooperating with arche noah. due to plant health restrictions and the complexity of handling planting material (seasonality, storage, shipping), the assortment of available varieties is less dynamic compared to seed crops. conserving and managing pgrfa in austria, there are approximately 150 active seed savers in the network of arche noah, contributing through growing, multiplying and collecting varieties. some of them manage their private collection of varieties (with or without links to the arche noah seed archive), but many engage in coordinated conservation activities of the organization. as ‘guardians’, they test varieties of the seed archive in their location and collect data. depending on season, location and personal preferences, they contribute new or confirm previous observations, and thus, add to the wealth of knowledge and perspectives regarding crop diversity. these seedsaver programmes cover both seeds and tubers as well as fruit crops. seed guardianship can be permanent or temporary (alternating varieties annually). fruit guardians are designated permanently with a minimum number of ten trees per location. to facilitate the exchange of plant material and contacts among seed savers and the broader public, arche noah digitalized the former seeds handbook to an online private database where seed and fruit tree savers can indicate their varieties via text and photos. 158 bocci et al genetic resources (2025), (s2), 147–161 table 4. seed marketing lines of arche noah (2024 status) line proven for home garden rare vegetables treasures from the seed archive distribution start year 2009 2014 1998 main target group beginner and advanced home gardeners advanced home gardeners advanced and professional home gardeners criteria for taking into the collection robust varieties of well-known vegetable crops, with reliable yield and good taste lesser-known vegetable crops, underutilized history, traits, utilization and/or special usage properties flexibility consistent collection, 1–3 varieties change per year consistent collection, 1–3 varieties change per year annually or biennially changing collection variety registration registration usually as ”varieties bred for cultivation under special conditions” usually, no registration necessary if species are not listed no registration necessary subject to restrictions and quantity limit set out in the austrian seed regulation 2006 number of varieties 53 18 approx. 100 varieties also sold to retailers 31 12 0 limited quantities due to legal restrictions figure 3. sale of arche noah seeds at the annual seed fair in wien (austria). genetic resources (2025), (s2), 147–161 community seedbanks in europe 159 farmers multiply seeds in larger quantities for arche noah’s seed sale. some of them run farms specialized in vegetable seed production, and others are biodiverse farms selling vegetables or other produce. together with arche noah, they carry out maintenance breeding of seed archive materials by positive or negative mass selection and selection of elite plants. the aims are to maintain the phenotype and to maintain or improve plant health and sensory quality. in addition to this partnership for seed multiplication, arche noah licences a label to organic farms which produce and sell rare varieties. in 2024, 32 farms registered for this label, the so-called arche noah diversity farms. some of the partner farmers of arche noah not only multiply, produce and sell rare varieties, but actively engage in further developing varieties by crossbreeding and selection. in 2010, the working group bauernparadeiser, a participatory breeding group on tomatoes, was founded on the initiative of farmers due to a lack of organically bred tomato varieties to suit the needs of direct marketing. conventional tomato breeding mainly breeds hybrids which neither can be reproduced on the farm nor adapted to local conditions, and sensory quality is often neglected. currently, the group consists of 12 organic austrian farmers, three research institutions and arche noah. the goal is the development of tasty and reproducible (open-pollinating) tomato varieties, featuring resistance to plant pathogens, by means of crossing heirloom and modern varieties. the group works, first, on varieties for greenhouse cultivation, resistant to the fungal pathogen cladosporium fulvum, to tobacco mosaic viruses and common root diseases and, second, on varieties for outdoor production, resistant to the fungal pathogens phytophthora infestans, early blight (alternaria spp.) and septoria leaf spot (septoria lycopersici), and less susceptible to fruit cracking. since 2020, arche noah has coordinated participatory screening and breeding activities within other vegetable crops, such as sugar pea and winter radish. networks apart from previously mentioned partners in the multiplication and marketing of plant material, arche noah is a partner in various research projects, being wellconnected with national universities and research stations. the latter are permanent partners in screening and breeding activities. there is loose contact with several other vegetable breeders, mainly in austria and germany. further, arche noah is an active member of the balkan seed network association. the organization was founded in 2021 by 16 organizations and institutions active throughout southeastern europe. the purpose of the network is to increase the conservation and sustainable use of pgrfa in agriculture. activities aim at stimulating resilient food systems and establishing a paradigm of collaboration within the wider balkan region, which has historically been shaped by conflict. the network consists of seed savers, breeders, scientists, farmers, gardeners, associations, organizations, research institutes and educational institutions. in addition, being an active member of the balkan seed network association, arche noah has been supporting seed savers organizations in southeastern europe by awarding small-scale grants, provided by foundations. perspectives arche noah calls itself ‘the association for preserving and developing the diversity of cultivated plants’ and cooperation with diverse network partners is extremely relevant. arche noah considers both preserving and developing as equally important activities. regarding conservation, arche noah makes increased use of longterm conservation at sub-zero temperatures of accessions, to enhance the capacities to study and distribute the most valuable accessions for use. the participatory breeding activities account for the necessity to let the accessions adapt and improve according to the needs of home gardeners and farmers. arche noah does not intend to be a sole breeding organization by any means, but rather a motivator and catalyst for organic breeding in austria. since breeding and cultivating diversity also require suitable frameworks, arche noah wants to create the appropriate awareness and the political foundations so that the development and marketing of diverse seeds does not only happen in niches but can also be economically successful on a broader scale. under the prevailing market economy conditions, diversity is a massive business disadvantage. it would therefore be naive to expect private and for-profit companies to do this work. however, since the preservation and further development of diverse seeds represent an indispensable basis for humanity’s livelihood, it cannot be left solely to the dynamics of the market. arche noah therefore claims that conserving and breeding for diversity should become part of public services and corresponding programmes should largely be publicly financed. conclusions the three cases presented here show that csbs in europe can maintain and manage thousands of varieties, landraces and populations within broad networks of different actors such as private gardeners, farmers and horticulturists. they operate in diverse, decentralized agricultural and climatic environments. their activities can be framed as community biodiversity management, and they focus not only on mere conservation but also on the dynamic management of pgrfa. this allows evolutionary and adaptive processes to happen. all the described csbs are well connected to the local/regional communities they are operating in. because they provide facilitated access to pgrfa, mostly open-pollinated varieties free of intellectual property rights, they are an excellent partner for agricultural movements that advocate for the enhancement of diversity in farming systems. they can be an excellent partner for researchers too, because they can act as bridges between scientific and tacit knowledge and help scale up interesting and crucial ideas for the future of our seed and food system. 160 bocci et al genetic resources (2025), (s2), 147–161 moreover, csbs can play an important and complementary role with regard to the ex situ system. in fact, they can be considered as an intermediary between farmers and the genebanks, receiving small samples from the banks and multiplying them before distributing them to farmers. propagation activities of the csbs, in collaboration with their networks, offer the opportunity to provide a larger quantity of seeds to the final users. very often the small quantities of seed coming from genebanks are considered as an obstacle by farmers, who do not necessarily know that providing larger quantities is not the mandate of these institutions. the multiplication and regeneration done by csbs can provide useful information about the agronomic value of pgrfa, which leads to a better understanding and knowledge of the variety or landrace itself. these processes, done by the csb members, operate in very diverse agricultural and horticultural systems, under different climatic conditions and within different social and economic contexts. this opens opportunities to collect site-specific information on how pgrfa perform and could help in coping with climate change and other challenges in the future, providing knowledge that is also useful for future breeding for diversity efforts. as described by the three case studies, the turning points for csbs are at least partially related to a change in the legal system. these changes allowed rsr to market the seeds of populations, psr to market the seeds of niche varieties and arche noah the ones of conservation varieties. this means that the operativity of such organizations is concretely impacted by the legal and political environment in which they are embedded, and which has historically been conceived to promote dus varieties while leaving diversity outside of the picture. as of the time of writing this article, the eu is reforming the rules on seed marketing, with a proposal released by the european commission in july 2023, voted by the parliament in april 2024 and revised by the council during 2025, before the final approval by the trilogue involving all three bodies. the proposal contains a series of derogations to the conventional system to allow more diversity and actors in the seed sector. for the first time, the concept of “dynamic management of diversity” by farmers is mentioned in a legal text and participatory plant breeding is defined as an activity which develops locally adapted varieties. moreover, an article is dedicated to the exchange between farmers, with the aim of creating a harmonized rule across europe with less space for different national interpretations. all these points need to be maintained in the final regulation, if an enabling environment is to be created in europe. only in this way the development of cbss, a relatively new and highly relevant actor in the pgrfa community, can be supported and the space for more diversity be created in our seed and food systems. author contributions conceptualization, rb, mgs, bb, hm, ma; methodology, rb, mgs, bb, hm, ma; validation, rb, mgs, bb, hm, ma; formal analysis, rb, mgs, bb, hm, ma; investigation, rb, mgs, bb, hm, ma; resources, rb, mgs, bb, hm, ma; data curation, rb, mgs, bb, hm, ma; writing-original draft preparation, rb, mgs, bb, hm, ma; writing-review and editing, rb, mgs; visualisation, rb, mgs; supervision, rb, mgs; project administration, rb, mgs; funding acquisition, rb, mgs, bb, hm, ma. all authors have read and agreed to the published version of the manuscript. the authors are grateful to gea galluzzi for her revision and proofreading. acknowledgements this study was supported by pnrr-pon funds within the university of turin ph.d. xxxvii cycle, 2022–2024 and the horizon project pro-grace ga101094738. conflict of interest statement the authors declare no 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(2015). community seed banks, origins, evolution and prospects. issues in agricultural biodiversity (bioversity international, routledge), 270p. doi: https://doi.org/10. 4324/9781315886329 https://doi.org/10.4324/9780203130599 https://doi.org/10.3390/horticulturae10080877 https://doi.org/10.3390/horticulturae10080877 http://data.europa.eu/eli/dir/2008/62/oj http://data.europa.eu/eli/dir/2008/62/oj http://data.europa.eu/eli/dir/2009/145/oj http://data.europa.eu/eli/dir/2010/60/oj http://data.europa.eu/eli/dec_impl/2014/150/oj http://data.europa.eu/eli/dec_impl/2014/150/oj http://data.europa.eu/eli/reg/2018/848/oj https://liberatediversity.org/wp-content/uploads/2021/05/manual3_eng_single_pages-3-1.pdf https://liberatediversity.org/wp-content/uploads/2021/05/manual3_eng_single_pages-3-1.pdf https://liberatediversity.org/wp-content/uploads/2021/05/manual2_web_single_pages-6.pdf https://liberatediversity.org/wp-content/uploads/2021/05/manual2_web_single_pages-6.pdf https://liberatediversity.org/wp-content/uploads/2021/05/manual1_web_single_pages-4.pdf https://liberatediversity.org/wp-content/uploads/2021/05/manual1_web_single_pages-4.pdf https://www.diversifood.eu https://doi.org/10.3390/su141711030 https://doi.org/10.3390/su141711030 https://faolex.fao.org/docs/pdf/aut72640.pdf https://faolex.fao.org/docs/pdf/aut72640.pdf https://doi.org/10.4324/9781315886329 https://doi.org/10.4324/9781315886329 introduction community seedbanks in europe three case studies rete semi rurali history turning points collaboration with institutions networks perspectives prospecierara history turning points development and monitoring sustainable use further development as a csb networks perspectives arche noah history multiplying accessions and making accessions available conserving and managing pgrfa networks perspectives conclusions author contributions acknowledgements conflict of interest statement review and position paper genetic resources (2025), (s2), 78–90 doi: 10.46265/genresj.aszo2413 https://www.genresj.org issn: 2708-3764 organization of plant biological resource centers for research in france: history, evolution and current status valérie bergheaud *,a, jean-marc audergon b, arnaud bellec c, anne delaunay a, jérôme duminild, stéphane dussertd, florence esnault e, emmanuel geoffriau f, brigitte gouesnard g, christophe jenny g,h, alain label i, philippe lashermesd, najate maghnaoui g,h, cécile marchal b, franciane nuissier j, nilda paulo-de-la-réberdiere g,h, aurélia priet k, valérie rieucau g,h, paule térès g,h and anne-françoise adam-blondon l a université paris-saclay, inrae, bap, f-78026 versailles, france b inrae, paca center, ugafl, f-84143 montfavet, france c inrae, cnrgv french plant genomic resource center, f-31320 castanet tolosan, france d diade, ird, université montpellier, cirad, f-34090 montpellier, france e inrae, institut agro, université de rennes, igepp, f-29260 ploudaniel, france f institut agro, université d’angers, inrae, irhs, sfr quasav, f-49045 angers, france g umr agap institut, université montpellier, cirad, inrae, institut agro, f-34090 montpellier, france h cirad, umr agap institut, cirad, montpellier, france i diagonal, inrae, f-29260 ploudaniel, france j inrae, ur astro, f-97170 petit-bourg, guadeloupe, france k inrae, ur4 (ur p3f), nouvelle-aquitaine-poitiers center, f-86600 lusignan, france l université paris-saclay, inrae, urgi, f-78026 versailles, france abstract: since their inception, the french academic organizations dedicated to agricultural research have developed plant collections in genebanks, often within a public–private framework, to support the study of plant traits and the development of new improved varieties. in addition, since the 2000s, a centre for genomic resources has also been established in france. over the last 20 years, this decentralized system, consisting of the academic genebanks and the centre for genomic resources, has been supported by a national coordination structure. the objectives were to align the network activities with the framework proposed by the organisation for economic co-operation and development (oecd) for biological resource centres and to foster collaboration with other national stakeholders involved in the conservation and characterization of plant genetic resources (pgr). in 2015, the network was named brc4plants and become part of the french national research infrastructure rare (www.agrobrc-rare.org), supported by the french ministry of research. this paper describes brc4plants, its users, services and cross-cutting activities. we also highlight its relations with its national and international stakeholders involved in the conservation and characterization of pgrs. brc4plants aims to be a key player in addressing societal and research challenges regarding agroecology, climate change mitigation and healthy food systems. keywords: plant genetic resources, genomic resources, brc, research infrastructure, ecpgr, genebanks citation: bergheaud, v., audergon, j., bellec, a., delaunay, a., duminil, j., dussert, s., esnault, f., geoffriau, e., gouesnard, b., jenny, c., label, a., lashermes, p., maghnaoui, n., marchal, c., nuissier, f., paulo-de-la-réberdiere, n., priet, a., rieucau, v., térès, p., adam-blondon, a. (2025). organization of plant biological resource centers for research in france: history, evolution and current status. genetic resources (s2), 78–90. doi: 10.46265/genresj.aszo2413. © copyright 2025 the authors. this is an open access article distributed under the terms of the creative commons attribution license (cc by 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. received: 30.10.2024 accepted: 24.12.2024 published online: 04.03.2025 https://www.genresj.org https://www.doi.org/10.46265/genresj.aszo2413 https://www.genresj.org https://www.doi.org/10.46265/genresj.aszo2413 genetic resources (2025), (s2), 78–90 plant biological resource centers in france 79 origins of brc4plants, the french network of plant biological resource centers (brc) for research since their inception, the french academic organizations for research in agriculture have built plant collections to support the study of plant traits and the development of new improved varieties. among the oldest academic french collections, the grapevine collection began to be established in the late 19th century by the college of agriculture of montpellier, to tackle the challenges of mildews and phylloxera that were devastating french viticulture (pouget, 1990). another emblematic example is the creation in the 1960s of the pro-mais french non-governmental organization grouping all the french maize breeders to facilitate collaborations with the academic sector, in particular to collect and conserve maize genetic resources (dallard et al, 2000). today, the conservation of biodiversity – particularly cultivated biodiversity or agrobiodiversity – is more crucial than ever and is key for adaptation to the accelerating global changes impacting the planet, human health and nutrition (pilling et al, 2020; ecpgr, 2021; lefèvre et al, 2024). the organization and evolution of the french activities dedicated to genetic resource conservation are described in roux-cuvelier et al (2021). here, we summarize the main steps and provide additional insights. in 1983, the french ministry of agriculture set up a coordination office for the conservation of genetic resources (plants and domestic animals) which operated until 2008 – the bureau de ressources génétiques (brg). the brg was a groupement d’intérêt scientifique, a french associative structure gathering under a memorandum of understanding the ministries of research, agriculture and environment and six french research institutes. it had three main mandates: the coordination of genetic resource management, the representation of france in international bodies, and the facilitation of genetic resource research. the brg was in particular mandated to implement a decentralized system for pgr conservation and to facilitate the establishment of commodity-specific and multi-stakeholders networks – including academic institutions, non-governmental organizations, regional organizations and private companies – to carry out this conservation. after 2008, the french government decided to merge brg with a newly created foundation, the fondation pour la recherche sur la biodiversité. in reality, coordination and facilitation – in particular for basic genetic resource conservation activities – have not been taken over by the fondation pour la recherche sur la biodiversité, which has focused on supporting the development of biodiversity knowledge. to address this gap, the managers of the plant collections from the main french research ∗corresponding author: valérie bergheaud (valerie.bergheaud@inrae.fr) institutes developed their own inter-institute networking and coordination activities. since the end of the 1990s, the management of plant biological resources dedicated to research and development activities (r&d) has been professionalized using the concept of biological resource center (brc, oecd organisation for economic co-operation and development, oecd (2001)) as a reference. in particular, academic brc managers actively contributed to the development of a french quality management norm dedicated to brcs, s96900:2011. later on, they contributed to the international norm dedicated to biobanking iso 20387:2018 and its technical specification (iso/ts 23105:2021) in order to improve the quality of their services, which include, among others, the conservation, documentation and provision to users of plant biological resources. a still growing number of french brcs dedicated to r&d are certified based on these norms and/or iso9001. these developments were supported by a french interresearch institute organization, infrastructures en biologie, santé et agronomie (ibisa, https://www.ibisa.net/) , aiming at labelling research facilities and funding their developments. in 2015, the informal network of plant brcs supported by french academic organizations joined four other brc networks on domestic animals, microbes, forest trees and environmental samples to set up a national infrastructure of resources for research in agriculture, rare (https://www.agrobrc-rare.org; figure 1) (tixier-boichard et al, 2024). rare is included in the french ministry of research’s roadmap and will soon need to reapply for this label, which provides access to funding and recognition. the plant network was named brc4plants: it currently gathers 21 brcs, including the french national center of genomic resources (cnrgv, table 1). the network spans the entire french territory, including overseas departments and regions, and conserves species cultivated in a wide range of climates (figure 2). the main missions of brc4plants’s brcs are to 1) ensure the proper maintenance of crop diversity in relation to the challenges faced by both biodiversity and agriculture (lefèvre et al, 2024) and 2) provide access to high-quality biological resources and associated data to the scientific community of plant biologists and breeders. brc4plants coordination activities aim to effectively support these initiatives through strategic discussions and decisions, training and facilitation. brc4plants has developed a formal governance structure that supports strategic decisions through a committee comprising representatives of the main funding institutes (inrae, the national research institute for agriculture, food and environment; cirad, the french agricultural research and cooperation organization working for the sustainable development of tropical and mediterranean regions, and ird, the french national research institute for sustainable development) and the coordinator of rare who meet twice a year. in addition, coordinated cross-cutting activities are organized mailto:valerie.bergheaud@inrae.fr 80 bergheaud et al genetic resources (2025), (s2), 78–90 both at the plant network level and the rare level. for instance, all brcs are supported by such internal transversal activities for their compliance with access and benefit sharing (abs) rules, the general data protection regulation and for their capacity to become certified iso9001. in 2020, rare established its international scientific advisory board comprising experts of all domains, which has met annually since then, along with an annual thematic scientific workshop. these two new annual events foster scientific strategic discussions across domains. rare facilitated the contribution of several networks to common projects such as the coordination support action of the h2020 ec programme, ‘genres bridge’ (grant agreement no 817580). the genres bridge project partners developed a genetic resources strategy for europe (genres bridge project consortium, ecpgr, erfp and euforgen, 2021) in collaboration with the three european networks for the conservation of genetic resources – the european cooperative programme for plant genetic resources (ecpgr), the european forest genetic resources programme (euforgen) and the regional focal point for animal genetic resources (erfp). brc4plants coordinated a strategic document on data management (adam-blondon et al, 2021). finally, rare has clarified the conditions necessary for a brc to join or leave the infrastructure. as a result, within some of the rare’s networks such as brc4plants, brcs are divided into two groups: full members of rare and those in progress towards membership. brc4plants is currently composed of 18 rare member brcs and three in-progress brcs (table 1). the difference between these two statuses is not yet significant as the only benefits obtained by brcs from rare derive from low-cost networking activities and it is not in the global interest to restrict access. however, the criteria of differentiation (certification of the quality management and/or ibisa label) might soon be important for getting access to some funding dedicated to brcs. after almost ten years of operation, this framework has reached its full maturity and its renewal on the roadmap is planned for 2025. brc4plants collections and services for the research community crop genetic and genomic resources have always been instrumental for research in plant genetics, plant biology and for fostering academic and socio-economic research and development partnerships. they have been intensively used to characterize genetic diversity, explore the evolutionary history of crops, develop advanced genomic tools, select panels of genetic resources for association studies, carry out pan-genome studies, develop prediction models and breeding strategies, screen for adaptive traits, and decipher molecular mechanisms with mutant collections (jourdan et al, 2015; allier et al, 2020; leuenberger et al, 2024; healey et al, 2024). most brcs and their collections are managed or comanaged by inrae, cirad and ird in partnership with technical institutes and higher education institutions (roux-cuvelier et al, 2021). as a whole, brc4plants maintains 214,918 accessions of a large range of model species, field crops, vegetables and fruit trees (table 1). an accession is defined as an entry in the collection and the unit of conservation. a crop variety can be represented by several accessions. different types of material (samples) can be conserved and distributed from a given accession: reproductive (e.g. seeds, in vitro plants, pollen, cuttings, etc.) or non-reproductive (e.g. leaf, dna, etc.). the collections are usually very diverse, in terms of accessions’ country of origin, phenotypes or genetic diversity (salinier et al, 2022). they include varieties, landraces and wild relatives but also material derived from breeding or research programmes (roux-cuvelier et al, 2021; salinier et al, 2022; esnault et al, 2025). figure 1. organization of the rare national research infrastructure (tixier-boichard et al, 2024), in five domain-specific networks of biological resource centres (brcs): crb-anim manages biological resources for domestic animals, brc4env manages biological resources and specimens in relation with various types of terrestrial environments (soils, lakes, pathogens, etc.), fbrc manages forest trees biological resources dedicated to research purposes, brc4microb manages microbial resources and brc4plants manages model and cultivated plant biological resources. g enetic resources (2025), (s2),78–90 plant biologicalresource centers in france 81 table 1. list of the biological resource centers that are members of brc4plants or with a status of candidate, their supporting institutes, the type of resources that they manage, the state of their contribution to national public–private networks dedicated to the conservation of genetic resources and to international working groups (2024). name main genera or species institute(s) french public–private networks international networks rare reference french plant genomic center (cnrgv) inrae member roux-cuvelier et al (2021) versailles arabidopsis stock center (vasc) arabidopsis thaliana inrae member ricou et al (2025) bracysol brassica oleracea, brassica napus, allium cepa aggregatum group, allium sativum, solanum tuberosum inrae vegetable crucifers, oilseed crucifers and potato networks ecpgr allium, brassica, potato member roux-cuvelier et al (2021); esnault et al (2025) coffea coffea spp. ird, cirad ecpgr cryopreservation member joët et al (2021) carrot and other vegetable apiaceae (carpia) daucus spp., chaerophyllum bulbosum, other apiaceae institut agro carrot and other daucus network (coordination) ecpgr umbellifer crops eva ishs carrot and other apiaceae member roux-cuvelier et al (2021) small grain cereals (cereales) triticum spp., hordeum spp., secale spp., triticosecale spp., avena spp. inrae small grain cereals network ecpgr wheat, avena, barley member debiton (2021) citrus citrus spp., poncirus spp., fortunella spp., clausena spp. inrae, cirad member roux-cuvelier et al (2021) forage and turf species (prairies) lolium spp., festuca spp., dactylis spp., medicago spp., trifolium spp. inrae forage and turf network ecpgr forages member sampoux et al (2025) pip fruit and rose (rosepom) malus spp., pyrus spp., cydonia spp., rosa spp. inrae pip fruits and rose networks ecpgr malus/ pyrus member roux-cuvelier et al (2021); feugey et al (2025) submitted seeds adapted to mediterranean and tropical conditions (gamét) oryza spp., sorghum spp., digitaria spp., gossypium spp., arachis spp., vigna unguiculata, zea spp., medicago spp. inrae, cirad promais network ecpgr maize member maghnaoui and prosperi (2017) continued on next page 82 bergheaud et al g enetic resources (2025), (s2),78–90 table 1 continued name main genera or species institute(s) french public–private networks international networks rare reference inrae solanaceae, lactuca and melon networks ecpgr cucurbits, solanaceae, leafy vegetable member salinier et al (2022) inrae promais network ecpgr maize candidate diaw et al (2017) conservatoire botanique national méditerranéen (cbnmed), inrae international olive council candidate marchal et al (2017); roux-cuvelier et al (2021) cirad member cirad, inrae member roux-cuvelier et al (2021) inrae ecpgr grain legumes member aubert et al (2023); carrillo-perdomo et al (1914) inrae prunus (in progress), juglans ecpgr prunus member roux-cuvelier et al (2021) vegetable germplasm (leg) french maize inbred lines genebank (mais-lig) olive trees (olivier) perennial plants in french guiana (ppg) tropical plants (plantrop) grain legumes (protea) prunus-juglans (pru-ju) sunflower and soy (helia-soja) solanum melongena, capsicum annuum, solanum lycopersicum, cucumis melo, lactuca sativa zea spp. olea europaea coffea spp., theobroma spp., hevea spp., aniba rosodora spp. musa spp., ananas spp., saccharum spp., mangiferea spp., dioscorea spp. vicia faba, lupinus spp., pisum spp. prunus armeniaca, prunus dulcis, prunus persica, prunus cerasus, prunus domestica, juglans spp. heliantus spp., glycine max inrae helianthus and soya candidate terzić et al (2020) vanilla of tahiti (vanira) vanilla spp. etablissement vanille de tahiti member roux-cuvelier et al (2021) vatel vanilla spp., tropical allium spp., roots and tubers, neglected vegetables cirad germination international network member roux-cuvelier et al (2021) grapevine biological resources center (vigne) vitis spp. inrae vitis network ecpgr vitis member roux-cuvelier et al (2021) genetic resources (2025), (s2), 78–90 plant biological resource centers in france 83 in addition, cnrgv develops and maintains plant genomic resources with a catalogue of more than 300 bacterial artificial chromosome (bac) libraries (around 35 million samples) from more than 60 different plant species. cnrgv provides the research community with a range of services to characterize genetic resources at the genomic level with cutting-edge technologies, in collaboration with other french national genomic facilities that together allow the production of high-quality reference genomes required for diversity analysis and association studies, and the detailed analysis of intraspecific diversity at loci carrying key genes. in 2022, brc4plants provided around 13,000 genetic resources accessions, over 100 bac clones or libraries and globally billed an important vol-ume of genomic services to a diversified panel of users: 49% to french public institutes, 37% to foreign public institutes, 13% to private companies or producers and 1% to schools, regional public structures, ngos etc. the introduction and distribution activities follow national and international regulations concerning sanitary issues and abs. the terms of access to brc’s services are available on the cnrgv website for genomic services (http s://cnrgv.toulouse.inra.fr/fr/services) and on the web portal of brc4plants for the other brcs (e.g. https:// florilege.arcad-project.org/fr/crb/bracysol/conditions-g enerales). the standard material transfert agreement (smta) of the international treaty on plant genetic resources for food and agriculture (itpgrfa) is used for species on annex 1 of itpgrfa. an inrae standard material transfer agreement, compliant with the convention on biological diversity (cbd) nagoya protocol is used for other species or for material developed by inrae (e.g. segregating population, pre-breeding material, etc.). in case of scarcity of the material for distribution, the priority has been so far to support research, development and training. brc4plants activities (of individual brcs as well as those coordinated by the network) are supported by academic organizations, within the framework of their research priorities, with permanent positions dedicated to the brc missions and a wide range of infrastructures, including robots, refrigerators, freezers (-20◦c and 80◦c), cryotanks, seed drying rooms, greenhouses, field plots for seed regeneration or plant conservation (vegetatively-propagated or perennial plants). the network as a whole also provides an important volume of communication activities that contribute to raising awareness of genetic resources to a large audience (e.g. pic et al (2017), new york times (poll, 2021), le monde (rosier, 2021)). finally, the network contributes to training and education on biodiversity conservation as well as on genomic approaches. brc4plants r&d activities brc4plants regularly updates its priorities in terms of r&d, which can be seen at two levels. first, r&d activities that aim at improving the efficiency of conservation and distribution of accessions. key areas include transversal issues such as management and legal compliance regarding phytosanitary and abs issues (tixier-boichard et al, 2024), fair (data are findable, accessible, interoperable and reusable) data management and automated data workflows between local databases and central information systems, andimproved cryopreservation methods (markovic et al, 2015). these activities also include local improvements of processes and equipment in brcs and are mainly funded by french research organizations, the french national research agency or sometimes european funds for regional development (feder). the second level includes r&d activities aiming at improving services dedicated to research projects using genetic resources to achieve their goals: development of new services, such as the development of new populations and core collections (esnault et al, 2017; terzić et al, 2020; salinier et al, 2022), new knowledge on biological resources, e.g. through their genomic or phenotypic characterization (antoine et al, 2023; mart́ınez-flores et al, 2020), and improved breeding strategies (sanchez et al, 2023). these r&d activities are supported by a diverse set of national and european calls and the applications are frequently driven by public and/or private researchers who are not brc members. calls dedicated to infrastructures at the national or international level can be an opportunity to work with other infrastructures. for instance, brc4plants has actively liaised with the european infrastructures emphasis (plant phenotyping) and elixir (bioinformatics for life sciences) to contribute to the development of a suite of guidelines and resources supporting fair-compliant management of plant genotyping and phenotyping data. this work was supported by several eu programme horizon 2020-funded projects: elixir-excelerate, grant agreement no. 676559 https://elixir-europe.org/about -us/how-funded/eu-projects/excelerate; agent, grant agreement no. 862613 https://www.agent-project.eu/ and elixir-converge, grant agreement no. 871075 https://elixir-europe.org/about-us/how-funded/eu-pr ojects/converge, and all the results can be accessed through the elixir portal of resources for data management (https://rdmkit.elixir-europe.org/). building on these resources, a national project currently gathers partners from three french national infrastructures, rare, the french institute of bioinformatics (https://w ww.france-bioinformatique.fr/) and inrae genomics ( https://inrae-genomics.hub.inrae.fr/) to develop a comprehensive service of genomic data management from data production to data submission to the european archives maintained by the european bioinformatics institute (embl-ebi). projects including the characterization of pgr or the development of pre-breeding populations can be funded by national calls dedicated to public–private partnerships. they often build on the trust developed in the public-private networks in which many of the brcs are involved (see below and table 1). 84 bergheaud et al genetic resources (2025), (s2), 78–90 figure 2. localization of the 21 brcs facilitated by brc4plants together with the number of accessions they manage. in general, they are single-located (green points) but two have several locations, pru-ju (purple points) and plantrop (blue points). they can also be funded by the european commission (e.g. h2020 g2p-sol project, grant agreement no. 677379, http://www.g2p-sol.eu/). brc4plants organizes training and dissemination activities to ensure that the outputs of the projects can be leveraged by all brcs. public–private partnerships half of the brcs collaborate with networks gathering different types of partners (seed companies, breeders, extension institutes, regional organizations for genetic resource conservation, ngos; table 1, figure 3 ). these collaborative networks, established in the 1990s, aim to pool efforts to manage and study mostly traditional genetic resources of a species or a related group of species. a model charter for these ‘genetic resources networks’ was developed in 2018 and updated in 2021 by brc4plants, inrae central services, the french seed association and the french ministry of agriculture to define the rules for material and data exchange within and out of the network. some of these networks have formal organizations (e.g. promäıs for maize, http://pr o-maize-corn.com/, the federation franc for the rose or a joint unit of inrae with the institut français de la vigne et du vin, ifv, for grapevine). the existence of such partnerships has made it possible to inventory and secure old french genetic material (e.g. dallard et al (2000); terzić et al (2020)). additionally, the grapevine-dedicated joint unit has been instrumental in exchanging technical expertise. ifv and inrae have also brought together all the other french regional extension services for viticulture and oenology to formally organize the development of grapevine varieties resistant to diseases and adapted to each french terroir (inra, 2018). inrae and ifv are promoting both traditional genetic resources (a renewal of interest for old varieties was observed in many places) and new resistant varieties under the brand entav-inra (www.entav-inra.fr). similarly, a convention between inrae and the four french potato breeders gathered within the association named acvnpt (association des créateurs de variétés nouvelles de pomme de terre) was signed in 1995. acvnpt provides financial support to inrae for the conservation and characterization of potato genetic resources maintained within the bracysol brc, and in return gets free access to the pre-breeding material generated by inrae within the framework of its research activities (kerlan et al, 2017; esnault et al, 2025). since 1995, inrae has proposed 994 pre-breeding clones, improved mainly for resistance to biotic stress. forty-one potato varieties, originating from these pre-breeding clones, have been registered. genetic resources (2025), (s2), 78–90 plant biological resource centers in france 85 figure 3. brc4plants in its ‘ecosystem’ of stakeholders at different levels. at the international level, brc4plants contributes to the activities of the european cooperative programme for plant genetic resources (ecpgr), deposits data in the catalogue of ecpgr, eurisco, and contributes to the deposition of collections of accessions in the multilateral system of the international treaty on plant genetic resources for food and agriculture (itpgrfa). at the national level, brc4plants: 1) is funded by the french ministry of research and follows its recommendations related to research infrastructures (enforced by brc4plants main funding institutes, inrae, cirad and ird) and 2) interacts with the french coordination on plant genetic resources conservation and contributes to the section of the technical committee for breeding (ctps) dedicated to plant genetic resources that are both under the governance of the french ministry of agriculture. the daily management of brc4plants collection is made in collaboration on one side with research units that were often at the origin of the collections and often on the other side with national public–private networks of partners that may also represent important users of those collections. the interactions with brc4plants stakeholders are represented by arrows and their results or purposes are specified in italics. brc4plants contribution to the french national coordination for plant genetic resources activities brc4plants is a self-organized, research-driven network of brcs. many other organizations are contributing to the conservation of pgr in france (duval et al, 2023): regional centres, ngos, the network of botanical conservatories, private companies, etc. the french ministry of agriculture is in charge of developing the french strategy for the conservation and use of pgr for food and agriculture. in particular, this strategy aims to contribute to fao’s global plans of action on genetic resources and to the itpgrfa (duval et al, 2023). to support this endeavour, the ministry established the french national coordination for pgr, which comprises a national support structure hosted within geves (group for the study and control of varieties and seeds) and a section of the french multistakeholder organization ctps (permanent technical selection committee). this coordination is responsible for implementing french policies on genetic resources (duval et al (2023); figure 3). brc4plants contributes to the ctps plant genetic resources section and facilitates, in collaboration with its funding bodies, the official recognition of brcs through ministerial acknowledgement. this recognition identifies them as managers of genetic resources and acknowledges their contribution to the french national collection of genetic resources (duval et al, 2023). so far, inrae and 86 bergheaud et al genetic resources (2025), (s2), 78–90 the etablissement vanille de tahiti have been officially recognized as managers of genetic resources and inrae is yearly increasing the official collection set under its responsibility in collaboration with its brcs (maa, 2022, 2024). the french national coordinator for plant genetic resources is also the french national coordinator for ecpgr (duval et al (2023); figure 3). brc4plants’ coordination and its brcs are actively contributing to ecpgr working groups (table 1) dedicated to crops as well as to cross-cutting themes. in collaboration with the national coordination, brc4plants deposits data in the european search catalogue for plant genetic resources (eurisco), managed by ecpgr, and contributes to the inclusion of accession collections in the itpgrfa multilateral system. in 2019, ecpgr launched the european evaluation network (eva) which leverages public–private partnerships at the precompetitive stage, to jointly generate standardized characterization and evaluation data on crop accessions present in european genebanks. these accessions are often poorly characterized and, consequently, underutilized. ecpgr provides the eva networks with standards and platforms for documentation and results sharing (kumar et al, 2024). so far, four french brcs are contributing to eva networks: crb gamét for maize (balconi et al, 2024), crb carpia for carrot (goritschnig et al, 2023) and crb-leg for pepper and lettuce. conclusions and perspectives the overarching goal of brc4plants is to facilitate the mobilization of its resources and services for research and innovation, with the aim of enhancing genetic diversity and beneficial interactions within agroecosystems. this supports the agroecological transition and the development of sustainable and healthy food systems. the network of brcs plays an important role as a source of genetic diversity to be screened for new traits (e.g. plant-plant or plant-microbe favourable interactions). brcs can also support the development of new plant materials for research on new breeding or cropping strategies. in this context, brc4plants faces several challenges that will need to be addressed through a mix of social, technical and scientific approaches: • how should brc4plants be organized to address new needs regarding species, material development or introduction of accessions, while ensuring these efforts remain feasible within a realistic level of funding and support? • how to increase in situ dynamic conservation activities and their integration with ex situ conservation? • how to deal with the threats associated with climate change, including the spread of new diseases, in particular for the conservation of perennial plants in orchards? • how to ensure that the variety of jobs necessary for the management and dissemination of genetic resources remains attractive and rewarding despite increasing legal and administrative complexity and responsibility? tackling these challenges requires the co-construction of strategies with brc4plants supporting research organizations, as well as with other stakeholders involved in the conservation and use of genetic resources, in particular with the national coordination on pgr. the current lack of full alignment or integration between research, agriculture and environment-driven national governance on pgr conservation is reducing the efficiency of efforts to coordinate the different stakeholders. it should be noted that this lack of integration is also a challenge at the european level and was identified as a major issue in the genetic resources strategy for europe developed in the frame of the genres bridge project (genres bridge project consortium, ecpgr, erfp and euforgen, 2021). nevertheless, at the national level, rare, as a national research infrastructure, is an effective platform for conveying messages through its participation in ministry-driven committees and initiatives, its governance committee, which includes representatives of its main supporting institutes, and its advisory committee, which includes members of the research community and other international infrastructures. rare can develop and present coordinated perspectives on issues common to all genetic resources domains (e.g. implementation of the french law on abs or phytosanitary risk management). in addition, brc4plants operates at the interface of a significant variety of stakeholders as illustrated in figure 3. this position enables it to codevelop responses and solutions with research organizations, different types of genetic resources users, and policymakers, such as national decision support systems dedicated to abs or diseases under regulation. another important benefit of these multi-stakeholder approaches is a more realistic distribution of responsibilities among research organization central offices, official agencies and individual brc managers. the contribution of brc4plants brcs to multistakeholder networks facilitates the development of species-specific collaborations aiming at characterizing and enhancing the use of pgr. however, a challenge now is to expand their diversity to include new players such as farmers, citizens, or industries, in order to develop integrated in situ and ex situ approaches for the management of genetic resources. this will also require an evolution in networking practices, including the structure of governing and advisory bodies (louafi et al, 2021). some brcs are currently involved in projects aiming at developing a common understanding of different stakeholders and expectations, and codeveloping common objectives in the context of the conservation and use of genetic resources. the french choice to organize pgr conservation in a fully decentralized way has some disadvantages in terms of visibility and requires a layer of coordination to avoid redundancy and identify and support common develgenetic resources (2025), (s2), 78–90 plant biological resource centers in france 87 opments and resources. however, it should be noted that nationally centralized genebanks in other countries primarily hold seed-propagated genetic resources and only a few vegetatively propagated species, whereas brc4plants manages many of the latter. moreover, the french academic decentralized network has a historical origin: brc collections were initially developed by individual researchers for their own breeding and research objectives, and were later centralized by crops by research institutes. since then, they have remained closely linked to research units that contribute to their development, characterization, funding and reputation within their specific species communities of interest. given the very low level of long-term funding for the basic operation and maintenance of collections, these tight links with research teams, various regional bodies and other stakeholders such as industry have been instrumental in raising funds for brcs. the decentralized organization also offers significant flexibility for including new partners with additional collections or new expertise and tools. additionally, it can provide backup solutions should any of the brcs face problems due for example to climatic and environmental changes (perennials) or changes in the local scientific and technical teams (all types of brcs). many of the challenges described above are also relevant in other countries (smith et al, 2021). on the other hand, the international level can provide cooperation and solutions. at the european level, the conservation of genetic resources is organized in a decentralized model by ecpgr, which fosters cooperation under the itpgrfa principles. brc4plants is already well involved in this framework. an ongoing project, pro-grace (https://www.grace-ri.eu/pro-gra ce), funded by the eu programme horizon europe (grant agreement no. 101094738) aims at extending or completing this framework through the creation of a pan-european research infrastructure on pgr. brc4plants is keen to contribute to such an infrastructure, including through its links with other national (e.g. metabohub for metabolomics, profi for proteomics and france genomics for genomics) and european infrastructures (emphasis for phenotyping and elixir for life science data) that could be leveraged in the context of the characterization and use of genetic resources. such a research infrastructure would strengthen the links of brcs with public and private researchers in to foster their contributions to addressing the current challenges of agriculture1. 1 see for instance the publications derived from three projects aiming at facilitating adaptation of different crops to climate change: sunrise for sunflower, https://anr.hal.science/search/in dex/?q=*&anrprojectreference s=anr-11-btbr-0005; the amaizing for maize, https://anr.hal.science/search/index/?q=*&rows=3 0&anrprojectreference s=anr-10-btbr-0001 or breedwheat for wheat, https://anr.hal.science/search/index/?q=*&rows=30&anrpro jectreference s=anr-10-btbr-0003 acknowledgements the network brc4plants thanks the research infrastructure agrobrc-rare, the french national research institute for agriculture, food and environment (inrae, biological resource centers for plants of agrobrcrare, brc4plants https://doi.org/10.17180/wn42-3j 20), the french agricultural research organization and international cooperation for sustainable development tropical and mediterranean regions (cirad), the french public research organization for equitable partnerships with the countries of the south and in french overseas territories (ird), the gis ibisa, institut agro and the alliance nationale de recherche pour l’environnement (allenvi) for supporting its activities. the writing of the paper was also supported by the horizon europe programme project ‘promoting a plant genetic resource community for europe (pro-grace)’, no. 101094738. authors contributions vb and afab drafted the paper, jma, ab, ad, jd, sd, fe, eg, bg, cj, al, pl, nm, cm, fn, np de la r, ap, vr and pt revised it. ap and eg contributed to the design of the figures. conflict of interest statement the authors declare that there are no conflicts of interest references adam-blondon, a. f., tixier-boichard, m., bozzano, m., goritschnig, s., sharrock, s., sturaro, e., van hintum, t., weise, s., and westergren, a. 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(2024). eclairages sur l’infrastructure de recherche rare (inrae ; cirad; ird; université paris-saclay; université de montpellier; université de rennes; agroparistech; institut agro), 15p. url: https://hal.science/hal-04750756. https://doi.org/10.1002/agj2.20761 https://doi.org/10.1002/agj2.20761 https://doi.org/10.1051/ocl/2020004 https://hal.science/hal-04750756 origins of brc4plants, the french network of plant biological resource centers (brc) for research brc4plants collections and services for the research community brc4plants r&d activities public–private partnerships brc4plants contribution to the french national coordination for plant genetic resources activities conclusions and perspectives acknowledgements authors contributions conflict of interest statement original article genetic resources (2025), 6 (12), 160–170 doi: 10.46265/genresj.unba8473 https://www.genresj.org issn: 2708-3764 received: 09.06.2025 | accepted: 08.10.2025 | published online: 17.11.2025 evaluation of red sanders (pterocarpus santalinus l.f.) germplasm for conservation and breeding abstract: pterocarpus santalinus l.f., an endangered species endemic to india’s eastern ghats, faces threats from illegal trade due to high demand and limited legal supply. field genebanks are essential for conserving genetic diversity and supporting sustainable use. this study evaluated a 12-year-old p. santalinus germplasm collection of 500 accessions, grown ex situ, for growth and heartwood traits. the survival rate was 80%, with notable variation in tree height (5.5–11.8m), girth at breast height (gbh, 26–78cm), clear bole height (0–6.2m), and heartwood core length (0–6.6cm), indicating substantial genetic diversity. early heartwood formation (< 12 years) occurred in 18.50% of accessions, earlier than the typical 15 years. superior accessions included s5r1-19 (6.6cm heartwood) and s7r1-4 (5.5cm), while s1r1-13, s5r4-20, and s1r3-18 exhibited desirable deep red heartwood. accessions from petbasheerabad showed high survival and heartwood yield, suggesting their value as elite seed sources. gbh positively correlated with heartwood length (r = 0.443), supporting its use as a selection trait. principal component analysis and clustering grouped accessions into three clusters: cluster 1 showed superior timber traits (high clear bole, low percentage of forking), whereas cluster 3 displayed less desirable traits. these findings aid in identifying elite accessions and developing conservation and breeding strategies. integration of molecular tools such as genome-wide association studies and transcriptomics is recommended to accelerate genetic improvement. keywords: red sanders, heartwood formation, germplasm evaluation, tree improvement, field genebank citation: shivaprasad, k. m., kumar, p. s., kishore, a., kumar, d. and pattanaik, s. (2025) “evaluation of red sanders (pterocarpus santalinus l.f.) germplasm for conservation and breeding”, genetic resources, 6(12), pp. 160–170. doi: 10.46265/ genresj.unba8473. © copyright 2025 the authors. this is an open access article distributed under the terms of the creative commons attribution license (cc by 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. kumbarahally murthigowda shivaprasada,*, pendela surath kumara, avula kishorea, dinesh kumara, swapnendu pattanaika,* agenetics and tree improvement division, indian council of forestry research and education (icfre)-institute of forest biodiversity, hyderabad, telangana, india-500100 * corresponding authors: : kumbarahally murthigowda shivaprasad (shivaprasadkm.ifb@gmail.com), swapnendu pattanaik (swapnen@ yahoo.com) introduction red sanders (pterocarpus santalinus l. f.) is a highly valuable timber species endemic to the southern part of the eastern ghats of india and is classified as endangered on the international union for the conservation of nature (iucn) red list of threatened species (ahmedullah, 2021). it is known for its characteristic timber of exquisite colour, beauty, and superlative technical qualities (arunakumara et al, 2011). the red-coloured heartwood is the economic part of the plant, and the wood has significant demand globally, primarily sought after for its application in crafting the traditional japanese musical instrument known as the 'shamisen'. additionally, wood sourced from red sanders is utilized in the fabrication of name seals, frames, carvings, furniture, and various traditional artefacts (arunakumara et al, 2011; pattanaik, 2024). further, red sanders wood possesses a significant insoluble or sparingly soluble red dye, consisting of approximately 16% of the santalin pigment, a principal colouring agent utilized notably in textile dyeing and in european medicine as a colouring agent. in the united states, it holds approval as a food dye for alcoholic beverages and is similarly sanctioned for use within europe, and classified as a spice extract rather than a conventional food colourant (arunakumara et al, 2011; new, 1981). additionally, https://doi.org/10.46265/genresj.unba8473 https://www.genresj.org https://doi.org/10.46265/genresj.unba8473 https://doi.org/10.46265/genresj.unba8473 mailto:shivaprasadkm.ifb%40gmail.com?subject= mailto:swapnen%40yahoo.com?subject= mailto:swapnen%40yahoo.com?subject= genetic resources (2025), 6(12), 160–170 red sanders germplasm evaluation 161 pterostilbene, a methyl ester derivative of resveratrol present in red sanders, offers a diverse array of promising pharmacological properties (schmidlin et al, 2008; seshadri, 1972). the exploitation of red sanders forests without commensurate restoration in the past has led to the present degraded state (ahmed and nayar, 1984). overexploitation prompted the union government in the 1980s to recommend the inclusion of red sanders in appendix ii of the convention on international trade in endangered species of wild fauna and flora (cites). subsequently, the species was listed in appendix ii of cites in 1995, and export of red sanders was prohibited in 2004. despite the complete ban on felling and trade of natural origin wood, the gap between demand and supply fuelled its illicit felling and smuggling (ahmedullah et al, 2019). sustained pressure on these natural resources has resulted in their widespread degradation, as evidenced by the fact that most of the standing crop is of coppice origin and that regeneration is poor or lacking at several locations of occurrence. the andhra pradesh forest department has estimated a global annual demand of approximately 3,000 metric tonnes (mt) of red sanders wood (nba, 2017), and reported substantial revenue from the auction of 5,489.51mt of seized wood between 2014 and 2019 (ahmedullah et al, 2019). in response to increasing international interest, the directorate general of foreign trade (government of india) revised its export policy in 2019 to permit the export of red sanders wood derived from cultivated sources, underscoring the urgent need to expand plantation-based production systems. despite red sanders being cultivated in various regions of andhra pradesh and other indian states, the wood produced in these plantations is typically of inferior quality compared to wood from natural populations. this quality gap limits the economic returns for farmers, even as global demand for premium-grade red sanders remains high (ahmedullah et al, 2019). the lack of genetically improved red sanders varieties forces farmers to rely on uncertified bulk seeds, resulting in considerable variability in key traits such as heartwood formation. furthermore, red sanders typically require 15–25 years to form heartwood, delaying economic returns (nba, 2017). to enhance productivity and profitability for farmers, it is essential to develop and supply superior planting material with improved wood traits. evaluation of available genetic resources is a critical step in identifying elite germplasm suitable for breeding and large-scale propagation. field genebanks play a vital role in the conservation and evaluation of such genetic resources, enabling long-term genetic improvement strategies. this study was undertaken to assess a red sanders germplasm assemblage for variation in growth and heartwood traits, with the goal of identifying superior accessions for use in future breeding and conservation programmes. materials and methods study area the present evaluation study was carried out in the field genebank located at icfre-institute of forest biodiversity (17.556911° n latitude, 78.446385° e longitude, and 542m above mean sea level). the original collection, consisting of 500 germplasm from eight different seed sources, was established in august 2012 for ex situ conservation of red sanders. the genebank was designed with a spacing of 5 × 4m, and the trees were maintained without pruning operations to allow natural branching patterns (figure 1). the details of genotypic origin and sample sizes are provided in table 1 and figure 2. figure 1. view of the red sanders field genebank at icfre-institute of forest biodiversity, hyderabad, india. figure 2. map showing the red sanders seed sources used for the red sanders field genebank genetic resources (2025), 6(12), 160–170162 shivaprasad et al table 1. geographic details of seed sources and corresponding number of plants established in the red sanders field genebank at icfreinstitute of forest biodiversity, hyderabad, india. longitude and latitude are expressed in decimal degrees. code seed source latitude longitude no. of germplasms no. of germplasm survived and evaluated s1 ahobilam 15.1408n 78.7344e 75 55 s2 k. v. bhavi 13.8917n 79.2341e 100 82 s3 red wood park 13.9462n 79.3528e 100 79 s4 mudireddipally 14.8062n 78.8096e 100 62 s5 petbasherabad 17.5132n 78.4736e 100 97 s6 gumudipoondi 13.4466n 80.1306e 10 10 s7 nayapakkam 13.2065n 79.8592e 10 10 s8 amirthi 12.7305n 79.0485e 5 5 evaluation of growth traits and heartwood formation germplasm evaluation was carried out in 2024 with ten morphological traits. trait measurements included tree height (m), measured from the base of the tree to the highest point of the crown using a measuring pole, and clear bole height (m), measured from the base of the tree to the first branching point using a measuring tape. girth at breast height (gbh) (cm) was recorded at 1.37m above the ground using a tape, while forking was assessed visually to record its presence or absence. the number of forked stems was counted, and the average gbh in forked trees (cm) was calculated by measuring the gbh of each forked stem and computing the average for trees with multiple stems. heartwood core length (cm) and sapwood length (cm) were determined from wood core samples extracted using an increment borer. the distinct heartwood and sapwood regions of the core were measured with a scale and expressed in cm, while the total core sample length (cm) was recorded as the combined length of heartwood and sapwood. heartwood colour was evaluated subjectively, and the wood samples were classified ocularly into three broad groups, viz., light red, medium red and deep red. these measurements were conducted systematically to ensure consistency and accuracy across all accessions, enabling detailed analysis of phenotypic variation. descriptive statistics descriptive statistical analysis was performed to summarize and compare the traits of 400 surviving germplasm accessions out of the 500 initially planted. measures such as mean, minimum and maximum values were calculated using the excel data analysis toolpak for an initial overview. frequency distributions and percentage pie charts were also plotted to illustrate key trends in survival rates and heartwood formation. subsequently, descriptive statistics for each seed source were analyzed independently, including the calculation of mean and standard deviation (sd) for all traits. to enhance visualization and facilitate comparisons among seed sources, the r programming (r core team, 2018) environment was utilized. packages such as dplyr and tidyr were employed for data preprocessing and aggregation, while ggplot2 was used to generate visual representations, including bar plots. these tools allowed for clear comparisons of trait variability and performance across seed sources. correlation to explore relationships among the traits, a correlation analysis was conducted. pearson correlation coefficients were computed for all trait combinations using r (r core team, 2018). the corrplot package was employed to visualize the correlation matrix, and rcolorbrewer was used to enhance the colour scheme, facilitating easier interpretation of trait interdependencies. the correlation plot provided insights into the strength and direction of relationships among the traits across seed sources. principal component analysis and boxplot principal component analysis (pca) was performed to reduce dimensionality while retaining maximum variance among tree growth traits. numeric traits were standardized using z-score normalization, and pca was conducted using the prcomp function in r (r core team, 2018). traits with absolute loadings above 0.3 were identified as major contributors. to classify trees into distinct groups, k-means clustering was applied to pca scores (pc1 and pc2), with the optimal number of clusters determined using the nbclust function based on the silhouette method. the resulting clusters were visualized using ggbiplot, with confidence ellipses highlighting group structures. all analyses were performed in r, utilizing packages such as ggplot2, ggbiplot, dplyr, nbclust, and rcolorbrewer for statistical analysis and visualization. results morphological diversity in red sanders accessions out of the 500 established germplasm lines, 400 survived and were assessed using ten morphological traits. the evaluation of the 12-year-old germplasm revealed an average tree height of 9.04m, with the tallest tree (11.8m) observed in accession s2r4-10 and the shortest (5.5m) in s4r1-7. the average clear bole height was 1.05m, with the highest (6.2m) recorded in s1r2-13. the number of forked stems ranged from 0 to 6, with 176 accessions exhibiting no forking. the accession s8r4-4 recorded the maximum of six forked stems. genetic resources (2025), 6(12), 160–170 red sanders germplasm evaluation 163 figure 3 shows the frequency distribution of forked stems among red sanders accessions, illustrating the variation in forking observed across the population. the average gbh was 45.00cm, with the highest (78.00cm) observed in s2r123 and the lowest (26.00cm) in s3r2-2. increment core samples had an average length of 7.58cm, with a maximum of 12.5cm in s2r1-23 and a minimum of 4.00cm in s3r22. the average sapwood length was 7.1cm, with the highest value (7.9cm) recorded in s5r2-2. heartwood formation was observed in 74 accessions (18.50%), with an average heartwood core length of 2.53cm. the maximum heartwood core length (6.6cm) was recorded in s5r1-19, followed by s7r4-4 (5.5cm), s6r4-3 (5.2cm), s5r4-20 (5.0cm), s3r423 (5.0cm) and s5r4-25 (4.5cm). the lowest heartwood core length (0.5cm) was observed in s4r1-19 and s3r1-17 (supplemental table 1). analysis of heartwood colour of 74 accessions revealed light red colour in 41 accessions (55.41%), a medium red colour in 19 accessions (25.68%), and a deep red colour in 14 accessions (18.92%) (figure 4a and b). a detailed list of the 74 heartwood-forming accessions, along with their heartwood colour and heartwood core length, is provided in table 2. figure 3. frequency distribution of forked stems in red sanders accessions figure 4. heartwood colour variation in red sanders germplasm (a). pie chart representing the distribution of different heartwood colour grades in the collection (b). genetic resources (2025), 6(12), 160–170164 shivaprasad et al table 2. list of the 74 heartwood-forming red sanders accessions and their heartwood colour and heartwood core length. accession heartwood core length (cm) heartwood colour accession heartwood core length (cm) heartwood colour s5r1-19 6.6 light red s5r4-18 2.3 deep red s7r1-4 5.5 medium red s5r4-21 2.3 light red s6r1-3 5.2 light red s5r3-5 2.2 light red s5r4-20 5 deep red s5r2-13 2.1 light red s3r4-23 5 light red s1r3-7 2.1 deep red s5r4-25 4.5 light red s5r1-9 2 light red s4r3-22 4.3 medium red s5r2-14 2 light red s5r3-19 4.3 light red s1r2-23 2 light red s2r1-23 4 medium red s2r2-23 2 light red s5r2-4 4 light red s4r4-2 2 medium red s5r4-2 4 medium red s4r4-17 2 medium red s3r4-21 4 medium red s3r4-14 2 light red s5r4-4 3.9 medium red s3r4-15 2 deep red s5r4-15 3.8 light red s1r3-24 1.9 light red s3r2-20 3.6 light red s5r1-21 1.8 light red s3r2-22 3.5 medium red s1r3-2 1.8 deep red s5r3-4 3.4 light red s3r1-5 1.7 deep red s1r2-2 3.3 medium red s2r3-22 1.7 light red s5r1-2 3.2 light red s5r3-20 1.6 medium red s4r3-18 3.2 medium red s3r2-25 1.5 light red s4r3-24 3.1 light red s3r4-24 1.5 medium red s4r4-6 3 medium red s1r1-17 1.4 deep red s5r4-13 3 medium red s4r3-7 1.4 light red s5r4-9 2.9 light red s1r3-4 1.4 medium red s5r1-15 2.8 light red s5r2-17 1.3 medium red s5r1-23 2.8 light red s5r2-12 1.2 light red s5r2-5 2.7 light red s3r3-6 1.2 light red s3r3-4 2.7 light red s1r2-21 1.1 medium red s5r3-6 2.7 light red s3r4-4 1.1 light red s1r1-20 2.6 light red s1r1-13 1 deep red s3r3-14 2.5 deep red s2r2-4 1 light red s1r3-8 2.5 light red s3r3-2 1 light red s5r1-12 2.4 light red s1r3-1 1 medium red s3r2-23 2.3 light red s1r3-14 1 deep red s4r3-3 2.3 light red s1r3-18 0.8 deep red s5r3-25 2.3 light red s3r1-17 0.5 deep red s5r4-3 2.3 deep red s4r1-19 0.5 deep red genetic resources (2025), 6(12), 160–170 red sanders germplasm evaluation 165 morphological diversity in red sanders seed sources the study evaluated eight seed sources based on survival rates, heartwood formation, and various growth and heartwood formation parameters in different accessions (table 3). survival rates varied significantly among sources, with gumudipoondi (s6), nayapakam (s7) and amirthi (s8) achieving 100% survival, whereas madireddipally (s4) exhibited the lowest survival rate (62%). heartwood formation also varied considerably, with petbasheerabad (s5) recording the highest heartwood formation (29.9%), followed by ahobilam (s1) (23.64%) and red wood park (s3) (20.25%), while k.v bhavi (s2) had the lowest heartwood formation (4.88%), and no heartwood formation was observed in amirthi (s8) (figure 5). growth traits revealed that amirthi accessions exhibited the greatest tree height (10.24m), while madireddipally accessions recorded the shortest (8.40m). petbasheerabad accessions demonstrated a favourable balance between growth and heartwood quality, with a high survival rate (97%) and consistent heartwood formation, indicating its potential as a reliable seed source. other traits, including clear bole height, heartwood core length, sapwood length, and gbh, exhibited substantial variation across seed sources. notably, amirthi and gumudipoondi accessions excelled in tree height and gbh. the observed variability highlights the importance of selecting genetically superior seed sources to enhance plantation productivity and optimize timber quality. these findings provide a scientific basis for identifying and promoting superior seed sources in breeding programmes aimed at improving plantation success and meeting commercial demands for high-quality wood products (table 3, figure 6). figure 5. frequency distribution of survival, forking and heartwood formation traits in red sanders seed sources correlation analysis among various traits in red sanders the correlation analysis revealed complex but statistically meaningful relationships among the recorded growth traits (figure 7). tree height showed a significant negative correlation with clear bole height (r = -0.314, p < 0.05) and significant positive correlations with the number of forking branches (r = 0.278, p < 0.05), average gbh (r = 0.334, p < 0.05), sapwood length ( r = 0.316, p < 0.05), and total core sample length (r = 0.435, p < 0.01). clear bole height exhibited a highly significant negative correlation with the number of forking stems (r = -0.796, p < 0.001) but only weak and mostly non-significant associations with other traits. the number of forking stems was negatively correlated with average gbh (r = -0.326, p < 0.05), while its correlations with heartwood core length (r = -0.077) and sapwood length ( r = 0.026) were weak and non-significant. average gbh showed strong and highly significant positive correlations with heartwood core length (r = 0.443, p < 0.01), sapwood length (r = 0.630, p < 0.001), and total core sample length (r = 0.916, p < 0.001). heartwood core length exhibited a significant negative correlation with sapwood length (r = -0.295, p < 0.05) and a significant positive association with total core sample length (r = 0.457, p < 0.01), whereas sapwood length was strongly and significantly correlated with total core sample length (r = 0.710, p < 0.001) (figure 7). these results underscore the role of clear bole height as an inverse determinant of branching, with its strongest relationship being the negative correlation with the number of forking stems. average gbh emerged as the most integrative trait, showing consistently strong and highly significant associations with both wood anatomical traits and core sample length, highlighting its utility as a key indicator for overall growth performance in tree improvement and breeding programmes. genetic resources (2025), 6(12), 160–170166 shivaprasad et al ta bl e 3. d es cr ip tiv e st at is tic s of m or ph ol og ic al a nd h ea rt w oo d fo rm at io n tr ai ts in r ed s an de rs co de se ed s ou rc e a cc es si on s pl an te d su rv iv in g ac ce ss io ns a cc es si on s w it h he ar tw oo d fo rm at io n tr ee he ig ht ( m ) cl ea r bo le he ig ht ( m ) n o. o f f or ke d st em s g ir th a t br ea st he ig ht ( cm ) h ea rt w oo d co re le ng th (c m ) sa pw oo dl en gt h (c m ) to ta l c or e sa m pl e le ng th ( cm ) s1 a ho bi lu m n or th , ru dr av ar am (a .p ) 75 .0 0 55 (7 3. 33 % ) 14 (2 5. 45 % ) m ea n 9. 43 1. 87 0. 62 48 .0 1 0. 43 7. 26 7. 76 sd 1. 30 1. 51 1. 06 10 .7 5 0. 83 1. 40 1. 70 m in 5. 70 0. 00 0. 00 27 .6 7 0. 00 4. 60 4. 60 m ax 11 .7 0 6. 20 4. 00 73 .0 0 3. 30 10 .2 0 11 .6 0 s2 k. v bh av i k od ur (a .p ) 10 0. 00 82 (8 2% ) 4 (4 .8 8% ) m ea n 8. 69 1. 13 1. 54 43 .9 6 0. 11 7. 32 7. 42 sd 1. 21 1. 47 1. 47 8. 74 0. 53 1. 39 1. 52 m in w 0. 00 0. 00 27 .0 0 0. 00 4. 20 4. 20 m ax 11 .8 0 4. 80 5. 00 78 .0 0 4. 00 11 .5 0 12 .5 0 s3 re d w oo d pa rk (a .p ) 10 0. 00 79 (7 9% ) 16 (2 0. 25 % ) m ea n 8. 90 1. 04 1. 53 45 .7 4 0. 46 7. 22 7. 68 sd 1. 15 1. 42 1. 44 9. 80 1. 06 1. 34 1. 58 m in 6. 20 0. 00 0. 00 26 .0 0 0. 00 4. 00 4. 00 m ax 11 .5 0 5. 00 5. 00 74 .0 0 5. 00 10 .5 0 11 .8 0 s4 m ad ir ed di pa lly , o ni pe nt a (a .p ) 10 0. 00 62 (6 2% ) 9 (1 4. 52 % ) m ea n 8. 40 1. 24 1. 11 43 .5 6 0. 35 6. 88 7. 23 sd 1. 29 1. 14 1. 39 8. 35 0. 95 1. 23 1. 32 m in 5. 50 0. 00 0. 00 26 .5 0 0. 00 4. 70 4. 70 m ax 11 .5 0 3. 10 4. 00 63 .0 0 4. 30 9. 70 10 .0 0 s5 pe tb as he er ab ad , h yd er ab ad ( t. s) 10 0. 00 97 (9 7% ) 29 (2 9. 90 % ) m ea n 9. 40 0. 48 2. 06 44 .4 6 0. 88 6. 85 7. 74 sd 1. 01 0. 90 1. 38 9. 75 1. 50 1. 51 1. 46 m in 6. 80 0. 00 0. 00 27 .0 0 0. 00 3. 60 4. 60 m ax 11 .7 0 3. 40 5. 00 76 .0 0 6. 60 10 .8 0 12 .0 0 s6 g um ud ip oo nd i (t .n ) 10 .0 0 10 (1 00 % ) 1 (1 0% ) m ea n 9. 97 1. 04 1. 30 47 .7 3 0. 52 7. 63 8. 15 sd 0. 55 1. 12 1. 49 6. 17 1. 64 1. 45 0. 78 m in 8. 60 0. 00 0. 00 38 .7 5 0. 00 4. 00 7. 10 m ax 10 .5 0 2. 40 4. 00 58 .0 0 5. 20 8. 90 9. 20 s7 n ay ap ak am (t .n ) 10 .0 0 10 (1 00 % ) 1 (1 0% ) m ea n 9. 73 0. 74 1. 50 46 .1 0 0. 55 7. 07 7. 62 sd 0. 62 0. 99 1. 35 8. 58 1. 74 1. 50 1. 37 m in 8. 90 0. 00 0. 00 33 .0 0 0. 00 4. 30 5. 00 m ax 10 .6 0 2. 50 3. 00 62 .0 0 5. 50 8. 90 9. 80 s8 a m ir th i ( t. n ) 5. 00 5 (1 00 % ) m ea n 10 .2 4 0. 00 3. 40 37 .9 3 0. 00 7. 14 7. 14 sd 0. 30 0. 00 1. 52 6. 91 0. 00 0. 97 0. 97 m in 9. 80 0. 00 2. 00 26 .0 0 0. 00 5. 70 5. 70 m ax 10 .5 0 0. 00 6. 00 44 .0 0 0. 00 8. 40 8. 40 genetic resources (2025), 6(12), 160–170 red sanders germplasm evaluation 167 principal component analysis among various traits in red sanders pca identified seven principal components (pcs) summarizing trait variance, with pc1 and pc2 explaining 40.85% and 28.86% of the variance, respectively, capturing a cumulative 69.71% of the total variation. pc3 contributed 18.20%, while pc4 explained 8.69%, and the remaining components (pc5, pc6 and pc7) collectively accounted for less than 4% of the variance (supplemental table 2). these results indicate that the first three components encapsulate most of the trait variation, underscoring the importance of total core sample length, gbh, forking, heartwood core length and sapwood core length in defining diversity. to determine the most influential traits for each principal component, a figure 6. bar plot representing mean and sd (standard deviation)  for growth-related traits in red sanders seed sources loading threshold of 0.3 was applied. traits exceeding this threshold were considered significant contributors to the observed variation. pc1 was primarily influenced by total core sample length (0.58), average gbh (0.55) and sapwood length (0.42), indicating their role in explaining the primary axis of variation. pc2 was dominated by clear bole height (-0.63) and the number of forked stems (0.66), suggesting that this axis captures trait variation driven by contrasting growth patterns. pc3 was significantly influenced by heartwood core length (0.78) and inversely by sapwood length (-0.60), reflecting their negative correlation (supplemental table 3). clustering analysis using the silhouette method in nbclust identified three optimal clusters. k-means clustering further revealed distinct groups: cluster 1 comprised trees with shorter tree height, higher clear bole height, and minimal genetic resources (2025), 6(12), 160–170168 shivaprasad et al forking; cluster 2 included taller trees with moderate clear bole height, higher gbh and moderate branching; while cluster 3 consisted of trees with significantly more forked stems and negligible clear bole height. the pca biplot further supported these clustering results. in the biplot, the black vectors represent the original quantitative traits, where the length and direction of each vector indicate the strength and direction of each trait’s contribution to the principal components. the coloured points correspond to individual accessions grouped into the three clusters, while the elliptical circles represent the 68% confidence limits for each cluster. the distinct separation of clusters along pc1 and pc2 axes reflects trait-based differentiation among populations. these findings highlight the genetic and phenotypic diversity within the seed sources and provide insights for selecting superior accessions for tree improvement programmes (figure 8). figure 7. correlation plot for various traits in red sanders. the circles represent the magnitude and direction of the pearson correlation coefficients between trait pairs. the color scale shows correlation values, where blue indicates positive correlations and red indicates negative correlations. the intensity of the color and the size of the circles correspond to the strength of the correlation. figure 8. principal component analysis biplot with clusters for various traits in red sanders. in the biplot, the black vectors represent the original traits, where the length and direction of each vector indicate the magnitude and direction of each trait’s contribution to the principal components. the coloured circles (ellipses) represent the 68% confidence intervals for the clusters, while the colours of the points correspond to the three identified groups of accessions. the separation of clusters along pc1 and pc2 reflects trait-based differentiation among populations. genetic resources (2025), 6(12), 160–170 red sanders germplasm evaluation 169 discussion the morphological and heartwood formation evaluation of red sanders germplasm provides valuable insights into the genetic variability and potential of this species for breeding and conservation. the high survival rate (80%) observed in the germplasm bank demonstrates the adaptability and resilience of red sanders to environments beyond its limited natural range, which is a crucial factor for its sustainable utilization in forestry programmes. diversity in growth morphological traits and heartwood formation parameters the observed variability in growth traits, such as tree height, gbh and clear bole height, reflects significant genetic diversity. the tallest accession, s2r4-10 (11.8m), and the shortest, s4r1-7 (5.5m), along with the wide range in gbh from 26.00cm to 78.00cm, demonstrate the population's growth potential. these findings align with the natural red sanders populations in the rajampet forest division, where tree heights range from 7.55m to 13.11m and gbh varies from 22.30cm to 81.82cm (senthilkumar et al, 2015). such diversity provides a valuable resource for selecting accessions for specific silvicultural and ecological objectives. the average tree height of 9.03m, exceeding the 8.52m average reported for 15–25-year-old red sanders plantations in gujarat (hegde, 2023) highlights the superior performance of certain germplasm lines. however, the lower average gbh (45.00cm) compared to the 61.21cm reported in gujarat's plantations and 49.84cm in a 20-year-old plantation (arun kumar et al, 2017) may be attributed to both genetic and environmental factors. the highest clear bole height, observed in s1r2-13 (6.2m), holds particular importance for timber production, where straight, knot-free logs are preferred. heartwood formation was observed in 18.25% of accessions in this 12-year-old field genebank, contrasting with previous reports that heartwood formation begins at 15 years (hegde, 2023; suresh et al, 2017). heartwood content increases significantly with age, as observed in studies reporting 70% heartwood formation in 20-yearold plantations and up to 97% in 45-year-old plantations (arunkumar, 2011). the occurrence of early heartwood formation in certain accessions highlights the strong genetic influence on this trait. accessions such as s5r1-19 (6.6cm heartwood core length) and s678r4-9 (5.5cm) show potential for breeding programmes aimed at improving heartwood production, enabling higher economic returns in shorter cultivation cycles. heartwood colour classification revealed a predominance of light red variants (54.79%), followed by medium red (26.03%) and deep red (19.18%) colours. this variation is crucial for meeting specific market demands and enhancing the economic value of red sanders plantations. seed source evaluation the significant differences in survival rates and growth traits among seed sources underscore the influence of genetic and environmental factors. superior survival rates in seed sources such as gumudipoondi, nayapakam and amirthi (100% survival) indicate their adaptability to environmental conditions. conversely, the lower survival rates observed in madireddipally (62%) warrant further investigation. the superior performance of accessions from amirthi (tree height of 10.24m) and petbasheerabad (high heartwood formation and survival rates) highlights their potential as elite seed sources for plantation programmes. trait correlations the correlation analysis provided critical insights into trait relationships, guiding breeding and management strategies. the negative correlation between clear bole height and the number of forking stems (-0.796) indicates trade-offs in stem architecture, where selecting for one trait may compromise the other. the strong positive correlations between average gbh and traits such as heartwood core length (0.443), sapwood length (0.630), and total core sample length (0.916) suggest that gbh can serve as an integrative indicator of overall tree performance. these findings align with previous studies reporting similar positive relationships between gbh and heartwood content in 20 and 45-year-old plantations (arun kumar et al, 2017). given the significant influence of age on heartwood development, selecting accessions with early heartwood formation can enhance breeding efficiency. such an approach maximizes genetic variability utilization at younger ages, increasing the likelihood of identifying superior accessions for economic gains. principal component analysis the pca results indicate that pc1 primarily represents growth-related traits, whereas pc2 reflects structural variations such as stem architecture and clear bole height. the clustering further highlights natural groupings among the trees, which could be linked to genetic and environmental factors influencing tree morphology. cluster 3 trees, with excessive branching and forked stems, might be less desirable for commercial timber purposes but could be valuable for conservation and biodiversity. in contrast, cluster 1 trees, which have minimal branching and a high clear bole, might be more suitable for timber production, as fewer knots improve wood quality. cluster 2 represents an intermediate growth pattern, possibly offering a balance between commercial and ecological benefits. this analysis highlights the multifaceted nature of trait variation and provides a robust framework for prioritizing traits in breeding programmes. implications for breeding and conservation the observed diversity in morphological and heartwood formation traits forms a robust foundation for breeding programmes targeting the improvement of red sanders. superior accessions such as s2r4-10 (tree height), s1r2-13 (clear bole height) and s5r1-19 (heartwood core length) should be prioritized in future breeding efforts. integrating morphological, genetic and environmental data will enhance the precision of selection strategies. the identified clusters provide valuable insights for breeding programmes, selection strategies and conservation planning. trees in cluster 1 may be prioritized for commercial plantations, while cluster 3 trees could be conserved for genetic diversity. future studies integrating genomic data with phenotypic clustering could further enhance the understanding of trait inheritance and selection strategies in red sanders breeding programmes. the application of molecular tools such as genome-wide genetic resources (2025), 6(12), 160–170170 shivaprasad et al association studies (gwas) and transcriptomics can further elucidate the genetic basis of key traits, enabling markerassisted selection for accelerated genetic improvement. from a conservation perspective, maintaining the genetic diversity observed in the evaluated germplasm is crucial to ensure the long-term sustainability and productivity of red sanders plantations. supplemental data supplemental table 1. morphological and heartwood formation traits data of red sanders accessions supplemental table 2. summary of principal components showing eigenvalues and percentage of variance explained supplemental table 3. principal component loadings of morphological and heartwood formation traits showing the contribution of each trait to the respective components in red sanders accessions acknowledgments the authors thank the director, icfre-institute of forest biodiversity, hyderabad, for his support. additionally, we sincerely acknowledge the technical assistance provided by mr sardar and mr b. suresh kumar. author contributions swapnendu pattanaik and kumbarahally murthigowda shivaprasad contributed to the study conception and design. material preparation, data collection and analysis were performed by kumbarahally murthigowda shivaprasad, pendela surath kumar, avula kishore and s. dinesh kumar. the first draft of the manuscript was written by kumbarahally murthigowda shivaprasad and swapnendu pattanaik and all authors commented on previous versions of the manuscript. all authors read and approved the final manuscript. conflict of interest statement the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential competing of interest. ethics statement this study did not involve any human participants or animals. the research was conducted on cultivated germplasm of pterocarpus santalinus maintained at the icfre–institute of forest biodiversity, hyderabad, india. no field sampling from wild populations was carried out, and no collection of plant material from protected or endangered habitats was involved. therefore, no specific permits or ethical committee approvals were required to conduct this research. references ahmed, m., nayar, m. 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(2017). variation in heartwood formation and wood density in plantation-grown red sanders (pterocarpus santalinus). in wood is good: current trends and future prospects in wood utilization. springer, pp. 139-151. doi: https://doi. org/10.1007/978-981-10-3115-1_14 https://www.genresj.org/index.php/grj/article/view/genresj.unba8473/suppdata303 https://www.genresj.org/index.php/grj/article/view/genresj.unba8473/suppdata303 https://www.genresj.org/index.php/grj/article/view/genresj.unba8473/suppdata303 https://doi.org/10.20324/nelumbo/v26/1984/74871 https://doi.org/10.20324/nelumbo/v26/1984/74871 https://dx.doi.org/10.2305/iucn.uk.2021-1.rlts.t32104a187622484.en https://dx.doi.org/10.2305/iucn.uk.2021-1.rlts.t32104a187622484.en https://doi.org/10.1007/978-981-10-3115-1_12 https://doi.org/10.1007/978-981-10-3115-1_12 https://doi.org/10.3839/jksabc.2011.076 https://doi.org/10.3839/jksabc.2011.076 https://www.r-project.org/ https://doi.org/10.1104/pp.108.126003 https://doi.org/10.1016/s0031-9422(00)88430-7 https://doi.org/10.1016/s0031-9422(00)88430-7 https://doi.org/10.1007/978-981-10-3115-1_14 https://doi.org/10.1007/978-981-10-3115-1_14 _hlk182239332 _hlk182239572 _hlk181960924 _hlk182239903 _hlk190171409 _hlk190777740 _hlk213245418 _hlk213245719 genebank report genetic resources (2025), (s2), 162–184 doi: 10.46265/genresj.augz3618 https://www.genresj.org issn: 2708-3764 history and future of industrial crop accessions preserved by crea-ci in bologna and rovigo, italy ilaria alberti a, manuela bagatta b, andrea del gatto c, massimo montanari b, daniela pacifico b and andrea carboni *,b a consiglio per la ricerca in agricoltura e l’economia agraria – centro di ricerca di cerealicoltura e colture industriali (crea-ci) – sede di rovigo, viale amendola 82, rovigo, 45100, italy b consiglio per la ricerca in agricoltura e l’economia agraria – centro di ricerca di cerealicoltura e colture industriali (crea-ci) – sede di bologna, via di corticella 133, bologna, 40128, italy c consiglio per la ricerca in agricoltura e l’economia agraria – centro di ricerca di cerealicoltura e colture industriali (crea-ci) – azienda sperimentale di osimo, via cagiata 90, osimo, 60027, italy abstract: the conservation and exploitation of industrial crops at the cereal and industrial crop centre of the council for research in agriculture and economics (crea-ci, the bologna and rovigo research centres) date back to the beginning of the 20th century and has led to the development of a germplasm bank containing 2,237 accessions. this collection reflects the multidisciplinary approach to the study of these crops and consists of wild relatives, traditional ecotypes and landraces collected in italy and europe, breeding lines and populations, as well as ancient and modern varieties. the main crops of this collection are sugar beet (beta vulgaris l., 381 accessions), flax (linum usitatissimum l., 283 accessions), hemp (cannabis sativa l., 90 accessions), potato (solanum tuberosum l., 45 accessions), sunflower (helianthus annuus l., 95 accessions), several species of the brassicales order (75 accessions), castor bean (ricinus communis l., 18 accessions) and grain legumes (1,250 accessions). this germplasm is maintained according to international standards; most of the accessions are stored in triple-layer vacuum bags and generally kept in two separate locations, at -20◦ to -25◦c and/or in a cold chamber under low temperature (5◦c) and low humidity, while the potato collection is maintained in vitro. each of these crops has been studied using different approaches, including genetic and genomic studies as well as chemical analyses. this article describes the genesis and the evolution of the collection preserved at crea-ci and how these plant genetic resources are fundamental to facing climate change, and ensuring global food security and environmental sustainability. keywords: agrobiodiversity, plant germplasm, ex situ conservation, industrial crops, genebank citation: alberti, i., bagatta, m., del gatto, a., montanari, m., pacifico, d., carboni, a. (2025). history and future of industrial crop accessions preserved by crea-ci in bologna and rovigo, italy. genetic resources (s2), 162–184. doi: 10.46265/genresj.augz3618. © copyright 2025 the authors. this is an open access article distributed under the terms of the creative commons attribution license (cc by 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. introduction global climate change’s effects on agricultural production are becoming increasingly evident (kumar et al, 2022). in addition to increasing drought and heat stress, factors such as overurbanization, deforestation, habitat destruction and soil depletion are responsible for ∗corresponding author: andrea carboni (andrea.carboni@crea.gov.it) the occurrence of pest and disease populations, creating unfavourable future conditions for agricultural production. in the context of a continuously growing global population, biodiversity conservation becomes a conditio sine qua non for guaranteeing food security and universal access to food – in other words, safeguarding our future (mccouch et al, 2013; fao, 2024). unfortunately, in the past, breeding efforts have focused only on improving a few species and crop traits, received: 06.12.2024 accepted: 20.03.2025 published online: 10.04.2025 https://www.genresj.org https://www.doi.org/10.46265/genresj.augz3618 https://www.genresj.org https://www.doi.org/10.46265/genresj.augz3618 mailto:andrea.carboni@crea.gov.it genetic resources (2025), (s2), 162–184 industrial crops conservation and use at crea-ci, italy 163 leading to genetic bottlenecks and a sharp reduction in crop biodiversity (reynolds and atkin, 2021). the changing environmental scenario poses an urgent need to modify the strategy for developing new plant varieties resistant to climate variations (pixley and cairns, 2023). plant genetic resources (pgr), i.e. the cultivated germplasm, particularly landraces, wild relatives and exotic germplasm, are essential sources of genetic variability, valuable crop traits and foreign alleles that can help mitigate abiotic and biotic stresses and a reduction in agricultural production caused by climate change (maxted et al, 1997; mercer and perales, 2010; lopes et al, 2015). the need for ex situ conservation is, therefore, undeniable, as it ensures that pgr can be utilized in future breeding and genetic improvement programmes. the first to draw attention to the importance of pgr for food security and to the danger of genetic erosion (loss of genetic diversity) was the russian scientist nikolai vavilov. vavilov himself described the importance of the mediterranean region, including italy, as a centre of origin of cultivated plants and biodiversity (vavilov, 1926, 1992). a significant part of the italian crop genetic diversity, accumulated over the centuries, has been lost due to genetic erosion, caused since the 1960s–1970s by the rapid spread of a few species and new and modern varieties. paradoxically, it is not incorrect to say that agriculture itself has been the main cause of the decline in crop genetic diversity (antonelli et al, 2020). this process accelerated enormously with the abandonment of mountainous and marginal areas. crea, the italian research council in agriculture and economics (i.e. the leading italian research organization dedicated to the agri-food supply chains, supervised by the italian ministry of agriculture, food sovereignty, and forestry, masaf), with its 12 different research centres, maintains a huge germplasm collection of 40,186 accessions, including cereals, vegetables, fruits, forages, industrial crops, forests and woody crops and medicinal plants. crea characterizes and preserves these pgr with various conservation strategies: seed banking, tissue culture and arboreta (vaccino et al, 2024). within crea, the research centre for cereals and industrial crops (crea-ci) is the one with the largest collection, with 16,469 accessions, and the number is constantly growing. among crea-ci research centres, the institutes of bologna and rovigo have historically dealt with the characterization and breeding of major and minor industrial crops since their foundation. the bologna and rovigo research centres are closely linked with two renowned italian agronomists, francesco todaro (1864–1950) and ottavio munerati (1875–1949), respectively, who, at the beginning of the last century, were key figures in the advancement of italian agriculture. francesco todaro, professor of agronomy and crop cultivation at the alma mater in bologna, in 1920 was the founder and first director of the istituto per l’allevamento vegetale dei cereali of bologna (institute of plant breeding for cereal cultivation) (regio decreto, 1920); in this research institute, now crea-ci bologna, he continued the characterization and breeding of cereals that had first begun at the royal station of agriculture in modena and after at the university of bologna (https://archiviostorico.unibo.it/system/27 /508/todaro francesco.pdf; felice (2011)). not only cereals, but also forage crops such as alfalfa (medicago sativa l.) and traditional fibre crops such as hemp have been the focus of this institute from the outset. over the decades, the number of agricultural species conserved grew in parallel with the number of researchers working in bologna. interest in grain legumes can be traced back to the 1960s, followed by other species, mainly sunflower, potato, flax, brassicaceae, castor bean, etc. the rovigo beet institute was founded in 1914 by professor ottavio munerati, who had already recognized in 1908 that the substantial contribution of sugar beets to technical, economic and social progress in agriculture required a significant genetic improvement programme, including this species in crop rotations (munerati, 1933). the current headquarters of the institute was built in 1951 with financing from the marshall plan. these funds were granted by american geneticists in recognition of professor munerati’s long-standing partnership with american research institutions (coons et al, 1955). the germplasm collection maintained in bologna and rovigo (table 1) fully reflects the multidisciplinary work of agronomists, breeders, geneticists, chemists, biochemists and phytopathologists who have contributed over more than 100 years of the centres’ history, and it now consists of 2,237 accessions. these are wild relatives, traditional ecotypes and local varieties collected in italy and europe, breeding lines and populations, as well as ancient or modern varieties, many of which developed in our research centres. table 1. crop accessions maintained at crea-ci centres of bologna and rovigo. crop no. of accessions beta spp. 381 brassicales order 75 castor bean 18 flax 283 grain legumes 1,250 hemp 90 potato 45 sunflower 95 total number 2,237 the vast majority of conserved accessions can be consulted within the european search catalogue for plant genetic resources (eurisco) (http://eurisco.ecpgr.or g/, weise et al (2017); kotni et al (2023)), but missing from the database are all breeding materials, pop164 alberti et al genetic resources (2025), (s2), 162–184 ulations, segregating progenies, lines under selection, and all accessions under phytopathological observation because, as far as possible, we try to conserve healthy material from the phytosanitary point of view. considerable effort goes into the conservation of the collection. since each crop species has unique characteristics and requirements, a specialized expert is assigned to oversee each crop. conservation, except for potato and some chemotypes of hemp, is mostly carried out in the form of seeds placed under vacuum-sealed, trilaminate aluminium bags and stored in low-temperature environments: (1) in cold chambers for the largest quantities of seed to be preserved (from 4◦ to 9◦c depending on the species and facilities available, with low humidity; in our cold rooms, over 500g per accession are stored for about five years); (2) in freezers (from -20◦ to -25◦c) for long-term preservation, up to ten years depending on the species and with a weight per sample of about 100 to 300g per accession. seed viability tests are carried out at the moment of storage and on a regular basis to monitor losses in viability during storage. in our experience, each species has different times of seed viability decline, depending on multiple factors. consequently, seed regeneration activities are planned according to the different needs of the various pgr. beta collection the seed collection consists of 381 accessions of the genus beta, stored under low temperature (5◦c) and low relative humidity (<10%) conditions. one hundred ten (110) of these accessions are pollinators, while 271 are ‘hybrids,’ including some crosses of particular interest for genetic and genomic studies between the two interfertile subspecies of the genus beta, vulgaris x maritima. the collection maintains many diploid accessions, 361, compared to only 20 tetraploids. some of the accessions are suited for autumn sowing, and several show resistance to rhizomania, cercospora leaf spot, nematodes, and rhizoctonia spp. (figure 1). pollinators, male sterile lines and o-type lines are well represented and have been the core of the breeding activity of the last years. the rovigo crea-ci collection’s main goal is to preserve the lines resulting from many years of genetic improvement work to develop beta cultivars appropriate for autumn sowing. autumn varieties must have certain specialized traits, such as tolerance to low temperatures and the capacity to survive the potentially harsh climate over the winter. reduced sensitivity to vernalization is another important trait that minimizes bolting when the crop resumes vegetative growth in spring. bolting, the first visible sign of reproductive transition in sugar beet, causes the mobilization of reserve sucrose from the roots, resulting in a loss of sugar content; additionally, the roots of bolted plants are more fibrous and of poor technological quality (biancardi, 1999). therefore, selection must be accurate, using more effective methods, including inbreeding. however, bolting resistance should not be pushed to excessively high levels to avoid depressing sucrose production and causing problems in seed multiplication. among the accessions, 8 tetraploid pollinators and 11 hybrids show identified traits of bolting resistance, with varying levels of productivity. another very important trait for sugar beet, well represented in the crea-ci collection, is resistance to rhizomania. this disease is caused by the beet necrotic yellow vein virus (bnyvv) transmitted and inoculated into the roots by the fungus polymyxa betae. selection has achieved significant milestones in the last 40 years, allowing high protection of crops with the use of resistant varieties. the first source of resistance to rhizomania was found at the end of the 1960s in one accession of italian origin, which also showed good resistance to cercospora bieticola. starting from 1977, using germplasm preserved in the rovigo beta collection, mass selections were carried out on various monogerm male-sterile (cms) lines and their maintainers (o-type), and in 1988 a pollinator (ro401) was released and subsequently exploited to create several commercial varieties. a recent study of b. vulgaris subsp. maritima populations, which ottavio munerati began collecting in the 1920s, discovered a significant association between hybrids vulgaris x maritima and resistance to rhizomania (biancardi et al, 2012). the b. vulgaris collection preserved at crea-ci, considering accessions with rhizomania resistance, consists of 44 tetraploid pollinators and 191 hybrids. the disease caused by the fungus c. bieticola is certainly today the main factor of productive and qualitative losses for beet cultivation in italy and worldwide. this fungus causes characteristic necrotic spots on the foliage, leading to rapid desiccation. the precise start date and basis of the selection for cercospora leaf spot disease at the rovigo research institute are unknown. although professor munerati left behind some important publications, much of the knowledge from his notes and field annotations was lost during world war ii; however, it is certain that by 1925 he had available disease-resistant lines whose seeds he made available for experimental trials in the usa (biancardi et al, 2012). the origin of this resistance is probably to be found in a progeny derived from crosses with the wild beet b. vulgaris subsp. maritima, which grew and still grows spontaneously along the po di levante embankment. subsequently, he started a breeding programme to eliminate some negative characters of b. vulgaris subsp. maritima, such as shallow root and tendency to annuity. given the state of knowledge at the time, it was a challenging work, but munerati managed to generate cultivable lines, albeit late-season, with increased sugar content and resistance to cercospora, drought, and curly top disease. genetic resources (2025), (s2), 162–184 165 figure 1. resistance traits distribution in the beta crea-ci collection of rovigo (beta vulgaris subsp. vulgaris x maritima) his results were so interesting that with the seeds sent to the usa, it was possible to improve yields in california, where curly top disease was rampant, as well as in colorado and michigan. notably, in 1946, the professor downplayed these results attributed to him, saying it was only a “modest contribution,” while after 90 years we now know that this was the only existing contribution to cercospora resistance. only in 2000, biancardi, a former director of the rovigo centre, and other researchers showed that resistance to cercospora is polygenic, relying on at least 4-5 gene pairs with effects that vary according to the level of infection (koch and jung, 2000; skaracis and biancardi, 2000). the crea-ci collection also includes, in addition to 17 tetraploid pollinators and 16 resistant hybrids, a certain number of accessions (20 pollinators and 12 hybrids) that combine rhizomania resistance with cercospora tolerance; these accessions are of particular interest in the perspective of ‘pyramiding’, i.e. stacking agronomically important genes in a single beet crop. to conclude this brief overview of the beta germplasm preserved at crea-ci in rovigo, it should also be noted the presence of other accessions that combine multiple resistance traits, particularly rhizomania resistance with nematode resistance (8 pollinators) and rhizoctonia resistance (2 pollinators and 18 hybrids). in recent years, efforts have been focused on pollinator seed reproduction with resistance to c. bieticola and on collecting wild material from the po delta area (figure 2). breeding activities are also underway, with a particular focus on developing hybrids resistant to water stress and heat waves that have characterized recent years. brassicales collection for almost 30 years, crea-ci in bologna has been conducting applied research on plants of the brassicaceae family which belongs to the brassicales order, characterized by the presence in the plant tissues of the glucosinolate-myosinase system, an effective defense strategy against many pathogens and insect pests (liu et al, 2021). glucosinolates are specific secondary metabolites which, after a pathogen attack, are hydrolyzed by the endogenous myrosinase enzyme and release breakdown products, among which are the isothiocyanates (itcs) with biocidal effects (lazzeri et al, 2004). there are over 140 glucosinolates identified (blažević et al, 2020), with different profiles distinguishing genera and species of brassicaceae (agerbirk et al, 2021), a rich source of biodiversity, distributed worldwide with about 372 genera and 4,000 species. the biofumigation technique, an environmentally friendly alternative to chemical fumigants, was developed by examining different brassicaceae species for their itcs biocidal properties as green manures in field applications (lazzeri et al, 2003; d’avino et al, 2004), displaying other environmental benefits, such as soil fertilization and biostimulant properties (lazzeri et al, 2013). furthermore, brassicaceae seeds are characterized by an oil content ranging from 10% to 45% of their dry mass and by a variable fatty acid composition providing tribological features for lipochemistry formulations (moser and vaughn, 2012). a seedbank was established over 20 years ago to conserve the germplasm of cultivated and wild industrial crops conservation and use at crea-ci, italy 166 alberti et al genetic resources (2025), (s2), 162–184 figure 2. a, collecting beta vulgaris subsp maritima on the po river delta; b, a b. vulgaris subsp. maritima plant. species, mainly of non-food brassicaceae, provided by germplasm banks or by seed companies in order to identify new plants with high-value green chemicals to be studied by agronomists and chemists. the collection currently includes 84 accessions. the species of the collection were characterized by evaluating: (1) their adaptability to cultivation in central-north italy, where our experimental fields are located, selecting for high biomass yield and hardiness; (2) their seed glucosinolate profile and content according to the eu official iso 9167-1 method, as described in the eu commission regulation no 1864/90 (ec, 1990), and based on the hplc (highperformance liquid chromatography) analysis; (3) their seed oil content and fatty acid composition, determined according to conte et al (1989); angelini et al (2015). each accession was duplicated at least once every five years to regenerate the seeds. to date, the brassicales collection includes 73 species of brassicaceae, 1 species of cleomaceae, and 3 species of resedaceae (see supplemental table 1 for a list of species). within brassicaceae, 53 are wild species; the remaining are mainly of brassica genus, currently cultivated and selected mostly for their high biomass yield, hardiness and specific glucosinolate content in seeds or epigeal tissues, to be used as biofumigant green manures (brassica juncea (l.) czern.) or as biofumigant meals and pellets with fertilizing and amendment properties (brassica carinata a. braun and brassica nigra (l.) w.d.j. koch) (lazzeri et al, 2013). the crea-ci collection contains varieties registered in the italian variety catalogue such as brassica carinata ‘isci 7’, brassica juncea ‘isci 99’ and ‘isci 20’, brassica juncea ‘isci100red’ and eruca sativa (l.) ‘cav. nemat’, included as components in patented biofumigant pellets, liquid foliar and root treatments (figure 3). an interspecific variation of the glucosinolate profiles among the species of our collection has been found, allowing us to characterize and identify most of them (agerbirk et al, 2021). the seed fatty acid analysis showed that in more than half of the species, primarily in the cultivated ones, a monounsaturated fatty acid is predominant, above all the erucic acid (c22:1), while in most of the wild species a polyunsaturated fatty acid, the alpha-linolenic acid (c18:3) is the most abundant (see supplemental table 1) (lazzeri et al, 2013). many species of our collection are potential multifunctional plants, exhibiting different functions often exploited in the past. some like barbarea verna (mill.) asch., diplotaxis erucoides (l.) dc., raphanus raphanistrum l., and rapistrum rugosum (l.) all. have traditionally been locally consumed as edible plants. others, such as barbarea vulgaris w.t. aiton, brassica montana pourr., diplotaxis muralis (l.) dc., diplotaxis tenuifolia (l.) dc., hesperis matronalis l., and sinapis arvensis l. are not only edible but have also been used as officinal plants (figure 4a and b). beyond their officinal values, isatis tinctoria l. and reseda luteola l. have been considered dyeing plants since mediaeval times (figure 4c). considering that many brassicaceae are melliferous (filipiak, 2024), during their field cultivation and characterization, we observed that some of them were selectively attractive to pollinators. our further interest was to obtain a preliminary visual estimate of the attractiveness of these species to honeybees and wild pollinators to identify the most visited. genetic resources (2025), (s2), 162–184 167 figure 3. a, inflorescences of brassica juncea ‘isci 100red’; b, brassica juncea ‘isci 99’ at full flowering; c, brassica juncea ‘isci 20’; d, flowers of eruca sativa ‘nemat’. figure 4. a, wild species of the collection (brassicaceae), at different flowering times during their field reproduction; b, sinapis arvensis l. (brassicaceae) at full flowering, a wild and indigenous species traditionally referred to as edible and officinal, melliferous and very attractive to pollinators; c, reseda luteola l. (resedaceae), at full flowering, a dyeing and officinal indigenous plant, a good melliferous species, attractive mainly to honeybees; d, inflorescences of reseda lutea l. (resedaceae) at full flowering visited by a honeybee. industrial crops conservation and use at crea-ci, italy 168 alberti et al genetic resources (2025), (s2), 162–184 we then distinguished them for their different flowering times to hypothesize a long-term supply of food resources. we focused on reseda lutea l., a rustic wild species from the resedaceae family, well adapted to extreme climatic conditions (figure 4d), and attractive to different pollinators. both these properties are fundamental for including a species in agroecological practices. the collected data about the diversity of glucosinolate and fatty acid content and profile indicate the great potential of brassicales germplasm to be used in more sustainable practices in agricultural systems offering a variety of environmental benefits, ranging from crop protection through biofumigation to increased soil fertility and agroecosystem resilience. flax collection since 1988 (eec directive 1272/88, ec (1988)), flax/linseed (linum usitatissimum l.) cultivation was favoured as an alternative crop introduced to face the eu deficit of oil for non-food uses (zanetti et al, 2013). since 1989, field research at the former experimental institute for industrial crops in bologna, now crea-ci, has evaluated agronomic practices for the reintroduction of this crop (cremaschi et al, 1995), while a flax and linseed germplasm collection has been established under the fao-funded ‘risorse genetiche vegetali [plant genetic resources]’ rgv programme (vaccino et al, 2024). the current germplasm collection comprises 283 accessions with worldwide origins (figure 5). given that flax/linseed breeding had been suspended in italy for decades, the evaluation of the available genetic materials was a key priority in identifying cultivars adapted to the italian climate. traditionally grown in autumn in southern italy until the middle of the last century, linseed has regained popularity also as a functional food due to its oil figure 5. the crea-ci collection includes the following accessions: 109 flax, 96 linseed, mostly ecotypes from southern italy, 68 unknown with uncertain suitability to the fibre or oil production and 10 designated as dual-purpose intended for both final outputs. and oil-derived products, opening up new commercial prospects. we therefore steered the research from flax genotypes towards linseed accessions and their seed oil content and fatty acid characterization. the seed stock is regenerated at least once every five years when morphological observations, using appropriate descriptors according to community plant variety office technical protocol (cpvo, 2014), and phenological characterizations are regularly scored in order to assess the accession adaptability to the growing environment. the germplasm was enriched by selected crosses between linseed varieties and ecotypes best performing in our area located in central-north italy. systematic observations between 1989 and 2022 resulted in a dataset encompassing up to 252 varieties, possibly the most significant source of knowledge on the adaptation of this crop in italy. this dataset was thoroughly examined, and summary data on oil content and fatty acid composition are shown in figure 6 (fila et al, 2024). the seed average oil content (soc) ranges from 35.4% to 47.9%, with a median value of 40.7%. the polyunsaturated fatty acid composition (pufa, linoleic + linolenic acid) of the seed oil varies between 59.9% and 71.5% with a median of 65.4%. the ratio between polyunsaturated and saturated acids (pufa/sfa) was in the 4.6–8.3 interval with a median of 6.2. high temperatures typically exerted a detrimental influence on seed yield and seed oil content, while the fatty acid composition remained almost unchanged. a higher variability was observed in the response to rainfall, which, depending on the accession, exhibited both positive and negative effects on seed yield and oil content. this variability influenced fatty acids, particularly the monounsaturated fraction, which was predominantly reduced by rainfall. linseed adaptation for autumn planting was studied by comparing south italian ecotypes rich in seed oil content and/or alpha-linolenic fatty acid (omega-3) with winter cultivars (figure 7b,c). autumn sowing, compared to spring sowing, increased seed yield by up to 79.4%, although oil content rose by only 1.6%. while saturated and monounsaturated fractions declined, the polyunsaturated fraction increased by a maximum of 13.1% (fila et al, 2024). one of the tested accessions, considered a spring accession, consistently showed an omega-3 seed content exceeding 60% in autumn sowing (tavarini and angelini, 2016). after a mass selection, it was registered as ‘pepita’ in the italian national variety list, the second cultivar of l. usitatissimum published by an italian breeder (figure 7a) a 3-year field trial compared cultivars and southern italian landraces and also evaluated climatic factors affecting linseed in our environment to identify genotypes suitable for quality-oriented dual-purpose cultivation for both seed/oil production and secondary fibres for the non-textile sector. the collection was effective in genetic resources (2025), (s2), 162–184 169 figure 6. distribution of accessions tested during 1989-2022 period in relation to seed oil content (soc % w/w), polyunsaturated fraction (pufa % w/w), and ratio of polyunsaturated to saturated fraction (pufa/sfa, elaborated from fila et al (2024)). figure 7. a, ‘pepita’, the new cultivar of linum usitatissimum adapted to autumn sowing, with high omega-3 seed content; b and c, different flowering times of several linseed accessions at the crea-ci experimental farm of anzola dell’ emilia (bologna); d, a 3-year field trial conducted in spring to identify accessions suitable for dual-purpose use. flax cultivars, with long stems (upper portion of the image) were compared to linseed southern italian landraces at different stages of stem maturity (lower portion of the image). industrial crops conservation and use at crea-ci, italy 170 alberti et al genetic resources (2025), (s2), 162–184 identifying a group of linseed accessions producing good seed yields (above 2t/ha), with a seed oil content of at least 40% and an alpha-linolenic content above 50% in northern italy, and yielding significant amounts of fibre (0.3–0.44t/ha) and straw (2.5–3.0t/ha) (figure 7d). based on the intended cultivation purpose, this study provides guidance for selecting the best-performing cultivars from the accessions tested (fila et al, 2018). the reintroduction of flax/linseed in italian environments as a low-input crop would be advantageous and appropriate for sustainable agricultural systems. this crop is easy to cultivate, requiring no specialized equipment, minimal water and chemical inputs, and having a short vegetative cycle. data collected suggest that flax may provide interesting outcomes in terms of variety of fibre, oil and fatty acid content, enabling harvest quality to be tailored to the intended use. winter linseed cultivation, traditionally practiced in the south, was demonstrated to be feasible even at the study site in the north, thus expanding options for designing crop rotations and improving yields. sunflower and castor bean collections the crea-ci experimental research unit in osimo, belonging to the bologna research centre and located in the marche region, preserves a large amount of sunflower and castor bean accessions resulting from breeding programmes conducted since the early 1980s. thanks to participation in the rgv/fao programme, in the last decade it was possible to undertake a serious recovery action which is essentially focused on two main objectives: (1) seed regeneration avoiding external pollen contaminations and using staggered sowings to elude the overlapping of flowering dates (figures 8 and 10), and a morpho-phenological characterization of the collection, using descriptors that were specifically implemented according to upov or national register of varieties standards. this multiplication activity began in 2011 with dedicated annual sowing and subsequent chemical analyses to measure oil content and fatty acid composition for both species. for the newly acquired lines, seeds were planted in a controlled environment to assess their phytosanitary status, adaptability and productivity, as well as to record their morpho-phenological characteristics. today, the collection maintains 95 accessions of sunflower and 18 of castor bean. accessions are catalogued and stored in vacuum-sealed trilaminate aluminium bags. bags are then stored in boxes and kept in a cold room at 4◦c to extend the viability of the seed batches produced each year. this helps maintain acceptable germinability for seed batches for 5–8 years. at the same time, long-term storage in a dedicated freezer at -20◦c is carried out. sunflower the starting material for sunflower breeding at crea-ci consisted of russian varieties of the vniimk, peredovik and cerneanka types. after self-fertilization, homogeneous base populations were selected and combinations of hybrids capable of improving yields in traditional sunflower-growing areas were generated (kovacik and skaloud, 1972; fick, 1975). pollinator maintainer lines (b) were selected, with the corresponding cytoplasmic male sterility (a) and other genes for the restoration of pollen fertility (r). since 1996, several f2 populations, extracted from commercial hybrids, have been established annually and used for the selection of new r, b and a lines. this material not only provided a relatively inexpensive source of genetic variability but has facilitated the breeding activity because it was no longer necessary to use parental and related wild materials (delgatto and laureti, 2002; laureti and delgatto, 2004). subsequently, a study of general and specific combining ability (gca and sca) of the breeding lines was carried out (serieys, 1994), to identify the testers for the following selection programmes, in addition to verifying the intrinsic value of experimental hybrids (laureti and delgatto, 2001). some genotypes showed interesting productive performances with shorter biological cycle duration (delgatto and laureti, 1998; laureti and delgatto, 2000; delgatto and laureti, 2002). nine of these genotypes (‘ausonia’, ‘esperia’, ‘kappa’, ‘sigma’, ‘tea’, ‘mito’, ‘gamma’, ‘elly’ and ‘lapo’) were inscribed in the national variety register (pirani et al, 1995) (see supplemental table 2 for in-depth descriptions). in 1997, a breeding programme for high-oleic (ho) varieties was initiated, starting from f3 populations provided by the sustainable agriculture institute of cordoba (spain) and from f2 commercial hybrid populations, with the aim to introgress the ho trait into conventional b and r lines. this allowed the identification of good individuals with interesting specific combinations laureti and delgatto (2001). in 2003, f2 populations were extracted from ho commercial hybrids with good productive performances. after the selection of ho maintainer lines and the corresponding male sterility delgatto and laureti (2006), some lines, suitable for establishing valid hybrid combinations and used as testers in future evaluations, were identified (delgatto and laureti, 2005; delgatto et al, 2005b,a). this breeding activity released hundreds of experimental hybrid combinations, and a programme was carried out in several localities to verify their agronomic value (delgatto and laureti, 2005). in 2004 one of these ho hybrid, ‘crono’, was registered in the national register of varieties (see supplemental table 3 for an in-depth description). the collection subsequently was enriched with differential lines with varying resistance to plasmopara halstedii: 9 of hungarian origin and 12 provided by the us department of agriculture-agricultural research service (usda) northern crop science lab in fargo. these accessions clearly distinguish the different races of the pathogen and provide a more comprehensive understanding of its spread in italy. genetic resources (2025), (s2), 162–184 171 figure 8. sunflower corollas of 25 different accessions of the crea-ci osimo collection. regarding the most recent activity, 91 sunflower lines were described, each accompanied by a significant photographic record, including images of the entire plant and close-ups of the inflorescence, as partially shown in figure 8. for each line, 23 descriptors were recorded on leaf, flower, seed, plant architecture, etc. (for a complete list of descriptors and results, see supplemental tables 4, 5, 6). in addition, analytical tests were carried out to: (1) measure oil content on dry matter using the nuclear magnetic resonance (nmr) method, and (2) create an acid profile of the extracted oil using a gas chromatographic analysis of the methyl esters of the fatty acids in the achene (supplemental table 7). a description of these analyses on a sub-sample of 53 accessions can be observed in figure 9. the collection shows considerable variability in terms of biological cycle length, overall and in its sub-phases, plant height, flowering and maturation times, as well as seed and oil production. in particular, the variability in oil content expression in the achene is quite remarkable, with frequent high values for the species, contrary to what might be expected from inbred lines. the fatty acid content also shows significant variability, demonstrating interesting potential for future applications. castor bean the starting material for breeding at crea-ci on castor bean is of us (‘mc nair 506’, ‘pacific’, ‘hale’, ‘dale’, ‘lynn’, ‘cnesl’), israeli (‘h22’) and french origin (‘hd 912’, ‘h531’, ‘hd 913’, ‘pronto’). all varieties introduced from abroad have shown an excessively long vegetative cycle in our climatic conditions. therefore, it was necessary to reduce the life cycle duration and select crops suitable for mechanized agriculture (laureti, 1981, 1982). an ideotype appropriate for the italian and european growing conditions was identified, with good productivity (more than 3.0t/ha) and, at the same time, a reduced size of the plant (about 1m). the development of hybrid cultivars required the selection of male-sterile (gynoecious) lines, and, over the years, 15 gynoecious lines were selected. at the same time, pollinator lines that would combine well with females were identified. applying the general and specific combining ability, it was possible to identify about 50 monoecious inbred lines adapted to the italian area and used to obtain experimental hybrid combinations (laureti, 1987). since 1985, several experimental hybrids have been created and undergone agronomic evaluation in repeated trials over several years in both dry and irrigated environments (laureti, 1995). after an initial varietal comparison, two high hybrids, ‘castore’ and ‘polluce’, were released, both with excellent productivity and earliness traits, and two other low hybrids, ‘riscio’ and ‘negus’, were identified (supplemental table 8) (laureti, 1998, 2002). in 2023, the collection was enriched with 17 lines from the usda germplasm bank (figure 10). industrial crops conservation and use at crea-ci, italy 172 alberti et al genetic resources (2025), (s2), 162–184 figure 9. oil content (% dry matter) and fatty acid composition (%) in 53 accessions of the osimo crea-ci collection. figure 10. characterization and regeneration of castor bean accessions at crea-ci osimo unit. genetic resources (2025), (s2), 162–184 173 a photographic portfolio was also established to highlight the key traits of the plant’s habitus and inflorescence. additionally, the material was characterized based on 20 morpho-phenological specific descriptors (supplemental table 9). further studies would be interesting for determining the fatty acid content of the seed, an aspect that has not yet been explored. hemp collection although some agronomic trials were carried out in bologna before world war ii, the establishment of the cannabis germplasm collection, currently maintained both in bologna and rovigo, began in the mid-1950s. in 1953, the consorzio nazionale produttori canapa (national hemp producers consortium) was established in bologna at the istituto di allevamento vegetale, now crea-ci. in those years, several study missions were conducted across europe, facilitating a rich exchange of plant material. initially sourced from germany, then from many other european countries, this exchange gave rise to the first nucleus of our collection (ranalli and casarini, 1998). the need to establish a collection of accessions from various origins and with wide genetic variability was prompted by the need to enrich the national varietal landscape, increase production performance and counterbalance the loss of hempgrowing areas. hemp is a naturally dioecious plant with predominantly anemophilous pollination. the traditional italian varieties selected and released in bologna have great intra-varietal genetic variability and can, therefore, be considered as populations (allavena, 1961; barbieri and tedeschi, 1968). the establishment of new improved varieties was not sufficient to reverse the decline of the crop, which was mostly driven by competition with cotton and synthetic fibres. the entire hemp supply chain almost disappeared during the 1970s and 1980s due to commercial and productive disinterest. however, the situation changed almost suddenly in the 1990s when renewed attention to the countless potential of this plant was observed, first in europe and then in italy (dicandilo et al, 2003). the research highlighted the new and different products derived from all components of the plant (stem, flower, seed) and their use in the most disparate production chains (pharmacological, automotive, food, and green building), thus bringing hemp back to the attention of the economic world not only as a fibre crop but as a multi-product plant that agronomically has interesting peculiarities due also to the low production inputs that its cultivation requires. collecting missions in various continents, as well as the exchange and purchase of seed from other european countries, where the study of this species had never been interrupted, revitalized the collection and reactivated subsequent studies and experimental activities (faeti et al, 1996; forapani et al, 2001; mandolino et al, 2002). at the time of writing, the cannabis sativa collection contains 90 accessions, both dioecious and monoecious, with different and multiple uses and genetically distinct chemotypes. this extraordinary ability to generate different uses of this species has led our research institute to activate two specific lines of research. one aims to characterize the germplasm for the production of terpenic substances for cosmetic, recreational and pharmaceutical uses (pacifico et al, 2008; grassi and partland, 2017; pieracci et al, 2021; menga et al, 2022), while the second line of research focuses on the characterization and development of hemp varieties for fibre/biomass or seed production in different italian production areas (dicandilo and liberato, 2002; ranalli and venturi, 2004). almost all of the materials in the collection are accessions from the latter industrial exploitation, and they are regenerated with funds from the ongoing rgv fao 2023/2025 programme (vaccino et al, 2024). the research on new germplasm material, along with its agronomic and chemical characterization, complements the activities undertaken. the major requirement in the management of a collection of allogamous species such as hemp is great attention during the regeneration process to avoid any possible contamination by external pollen. to achieve this goal, different isolation strategies are used: (1) physical barriers when the genetic variability of the accession is restricted (figure 11), i.e. the number of individuals of each population/variety is limited; (2) when seed regeneration is in very large fields, according to italian regulations (directive 2002/57/ce, ec (2002)) a distance of several kilometres between the different multiplications is required as the pollen of this species is very light and can fly over distances of up to a few kilometres. as breeders of various varieties registered in the eu plant catalogue, we cannot forget the obligation to carry out a conservative selection according to different distinctive bio-morphological parameters during multiplication. furthermore, molecular and/or chemical analyses are necessary to control the chemotype of the reproduced accession. since the 1990s, several varieties have been established for different industrial uses (see table 2). the latest born is the dioecious variety ‘felsinea’, which has recently been included in the ue catalogue of varieties (figure 12). this cultivar was selected from a historical accession and has two very important characters – high fibre/biomass production and a block in the pathway of cannabinoids synthesis. this feature allows the accumulation of the cannabigerol (cbg) cannabinoid, precursor to the synthesis of the two most common cannabidiol (cbd) and tetrahydrocannabinol (thc), thus reducing their synthesis. several of our improved cultivars/lines selected for pharmaceutical use are also characterized by different blocks during cannabinoid synthesis, and for this reason, they must undergo the most stringent control tests during their multiplication. industrial crops conservation and use at crea-ci, italy 174 alberti et al genetic resources (2025), (s2), 162–184 figure 11. small isolators used in the reproduction of very low genetic variety hemp lines. table 2. hemp cultivars selected by crea-ci and included in the european plant variety portal (eupvp) cultivar name sexual determination use carmagnola dioicious fibre/biomass cs dioicious fibre/biomass fibranova dioicious fibre/biomass fibrante (ex red petiole) dioicious fibre/biomass asso dioicious fibre/biomass codimono monoecious seeds/biomass carmaleonte monoecious seeds/biomass eletta campana dioicious fibre/biomass felsinea dioicious fibre/biomass figure 12. the new hemp cultivar ‘felsinea’. genetic resources (2025), (s2), 162–184 175 potato collection the crea-ci bologna has a potato (solanum tuberosum l.) collection maintained through in vitro culture. the conservation of potato germplasm has a primary goal – the maintenance of the health status of the propagating material, guaranteed by micropropagation of potato plantlets. for these reasons, for several decades, crea has been preserving and enriching the in vitro collection with traditional varieties, local ecotypes, clonal selections and wild species, although complete phenotypic characterization has not always been possible. currently, the collection consists of 45 potato genotypes (table 3). each genotype is preserved in quadruplicate in two climate-controlled rooms. over the years, the potato collection has provided also the basis for genetic improvement programmes with cross-breeding and field trials, obtaining new materials and varieties registered in the national variety register (rnv), characterized by the main useful traits for italian potato cultivation: early maturity, adaptability to southern environments and fresh consumption, prolonged dormancy, low tendency to sweetness, adaptability to industrial processing, and resistance to pests, among others. as a result of the breeding activity, in the last ten years, the bologna center has registered ten potato varieties in the rnv. a key role of the collection is also the preservation of recovering ecotypes from typical and marginal cultivation areas (parisi et al, 2022). often grown at high altitudes, italian potato ecotypes represent not only an important genetic pool to be preserved from the erosion typical of recent decades but also an economic opportunity for mountain communities. indeed, in the last 30 years, the establishment and spread of modern varieties have led to the loss of many potato ecotypes. since most ecotypes still cultivated locally are often multiplied without repeated virus-free plant purification processes, they exhibit viral deterioration with multiple infections, reduced plant vigour and low productivity. we recovered tubers from potato ecotypes in different regions of italy and conducted serological and molecular tests to determine the putative presence of phytoviruses (potato virus y (pvy), potato virus x (pvx), potato leafroll virus (plrv)). virus cleaning for ecotypes multiplied for decades on farm is a mandatory step before the insertion of these ecotypes in in vitro free stock plantlets collection. if the ecotypes test positive, a sanitation process must be conducted, which involves the extraction, insertion and maintenance of in vitro plants in the collection. these are subjected to meristem culture treatments and, if necessary, chemotherapy with ribavirin, regeneration, diagnostic checks and repeated treatments until complete sanitation is achieved. after regeneration, greenhouse acclimatization and tuberization, it is possible to produce healthy material. it is important to consider that, since the sanitation process can vary significantly in terms of duration depending on the quantity and quality of viruses detected in the serological investigation, the subsequent deployment of healthy tubers can take place from a few months to several years after the sanisation activity. in the last decade, crea-ci bologna contributed to the rediscovery, sanitation and multiplication of ten traditional ecotypes, including ‘bianca di starleggia’ and ‘rossa di starleggia’ (lombardy), ‘formazza’ (piedmont), ‘roti oigje’ (veneto), and ‘crispa di gavoi’ (sardinia). ‘ricciona’ (campania) was registered in 2012 in the national register of conservation varieties (rnvc), becoming the first italian potato ecotype to be listed in this specific register, aimed at regulating the reintroduction of local plant germplasm in the areas of origin, and its commercialization has also begun by the o.p. campania patate consortium. the genetic profiles of 27 local italian potato varieties, including those preserved in the crea-ci potato collection, were determined. their simple sequence repeat (ssr) profiles were compared among them, and with over 2,000 varieties belonging to eu common catalogue and sasa (science and advice for scottish agriculture) collection. using 12 ssr markers we were able to discriminate all varieties, excluding known mutants (e.g. cultivars ‘cara’ and ‘red cara’). indeed, it is necessary to distinguish ecotypes unambiguously from the most used varieties, such as ‘kennebec’, ‘vitellotte’ and ‘desiree’ in order to promote them properly and ensure their traceability (mandolino et al, 2015). the potato collection also includes a huge variability in the composition and concentration (pacifico et al, 2024) of secondary metabolites (mainly steroidal glycoalkaloids and phenols). these metabolites have been shown to play a role in increasing plant ability to cope with environmental challenges, due to their biocide activity reported on insects, bacteria and fungi. they are also associated with health-promoting features, serving as nutraceuticals and pharmaceuticals (calcio-gaudino et al, 2020), as well as additives for improving the shelflife of fresh-cut fruits (venturi and bartolini, 2019). among the genotypes collected in vitro, the solanum tuberosum x solanum berthaultii advanced hybrid line, ‘q115’, resulted particularly interesting as a putative source of genetic determinants of resistance to biotic stress, reduced or altered from the domestication. thirteen advanced 4x-breeding clones derived from ‘q 1156’ and ‘bionica’ crossing have been obtained at crea-ci bologna and some of those showed a good range of ptm (potato tuber moth, phthorimaea operculella zeller) resistance. this resistance was measured as mortality during the early stages of larval development due to their skin content in caffeic acid and α-chaconine (pacifico and musmeci, 2019). recently, from the biochemical characterization of peels of five potato genotypes present in the collection, we have demonstrated the great potential in the reuse or recovery of potato peel waste (ppw) from the agroindustrial potato processing. raw extracts from the peel of ‘lady claire’, a processing variety conserved in the crea-ci collection, proved to be the most suitable as a fungicide against fungal pathogens of cereals industrial crops conservation and use at crea-ci, italy 176 alberti et al genetic resources (2025), (s2), 162–184 table 3. overview of the potato collection at the crea-ci bologna accessions italian origin crea-ci selections anthocyanin rich carotenoids rich potato tuber moth tolerance 18 varieties 9 5 4 4 12 clones 12 12 2 2 2 15 ecotypes 15 45 36 17 6 6 2 figure 13. tubers of varieties and clones with different levels, distributions and types of anthocyanin content of skin and flesh. (fusarium graminearum and fusarium verticillioides). its effectiveness is mainly due to the activity of the phenolic fraction, which inhibited the tested fungi by up to 30% (pacifico et al, 2024). during the last decade, peel and flesh-pigmented potatoes (anthocyanins rich and carotenoids rich; figure 13) were obtained, included in the collection and used in different research activities (pacifico, 2018). recently, some commercial potato varieties (‘bleuet’; purple skinned and fleshed tubers; ‘desiree’, red-skinned and yellow-fleshed tubers and ‘kennebec’, yellow-skinned and white-fleshed tubers), one advanced hybrid line (‘98-11-1’, purple parti-coloured skinned and fleshed tubers) and two italian traditional ecotypes (‘bianca di starleggia’, yellow-skinned and whitefleshed tubers, and ‘rossa di starleggia’, red-skinned and yellow-fleshed tuber) were also tested at different altitudes grown either at the experimental farm of crea, located in budrio (bologna area, 25 m.a.s.l.) and at starleggia (campodolcino, valchiavenna, 1,560 m.a.s.l.). preliminary results showed that the up-land environment influences the potato nutritional profile (pacifico et al, 2022) and that potato antioxidant and antiinflammatory compounds, such as anthocyanins, could have a preventive effect against lps-induced inflammation in thp1 macrophages (toccaceli et al, 2023) recently, from the biochemical characterization of peels of five potato genotypes present in the collection, we have demonstrated the great potential in the reuse or recovery of potato peel waste (ppw) from the agroindustrial potato processing. raw extracts from the peel of ‘lady claire’, a processing variety conserved in the crea-ci collection, proved to be the most suitable as a fungicide against fungal pathogens of cereals (fusarium graminearum and fusarium verticillioides). its effectiveness is mainly due to the activity of the phenolic fraction, which inhibited the tested fungi by up to 30% (pacifico et al, 2024). during the last decade, peel and flesh-pigmented potatoes (anthocyanins rich and carotenoids rich; figure 13) were obtained, included in the collection and used in different research activities (pacifico, 2018). recently, some commercial potato varieties (‘bleuet’; purple skinned and fleshed tubers; ‘desiree’, red-skinned and yellow-fleshed tubers and ‘kennebec’, yellow-skinned and white-fleshed tubers), one advanced hybrid line (‘98-11-1’, purple parti-coloured skinned and fleshed tubers) and two italian traditional ecotypes (‘bianca di starleggia’, yellow-skinned and whitegenetic resources (2025), (s2), 162–184 177 fleshed tubers, and ‘rossa di starleggia’, red-skinned and yellow-fleshed tuber) were also tested at different altitudes grown either at the experimental farm of crea, located in budrio (bologna area, 25 m.a.s.l.) and at starleggia (campodolcino, valchiavenna, 1.560 m.a.s.l.). preliminary results showed that the up-land environment influences the potato nutritional profile (pacifico et al, 2022) and that potato antioxidant and antiinflammatory compounds, such as anthocyanins, could have a preventive effect against lps-induced inflammation in thp1 macrophages (toccaceli et al, 2023) grain legumes collection the research activity on grain legumes at the creaci bologna can be traced back to the 1960s with the first studies on phaseolus vulgaris l. breeding has always attempted to address problems associated with biotic stress, while also improving drought and hightemperature tolerance, quality, and optimizing the crop for various end-uses, including fresh consumption, dry grain production and the freezing industry. more than 40 common bean and pea varieties have been released over the past 50 years, and they have long served as italian standard varieties (ranalli, 1999; ranalli and parisi, 2000; ranalli et al, 2004). the grain legume collection at crea-ci currently consists of 1,250 accessions with 27 species and 42 countries of origin represented (figure 14). the most numerous species are common bean with 1,115 accessions, followed by chickpea with 30, runner bean with 23, lentil with 21. in the common bean collection, accessions can also be distinguished by their biological status: wild (285 accessions) and domesticated (830), traditional landraces (551) and modern cultivars (279); but also, further sub-clusters according to the type of product (snapbeans, borlotto and cannellino beans, black beans, kidney beans, etc.). as for the countries of origin, the most represented, in terms of the number of accessions, are: guatemala (261), italy and mexico (201), spain (119), usa (76), colombia (61) and portugal (39). over the past five years, the number of italian landraces increased as a result of collecting trips to farms and local markets as well as the collaboration of regional institutions (e.g. in liguria and calabria, see figure 15). the iberian peninsula has also substantially contributed to the collection, and together with italy is the second largest centre of differentiation for this species since repeated crosses between andean and mesoamerican accessions have been shown to occur, facilitating the development of new genotypic and phenotypic diversity (santalla et al, 2002; angioi et al, 2010). the last 25 years have seen a significant increase in the collection size, as marker-assisted selection (mas) has facilitated characterization and accelerated prebreeding. in addition, more cutting-edge techniques allowed the study of germplasm of different origins and with much wider genetic variability (rodriguez et al, 2016). a significant example was the breeding initiative aimed at introducing different resistant sources for rootknot nematodes (rkn) in common bean. following the collection and characterization of resistant and tolerant accessions, markers associated with these resistances and new improved varieties and lines were developed (delbianco et al, 2004; carboni et al, 2004, 2005; delbianco and carboni, 2006; divito et al, 2007; delbianco et al, 2007; parisi et al, 2007). after a genomic analysis conducted on over 400 wild and 400 domesticated accessions of mesoamerican and andean genepools, three wild and five cultivated accessions showing resistance were found. a subsequent research project involved extensive phenotypic and genetic characterization of 192 genotypes, mostly landraces, with over 40 bio-morphological descriptors across two environments over two years. the data collected showed a high level of genetic diversity, especially for characters associated with flowering and 100-seed weight. a subsequent genome-wide association study (gwas) enables: (1) the definition of the genetic structure of european germplasm, (2) the identification of markers and favourable alleles in genotypes that perform better under various environmental conditions, and (3) the identification of seven snps associated with flowering character control (caproni et al, 2019; raggi et al, 2019). a distinguishing characteristic of the leguminosae family is that it is a valuable source of plant protein, and the collection is constantly analyzed to determine the total protein content of the seeds (figure 16). these boxplots are based on the average values of at least two to three reproduction cycles for every accession and the data are summarized by different bean cluster or sub-cluster of commercial type. in particular, the first three boxplots mesoamerica genepool (mg), andean genepool (ag) and european landraces (el) were calculated on homogeneous groups in number, 180 accessions each. the fourth boxplot, italian and european cultivar (ie) could be subdivided according to different categories of market end-products: borlotto type with 28 accessions, yellow romano type with 11 and snapbean type with 40. the mg’s distribution is significantly more homogeneous than that of the ag group, and the south american group’s 75th percentile is even lower than the 25th percentile of the mg group. the graph also demonstrates that the higher protein content is found in the mg group, with a maximum of 35.62% dry matter, and in the el group. the ie group has a protein content distribution similar to that of the ag group. a closer look at the ie group reveals that three distinct subclusters show different behaviours: the borlotto type exhibits very low protein production values; the yellow romano type appears to show less genetic variability and a strong correlation between pod phenotype and high protein production; and the industrial crops conservation and use at crea-ci, italy 178 alberti et al genetic resources (2025), (s2), 162–184 figure 14. a, species and b, countries of origin of the accessions maintained in the grain legume collection. figure 15. examples of italian agrobiodiversity maintained in the grain legume collection: a, b and c are ligurian landraces (respectively ‘pisello nero di l’ago’, ‘fagiolana di torza’, ‘fagiolo di mangia’); d, e and f are calabrian landraces (‘russa janca’, ‘capomacchia’, ‘cocò gialla’). snapbean type is intermediate and different from the other two. it is interesting to note that traditional breeding, focused in the past mainly on resistance genes or macromorphological traits of seed and pod quality, never evaluated the protein production trait. this bottleneck is evident comparing the borlotto type with the other two classes of beans (yellow romano and snapbean). in the case of borlotto, the selection was traditionally carried out by looking at the brightness of the red colour of seed and pod, according to the preference of the italian consumers or the freezing industry. yellow romano type and snapbean, although not selected for seed production, show significantly higher protein yields when harvested as dry seed than the borlotto type. this is an example of how a well-characterized germplasm collection can become an essential tool for correcting unwitting genetic drift. conclusions more than 20 years ago, when the italian ministry of agriculture, prompted by visionary colleagues such as professor carlo fideghelli, called for action to better preserve the germplasm accumulated within its research genetic resources (2025), (s2), 162–184 179 figure 16. box-plots of protein content (%dm, dry matter percentage) in different clusters of the crea-ci common bean collection. institutes, the scientific community initially struggled to understand the reason for this initiative. for most of the older staff, who may have perceived the duty of sharing materials as a risk of losing possession of ‘their’ pgr and the associated knowledge, training activities and the need to regenerate stored accessions initially seemed disconnected from their daily research activity. this process was gradual but necessary, making it possible to establish a ‘dispersed’ germplasm bank of 40,186 accessions (considering all crea collections) and to create a network and a critical mass that is now more aware and active in germplasm preservation and exchange (vaccino et al, 2024). however, such a radical change in perspective has not always been linear. the diverse educational background of those working with pgr in bologna and rovigo is indicative of biodiversity itself: we are agronomists, geneticists, biologists and biotechnologists with distinct specializations. yet, perhaps because of our different approaches to pgr, we have developed a level of teamwork that was not so obvious at the beginning but is now accelerating in an unexpected, engaging and more conscious way. the 2,237 accessions preserved in bologna and rovigo are the result of the work of many researchers over more than 100 years. this collection functions as a living organism, having experienced challenges over the course of its history: losses from different causes followed by frequent renewals. these pgr are well characterized from different points of view: from phytopathological tests to the latest and innovative chemical and genomic analyses; from traditional agronomic trials to frontier pharmacological, nutraceutical, medical, food and industrial uses. these accessions aim to provide pollinating insects with food support or to supply essential secondary metabolites in an emerging ecologically friendly and sustainable agriculture. they are the outcome of selection aimed at mitigating the effects of climate change, which is forcing us to deal with drought and extreme temperatures as well as new biotic stresses. the next crucial steps include adding further characterization data to the passport data that identify each accession anglin et al (2018); kumar et al (2024) as well as a greater openness to exchange pgr. until now, these resources have only been made available through scientific collaboration agreements. expanding access will be a key challenge in the coming years. supplemental data supplemental table 1. description of the brassicales collection: status, major glycosinolates, major fatty acid. supplemental table 2. sunflower hybrids registered in the national variety register. supplemental table 3. sunflower hybrids with high oleic acid content in the national variety register. industrial crops conservation and use at crea-ci, italy https://www.genresj.org/index.php/grj/article/view/genresj.augz3618/suppdata254 180 alberti et al genetic resources (2025), (s2), 162–184 supplemental table 4. descriptive traits recorded on 46 sunflower lines in 2021. supplemental table 5. descriptive traits recorded on 25 sunflower lines in 2022. supplemental table 6. descriptive traits recorded on 22 sunflower restorer in 2022. supplemental table 7. oil content and analysis of the fatty acid spectrum contained in sunflower lines. supplemental table 8. description of the hybrids produced at osimo crea-ci unit. supplemental table 9. descriptive traits recorded on 14 castor bean lines in 2021. acknowledgements it would take an enormous amount of space to name all the people who contributed to crea-ci in bologna and rovigo over the last 100 years, and we would run the danger of overlooking some of them. we are profoundly grateful to each and every one of them. however, we must not forget our current colleagues. in alphabetical order: cristina baldin, virna benazzi, matteo carloni, nerio casadei, mauro colombo, michele diozzi, stefano fanin, gianni fila, lorena malaguti, claudia maestrini, lorella mangoni, vincenza milito, anna moschella, federica nicoletti, bruno parisi, sandro pieri, daniele sanna. if the collection is so alive, it is also because of them. thank you! it is also impossible to list all the projects that have contributed to the collections. however, we must mention the programme funded by the masaf: ‘rgv/fao national programme for conservation, characterization, use, and enhancement of plant genetic resources for food and agriculture,’ which has allowed our crea research centres to reorganize the germplasm and promote collection regeneration for the past two decades. authors contribution andrea carboni was responsible for the initial conception and design of the article, and wrote the draft of the introduction, discussion and conclusion. each of the authors described the status of their collection for their respective species and can be contacted via e-mail for enquiries: ilaria alberti (ilaria.alberti@crea.gov.it) for sugar beet, manuela bagatta (manuela.bagatta@crea.gov.it) for brassicales and flax, andrea carboni (andrea.carboni@crea.gov.it) for grain legume, andrea del gatto (andrea.delgatto@crea.gov.it) for sunflower and castor beans, massimo montanari (massimo.montanari@crea.gov.it) for hemp, and daniela pacifico (daniela.pacifico@crea.gov.it) for the potato collection. all authors read and approved the final manuscript. conflict of interest statement the authors declare that they have no conflicts of interest. references agerbirk, n., hansen, c. c., et al. 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(2013). challenges and opportunities for new industrial oilseed crops in eu-27: a review. ind. crops prod 50, 580–595. doi: https://doi.org/10.1016/j.indcrop. 2013.08.030 https://doi.org/10.3390/agronomy14061263 https://doi.org/10.3390/agronomy14061263 https://doi.org/10.3390/app9122431 https://doi.org/10.1093/nar/gkw755 https://doi.org/10.1093/nar/gkw755 https://doi.org/10.1016/j.indcrop.2013.08.030 https://doi.org/10.1016/j.indcrop.2013.08.030 introduction beta collection brassicales collection flax collection sunflower and castor bean collections sunflower castor bean hemp collection potato collection grain legumes collection conclusions supplemental data acknowledgements authors contribution conflict of interest statement original article genetic resources (2025), 6 (12), 171–193 doi: 10.46265/genresj.slta9371 https://www.genresj.org issn: 2708-3764 received: 15.03.2025 | accepted: 18.08.2025 | published online: 24.11.2025 biochemical characteristics of bread wheat genotypes related to ssr markers in moisture stress conditions abstract: wheat is one of the oldest and most important staple crops worldwide, facing various biotic and abiotic stresses that affect its productivity. this study examines microsatellite markers related to grain yield, biochemical traits and drought tolerance indices in 25 wheat genotypes. the experiment was set up based on the randomized complete block design with three replications under rainfed and irrigated conditions. combined variance analysis revealed significant differences among genotypes. principal component analysis identified drought-tolerant genotypes (6, 10, 15, 18, 13, pishtaz) linked to superior yield, stress indices, and antioxidant activity under rainfed conditions. polymorphic ssr markers revealed key associations: xcfd168 with catalase, xgwm350 with ascorbic peroxidase (both under rainfed conditions), and xgwm136 with yield in irrigated conditions and multiple stress indices. marker xgwm410(a1) was associated with yield in both environments, catalase in irrigated conditions, and multiple indices. marker xgwm2(a2) was linked to yield in irrigated conditions, ascorbic peroxidase in rainfed conditions, and abiotic tolerance index, while xgwm124(a2) was associated with yield, superoxide dismutase in rainfed conditions, and multiple indices. the study identifies these genotypes as top candidates for drought tolerance due to their high yield and optimal biochemical responses under stress. furthermore, key molecular markers – xcfd168, xgwm350, xgwm136, xgwm124(a2), xgwm410(a1), and xgwm2(a2) – associated with biochemical and yield traits are prioritized for marker-assisted selection to enhance drought tolerance and yield stability in breeding programmes. keywords: antioxidant enzymes, bread wheat, genetic variation, molecular marker citation: bavandpouri, f., farshadfar, e., cheghamirza, k. and farshadfar, m. (2025) “biochemical characteristics of bread wheat genotypes related to ssr markers in moisture stress conditions”, genetic resources, 6(12), pp. 171–193. doi: 10.46265/ genresj.slta9371. © copyright 2025 the authors. this is an open access article distributed under the terms of the creative commons attribution license (cc by 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. fatemeh bavandpouri*,a, ezatollah farshadfara, kianoosh cheghamirzaa and mohsen farshadfarb adepartment of plant production and genetics engineering, faculty of agricultural sciences and engineering, razi university, kermanshah, iran bforests and rangelands research department, kermanshah agricultural and natural resources research and education center, (areeo), kermanshah, iran * corresponding author: fatemeh bavandpouri (f.bavandpori@yahoo.com) introduction wheat (triticum aestivum l.) contributes to approximately one-third of the global food supply. the food and agriculture organization of the un (fao) estimates that by 2050, an annual production of around 840 million tonnes of wheat will be required (ma et al, 2022). however, wheat production is increasingly affected by various biotic and abiotic stresses that reduce crop yield and productivity. among these, drought stress stands out as a major abiotic challenge, posing a significant threat to global food security, especially in the context of climate change (sunil kumar et al, 2023). as a result, there is a critical need to identify and cultivate drought-tolerant, high-yielding genotypes to ensure sustainable food production and meet the demands of a growing global population (galal et al, 2023). drought stress in wheat triggers morphological, physiological, biochemical and molecular changes (gupta et al, 2024; rashid et al, 2022). utilizing selection factor indicators can significantly improve the identification of genotypes that perform well in both optimal and stress conditions. a promising strategy to enhance wheat drought tolerance is to improve its antioxidant defense mechanisms (gupta et al, 2024). antioxidant enzymes are critical in protecting plants from oxidative damage caused by various environmental stresses. molecular markers associated with biochemical parameters can significantly expedite the identification of tolerant genetic materials in breeding programmes. the https://doi.org/10.46265/genresj.slta9371 https://www.genresj.org https://doi.org/10.46265/genresj.slta9371 https://doi.org/10.46265/genresj.slta9371 mailto:f.bavandpori%40yahoo.com?subject= genetic resources (2025), 6(12), 171–193172 bavandpouri et al simple sequence repeat (ssr) marker system is highly effective for detecting significant marker–trait associations in wheat germplasm (pour-aboughadareh et al, 2022). ssrs, also known as microsatellites, are short, tandemly repeated dna sequences (typically 1–6 nucleotides in length) that are distributed genome-wide, exhibiting high polymorphism due to replication slippage in non-coding regions (ellegren, 2004). because of their multi-allelic nature, co-dominant inheritance, uniform genomic distribution and simple detection methodology, these markers are widely favoured for assessing genetic variation and analyzing population structures (jabari et al, 2023; ahmed et al, 2024). exploring the genetic foundations of quantitative traits in crops and understanding the relationship between dna polymorphisms and phenotypic variations are essential for plant breeding programmes. identifying quantitative trait loci (qtls) linked to drought tolerance through marker-assisted selection is particularly important for crop improvement and represents a valuable strategy for boosting wheat yield (zhao et al, 2023). multivariate regression analysis (mra) offers a fast and effective approach for establishing the association between traits and markers. a significant benefit of mra is its capacity to pinpoint loci associated with quantitative traits. furthermore, this method is both time-efficient and costeffective (vaillancourt et al, 2008) and does not require the creation of specialized populations for mapping. the genetic diversity of 18 wheat genotypes was evaluated for drought tolerance using 25 microsatellite markers alongside morpho-physiological traits. findings revealed that integrating these two approaches enhanced the efficiency of the screening process and provided more reliable outcomes for improving drought tolerance in wheat (ahmed et al, 2023). a study investigating morphological, biochemical and genetic diversity for diagnosing salt tolerance in 18 wheat genotypes using ssr markers highlighted significant findings. the stepwise regression analysis emphasized the importance of root dry matter, relative turgidity and their respective contributions to shoot dry matter. out of 23 ssr primers analyzed, 17 exhibited polymorphisms (al-ashkar et al, 2020). an association analysis performed on wild relatives of wheat in drought stress conditions, using 24 ssr markers, identified eight and nine significant marker-trait associations (mtas) in control and drought stress conditions, respectively. notably, two mtas were consistently observed in both growth conditions (pour-aboughadareh et al, 2022). a study on iranian wheat varieties and landraces employed agronomic traits and drought tolerance indices to identify significant snp loci associated with drought-tolerance characteristics. the findings revealed that association mapping based on multiple drought tolerance indices can be highly effective in identifying critical markers for drought tolerance and uncovering linked gene networks (rabieyan et al, 2023). additionally, a research evaluation combining genetic and phenotypic analyses was conducted to identify droughttolerant bread wheat genotypes using multivariate analysis techniques, including stepwise multiple linear regression. the results demonstrated that ssr markers were associated with nine agro-physio-biochemical traits, highlighting their utility as a valuable tool in the selection process for drought tolerance (sallam et al, 2024a). despite these advances, very few studies have explored the association between molecular markers and biochemical traits whose activity increases in drought stress. biochemical traits, like the accumulation of proline or antioxidants, are the measurable physiological responses of a plant to stress. molecular markers are dna sequences that can pinpoint the specific genes or genomic regions controlling these biochemical pathways (oguz et al, 2022). therefore, this research aimed to: (1) characterize bread wheat genotypes in terms of biochemical traits, grain yield, and drought tolerance indices, (2) analyze the impact of drought stress on wheat traits to enhance yield and drought tolerance, (3) evaluate the genetic diversity of wheat genotypes for drought tolerance using studied traits and ssr markers, and (4) investigate the association between the studied traits and indices with ssr markers and identify informative markers associated with grain yield, biochemical traits, and drought tolerance indices in wheat in both rainfed and irrigated conditions. materials and methods field experiment twenty-five bread wheat genotypes were evaluated, including two cultivars (pishtaz and pishgam) as controls, and 23 accessions of bread wheat (table 1). the genotypes were sourced from the genebank of karaj seedling and seed breeding research institute, iran. field experiments were conducted during the 2018–2019 growing season using a randomized complete block design with three replications under rainfed and irrigated conditions in a cold mediterranean climate (34°21'n, 47°9'e; altitude: 1,319m; mean annual rainfall: 430–460mm) in iran. each experimental plot consisted of five rows, each with a length of 2m, row spacing of 23cm, and a planting density of 400 seeds per square metre. the planting date (14 november 2018) coincided with the first irrigation, but no irrigation was provided to the rainfed plots during the growth period. the total rainfall during the experimental year was 401.51mm. for the irrigated treatment, three additional irrigations were applied: the first on 15 may, at the heading stage (50% spike emergence). the second in late may, after full spike emergence. the third on 14 june, during the seed milking stage. no chemical fertilizers were applied during the experiment, and weeding was performed manually. molecular experiment for molecular evaluation of the studied genotypes, 20 pairs of ssr markers were utilized. dna was extracted from twoto three-week-old seedlings grown from seeds using the cetyltrimethylammonium bromide (ctab) method, based on the modified protocol of doyle and doyle (1987), in bulk. genomic dna was extracted from 50 mg of cryogenically homogenized tissue. samples were suspended in 800μl extraction buffer (100ml containing: 4g ctab, 16.36g nacl, 3.15g tris-hcl, 1.48g edta, and 400μl β-mercaptoethanol; ph 8.0) and incubated at 65°c for 30 min. after adding 800μl chloroform-isoamyl alcohol (24:1), samples were vortexed for 60 min, centrifuged (13,000 × g, 15 min), and the aqueous phase was transferred to fresh tubes. this phase was mixed with 500μl cold isopropanol and held at -20°c for 2 hours. subsequent centrifugation (13,000 × g, 15 min) yielded dna pellets, which were washed twice with 500μl cold 80% ethanol (brief centrifugation, supernatant removal). pellets were air-dried and resuspended in 100μl nuclease-free h2o. extracted genomic dna was evaluated by 0.8% agarose gel electrophoresis. genetic resources (2025), 6(12), 171–193 biochemical traits and ssr markers in wheat 173 table 1. list of bread wheat planting materials used for the study. ir, iran; us, united states of america. genotype number genotype number and name origin 1 wc-4924 kalat, ir 2 wc-4582 kermanshah, ir 3 wc-4592 kermanshah, ir 4 wc-47341 montana, us 5 wc-4965 kashan, ir 6 wc-4840 sarakhs, ir 7 wc-4958 badranloo, ir 8 wc-47399 bulgaria 9 wc-4600 kermanshah, ir 10 wc-4987 unknown, ir 11 wc-47615 mexico 12 wc-4612 kordestan babrar, ir 13 wc-5001 unknown, ir 14 wc-4994 unknown, ir 15 wc-47638 peru 16 wc-47583 canada 17 wc-47522 mexico 18 wc-47569 minnesota, us 19 pishtaz pishtaz, ir 20 pishgam pishgam, ir 21 wc-47640 minnesota, us 22 wc-47467 mexico 23 wc-4553 kerend, ir 24 wc-4583 kermanshah, ir 25 wc-4554 kerend, ir polymerase chain reaction (pcr) was conducted in three temperature-dependent steps. dna samples diluted to 10ng/ µl were amplified using 20 primer pairs (primer sequences are provided in table 2). pcr was performed in 20μl reaction volumes using a bio-rad thermocycler. the pcr products were electrophoresed on a 3% agarose gel in 1x tbe buffer, stained with 10μl safe stain. dna bands were visualized using a quantum st4 gel documentation system. as not all samples were loaded on the same gel, a 100–1500bp dna size marker (producing 11 bands) was included. band presence was scored as ‘1’ and absence as ‘0’, compiling the data into a matrix. alleles detected in genotypes were designated a, b, c, and d for each marker. this matrix served as the foundation for subsequent statistical analyses based on electrophoretic band pattern. biochemical enzyme assays measurement methods of the biochemical traits were carried out as follows. extraction buffer preparation: a 200ml tris-hcl extraction buffer (ph 8.0) was prepared by dissolving 2.428g of tris and 0.2g of pvp in 40ml of distilled water. the solution’s ph was then adjusted to 8.0 using hcl, and the final volume was brought to 200ml with distilled water. the prepared buffer was stored at 4°c, protected from light with aluminium foil. enzyme extraction: flag leaf samples were ground into a fine powder in liquid nitrogen. subsequently, 250mg of the homogenized powder was combined with 1ml of the precooled extraction buffer in a 2ml microtube. the mixture was vortexed for 30 seconds and then incubated at 4°c for 12 hours. during this incubation period, the samples underwent two additional 30-second vortexing steps at 2-hour intervals. following incubation, the homogenate was centrifuged at 13,000 × g for 15 minutes at 4°c. the resulting clear supernatant was carefully collected for the subsequent analysis of soluble protein content and antioxidant enzyme activities (ramachandra reddy et al, 2004). meanwhile, the biotek powerwave xs2 microplate reader was used to measure biochemical traits. peroxidase activity (pod) was assayed according to the method of chance and maehly (1995) with slight modifications by combining 6.6μl of diluted enzyme extract (1:4) with 200μl of substrate solution [408.71μl guaiacol + 78.3μl 0.9 m h2o2 in 50mm potassium phosphate buffer (ph 7.0)]. after a 15-minute incubation, absorbance was measured at 470nm every 30s. superoxide dismutase activity (sod) was assayed following the method of beauchamp and fridovich (1971). the assay solution consisted of 50 mm potassium phosphate buffer (ph 7.8), 12.26mg nitroblue tetrazolium (nbt), 387.92mg l-methionine, 1mm edta, and 0.04mm riboflavin (stored in light-protected containers). for the assay, 196, 197, 198, and 199μl of extraction buffer were mixed with 4, 3, 2, and 1μl of diluted enzymatic extract (1:4), respectively, to achieve 200μl reaction mixtures. these mixtures were transferred to a 96-well microplate, followed by addition of 10μl riboflavin solution under dark conditions. after 30 min illumination in a light chamber, absorbance was measured at 560nm. catalase activity (cat) was assayed according to the method of sinha (1972) with slight modifications. the assay was performed by combining 1.5μl of diluted enzymatic extract (1:4) with 150μl of 50 mm phosphate buffer (ph 7.0). the reaction was initiated by adding 75μl of 0.32 mm hydrogen peroxide solution. at timed intervals (2, 4, 6 and 8 min post-initiation), 62μl of dichromate reagent was rapidly added to each tube with immediate vortexing. tubes were transferred to a preheated 95°c water bath for 10 min. after chromogenic development (green-to-yellow gradient), samples were centrifuged at (10,000g, 5 min), and the supernatant absorbance was measured at 570 nm. ascorbic peroxidase activity (apx) was assayed according to the method of nakano and asada (1981). the reaction was initiated by adding 50μl of the enzymatic extract to 1ml of an assay solution containing 50 mm potassium phosphate buffer (ph 7.0), 0.1 mm edta, 0.5 mm ascorbic acid (asa), and 0.15 mm hydrogen peroxide (h2o2). absorbance at 290 nm was recorded every 10 s for 1 min. soluble protein concentration (protein) was determined using the method of bradford (1976). for the assay, 1μl of the extracted sample was mixed with 200μl of freshly prepared coomassie brilliant blue g-250 dye reagent. after 15 min incubation, absorbance at 595nm was measured, with dye reagent as the blank. protein concentration was calculated from a bovine serum albumin (bsa) standard curve (01,500μg/ml). genetic resources (2025), 6(12), 171–193174 bavandpouri et al table 2. ssr primers used to assess the genetic diversity of bread wheat genotypes. referencesband sizegc%tm (5'-3') sequence nameno. haque et al (2020); bavandpouri et al (2025) 150bp47.6575' acctcatccacatgttctacg 3'xgwm350-7d-f1 64.75' gcatggataggacgccc 3'xgwm350-7d-r rosewarne et al (2013); halder et al (2023) 100bp30505' aatttcaaaaaggagagaga 3'xgwm334-6a-f2 305' aacatgtgtttttagctatc 3'xgwm334-6a-r ahmed et al (2020); el-rawy and hassan (2021) 100bp55.6585' caatcatttccccctccc 3'xgwm155-3a-f3 36.45' aatcattggaaatccatatgcc 3'xgwm155-3a-r el-rawy and hassan (2021); el-demery et al (2022); firouzian et al (2023) 150bp31.8565' atggcataatttggtgaaattg 3'xgwm577-7b-f4 36.45' tgtttcaagcccaacttctatt 3'xgwm577-7b-r batool et al (2018); ilyas et al (2020) 200bp5552.55' agtggctgggagagtgtcat 3'xgwm70-6b-f5 61.65' gcccattaccgaggacac 3'xgwm70-6b-r islam et al (2012); ahmed et al (2024) 180–200bp45585' acggcgagaaggtgctc 3'xgwm642-1d-f6 64.75' catgaaaggcaagttcgtca 3'xgwm642-1d-r budak et al (2013); kaur et al (2016); khan et al (2021); bavandpouri et al (2025) 250bp57.9525' gacagcaccttgccctttg 3'xgwm136-1a-f7 52.65' catcggcaacatgctcat 3'xgwm136-1a-r amalova et al (2024); bavandpouri et al (2025) 200bp61.157.55' gccatggctatcacccag 3'xgwm124-1b-f8 455' actgttcggtgcaatttgag 3'xgwm124-1b-r choudhary et al (2016); kumari et al (2025) 150bp4558.55' tgttgcggatggtcactatt 3'xgwm265-2a-f9 52.45' gagtacacatttggcctctgc 3'xgwm265-2a-r maccaferri et al (2011); naroui rad et al (2012) 250bp61.6515' gcttgagaccggcacagt 3'xgwm410-2b-f10 555' cgagaccttgagggtctaga 3'xgwm410-2b-r ahmed et al (2020); el-rawy and hassan (2021) 200bp5050.65' tgcagtggtcagatgtttcc 3'xgwm165-4b-f11 455' cttttctttcagattgcgcc 3'xgwm165-4b-r mallick et al (2022a)250bp4052.55' gctgatgcatataatgctgt 3'xgwm4-4a-f12 47.65' cactgtctgtatcactctgct 3'xgwm4-4a-r islam et al (2012); heidari et al (2024) 100bp4550.75' ggttttctttcagattgcgc 3'xgwm192-5d-f13 47.65' cgttgtctaatcttgccttgc 3'xgwm192-5d-r mallick et al (2022a)100bp26.146.75' tcaaaacataaatgttcattgga 3'xgwm233-7a-f14 40.95' tcaaccgtgtgtaattttgtcc 3'xgwm233-7a-r ahmed et al (2020); kumari et al (2025) 250bp5049.45' ctgcaagcctgtgatcaact 3'xgwm2-3d-f15 355' cattctcaaatgatcgaaca 3'xgwm2-3d-r ahmed et al (2020); mallick et al (2022a) 200bp57.959.55' tgccctgtccacagtgaag 3'xcfd5-5b-f16 455' ttgccagttccaaggagaat 3'xcfd5-5b-r ahmed et al (2020); mallick et al (2022a) 250bp5550.65' tcagtgggcaagctacacag 3'xgwm129-5a-f17 44.45' aaaacttagtagccgcgt 3'xgwm129-5a-r khan et al (2021); bavandpouri et al (2025) 250bp45565' cttcgcaaatcgaggatgat 3'xcfd168-2d-f18 505' ttcacgcccagtattaaggc 3'xcfd168-2d-r khan et al (2021); mallick et al (2022b) 220–230bp50545' gagtcctgatgtgaagctgttg 3'xgwm234-5b-f19 555' ctcattggggtgtgtacgtg 3'xgwm234-5b-r ahmed et al (2020); mallick et al (2022a) 100bp47.6595' ggagtcacacttgtttgtgca 3'xgwm33-1a-f20 45.55' cactgcacacctaactacctgc 3'xgwm33-1a-r genetic resources (2025), 6(12), 171–193 biochemical traits and ssr markers in wheat 175 differences were observed across various irrigated conditions for all characteristics. genotypes showed significant variation for all traits except soluble protein. furthermore, the genotype-by-irrigated interaction effect was significant for most biochemical traits, except for grain yield and malondialdehyde. the mean comparison (mean of three replications) of genotypes based on the studied traits in rainfed and irrigated conditions, presented in the form of a bar graph, is as follows. genotype 10 showed the highest grain yield under rainfed and irrigated conditions (figure 1, chart gy) with values of 424.73 and 565.75, respectively. the maximum peroxidase (pod) activity in rainfed and irrigated conditions was observed in genotype 6 (0.49) and genotype 18 (0.34), respectively (figure 1, chart pod). for superoxide dismutase (sod), the highest values in rainfed and irrigated conditions belonged to genotype 15 (1.02) and genotype 12 (0.64), respectively (figure 1, chart sod). catalase (cat) activity was most significant in genotype 12 (3.01) in rainfed conditions and genotype 24 (1.56) in irrigated conditions (figure 1, chart cat). the highest soluble protein content was found in genotype 14 (112.03) in rainfed conditions and genotype 12 (167.09) in irrigated conditions (figure 1, chart protein). proline (pc) levels were highest in genotype 15 (10.14) in rainfed conditions and genotype 8 (7.24) in irrigated conditions (figure 1, chart pc). the maximum ascorbic peroxidase (apx) activity was recorded for genotype 6 (418.12) in rainfed conditions and genotype 15 (263.35) in irrigated conditions (figure 1, chart apx). finally, the highest malondialdehyde (mda) values in both conditions were observed in genotypes 23 and 24 (0.45) in rainfed conditions and genotype 23 (0.42) in irrigated conditions (figure 1, chart mda). complete information on the comparison of the mean genotypes for each trait is shown in table 5. assessment of broad-sense heritability and genetic gain of studied traits in rainfed and irrigated conditions the estimation of broad-sense heritability and genetic gain for grain yield and biochemical traits under rainfed conditions is summarized in table 6. in rainfed conditions, the average broad-sense heritability and genetic gain for grain yield were 0.278 and 16.08%, respectively. almost all biochemical traits exhibited heritability above 0.90, including pc (0.998), sod (0.997), cat (0.983), and apx (0.972). among these, pc showed the highest heritability. for genetic gain, cat (92.022%), sod (89.91%), apx (67.62%), and pc (63.28%) were most significant, with cat ranking highest. under irrigated conditions, heritability and genetic gain for grain yield were 0.604 and 33.20%, respectively. the traits cat (0.997), protein (0.989), pc (0.979), sod (0.971), apx (0.966) and pod (0.929) all demonstrated high heritability (> 0.90), with cat showing the highest value. also, cat exhibited the most significant genetic gain (133.7%), followed by sod (86.9%), pc (83.19%), apx (80.39%). in both conditions, the mda trait showed the lowest heritability and genetic gain. malon-dialdehyde (mda) was determined according to the method of heath and packer (1968). briefly, 0.25g of wheat leaves were homogenized in 500μl ice-cold 1.0% (w/v) trichloroacetic acid (tca) using a porcelain mortar. the homogenate was centrifuged at 10,000 × g for 5 min at 4°c. subsequently, 250μl of the supernatant was reacted with 1ml of thiobarbituric acid (tba) reagent [0.5% (w/v) tba in 20% (w/v) tca]. the mixture was incubated at 95°c for 30 min in a water bath, then immediately cooled on ice and centrifuged again (10,000 × g, 10 min, 4°c). a 200μl aliquot of the resulting chromogenic supernatant was transferred to a 96-wall microplate. absorbance was measured at 532 and 600nm. proline concentration (pc) was determined according to the method of bates et al (1973). briefly, 0.05g of fresh leaf tissue was homogenized in 1ml of ice-cold 3% (w/v) sulfosalicylic acid using a pre-chilled mortar. the homogenate was centrifuged at 4,000 × g for 15 min (4°c). a 10μl aliquot of the resulting supernatant was then reacted with 200μl of acid-ninhydrin reagent [1.25 g ninhydrin in 30ml glacial acetic acid + 20ml 6 m phosphoric acid] and 200μl of glacial acetic acid. tubes were incubated at 95°c for 60 min, immediately cooled on ice for 5 min, and then mixed with 400μl toluene via 30-second vortexing. after a 20-minute phase separation at 25°c, the upper toluene layer was transferred to a 96well microplate. absorbance was measured at 520nm with pathlength correction. proline concentration was determined from a standard curve (0-20μg/ml). statistical analysis combined variance analysis based on the data obtained from the evaluation of 25 genotypes, including two cultivars and 23 accessions, was performed to determine the contribution of the main effects of genotype, irrigated conditions, and their interaction using sas 9.1.3 software. a comparison of mean genotypes by the least significant difference (lsd) test was performed. a bar graph related to the mean comparison was drawn in excel. pca was calculated based on the means of traits and genotypes. principal components analysis was carried out using the minitab16 software, and correlations between the studied traits and indicators were analyzed using the “corrplot” package in r-studio version 4.5 (r core team, 2025). to analyze the differences among the studied genotypes using ssr molecular markers, analysis of molecular variance (amova) was performed by genalex software version 6.502. the association between ssr markers, field-measured traits, and biochemical traits was analyzed using stepwise multiple regression in spss 26 software. each quantitative trait was treated as a dependent variable, while the ssr markers served as independent variables. the studied traits and indices were measured in the field and molecular experiment section, as shown in table 3. results analysis of combined variance and mean compression the combined analysis of variance for grain yield and biochemical characteristics is presented in table 4. significant genetic resources (2025), 6(12), 171–193176 bavandpouri et al table 3. measurement methods of the studied traits and indices. yi: yield of a genotype under irrigated conditions; yr: yield of a genotype under rainfed conditions; : mean yield of all genotypes under irrigated conditions; : mean yield of all genotypes under rainfed conditions; : genotypic variance; : phenotypic variance; : overall mean of the trait; tcp: trait changes percentage; mtic: mean of the trait under irrigated conditions; mtrc: mean of the trait under rainfed conditions. the pic index for ssr markers was calculated based on allele frequency at each locus across all genotypes. in the calculation of the rp index, pi refers to the proportion of genotypes that possess a particular band. cov (x1x2): covariance between variables x1 and x2. v(x1): variance of one trait (x1). v(x2): variance of other trait (x2). traits and indices measurement method and formulas gy: grain yield the grain weight from three 1m sections of the middle rows per plot. ati: abiotic tolerance index (moosavi et al, 2008) sspi: stress susceptibility percentage index (moosavi et al, 2008) tol: tolerance (rossielli and hamblin, 1981) mp: mean productivity (rossielli and hamblin, 1981) gmp: geometric mean productivity (fernandez, 1992) hmp: harmonic mean productivity (fernandez, 1992) sti: stress tolerance index (fernandez, 1992) ssi: stress susceptibility index (fischer and maurer, 1978) pev: press evaluation (bouslama and schapaugh, 1984) rdy: relative decrease in yield (emre et al, 2011) h2b.s , gg: broad-sense heritability and genetic gain (kearsey and pooni (1996) and the glm manova analysis in sas 9.3.1 software) correlation (miller et al, 1958) tcp%: percentage of changes in the irrigated environment compared to rainfed for traits (nourmand-moaied et al, 2001) polymorphic percentage (mohammadi and prasanna, 2003) the number of polymorphic bands is divided by the total number of amplified bands and multiplied by 100. pic: polymorphic information content index (anderson et al, 1993) mi: marker index (kumar et al, 2009) the number of polymorphic bands was multiplied with the pic value. emr: effective multiplex ratio index (kumar et al, 2009) this index was obtained by multiplying the percentage of polymorphic loci by the number of polymorphic loci. rp: resolving power (altintas et al, 2008) ( )i r r i i r y yati y y y y    − = × ×      100 2 i r i y ysspi y − = ×   i rtol y y= − ( ) / 2r imp y y= + ( )r igmp y y= × 2 i r i r y yhmp y y × = + 2 i r i y ysti y × = ( )( ) ( )( )1 / / 1 /r ir issi y y y y= − − 1 r i ypev y   = −     ( )100 /100r irdy y y= − × 2 2 2.06. .100g pgg x δ δ           = 2 2 . 2 g b s p h δ δ = ( ) ( ) ( ) ( ) 1 2 1 2 1 2 cov x x r x x v x v x = 100mtic mtrctcp mtic − = × 21pic pi= −∑ ( )1 2 0.5 rp ib ib pi = ∑ = − × −   genetic resources (2025), 6(12), 171–193 biochemical traits and ssr markers in wheat 177 table 4. analysis of combined variance in both rainfed and irrigated conditions for grain yield and biochemical characteristics in 25 bread wheat genotypes. ns, not significant; *, significant at 5% probability level; **, significant at 1% probability level; s.o.v, source of variations; error 1, is nesting the replication in the irrigated factor; error 2, is the total error of the experiment. s.o.v df grain yield peroxidase activity superoxide dismutase activity catalase activity soluble protein proline content ascorbic peroxidase malondialdehyde irrigated 1 470283.30** 0.37** 2.58** 33.22** 32924.41** 272.49** 489561.60** 0.05** error 1 4 21490.83 0.0003 0.0002 0.0004 11.35 0.02 178.19 0.001 genotype 24 28250.64** 0.02** 0.17** 1.42* 1416.56ns 16.07** 21384.36* 0.005** genotype× irrigated 24 6237.08ns 0.006** 0.05** 0.63** 855.42** 5.44** 8793.39** 0.001ns error 2 96 4496.36 0.001 0.0003 0.005 25.39 0.04 153.87 0.001 (c.v) % 19.74 8.89 4.08 6.28 5.07 3.44 6.39 10.07 figure 1. bar graphs related to the comparison of mean genotypes in rainfed and irrigated conditions. gy, grain yield; pod, peroxidase activity; sod, superoxide dismutase activity; cat, catalase activity; protein, soluble protein; pc, proline concentration; apx, ascorbic peroxidase activity; mda, malon-dialdehyde. genetic resources (2025), 6(12), 171–193178 bavandpouri et al t ab le 5 . m ea n co m pa ri so n of b re ad w he at g en ot yp es u nd er r ai nf ed a nd ir ri ga te d co nd iti on s fo r th e st ud ie d tr ai ts . g , g en ot yp e; r , r ai nf ed ; i , i rr ig at ed . g g ra in y ie ld (g m 2 ) pe ro xi da se a ct iv it y (µ m ol m in -1 m g-1 pr ot ei n) su pe ro xi de d is m ut as e ac ti vi ty (µ m ol g -1 fw ) ca ta la se a ct iv it y (µ m ol m in -1 m g-1 pr ot ei n) so lu bl e pr ot ei n (m g g-1 f w ) pr ol in e co nt en t (m g g-1 f w ) a sc or bi c pe ro xi da se (µ m ol m in -1 m g-1 pr ot ei n) m al on -d ia ld eh yd e (µ m ol g -1 fw ) r i r i r i r i r i r i r i r i g 1 23 9. 38 32 2. 20 0. 32 0. 24 0. 79 0. 37 1. 48 0. 90 80 .0 6 98 .0 6 8. 38 2. 39 31 6. 01 15 1. 37 0. 37 0. 35 g 2 23 8. 40 52 6. 59 0. 27 0. 19 0. 32 0. 11 2. 24 0. 31 66 .0 9 16 2. 09 4. 88 3. 03 21 3. 84 11 3. 37 0. 41 0. 38 g 3 26 2. 82 35 5. 60 0. 28 0. 26 0. 59 0. 29 1. 17 0. 12 69 .7 6 10 3. 06 6. 07 3. 60 25 1. 30 23 4. 07 0. 39 0. 37 g 4 20 8. 83 30 4. 55 0. 41 0. 15 0. 55 0. 20 0. 82 0. 15 75 .4 9 15 1. 33 9. 23 5. 67 32 0. 57 54 .8 7 0. 32 0. 32 g 5 26 1. 51 35 3. 34 0. 29 0. 17 0. 39 0. 35 0. 88 0. 53 75 .0 9 12 0. 79 3. 27 1. 53 27 6. 49 17 5. 16 0. 36 0. 33 g 6 34 2. 69 44 2. 37 0. 49 0. 32 0. 70 0. 33 1. 80 0. 15 68 .9 6 10 1. 36 4. 02 2. 14 41 8. 12 18 8. 34 0. 39 0. 34 g 7 27 7. 26 43 1. 71 0. 38 0. 24 0. 53 0. 27 0. 80 0. 33 10 0. 46 10 6. 23 8. 72 3. 23 18 7. 76 13 3. 83 0. 34 0. 34 g 8 33 8. 53 35 8. 07 0. 28 0. 26 0. 80 0. 38 1. 27 0. 45 85 .6 9 11 6. 39 8. 86 7. 24 26 3. 08 18 9. 28 0. 34 0. 32 g 9 27 5. 12 41 0. 24 0. 37 0. 22 0. 55 0. 33 2. 71 1. 41 10 1. 79 11 0. 73 7. 59 3. 71 25 0. 79 14 3. 65 0. 34 0. 31 g 10 42 4. 73 56 5. 75 0. 28 0. 28 0. 90 0. 42 1. 47 0. 42 89 .4 3 95 .6 9 8. 08 4. 62 27 6. 15 16 1. 90 0. 36 0. 32 g 11 20 1. 89 31 7. 21 0. 34 0. 25 0. 34 0. 19 0. 61 0. 25 90 .9 9 13 1. 69 3. 93 2. 75 17 6. 85 13 3. 93 0. 39 0. 34 g 12 23 6. 94 29 8. 98 0. 28 0. 14 0. 70 0. 64 3. 01 0. 43 10 5. 99 16 7. 09 4. 96 3. 97 13 8. 82 10 3. 03 0. 38 0. 36 g 13 30 9. 49 50 8. 45 0. 36 0. 31 0. 98 0. 48 1. 73 0. 79 78 .2 7 95 .7 9 5. 05 4. 65 11 3. 31 11 0. 31 0. 41 0. 35 g 14 26 3. 96 48 2. 62 0. 26 0. 16 0. 56 0. 21 1. 22 0. 92 11 2. 03 12 6. 23 7. 55 3. 71 27 1. 23 15 3. 73 0. 39 0. 32 g 15 37 2. 95 48 2. 01 0. 34 0. 23 1. 02 0. 32 2. 58 1. 22 67 .4 3 76 .7 3 10 .1 4 4. 28 30 4. 86 26 3. 35 0. 42 0. 40 g 16 19 0. 15 21 4. 21 0. 42 0. 24 0. 37 0. 23 2. 26 0. 89 68 .9 3 12 8. 49 5. 88 5. 13 23 2. 76 13 7. 50 0. 39 0. 37 g 17 31 9. 10 35 4 0. 27 0. 25 0. 93 0. 43 1. 61 0. 86 81 .9 6 84 .1 9 7. 09 5. 86 37 0. 74 23 8. 03 0. 44 0. 36 g 18 35 4. 27 54 4. 26 0. 39 0. 34 0. 41 0. 22 0. 91 0. 89 75 .8 6 10 0. 53 6. 39 5. 09 27 6. 11 11 5. 07 0. 38 0. 36 c1 9 31 8. 70 49 2. 24 0. 48 0. 28 0. 53 0. 39 1. 22 0. 88 65 .5 9 76 .0 6 7. 91 1. 63 38 2. 05 96 .4 6 0. 39 0. 39 c2 0 28 5. 14 38 4. 01 0. 35 0. 26 0. 56 0. 35 2. 76 0. 60 87 .5 3 96 .6 6 2. 75 2. 06 33 3. 49 12 1. 35 0. 36 0. 35 g 21 30 3. 47 31 3. 97 0. 27 0. 22 0. 20 0. 17 2. 03 0. 58 94 .9 3 14 4. 13 8. 55 6. 72 18 4. 70 71 .2 0 0. 34 0. 31 g 22 19 7. 36 33 1. 92 0. 41 0. 26 0. 26 0. 16 0. 84 0. 14 94 .5 3 12 4. 46 8. 07 2. 22 98 .2 2 77 .4 2 0. 40 0. 34 g 23 24 0. 30 40 3. 74 0. 33 0. 26 0. 56 0. 17 1. 87 1. 13 87 .1 9 11 6. 59 6. 76 5. 43 30 0. 27 10 9. 35 0. 45 0. 42 g 24 29 2. 04 30 4. 24 0. 37 0. 29 0. 40 0. 37 1. 76 1. 56 88 .3 3 10 6. 69 5. 56 4. 57 16 7. 80 79 .1 0 0. 45 0. 34 g 25 29 3. 72 39 1. 08 0. 30 0. 21 0. 14 0. 14 0. 55 0. 14 10 1. 81 11 3. 89 10 .0 2 7. 09 15 5. 52 68 .7 4 0. 43 0. 39 ls d 5 % 11 1. 12 10 9. 03 0. 06 0. 02 0. 02 0. 04 0. 16 0. 04 10 .9 4 4. 14 0. 19 0. 39 23 .5 0 16 .6 5 0. 06 0. 06 genetic resources (2025), 6(12), 171–193 biochemical traits and ssr markers in wheat 179 analysis of trait-index correlations in wheat genotypes under rainfed and irrigated conditions the correlation patterns between studied traits and drought tolerance indices revealed distinct profiles across conditions. yield in irrigated conditions (yi) showed strong positive correlations (p < 0.01) with rainfed yield (yr; 0.71) and the indices sti (0.92), mp (0.96), gmp (0.93), hmp (0.91), ssi (0.52), tol (0.78), ati (0.90), sspi (0.78), and pev (0.52), but exhibited a significant negative association with rdy (-0.92). similarly, yr demonstrated strong positive correlations with sti (0.91), mp (0.88), gmp (0.91), and hmp (0.94) (p < 0.01), while having a negative correlation with rdy (-0.91). in irrigated environments, pod activity correlated positively (p < 0.05) with its rainfed counterpart (0.44) and the indices sti (0.44), mp (0.41), gmp (0.43), and hmp (0.44), yet displayed a negative relationship with rdy (-0.44). sod enzyme activity showed high consistency between the two conditions (irrigated vs. rainfed: 0.68; p < 0.01). rainfed sod further correlated positively with sti (0.43), mp (0.41), gmp (0.43), and hmp (0.44) (p < 0.05) but negatively with rdy (-0.43). cat activity also followed this pattern with significant concordance between irrigated and rainfed conditions (0.44; p < 0.05). for pc in irrigated conditions, inverse correlations appeared with sti (-0.50), mp (-0.48), gmp (-0.51), and table 6. estimation of broad-sense heritability and genetic gain for grain yield and biochemical characteristics in bread wheat genotypes in rainfed and irrigated conditions. gy, grain yield; pod, peroxidase activity; sod, superoxide dismutase activity; cat, catalase activity; protein, soluble protein; pc, proline concentration; apx, ascorbic peroxidase activity; mda, malon-dialdehyde. conditions traits mean h2 bs gg rainfed gy 283.75 0.278 16.08 pod 0.340 0.750 28.74 sod 0.560 0.997 89.91 cat 1.58 0.983 92.022 protein 84.57 0.791 28.044 pc 6.79 0.998 63.28 apx 251.23 0.972 67.62 mda 0.390 0.250 6.82 irrigated gy 395.75 0.604 33.203 pod 0.240 0.929 42.18 sod 0.300 0.971 86.9 cat 0.640 0.997 133.7 protein 114.2 0.989 42.932 pc 4.09 0.979 83.19 apx 136.98 0.966 80.39 mda 0.350 0.250 5.373 hmp (-0.53) (p < 0.05/p < 0.01), in contrast to its positive linkage with rdy (0.50; p < 0.05). pc also aligned with its rainfed equivalent (0.51; p < 0.01) and showed negative associations with ssi (-0.47) and pev (-0.47) (p < 0.05). apx and mda activities maintained significant consistency between the two conditions (apx: 0.46, mda: 0.69; p < 0.05/p < 0.01). inter-index correlations revealed tightly coupled networks: sti exhibited near-perfect positive alignment with mp (0.99), gmp (0.99), and hmp (0.99) (p < 0.01), moderate ties to ati (0.69), tol (0.49), and sspi (0.49) (p < 0.05), and a complete inverse correlation with rdy (-1.00; p < 0.01). the mp, gmp, and hmp indices showed nearly identical mutual relationships (0.99–1.00; p < 0.01) and positive associations with ati (0.64–0.74), tol (0.44–0.56), and sspi (0.44– 0.56) (p < 0.05/p < 0.01), while uniformly opposing rdy (-0.99 to -1.00; p < 0.01). ssi correlated strongly with pev (1.00), tol (0.93), sspi (0.93), and ati (0.80) (p < 0.01). tol demonstrated positive linkages with sspi (1.00), ati (0.96), and pev (0.93) (p < 0.01) but a negative correlation with rdy (-0.49; p < 0.05). ati correlated positively with sspi (0.96) and pev (0.80) (p < 0.01) and negatively with rdy (-0.69; p < 0.01). finally, sspi and pev shared a strong positive correlation (0.93; p < 0.01), while sspi was inversely associated with rdy (-0.49; p < 0.05) (figure 2). genetic resources (2025), 6(12), 171–193180 bavandpouri et al figure 2. heatmaps of pearson’s correlation coefficients between the studied characteristics and drought tolerance indices in 25 wheat genotypes in rainfed and irrigated conditions. yi, yield in irrigated conditions; yr, yield in rainfed conditions; i, irrigated; r, rainfed; pod, peroxidase activity; sod, superoxide dismutase activity; cat, catalase activity; protein, soluble protein; pc, proline concentration; apx, ascorbic peroxidase activity; mda, malon-dialdehyde; sti, stress tolerance index; mp, mean productivity; gmp, geometric mean productivity; hmp, harmonic mean productivity; ssi, stress susceptibility index; tol, tolerance; ati, abiotic tolerance index; sspi, stress susceptibility percentage index; pev, press evaluation; rdy, relative decrease in yield. negative and positive correlations are indicated by red and blue cells, respectively. color darkness scales with correlation strength (ǀ*r*ǀ) (significance: *r* ≥ 0.40 at *p* < 0.05*; *r* > 0.50 at **p* < 0.01). genetic resources (2025), 6(12), 171–193 biochemical traits and ssr markers in wheat 181 principal components analysis and biplot graphic display based on drought tolerance indices and studied traits in rainfed conditions pca is calculated based on the mean of traits and genotypes. the results of both are shown in figure 3. the pca result for traits is presented in table 7. it demonstrates that the first four components, with eigenvalues greater than one, contributed the most to explaining the variance in the dataset. specifically, the first component explained 42.303% of the variance. the second component accounted for 21.78%. the third component contributed 9.612%. the fourth component explained 7.353%. together, these four components explained 81.05% of the variance. the first component was characterized by positive and high coefficients for the grain yield trait and the mp, gmp, hmp, sti, ati, tol and sspi indices, as well as negative and high coefficients for the rdy index. this component was labelled the drought-tolerant pca. the second component had positive and high coefficients for the ssi, pev, tol, sspi and ati indices, along with negative and high coefficients for grain yield and the superoxide dismutase enzyme. this figure 3. biplot diagram of principal components analysis for drought tolerance indices and studied traits of wheat genotypes in rainfed conditions. component was referred to as the drought-stress pca. the third component showed positive and high coefficients for the soluble protein and proline traits, while having negative and high coefficients for the peroxidase and ascorbic peroxidase enzymes. the fourth component was defined by positive and high coefficients for the catalase enzyme activity and malondialdehyde traits, and negative and high coefficients for proline and peroxidase enzyme. according to the data, a biplot of the first two principal components was generated to analyze the traits and indicators under investigation. based on the biplot (figure 3), genotypes 10, 15, 6, 18, 13, and the pishtaz cultivar, which were positioned near the vectors corresponding to the most effective drought tolerance indicators (mp, sti, gmp and hmp), demonstrated high yields in rainfed and irrigated conditions. furthermore, in rainfed conditions, traits such as grain yield, superoxide dismutase activity, ascorbic peroxidase activity, proline content, malon-dialdehyde levels, and catalase enzyme activity were consistent with group a genotypes (those with high yield in both rainfed and irrigated conditions). conversely, genotypes 22, 11, 4, 1, 5, 3, 12 and 16 exhibited the lowest levels of drought tolerance based on the selected indices, particularly the rdy index. genetic resources (2025), 6(12), 171–193182 bavandpouri et al determination of the genetic variability of wheat genotypes based on ssr markers after evaluating 20 primer pairs across 25 bread wheat genotypes, 16 primers exhibiting high levels of polymorphism. 33 out of 35 total bands, showed high polymorphism, (93.75%). on average, each primer produced 2 bands, with a mean polymorphism of 2 bands per primer. the highest number of alleles was detected with primer xgwm136 (five). the primers xgwm155, xgwm234, xcfd168, xgwm577, xgwm642 and xcfd5 exhibited the highest polymorphic information content indices. among the molecular indices assessed, the highest marker index values were identified for primers xgwm136, xcfd168 and xgwm350. the primers xgwm136, xgwm350, xcfd168 and xgwm165 recorded the highest effective multiplex ratio. regarding resolving power, the primers xgwm4, xcfd168 and xgwm350 showed the highest values (table 8). the ssr markers banding pattern generated by the xgwm2, xgwm124, xgwm4, and xcfd5 primers for the wheat genotypes examined in this study is illustrated in figure 4a–d. table 7. principal components analysis of 25 wheat genotypes in rainfed conditions. gy, grain yield; pod, peroxidase activity; sod, superoxide dismutase activity; cat, catalase activity; protein, soluble protein; pc, proline concentration; apx, ascorbic peroxidase activity; mda, malon-dialdehyde; sti, stress tolerance index; mp, mean productivity; gmp, geometric mean productivity; hmp, harmonic mean productivity; ssi, stress susceptibility index; tol, tolerance; ati, abiotic tolerance index; sspi, stress susceptibility percentage index; pev, press evaluation; rdy, relative decrease in yield. traits and indices component 1 component 2 component 3 component 4 gy 0.266 -0.330 0.085 -0.030 pod 0.008 0.070 -0.494 -0.344 sod 0.138 -0.234 -0.124 0.170 cat -0.010 -0.153 -0.135 0.604 protein -0.069 0.097 0.592 0.077 pc 0.034 -0.092 0.345 -0.489 apx 0.097 -0.178 -0.469 -0.197 mda 0.036 0.005 -0.064 0.441 sti 0.341 -0.155 0.052 -0.023 mp 0.349 -0.120 0.064 -0.002 gmp 0.342 -0.151 0.064 -0.010 hmp 0.334 -0.182 0.063 -0.018 ssi 0.179 0.429 -0.044 -0.001 tol 0.270 0.332 -0.016 0.050 ati 0.318 0.227 -0.009 0.055 sspi 0.270 0.332 -0.016 0.050 pev 0.179 0.429 -0.044 -0.001 rdy -0.341 0.155 -0.052 0.023 eigenvalues 7.61 3.92 1.73 1.33 % of variance 42.303 21.78 9.612 7.353 cumulative % 42.303 64.08 73.691 81.05 molecular variance analysis the molecular variance analysis (amova) for the ssr markers is presented in table 9. accordingly, a significant difference between the groups was observed at the 5% probability level. the proportion of variance attributed to intergroup differences was 10%, while intragroup variance accounted for 90%. investigating the relationship of studied characteristics and indices with ssr markers the critical step in this process is assessing the efficiency of linkage markers associated with quantitative traits and identifying informative markers. to pinpoint alleles influencing grain yield, biochemical traits and drought tolerance indices in wheat genotypes under irrigated and rainfed conditions, an association analysis was conducted. this analysis examined the relationship between eight measured traits and ten indices (as dependent variables) and the molecular markers under study (as independent variables) using stepwise multiple regression analysis (table 10, table 11 and table 12). the relationship with ssr markers was analyzed exclusively for characteristics that were statistically significant in the variance analysis. genetic resources (2025), 6(12), 171–193 biochemical traits and ssr markers in wheat 183 table 8. molecular characteristics of more effective ssr primers in bread wheat genetic diversity in the present study. marker no. of polymorphic bands polymorphic information content marker index effective multiplex ratio resolving power xgwm350 3 0.337 1.011 3 3.12 xgwm155 1 0.499 0.499 1 0.96 xgwm577 2 0.467 0.934 2 1.60 xgwm642 1 0.461 0.23 0.5 0.72 xgwm136 5 0.352 1.76 5 2.32 xgwm165 3 0.324 0.973 3 2.96 xgwm4 2 0.211 0.422 2 3.52 xcfd5 2 0.442 0.883 2 1.76 xcfd168 3 0.489 1.466 3 3.20 xgwm234 1 0.493 0.493 1 0.88 figure 4. patterns of some ssr markers used in the present study in wheat genotypes. a, xgwm2 primer; b, xgwm124 primer; c, xgwm4 primer; d, xcfd5 primer. table 9. molecular variance analysis (amova) of wheat genotypes. *, significant at 5% probability level. predicted group source of variation df ss ms estimated variance percentage of total variance φpt 4 among groups 3 25.85 8.62 0.68 10 0.039* within groups 21 126.83 6.04 6.04 90 total 24 152.68 6.72 100 genetic resources (2025), 6(12), 171–193184 bavandpouri et al grain yield and biochemical characteristics in rainfed conditions the analysis identified two markers, xgwm124(a2) and xgwm410(a1), as significantly related to yield in rainfed conditions, explaining 34% of the variation (table 10). additionally, the marker xgwm124(a2) showed a strong correlation with superoxide dismutase enzyme activity in rainfed conditions, accounting for 12% of the variation. the catalase enzyme activity was notably associated with the marker xcfd168(a3), contributing 16% to the observed variance. similarly, the ascorbic peroxidase enzyme activity displayed significant associations with three markers (xgwm2(a1), xgwm234(a1), and xgwm350(a2)), collectively explaining 52% of the variance. moreover, a single locus amplified by the marker xgwm155(a1) was significantly associated with the malon-dialdehyde trait, accounting for 18% of the total variation. overall, eight gene loci were identified as being associated with yield and biochemical characteristics in rainfed conditions. notably, the xgwm124(a2) marker was shared between grain yield and the superoxide dismutase enzyme activity, highlighting its importance. grain yield and biochemical characteristics in irrigated conditions the analysis revealed that grain yield was significantly correlated with seven amplified loci, including xgwm577(a2, a1), xgwm136(a3, a4), xgwm265(a1), xgwm410(a1), and xgwm2(a2) (table 11). among these, the loci xgwm136(a3), xgwm577(a2), and xgwm410(a1) demonstrated the most significant and positive effects. the marker xcfd5(a2) was significantly associated with the superoxide dismutase trait in irrigated conditions, explaining 17% of the variation. for the catalase enzyme activity, the marker xgwm410(a1) contributed 13% to the total variance. altogether, nine gene loci were identified as being linked to yield and biochemical characteristics in irrigated conditions. notably, the xgwm410(a1) marker was shared between grain yield and the catalase enzyme activity, underlining its importance. table 10. markers association with grain yield and biochemical characteristics in rainfed conditions. *, significant at 5% probability level; **, significant at 1% probability level; † a1, a2, a3, a4, and a5 are the average alleles 1, 2, 3, 4, and 5, respectively. traits marker† regression coefficient (b) standard error (se) t-value significance level r2 adjusted r2 grain yield constant 320.44 17.871 17.931 ** 0.397 0.342 xgwm124(a2) -68.482 20.944 -3.27 ** xgwm410(a1) 52.57 22.02 2.39 * ascorbic peroxidase constant 84.99 43.78 1.94 ns 0.583 0.523 xgwm2(a1) 120.18 30.36 3.96 ** xgwm234(a1) -80.304 24.72 -3.25 ** xgwm350(a2) 114.60 44.99 2.55 * malon-dialdehyde constant 0.402 0.009 43.67 ** 0.217 0.183 xgwm155(a1) -0.034 0.013 -2.53 * catalase activity constant 1.24 0.197 6.27 ** 0.193 0.158 xcfd168(a3) 0.62 0.264 2.35 * superoxide dismutase activity constant 0.716 0.086 8.34 ** 0.161 0.124 xgwm124(a2) -0.212 0.101 -2.1 * genetic resources (2025), 6(12), 171–193 biochemical traits and ssr markers in wheat 185 table 11. markers association with grain yield and biochemical characteristics in irrigated conditions. *, significant at 5% probability level; **, significant at 1% probability level; † a1, a2, a3, a4, and a5 are the average alleles 1, 2, 3, 4, and 5, respectively.. traits marker† regression coefficient (b) standard error (se) t-value significance level r2 adjusted r2 grain yield constant 374.72 12.76 29.37 ** 0.885 0.838 xgwm577(a2) 145.7 19.04 7.653 ** xgwm136(a3) 171.744 33.4 5.143 ** xgwm265(a1) -122.541 26.084 -4.7 ** xgwm410(a1) 89.64 18.29 4.902 ** xgwm2(a2) -62.8 18.43 -3.41 ** xgwm577(a1) -58.85 16.294 -3.612 ** xgwm136(a4) -79.04 29.13 -2.713 * superoxide dismutase activity constant 0.233 0.036 6.513 ** 0.206 0.171 xcfd5(a2) 0.113 0.046 2.44 * catalase activity constant 0.55 0.089 6.13 ** 0.163 0.126 xgwm410(a1) 0.39 0.183 2.12 * drought tolerance indices the analysis identified a significant correlation between the ati index and six amplified loci: xgwm136(a3, a4), xgwm577(a2), xgwm2(a2), xgwm410(a1) and xgwm265(a1), collectively explaining 84% of the total variance (table 12). the tol and sspi indices were significantly associated with the markers xgwm136(a3, a4), xgwm265(a2), xgwm577(a2) and xgwm165(a2), accounting for 72% of the variation. additionally, the mp, gmp and hmp indices demonstrated strong associations with three loci amplified by the markers xgwm124(a2), xgwm410(a1) and xgwm165(a1), explaining 57%, 57% and 56% of the total variation, respectively. the ssi and pev indicators were significantly linked to the markers xgwm136(a3, a4) and xgwm265(a2), accounting for 51% of the variance. furthermore, the sti and rdy indices showed significant associations with two loci amplified by the markers xgwm124(a2) and xgwm410(a1), each explaining 48% of the variation. among these, the xgwm410(a1) marker exhibited the most substantial positive effect on the sti index, while the xgwm124(a2) marker had the strongest impact on the rdy index. overall, 35 gene loci were identified for the drought tolerance indicators, with 10 gene loci being common across all measured indices. discussion significant differences in most of the studied characteristics highlighted the genetic diversity among wheat genotypes. this diversity suggests the potential to select superior cultivars based on grain yield and biochemical characteristics in rainfed and irrigated conditions. in addition, based on the percentage of changes in the irrigated environment compared to rainfed (tcp%), grain yield and soluble protein increased under irrigated conditions and decreased with stress. but on the other hand, the activity of peroxidase, superoxide dismutase, catalase, proline content, ascorbic peroxidase and malondialdehyde increased with stress, and the increase in the activity of these biochemical compounds aligns with enhanced stress resistance and reduced stress-induced damage. therefore, the presence of a better antioxidant enzyme system, as evidenced by higher pod, sod, cat and apx activities in drought-tolerant wheat genotypes, could indicate that these genotypes are more efficient in removing superoxide anions produced in plants due to drought stress. similarly, saedmoucheshi et al (2019) reported significant differences among genotypes for all yield and biochemical traits in triticale under regular irrigation and drought stress conditions. furthermore, they observed significant increases in proline, malondialdehyde, protein content and antioxidant enzyme activities in response to drought stress, which aligns with the findings of this study. in a study by pour-aboughadareh et al (2022) evaluating biochemical traits in wild relatives of wheat under drought stress, anova results revealed significant variations across growth conditions, except for dry matter in control and drought stress environments. additionally, the activities of all antioxidant enzymes increased compared to the control conditions, which is consistent with current research. genetic resources (2025), 6(12), 171–193186 bavandpouri et al table 12. markers association with drought tolerance indices of wheat genotypes. *, significant at 5% probability level; **, significant at 1% probability level; †, a1, a2, a3, a4, and a5 are the average alleles 1, 2, 3, 4, and 5, respectively. sti, stress tolerance index; mp, mean productivity; gmp, geometric mean productivity; hmp, harmonic mean productivity; ssi, stress susceptibility index; tol, tolerance; ati, abiotic tolerance index; sspi, stress susceptibility percentage index; pev, press evaluation; rdy, relative decrease in yield. indices marker† regression coefficient (b) standard error (se) t-value significance level r2 adjusted r2 ati constant 21398.56 2357.34 9.08 ** 0.881 0.841 xgwm136(a3) 38568.94 6801.55 5.67 ** xgwm577(a2) 23254.62 3825.74 6.08 ** xgwm2(a2) -18557.49 3870.85 -4.79 ** xgwm410(a1) 16573.71 3837.98 4.32 ** xgwm265(a1) -17499.84 5480.78 -3.19 ** xgwm136(a4) -17941.44 5977.63 -3 ** sspi constant 17.01 2.07 8.23 ** 0.775 0.716 xgwm136(a3) 26.8 4.25 6.31 ** xgwm136(a4) -13.24 3.5 -3.79 ** xgwm265(a2) -4.67 2.1 -2.22 * xgwm577(a2) 5.81 2 2.91 ** xgwm165(a2) -5.56 2.22 -2.5 * tol constant 134.65 16.35 8.24 ** 0.775 0.716 xgwm136(a3) 212.14 33.63 6.31 ** xgwm136(a4) -104.84 27.68 -3.79 ** xgwm265(a2) -36.93 16.6 -2.23 * xgwm577(a2) 46 15.81 2.91 ** xgwm165(a2) -43.99 17.59 -2.502 * mp constant 393.77 17.393 22.64 ** 0.619 0.565 xgwm124(a2) -87.581 20.85 -4.201 ** xgwm410(a1) 61.89 21.85 2.833 ** xgwm165(a1) -72.78 34.633 -2.101 * gmp constant 386.27 16.91 22.85 ** 0.619 0.565 xgwm124(a2) -84.36 20.27 -4.163 ** xgwm410(a1) 59.544 21.24 2.804 * xgwm165(a1) -73.03 33.67 -2.17 * hmp constant 378.97 16.64 22.78 ** 0.613 0.558 xgwm124(a2) -81.242 19.94 -4.074 ** xgwm410(a1) 57.29 20.89 2.742 * xgwm165(a1) -73.24 33.13 -2.211 * ssi constant 1.11 0.121 9.16 ** 0.575 0.514 xgwm136(a3) 1.36 0.262 5.2 ** xgwm136(a4) -0.824 0.231 -3.56 ** xgwm265(a2) -0.309 0.147 -2.1 * pev constant 0.314 0.034 9.16 ** 0.575 0.514 xgwm136(a3) 0.386 0.074 5.2 ** xgwm136(a4) -0.233 0.065 -3.57 ** xgwm265(a2) -0.087 0.042 -2.1 * sti constant 0.968 0.081 11.89 ** 0.526 0.483 xgwm124(a2) -0.416 0.095 -4.36 ** xgwm410(a1) 0.294 0.1 2.93 ** rdy constant -1416.28 127.5 -11.11 ** 0.526 0.483 xgwm124(a2) 650.93 149.42 4.36 ** xgwm410(a1) -460.43 157.09 -2.93 ** genetic resources (2025), 6(12), 171–193 biochemical traits and ssr markers in wheat 187 in a study on 20 bread wheat cultivars under water stress and non-stress conditions, water stress caused a significant 54.9% reduction in grain yield and reductions in all studied traits except grain protein content (al-naggar et al, 2020), contrasting with the present study regarding protein content. similarly, firouzian et al (2023) reported that stress reduced yield components, physiological traits, and ultimately decreased grain yield by about 25% in bread wheat, while, in the present study, drought stress reduced grain yield by 28.3%. in a study investigating terminal heat stress effects on wheat cultivars, variance analysis of phenological traits, grain yield and biochemical traits showed significant variations in genotypes, environments, and genotype × environment interactions (for grain yield, sod, pod, apx, cat and proline) (kumar et al, 2023a). in the present study, variance analysis also revealed significant variations for grain yield and all biochemical traits in the environment effect, for all traits except soluble protein in the genotype effect, and for all traits except grain yield and malon-dialdehyde in the interaction effects. similarly, in a study by mkhabela et al (2019) investigating drought-tolerant wheat genotypes under drought stress and non-stress conditions, the effects of genotype, stress condition, and genotype × stress condition interaction were significant for the tested traits, indicating differential genotypic responses to selection, in agreement with this study. estimating heritability helps plant breeders identify elite genotypes (farshadfar, 2010). likewise, genetic advancement reflects the mean genotypic value relative to the parental population and serves as an indicator of the genetic gain achieved through selection (kumar et al, 2023b). high broad-sense heritability suggests that the trait is minimally influenced by environmental factors. however, modifying such a trait may be less beneficial, as broadsense heritability encompasses the total genetic variance, including additive (fixable), dominance and epistasis (nonfixable) variances. on the other hand, high genetic advance or genetic gain indicates that the trait is primarily governed by additive genes, making selection a practical approach for improvement. conversely, low genetic advance or gain suggests that the trait is controlled by non-additive genes, in which case heterosis breeding would be a more effective strategy (farshadfar, 2010; kaur et al, 2023). traits with heritability (h² > 60.0%) and genetic gain (gg > 20.0%) indicate that the observed variation is predominantly due to genetic factors, thereby making these traits reliable candidates for selection (faysal et al, 2022; kaur et al, 2023), which in the present study also included most biochemical traits with heritability above 90% and genetic gain above 30%. this indicates the high influence of genetic factors and aligns with the aforementioned findings. in summary, this study showed that broad-sense heritability and genetic gain for catalase, superoxide dismutase activity, proline content and ascorbic peroxidase activity were high under both irrigated and rainfed conditions, and were lowest for malon-dialdehyde. therefore, it is recommended to use these traits as ideal criteria, along with yield, to select high-yielding genotypes in breeding programmes. in research on bread wheat genotypes, moderate heritability values and high genetic gain for grain yield were recorded, suggesting these traits are promising targets for improvement through favorable selection (amare, 2023). in the present study, moderate broad-sense heritability and genetic gain were obtained for grain yield. similarly, in the study by saed-moucheshi et al (2019), grain yield showed heritability values of 32.14% and 29.62% under normal irrigation and drought stress conditions, respectively, indicating environmental influence. additionally, sod and mdh showed the highest heritability under both conditions, while in the present study, cat, sod, pc and apx showed the highest heritability, and mda the lowest heritability in both environmental conditions. the heritability of grain yield was 27.8% and 60.4% in rainfed and irrigated conditions, respectively. in studies by shah et al (2019) on bread wheat under rainfed conditions and sallam et al (2024b) on bread wheat under heat stress, the traits grain protein content, proline and catalase, respectively, showed high heritability and genetic gain, consistent with the study. additionally, various researchers have utilized broad-sense heritability (li et al, 2023; sowadan et al, 2024) and genetic gain (yusuf et al, 2021; dukamo et al, 2023) to examine genetic variability and identify suitable traits for breeding programmes, aligning with the study’s findings regarding the importance of these parameters. these indices are used to calculate the level of drought tolerance in plants. different indices are designed based on different traits that are related to grain yield. these indices are used in different agronomic, biochemical, molecular and even cytogenetic categories to select the best genotype. the correlation heatmaps were created to analyze the relationships between studied traits and drought tolerance indices in rainfed and irrigated conditions. based on the nature of the indicators, it was observed that most of the studied traits showed a highly significant correlation with indices such as sti, mp, gmp, hmp and rdy. these indicators were identified as the most effective for selecting drought-tolerant and high-performing genotypes. additionally, these indices had a strong influence on the first principal component, which was identified as the drought tolerance component. similar to the present study, reddy et al (2023), used correlation heatmaps to examine the relationship between phenotypic traits and drought tolerance indices such as sti, mp, and gmp. also, in the study by giovenali et al (2023), pearson correlation coefficients were analyzed using heatmaps to investigate the relationships between yield-related traits, physiological parameters and biochemical parameters, and significant positive and negative correlations were obtained. in a related study, correlation heatmaps were employed to explore the relationships between phenological, physiological and biochemical variables in optimal conditions, heat stress conditions, prolonged heat stress conditions, and a combined environment. in optimal conditions, the correlation between seed yield and the apx and cat traits was positive but not statistically significant. in heat stress conditions, a positive and significant correlation was observed between seed yield and the traits proline and sod. when heat stress was prolonged, the correlation between seed yield and cat became negative and was not significant. however, in high-temperature conditions, seed yield demonstrated a positive and significant correlation with proline, sod and pod, while its relationship with apx remained positive but non-significant (kumar et al, 2023a). in the present study, cat and apx did not show statistically significant correlations with any of the drought tolerance indices. under irrigation conditions, there was a significant negative correlation between pc and the ssi and pev indices. there was also a significant positive correlation between pod and the sti, mp, gmp and hmp indices, and a genetic resources (2025), 6(12), 171–193188 bavandpouri et al significant negative correlation with the rdy index. under rainfed conditions, the correlation of sod with the sti, mp, gmp and hmp indices was positive and significant, and with the rdy index was negative and significant. principal component analysis (pca), a multivariate statistical method, serves as an efficient approach to data reduction by identifying strong correlations among variables to derive clear conclusions. in this study, pca and biplot visualization were applied to analyze traits across 25 bread wheat genotypes under rainfed conditions. the first two principal components (pc1 and pc2) accounted for 64.08% of the total variation in drought tolerance indices and studied traits. biplot visualization revealed considerable genetic diversity among genotypes in response to drought stress. these findings align with existing literature: pouraboughadareh et al (2022) reported pc1 and pc2 explaining 64.52% of biochemical variation in wild wheat species under drought (pc1 = 47.86%; pc2 = 16.66%). sallam et al, (2024a) identified four principal components (eigenvalues >1) capturing 89.79% of variance across 30 agro-physiobiochemical traits. pc1 correlated with 24 traits (e.g. grain yield, catalase, peroxidase, superoxide dismutase and proline), pc2 with five traits (e.g. soluble protein), pc3 showed no significant associations, and pc4 linked to glycine betaine. similarly, in the present study, the first four principal components explained 81.05% of total variance: pc1 (grain yield and drought-tolerance indices), pc2 (drought-stress indices), pc3 (soluble protein and proline), and pc4 (catalase activity and malon-dialdehyde). of the 20 ssr markers tested, 16 showed significant polymorphism. genetic diversity assessment of bread wheat genotypes utilizing ssr markers revealed xcfd168, xgwm350 and xgwm136 as fully polymorphic (100%). these markers demonstrated the highest allele counts and superior performance across key genetic indices: polymorphism information content (pic), marker index (mi), effective multiplex ratio (emr), and resolving power (rp) (table 8). consequently, these represent optimal candidates for advanced wheat genetic analyses. notably, the significant discriminatory power achieved with limited primer sets confirms that highly polymorphic ssrs efficiently differentiate both individual accessions and population subgroups. these findings corroborate prior research identifying the same three markers as exceptionally informative. for instance, the marker xgwm136 was similarly highlighted by budak et al (2013) and kaur et al (2016), while khan et al (2021) identified xgwm136 and xcfd168, and haque et al (2020) emphasized xgwm350, collectively supporting their utility as reported in this study. in genomic diversity research on bread wheat using ssr markers, markers xgwm136, xcfd168, xgwm2, xgwm155, xcfd5, xgwm165, xgwm33 and xgwm129 were used (ahmed et al, 2020), consistent with the marker selection in this study. additionally, research on bread wheat genetic diversity revealed high pic and marker index, showing greater diversity in the a and b genomes compared to the d genome (feltaous, 2019). in the present study, more diversity was observed in the b genome, followed by d and a. in another study, 17 bread wheat genotypes evaluated with 16 ssr markers showed only 11 markers with high polymorphism and reproducibility (kara et al, 2020). similarly, a study assessing the genetic diversity and population structure of wheat genotypes employed ten ssr markers to characterize diversity across 22 genotypes (hassan et al, 2025). here, 25 bread wheat genotypes examined with 20 ssr markers revealed 16 with significant polymorphism. regarding pic values and marker utility, these findings align with prior reports. for example: in ssr evaluation of bread wheat, the pic ranged from 0.276 to 0.541 (average: 0.384), using primers xgwm192 and xgwm642 (islam et al, 2012). three subsequent studies (el-rawy and hassan, 2021; ahmed et al, 2024; bavandpouri et al, 2025) reported a pic range of 0.20–0.50 (average: 0.33). el-rawy and hassan (2021) utilized primers xgwm165, xgwm155, and xgwm577, with xgwm577 showing superior performance. bavandpouri et al (2025) introduced three markers – namely xcfd168, xgwm350, and xgwm136 – as the most significant; while ahmed et al (2024) highlighted xgwm642. in a separate analysis of ten bread wheat genotypes using ten ssr markers, kumari et al (2025) detected 64 polymorphic bands, where alleles per locus ranged from 1 to 4 (highest for xgwm2 and xgwm265). in the present study, 33 out of 35 bands were polymorphic. the highest number of alleles (five) was observed for primer xgwm136, while the lowest number (two alleles) was recorded for primers xgwm155, xgwm410 and xgwm234. collectively, these findings confirm ssr markers as reliable indirect selection tools for more efficient cultivar improvement. genetic structure within populations is commonly analyzed through variance analysis, where the variance between and within groups is determined based on the genetic distances among individuals. amova is particularly effective in partitioning variance in wild species and among groups of cultivars originating from different regions (farshadfar, 2023). the results of amova revealed that the observed grouping of bread wheat genotypes could, to some extent, be explained by the diversity in ssr marker bands. the φpt statistic is employed as a criterion to test the assumption of population differentiation at the relevant level. in this experiment, amova indicated that the φpt statistic was low due to the high genetic diversity observed within the populations. similar findings were reported in a study investigating the genetic diversity of bread wheat using issr and ssr markers, where amova for both types of markers revealed that genetic variation within species surpassed the genetic diversity among them (jabari et al, 2023). additionally, in another study, amova results demonstrated that 19% of the total genetic variation occurred among subpopulations, while the remaining 81% was attributed to individual differences within each subpopulation (sowadan et al, 2024). in drought tolerance research, pinpointing qtls linked to drought-responsive traits is pivotal for deciphering their genetic mechanisms (sallam et al, 2019). to identify relevant ssr markers, regression analysis was conducted between grain yield and biochemical traits under rainfed and irrigated conditions, with ten drought tolerance indices as dependent variables and marker gene locations as independent variables. results revealed significant trait–primer relationships. a key advantage of this multivariate regression approach is its efficiency in qtls detection, reducing time and cost while eliminating the need for mapping populations (ruan et al, 2009). this study specifically aimed to identify alleles correlated with grain yield and biochemical traits as informative markers. outcomes supporting this objective are detailed in table 10, table 11 and table 12. critical associations include: xcfd168 marker showing strong correlation with catalase activity under rainfed conditions; xgwm350 linked genetic resources (2025), 6(12), 171–193 biochemical traits and ssr markers in wheat 189 to ascorbic peroxidase activity (rainfed); and xgwm136 associated with yield under irrigated conditions and ati, tol, sspi, ssi and pev indices. notably, xgwm410(a1) correlated with yield in both environments, catalase activity under irrigation, and multiple indices; xgwm2(a2) tied to irrigated yield, rainfed ascorbic peroxidase activity, and ati; while xgwm124(a2) demonstrated associations with yield, rainfed superoxide dismutase activity, and several indices. in a study investigating associations between biochemical traits and stress tolerance indices in wheat under drought using 24 ssr markers, two markers were linked to apx and three to cat in control conditions, whereas under drought stress, two markers associated with apx and one with pod, alongside two markers significantly correlated with the sti index. these results suggest genomic regions governing growth and developmental characteristics across conditions (pour-aboughadareh et al, 2022). comparatively, in the current study, biochemical trait analysis revealed one marker correlated with sod and one with cat under irrigation, while under rainfed conditions, one marker associated with sod, one with cat, three with apx and one with mda. regarding sti index, consistent with pour-aboughadareh et al (2022), two markers showed significant correlations. in a linkage mapping study for photosynthesis and yield traits under moisture stress and drought indices (ssi and sti) in winter bread wheat, 28 linkages were identified for drought tolerance indices, with one marker consistently associated across two seasons (saeed et al, 2017). here, 35 linkages emerged for drought indices, four of which were associated with both ssi and sti. el-rawy and hassan (2021) reported ssr markers xgwm260 and xgwm573 as specific to drought-tolerant bread wheat genotypes (low dsi values), suggesting marker-trait associations for drought tolerance. in contrast, our study identified xgwm136 and xgwm265 as significantly associated with ssi. negisho et al (2022) detected 184 marker-trait associations (mtas) for drought indices in ethiopian durum wheat, with six mtas (on chromosomes 2b, 3b, 4a, 5b and 6b) positively affecting gygmp. notably, 41 mtas (22.28%) associated with ≥ 2 indices, of which 16 (39.02%) linked to gmp and sti. similarly, we identified an mta positively affecting gmp on chromosome 2b, along with 35 gene loci for drought indices –10 (28.57%) common to all indices, with 30% of stable mtas associated with gmp and sti. across these studies, a positive regression coefficient indicates that selecting genotypes harbouring such alleles may enhance yield and drought tolerance. the association between individual markers and multiple traits may arise from pleiotropic effects or overlapping qtls influencing diverse characteristics. primers such as xgwm136, xgwm234 and xcfd168 – previously used to investigate grain yield and agronomic traits relationships via ssr markers with potential for heat-tolerance breeding (khan et al, 2021) – were similarly employed in this study, with xcfd168 and xgwm136 emerging as superior markers. consistent with our findings, numerous studies report significant yield-marker associations: maccaferri et al (2011) established xgwm410–yield relationships; amalova et al (2024) documented correlations between grain yield and xgwm124. bavandpouri et al (2025) reported significant relationships between xgwm265 and grain yield under irrigated conditions, and between xgwm410, xgwm577, and xgwm124 markers and grain yield under both rainfed and irrigated conditions, while eldemery et al (2022) and firouzian et al (2023) observed xgwm577–yield linkages under heat stress. concurrently, xgwm165 – also utilized here – showed notable associations with sspi, tol, mp, gmp, and hmp indices, aligning with our results and collectively reinforcing these outcomes. ultimately, this research confirms that molecular markers exhibiting strong regression coefficients for biochemical traits and drought tolerance indicators offer breeders actionable insights. such markers enable selection of environmentally stable qtls linked to yield and drought tolerance, accelerating the development of superior genotypes. moreover, the identified mtas hold direct utility in wheat breeding programmes targeting drought stress, particularly for marker-assisted selection and gene pyramiding strategies. conclusion molecular markers linked to biochemical traits can accelerate the identification of drought-tolerant germplasm, enhancing breeding efficiency. significant genotypic variance confirmed substantial genetic diversity and differential drought stress responses. key biochemical traits – catalase (cat), superoxide dismutase (sod) activity, proline content (pc), and ascorbate peroxidase (apx) activity – exhibited high heritability (> 90%) and genetic advance (> 30%) under both irrigated and rainfed conditions, unlike malondialdehyde (mda). thus, these traits are recommended for selecting high-yielding genotypes. principal component analysis (pca, 64.08% variance explained) and correlation identified stress tolerance index (sti), mean productivity (mp), geometric mean productivity (gmp), harmonic mean productivity (hmp), and relative decrease in yield (rdy) as the most effective drought tolerance indices, strongly correlated with grain yield and biochemical traits. genotypes 6, 10, 15, 18, 13, and pishtaz demonstrated superior drought tolerance, high yield potential, and optimal biochemical performance (sod, apx, pc, mda, cat) under stress. among 20 ssr markers, 16 showed significant polymorphism. markers xcfd168 (rainfed-cat), xgwm350 (rainfedapx), xgwm124(a2) (yield, rainfed-sod, multiple indices), xgwm136 (irrigated yield, ati, tol, sspi, ssi, pev), xgwm410(a1) (yield in both environments, irrigated-cat, multiple indices), and xgwm2(a2) (irrigated yield, rainfedapx, ati) exhibited significant trait associations. these markers are strongly recommended for marker-assisted breeding to improve yield and drought tolerance. acknowledgments we gratefully acknowledge the administration of razi university, kermanshah, for providing the laboratory and field facilities necessary to conduct this research. we also extend our thanks to the karaj seed and plant improvement institute for supplying the plant seeds. it is important to note that while razi university provided the research infrastructure, it did not provide specific funding for the publication of this article. genetic resources (2025), 6(12), 171–193190 bavandpouri et al author contributions conceptualization: ezatollah farshadfar, kianoosh cheghamirza; data curation: fatemeh bavandpouri; methodology: fatemeh bavanpouri; formal analysis: fatemeh bavandpouri, mohsen farshadfar; investigation: ezatollah farshadfar; funding acquisition: ezatollah farshadfar, mohsen farshadfar; project administration: fatemeh bavanpouri, kianoosh cheghamirza; visualization: fatemeh bavanpouri, mohsen farshadfar; supervision: ezatollah farshadfar, kianoosh cheghamirza; resources: fatemeh bavandpouri, mohsen farshadfar; writing – original draft preparation: fatemeh bavanpouri; writing – review and editing: fatemeh bavandpouri, ezatollah farshadfar. all authors have read and agreed to the published version of the manuscript. conflict of interest statement the authors have declared that no competing interests exist. data availability statement the datasets collected and analyzed for this study are available upon reasonable request. references ahmed, h. g. m. d., kashif, m., rashid, m. a. r., sajjad, m., and zeng, y. 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(2025) “egg production characteristics of several bulgarian chicken breeds”, genetic resources, 6(12), pp. 120–129. doi: 10.46265/genresj.qniv2785. © copyright 2025 the authors. this is an open access article distributed under the terms of the creative commons attribution license (cc by 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. hristo lukanova, ivelina pavlovab,*, atanas gencheva, todor petrova atrakia university, faculty of agriculture, department of animal husbandry non-ruminant animals and special industries, student campus, stara zagora, bulgaria btrakia university, faculty of veterinary medicine, department of general livestock breeding, student campus, stara zagora, bulgaria * corresponding author: ivelina pavlova (ivelina.hristova@trakia-uni.bg) introduction global poultry production has steadily increased, driven by rising demand for affordable and accessible animal protein sources (oecd-fao, 2017). according to oecd-fao (2024), of the approximately 354 million tonnes of meat produced worldwide in 2023, around 139 million tonnes were from poultry, making it the leading category with nearly 40% of the total production. the majority of this poultry meat production is chicken, with about 103.5 million tonnes produced in 2023 (usda fas, 2024). a similar trend is observed in egg production, with approximately 97 million tonnes produced in 2023, of which around 94% were chicken eggs (fao, 2024). regionally, the eu produced about 13.3 million tonnes of poultry meat (eurostat, 2024) and 6.7 million tonnes of eggs (ec, 2023a) in 2023. this highlights the dominant role of domestic chickens in both global and regional poultry farming. in bulgaria, poultry farming is one of the most advanced livestock sectors, with poultry meat accounting for over half of the country’s total meat production (genchev and lukanov, 2025). parallel to the positive development of the poultry sector, a negative trend is observed regarding the preservation of genetic diversity in domestic chickens (malomane et al, 2019). in modern industrial poultry farming, highly productive lines from just a few chicken breeds are used (teneva et al, 2015; preisinger, 2021) out of the vast number of breeds known worldwide. in addition to productive purposes, the domestic chicken serves a variety of other functions in human life (ornamental, exhibition, sporting, etc.), which form the foundation for the breed diversity observed within the species https://doi.org/10.46265/genresj.qniv2785 https://www.genresj.org https://doi.org/10.46265/genresj.qniv2785 mailto:ivelina.hristova%40trakia-uni.bg?subject= genetic resources (2025), 6(12), 120–129 egg production of bulgarian chicken breeds 121 (lukanov, 2017a). historically, numerous attempts have been made to classify chicken breeds based on factors such as origin, purpose, plumage, body size and other characteristics (bdrg, 2006; roberts, 2008; apa, 2023; kochish et al, 2023). among these, the combined classification appears to be the most comprehensive. it categorizes chicken breeds into the following groups: meat breeds, egg-laying breeds, dual-purpose breeds, fighting breeds and ornamental breeds (including long-tailed, long-crowing, true bantams, miniature breeds and other ornamental varieties)(lukanov, 2017a). the preservation of breed and genetic diversity in domestic chickens globally is predominantly attributed to the efforts of hobbyist poultry breeders, in conjunction with national poultry genetic centres, where such institutions are present (teneva et al, 2015; pavlova and lukanov, 2024). in bulgaria, a total of ten chicken breeds are recognized, seven of which are of standard body size, and three are true bantams (lukanov, 2023). birds that do not exhibit signs of dwarfism are considered standard breeds, whereas those that do are classified as bantams. these include the katunitsa chicken, black shumen chicken, stara zagora red chicken, struma chicken, southwest bulgarian chicken, bulgarian longcrower, rhodope painted chicken, bregovska dzhinka, struma bantam, and southwest bulgarian dzhinka (lukanov and pavlova, 2021; pavlova and lukanov, 2024). among these, the struma chicken (sch), rhodope painted chicken (rpch), southwest bulgarian chicken (swbch) and bulgarian longcrower (bl) share a similar geographical origin in southwestern bulgaria (lukanov, 2023). the referenced study has investigated the incubation characteristics of eggs from these four breeds, while another has examined the exterior traits of these local bulgarian chicken breeds (pavlova and lukanov, 2023). all four breeds are of standard size (standard chicken breeds), with three of them (swbch, sch and bl) being typical ornamental breeds, while rpch can be classified as a dual-purpose breed. egg production is one of the most important economic factors in the poultry industry (el-sabrout et al, 2022), as it is essential for both table egg production and hatching eggs. in this context, the traits that characterize egg productivity are significant for various branches of poultry farming, including backyard and ornamental poultry. to date, there has been no assessment of rpch, swbch, bl and sch egg-laying productivity, which would reveal their potential in this area. in this context, the aim of the present study was to investigate and analyze the egg productivity of rpch, swbch, bl and sch breeds. material and methods experimental design the study was conducted from september 2022 to november 2023 at the experimental station of the poultry science section, faculty of agriculture, trakia university, stara zagora, bulgaria. for the purposes of the study, hatching eggs were collected for incubation from various breeders of the tested breeds as follows: 276 hatching eggs from the rpch breed (6 farms, 6 breeding groups), 366 from the bl breed (4 farms, 7 breeding groups), 254 from the swbch breed (5 farms, 5 breeding groups), and 180 from the sch breed (2 farms, 5 breeding groups). the resulting chicks were reared at the same experimental station until the beginning of the trial. the study included typical representatives of the rhodope painted chicken (rpch), southwest bulgarian chicken (swbch), bulgarian longcrower (bl), and struma chicken (sch) breeds, all at the same initial age of 140 days. four groups of 25 pullets each were formed, corresponding to the four breeds and designated as rpch, swbch, bl and sch. the study covered one laying cycle, from the onset of egg production (20% laying rate) to the start of natural molting, lasting 52 weeks in rpch, 49 weeks in swbch, 52 weeks in bl, and 40 weeks in sch. the differences in the test period are due to variations in age at sexual maturity among the breeds. experimental bird management the birds were housed on a deep litter system in a semienclosed facility divided into four pens, each measuring 2.5 × 4m, with an initial density of 2.5 birds per m². each group’s housing was fitted with natural light openings of identical size, providing an approximate individual area of 3m² and allowing continuous exposure to diffused natural daylight throughout the full natural photoperiod. perches were provided at one end of the pen, ensuring a minimum perch space of 20cm per bird. a nest was provided for every five hens (a module of five individual nests in each pen). the facilities were equipped with manually refillable pan feeders and automatic cup drinkers, appropriately adjusted to the number of birds in each pen (genchev and lukanov, 2025). feeding was ad libitum with a balanced compound feed in two phases: pre-laying and laying phase (table 1). feed consumption was recorded daily by weighing the feed residue remaining 24 hours after the feed was supplied, and was calculated as the average daily feed intake (adfi) per bird. eggs were collected regularly throughout the period of highest laying activity, from morning until early afternoon, with an additional collection in the late afternoon. the total number of eggs collected per day was considered the daily yield and was used to calculate daily egg production. following collection, the eggs were weighed to calculate the average daily egg weight. climate control was not implemented due to the facility's specifics and the birds' management system. temperature (instantaneous, minimum and maximum) was monitored using a digital thermometer (tfa dostmann ltd.) installed in the ‘birds' room’, away from direct sunlight. the minimum recorded temperature during the entire period was -6.6°c on 11 february 2023, while the maximum was 38.5°c on 4 august 2023. the presented data on ambient temperature refer to the average daily values recorded by the stara zagora meteorological station. for the purposes of the study, a lighting programme with additional artificial lighting was used, similar to those applied in intensive poultry farming in open-house systems. in our conditions, the birds were housed with a natural day length of approximately 12 hours. additional artificial lighting was applied for ten days (until they reached 150 days of age) to gradually extend the day length to 14 hours by the end of these ten days. at 157 days of age, the day length was further increased by one hour, reaching 15 hours, with nine hours of darkness. two weeks later (171 days of age), the day length was increased by one more hour, reaching 16 hours of daylight and eight hours of darkness. by the end of the test period, a day length of 16 hours was maintained. the extension of the photoperiod was accomplished by delaying the onset of the dark phase, with artificial lighting provided following the end of the natural daylight period. genetic resources (2025), 6(12), 120–129122 lukanov et al table 1. nutritional composition of the compound feed used. *, time of first egg production in the group component pre-lay phase (140 days of age – maturity*) laying phase (whole egg-laying period) metabolized energy, mj/kg 11.6 11.5 crude protein, % 17.5 17.0 lysine, % 0.75 0.8 methionine, % 0.36 0.35 calcium, % 2.0 3.8 av. phosphorus 0.43 0.38 egg production data collection the following traits were analyzed: age of sexual maturity (at 20% laying rate)(days); average daily feed intake (g); daily egg production and culled eggs, number; daily egg weight (g); livability for the entire production period (including culled birds) (%). the following productive parameters were calculated: egg number per hen-housed; egg-laying intensity (laying rate) (%); feed conversion ratio (per kg of eggs); feed conversion per egg (g feed per egg). the egg production efficiency index (epei) was calculated by using the formula (lukanov et al, 2023): epei = [(l × demp)/fcr)] × 100, where: l is livability for the period (%), demp is daily egg mass produced (kg), and fcr is the feed conversion ratio (kg/kg egg mass). demp = (chdep x aew)/t, where: chdep is the cumulative hen-day egg production for the period (number), aew is the average egg weight (kg), and t is the period (days). statistical analysis statistical analyses were conducted using the ibm® spss® statistics software package (version 26). a one-way analysis of variance (one-way anova) was applied to assess inter-group differences. the following statistical parameters were calculated for data analysis and interpretation: mean value (x̄) and standard error of the mean (sem). data are expressed as mean ± sem. inter-group differences were considered statistically significant at p < 0.05, based on the lsd post hoc test, provided that the assumptions of normality (shapiro–wilk test; n < 50) were met and the anova model was significant (f-test, p < 0.05). microsoft excel 16.0 (2018, windows version) was used for the graphical presentation of the results. results figure 1 illustrates the changes in egg-laying intensity over the entire productive period for the tested groups of hens. peak values of 77.1% laying intensity for rpch group were recorded during the 24th productive week, with an average weekly ambient temperature of 10.4°c. figure 1. egg-laying intensity recorded throughout the entire productive period. rpch, rhodope painted chicken; swbch, southwest bulgarian chicken; bl, bulgarian longcrower; sch, struma chicken. genetic resources (2025), 6(12), 120–129 egg production of bulgarian chicken breeds 123 in contrast to rpch, the swbch group showed a significantly delayed onset of productive maturity, reaching 20% laying rate at 179 days of age. this breed was characterized by a slow increase in laying performance, with the 50% threshold commonly used in industrial poultry production being reached only at 217 days of age. in this group, a sharp increase in laying intensity was observed after the 15th production week, reaching a peak value of 83.9% in the 26th week. a high laying rate was maintained until approximately the 32nd production week, after which the curve showed a marked decline. the swbch group also exhibited a shorter productive period. by week 49, the average weekly egg production had declined to 26.5%, with the majority of birds already undergoing molt. in the bl group, 20% laying intensity was reached at 179 days of age, while the threshold of 50% was attained at 215 days. as with the other studied breeds, a typical laying curve characteristic of intensive poultry systems was not observed. a laying intensity of approximately 70% or higher was maintained between the 18th and 28th productive weeks, corresponding to ambient temperatures favourable for the species. peak average weekly laying performance reached 78.6% during the 26th productive week. unlike swbch birds, bl hens showed no sharp temperature-induced decline. a more substantial decrease was recorded only after the 48th productive week, likely linked to the onset of molting in some individuals and a gradual reduction in egg production within the group, declining to 30.6% by the 52nd productive week. the sch group was identified as the slowest-maturing among all the studied breeds, reaching a 20% laying rate at 250 days of age, with the first egg being laid slightly earlier, at 232 days. the genetic background, combined with the natural rearing conditions, is directly associated with the observed short productive period in the sch group, which lasted for 40 weeks. when analyzing the dynamics of the trait change over the testing period, no significant differences in the laying curve are observed compared to the rpch and bl groups. the later sexual maturity of the birds is also linked to the fact that the first half of the productive period occurred under more favourable ambient temperatures, which likely contributed to the relatively rapid achievement of peak production. for the sch group, peak egg production values can be considered those above 50%, sustained between the 8th and 23rd productive weeks. the highest weekly average values for this trait were recorded in the 13th productive week (66.7%). similar to the other groups, a decline in productivity is observed in parallel with the increasing age of the birds and ambient temperatures, with the dynamics of this decline being comparable to those of rpch and bl groups. the change in egg weight during the productive period in the rpch group followed a typical growth curve with increasing age (figure 2). at the start of the productive period, the average egg weight was 41.8g, gradually rising to the threshold of 53g by the 13th week. as the ambient temperature increased, a significant decline in laying intensity was observed, which coincided with the final third of the productive period. in terms of changes in swbch egg weight, no significant variation was observed throughout the entire laying period. in comparison to rpch, which exhibited a 33.2% difference between the minimum and maximum average weekly egg weight, the variation in swbch was considerably lower, amounting to only 17.9%. at the beginning of the production cycle, the average weekly egg weight was 45.5g, reaching approximately 50g within 5–6 weeks. peak values were recorded in the middle of the laying period (weeks 17–29), coinciding with favourable ambient temperature conditions. unlike rpch, the swbch group was characterized by a significantly lower egg weight, almost entirely falling within the s size category (< 53g) (ec, 2023b). the egg weight in the bl group showed a rate of change throughout the productive period similar to the rpch group, with the difference between the minimum and maximum weekly averages being 32.3%. in this breed, the optimal egg weight of 53g was reached by the 12th productive week and was maintained within the range of 53–62g until the end of the testing period. the highest weekly average egg weight was 62.2g, recorded in the 50th productive week. figure 2. average egg weight recorded throughout the entire productive period. rpch, rhodope painted chicken; swbch, southwest bulgarian chicken; bl, bulgarian longcrower; sch, struma chicken. genetic resources (2025), 6(12), 120–129124 lukanov et al the sch egg weight exhibited the least fluctuation when compared to the reported minimum and maximum weekly average values throughout the entire productive period across all tested groups, with a difference of only 14.33%. this breed also showed the highest initial weekly average weight, which was 53.04g in the first productive week. the maximum weekly average egg weight was recorded in the 38th productive week, at 61.9g. in contrast to all other breeds included in the experiment, only the struma chicken maintained egg weights throughout the entire productive period that consistently fell within the m weight category (53–62g) (ec, 2023b), based on the weekly average. patterns of average daily feed intake were generally similar among the four experimental groups, although rpch exhibited more pronounced fluctuations up to approximately mid-lay (figure 3). the highest intakes were recorded during the first half of the productive cycle, with rpch consistently showing the greatest values (peaking above 160 g/day), followed by swbch, bl and sch. as average daily ambient temperatures increased in late spring and summer, feed intake gradually declined across all groups, remaining at lower levels until the onset of molting or the end of the experimental period. the sch group maintained the most stable intake pattern throughout the cycle, with only minor variations relative to seasonal changes in temperature. the calculated epei for each of the four breeds included in the study is presented in figure 4. this index reflects the efficiency of producing both table and hatching eggs. lower epei values indicate less efficient production. notably, the rpch group recorded the highest efficiency (epei = 65.08), clearly distinguishing itself from the other breeds. the second-highest value was observed in the bl group, although it was 32.6% lower than that of rpch. the least efficient performance was observed in the swbch and sch groups, with epei values of 28.84 and 29.89, respectively. liveability is one of the main parameters influencing the epei. over the entire production period, the overall liveability of hens from the four groups was identical at 80%, with five birds per group lost due to mortality or culling, predominantly during the final stage of the laying period. figure 3. average daily feed intake recorded throughout the entire productive period. rpch, rhodope painted chicken; swbch, southwest bulgarian chicken; bl, bulgarian longcrower; sch, struma chicken. figure 4. egg production efficiency index (epei) calculated for each tested breed. sch, struma chicken; bl, bulgarian longcrower; swbch, southwest bulgarian chicken; rpch, rhodope painted chicken. genetic resources (2025), 6(12), 120–129 egg production of bulgarian chicken breeds 125 table 2 summarizes the results regarding the key parameters related to egg production in the four studied breeds. as previously noted, the rpch group exhibited the earliest onset of sexual maturity, while the sch group was the latest. the remaining two groups (bl and swbch) can be classified as intermediate in terms of sexual maturity. among the studied groups, rpch exhibited the highest productivity, with a total of 223.9 eggs laid over a 52-week production period, corresponding to a laying rate of 57.4%. in contrast, the sch group showed the lowest performance, producing 123.9 eggs − 44.7% less than rpch − which equates to a laying rate of 33.9%. the remaining two groups, bl and swbch, displayed intermediate levels of productivity, with the bl group showing a higher production potential, recording 191.6 eggs during the same 52-week period and a laying rate of 52.5%. according to the data presented in table 2, the breed with the highest egg weight was sch (58.0± 0.54g), while the lowest was observed in swbch (50.9 ± 0.68g) (p < 0.001). the other two groups (bl and rpch) showed mean values similar to the sch group, and their differences from the swbch group were also statistically significant (p < 0.05) considering the average egg weight and the number of eggs produced by the hen-housed, it can be summarized that the highest total egg mass per productive cycle was achieved by the rpch group (approximately 12.7kg), followed by the bl group (10.73kg), swbch (8.49kg) and sch (7.18kg). table 2 presents two expressions of feed converting efficiency, represented as the feed conversion ratio for producing one kilogram of egg mass or the feed required to produce a single egg. in this study, the most cost-effective feed conversion was observed in the rpch group (244.1±9.2g of feed required to produce one egg and 4.34±0.18kg of feed required to yield one kilogram of eggs). conversely, the least efficient feed conversion was observed in the swbch group (328.3±47.95g of feed required to produce one egg and 6.55±1.04kg of feed required to yield one kilogram of eggs). discussion age at sexual maturity is a major factor considered in selection for egg-laying poultry, as it represents an important reproductive trait (xu et al, 2011; liu et al, 2019; genchev and lukanov, 2025). giesbrecht and nordskog (1963) used the 20% level as the lowest point with reliable data when estimating age at sexual maturity, while suggesting 50% as the optimal threshold. some authors propose an even lower threshold – such as a 10% laying rate – when evaluating the onset of maturity in indigenous chicken breeds (schreiter and freick, 2023). hens from the rpch group reached 20% laying rate at 157 days of age, with the age for reaching 50% laying rate, considered a benchmark in productive poultry farming (genchev and lukanov, 2025), being 162 days. compared to the age of sexual maturity in modern highperformance laying hens (around 140–150 days), rpch is relatively close, differing by approximately two weeks. when compared to purebred chickens that have not undergone targeted, scientifically based selection, rpch ranks among early-maturing breeds, reaching sexual maturity at approximately 4.5 to 5.5 months (lukanov, 2017a). the spanish breed asturian painted chicken, which is somewhat similar in exterior to rpch but slightly larger and more massive (pavlova, 2024), shows official data indicating a later maturation age of around 7 months (mapa, 2025). according to the 20% laying intensity and the threshold of 50% in the bl group, they are classified as a typical mediummaturing breed (lukanov, 2017a). the late onset of sexual maturity, like in the sch group, is characteristic of many large chicken breeds (lukanov, 2017a). the curve representing the laying intensity of the tested groups of hens does not follow the typical shape observed in hens raised under controlled microclimatic conditions. this is related to the rearing method, where the birds are kept in natural temperature conditions. in adult birds, the thermoneutral zone can be broadly defined, starting from table 2. egg production parameters of the tested bulgarian chicken breeds. means ± sem followed by the same letter are not significantly different at p < 0.05. rpch, rhodope painted chicken; swbch, southwest bulgarian chicken; bl, bulgarian longcrower: sch, struma chicken; sm, sexual maturity (at 20% egg-laying intensity); adfi, average daily feed intake; amepr, average monthly egg production rate; hhep, hen-housed egg production; aeli, average egg-laying intensity; aew, average egg weight; fcr, feed conversion ratio; fce, feed conversion per egg; sem, standard error of the mean. breed sm, days adfi, g amepr, eggs hhep, eggs aeli, % aew, g fcr, kg/kg fce, g/egg rpch n = 25 157 138.5±3.67 abc 17.2±0.62 a 223.9 57.4±2.08 a 56.7±1.84 a 4.34±0.18 ab 244.1±9.2 a swbch n = 25 191 129.0±1.67 ad 13.9±1.56 166.6 46.3±5.19 ab 50.9±0.68 abc 6.55±1.04 a 328.3±47.95 bl n = 25 179 127.1±2.85 b 14.7±1.22 191.6 52.5±4.08 c 56.0±1.68 b 5.45±0.98 291.2±38.56 sch n = 25 250 122.1±2.52 cd 12.4±0.89 a 123.9 33.9±2.98 abc 58.0±0.54 c 5.35±0.41 b 308.8±22.49 a anova (p-value) < 0.001 < 0.05 < 0.05 < 0.01 < 0.05 < 0.05 genetic resources (2025), 6(12), 120–129126 lukanov et al 16°c (poku et al, 2024) and reaching up to 29.9°c (ribeiro et al, 2020), with an optimal range of 18°c to 22°c at a relative humidity of 50–75% (kamanli et al, 2015). temperature fluctuations have a significant impact on hens raised under such conditions, reflected in substantial variations in the laying curve (gerzilov, 2011). a similarly negative effect of high daily temperatures on egg production has been reported by other authors (gerzilov, 2011; yoshida et al, 2011; kim et al, 2024). this is mainly explained by heat stress, mediated by the reduced feed consumption of the birds (getabalew and negash, 2020). under natural rearing conditions typical of the temperate climate in bulgaria, birds are exposed not only to heat stress, mainly during the summer months (july, august and june), but also to cold stress, especially during the winter months (december, january and february). cold stress is recognized as an environmental and managemental challenge, particularly in regions where temperatures regularly fall below 18°c (kim et al, 2023). similar to heat stress, birds exposed to temperatures lower than the thermoneutral zone exhibit negative parameters related to egg production (torki et al, 2015; li et al, 2020; kim et al, 2023). the number of eggs produced by an individual hen is a critical parameter in the selection process in modern poultry farming, while hen-housed egg production serves as a key indicator of the laying performance at the group level (liu et al, 2019). the egg production capacity of local breeds, compared to modern results from high-productivity strains used in industrial poultry farming, shows a striking difference, especially over an extended productive period, which is commonly applied to modern egg-laying hens (el-sabrou et al, 2022). it should be noted that modern egg-laying hybrids demonstrate this capacity under optimal rearing and feeding conditions. in contrast, local breeds are better adapted to the environment, i.e. when raised under uncontrolled conditions with limitations in optimal nutrition. this makes them valuable as a genetic reserve, including for potential inclusion in future breeding programmes (chebo et al, 2022). the results obtained in this study position rpch and bl as breeds with high genetic potential for egg production, when compared to other purebred chickens (bdrg, 2006; henning et al, 2017; lukanov, 2017a; schreiter and freick, 2023). comparing rpch with the data presented for the asturian painted chicken (average of 160 eggs; mapa, 2025), it can be said that the former shows significantly higher potential in terms of laying capacity. the egg production recorded for sch over the productive period is comparable to that of other large ornamental chicken breeds, such as brahma, cochin and orpington (hrnčár et al, 2015). modern commercial laying hens produce eggs with an average weight of 62–65g over the entire laying period (genchev and lukanov, 2025). globally, however, average egg weight tends to be slightly lower, around 60–61g, with regional preferences influencing egg size (thiruvenkadan et al, 2010). the detrimental impact of elevated ambient temperatures on egg weight in domestic hens has been thoroughly documented by bennion and warren (1933). similar to egg production, egg weight is adversely affected by heat stress and reduced feed intake of the birds (kilic and simsek, 2013). the rpch breed, identified in the study as having the highest laying performance, produced eggs with a lower average weight compared to the larger asturian painted chicken, which reaches an average of 65g (mapa, 2025). the low egg weight observed in swbch corresponds to the lower range of the trait reported for the breed by pavlova (2024), namely 50–55g. regarding egg weight, the bulgarian longcrower exhibits typical values reported for other balkan long-crowing chicken breeds (lukanov, 2012; różewicz and kaszperuk, 2018). the turkish breed denizli, which is believed to be close to the balkan long-crowing breeds (lukanov, 2017b), is reported in various sources to have a lower average egg weight of 50–52g (fidan and nazlıgül, 2012; özdemir et al, 2013) to values similar to those of bl (bdrg, 2006; özdoğan et al, 2007; kaya and yıldız, 2014). in a review focused on bulgarian chicken breeds, a lower egg weight range (50–55g) was suggested for bl; however, this estimate was based on preliminary assumptions rather than comprehensive research (lukanov et al, 2021). according to lukanov (2023), both rpch and bl exhibited slightly greater egg weights, likely attributable to the advanced age of the hens, including those in their second productive cycle. the egg weight reported by hrnčár et al, (2015) for three large ornamental breeds (brahma, cochin and orpington) is significantly lower than that of sch. the recorded egg weight in the struma chicken breed is consistent with the findings of a more recent study (lukanov, 2023) and higher than the average values reported for the breed prior to these detailed investigations (lukanov, 2012; teneva et al, 2015; lukanov, 2017a). comparison with the other large native bulgarian breed – the katunitsa chicken – shows that the latter has higher egg weight (gerzilov et al, 2015) and reaches sexual maturity significantly earlier (nikolov and gerzilov, 2011). the average egg weight observed in all four tested bulgarian chicken breeds is lower compared to that of commercial layers subjected to targeted selection for traits related to egg production, including egg weight. nevertheless, the two breeds demonstrating the highest laying potential – rpch and bl – exhibited relatively high egg weights when compared to many other local breeds. as no focused selection for this trait has been applied to these populations, their current performance suggests a promising potential for further genetic improvement in this direction. the egg production efficiency index (epei) is a dimensionless indicator combining liveability, egg production and feed conversion into a single score, with higher values indicating better overall production efficiency. the calculated epei is significantly lower in all four studied breeds compared to the modern laying hybrids, in which the reference value is approximately 230 (lukanov et al, 2023). these differences can be explained by the lower egg production parameters observed in the local chicken breeds included in the present study, as well as by the higher proportion of culls (which negatively affects liveability), compared with the optimal values reported for commercial hybrids in the cited study. the reduced efficiency in sch and swbch groups can be largely explained by the limited laying performance in the sch group and the lower average egg weight recorded in the swbch. feed conversion is an important economic trait that reflects the efficiency of converting feed into finished production (eggs), primarily determined by the feed conversion ratio (fcr) (li et al, 2024). it is well known that feed constitutes a significant portion of the production cost in egg-laying poultry farming (farooq et al, 2002; thiruvenkadan et al, 2010), with considerable variation observed depending on the farming practices applied (kato et al, 2022). the results genetic resources (2025), 6(12), 120–129 egg production of bulgarian chicken breeds 127 obtained regarding feed conversion are comparable to those reported for epei. when comparing the feed transformation efficiency of the four experimental groups of hens with the current performance levels of modern whiteand brown-egg laying hybrids, it is evident that the experimental groups lag considerably behind. even the best-performing group, rpch, exhibited approximately twice the values for both types of fcr compared with those of modern commercial laying (churchil and suresh, 2021; genchev and lukanov, 2025). studies involving various non-commercial chicken breeds report variable fcr values, ranging from those similar to modern laying hybrids (besari et al, 2017) to higher values similar to our results (lukanov et al, 2016; phuong and nha, 2024), or even more striking differences in some lowperforming breeds (nguyen van et al, 2020). conclusion the results of the study indicate that the rhodope painted chicken (rpch) breed demonstrates the highest potential in terms of egg production characteristics, followed by the bulgarian longcrower (bl). both breeds are distinguished by early maturity, with this trait being particularly pronounced in rpch. egg production in these two indigenous breeds is above average compared to other purebred chickens that are not part of industrial poultry farming. due to their high egg production, egg weight and low feed consumption compared to the other three tested breeds, the rpch group demonstrates the most efficient egg production. the two other tested breeds, the struma chicken and the southwest bulgarian chicken, display less favourable characteristics in terms of egg productivity. the good egg production and attractive exterior of the rpch and bl breeds provide strong grounds for their significant potential in amateur and backyard poultry farming. acknowledgements the authors would like to express their sincere gratitude to all those who contributed to this work, especially the breeders of the studied chicken breeds. we also wish to acknowledge the financial support from project 4af/22 ‘study on the egg productivity of some bulgarian chicken breeds’. author contributions hristo lukanov and ivelina pavlova contributed equally to the conception and design of the study, the execution of the experimental work, data analysis, and manuscript preparation. atanas genchev participated in the development and optimization of the experimental methodology and provided technical support during data collection. todor petrov was responsible for monitoring the productivity parameters. conflict of interest statement the authors declare that there is no conflict of interest. ethics statement all experimental procedures involving animals were carried out in accordance with the relevant institutional and national regulations for the ethical treatment of animals. since the study involved only routine productivity assessment under normal housing conditions, no specific ethical approval was necessary. references apa. 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(2011). effect of high environmental temperature on egg production, serum lipoproteins, and follicle steroid hormones in laying hens. the journal of poultry science, 48(3), 207–211. doi: https:// doi.org/10.2141/jpsa.010126 https://doi.org/10.1787/agr_outlook-2017-en https://doi.org/10.1787/agr_outlook-2017-en https://doi.org/10.1787/4c5d2cfb-en https://doi.org/10.4081/ijas.2013.e49 https://doi.org/10.59913/dagr.2024.17293 https://doi.org/10.59913/dagr.2024.17293 https://doi.org/10.5513/jcea01/25.2.4204 https://doi.org/10.1007/s11250-024-04242-1 https://doi.org/10.1007/s11250-024-04242-1 https://doi.org/10.1016/j.jtherbio.2020.102678 https://doi.org/10.1016/j.jtherbio.2020.102678 https://doi.org/10.1016/j.japr.2023.100359 https://doi.org/10.1017/s2078633615000016 https://doi.org/10.1017/s2078633615000016 https://doi.org/10.1017/s0043933910000553 https://doi.org/10.1017/s0043933910000553 https://doi.org/10.1007/s00484-014-0928-z https://doi.org/10.1007/s00484-014-0928-z https://www.fas.usda.gov/sites/default/files/2024-07/livestock_poultry.pdf https://www.fas.usda.gov/sites/default/files/2024-07/livestock_poultry.pdf https://doi.org/10.1186/1471-2156-12-33 https://doi.org/10.2141/jpsa.010126 https://doi.org/10.2141/jpsa.010126 original article genetic resources (2025), 6 (12), 194–204 doi: 10.46265/genresj.kwvh7038 https://www.genresj.org issn: 2708-3764 received: 10.09.2025 | accepted: 22.10.2025 | published online: 10.12.2025 seed germinability of stylosanthes spp. (fabaceae) accessions under water stress conditions abstract: this study aimed to evaluate and compare the germinative performance of seeds from accessions of stylosanthes spp. held in the forage germplasm bank of the state university of feira de santana (bgf-uefs), brazil, under water stress conditions during the initial phases of germination. germination tests were conducted using seeds from six accessions subjected to different osmotic potentials (0.0mpa – distilled water, -0.2, -0.4, -0.6 and -0.8mpa) prepared with polyethylene glycol 6000 (peg 6000). the experimental design was completely randomized, using 25 seeds per replicate for each treatment. the seeds were evaluated over a period of 5 days under water stress conditions, followed by an additional 5-day recovery period in distilled water for the seeds remaining from the -0.8mpa treatments. the following variables were measured: germination percentage (g%), mean germination time (mgt), germination speed index (gsi), and germination recovery (gr). the results indicated an interaction between factors affecting the germinative behaviour of the stylosanthes spp. accessions for all variables. the genotypes showed significant reductions in g%, with accessions bgf 12-014, bgf 10-018 and bgf 10-029 exhibiting the best performance under the most severe osmotic potential (-0.8mpa). mgt and gsi were also significantly affected by increased water stress. accessions bgf 12-014, bgf 10-018 and bgf 10-029 were the most promising based on their germinative performance under water stress conditions simulated with peg 6000 during the early germination phase. keywords: forage legume, polyethylene glycol 6000, osmotic potential, germination, water deficit. citation: oliveira dos santos, v., alves da silva, a., de jesus santos, r., caldas de oliveira, u., neves do nascimento, m., and pelacani, c. r. (2025) “seed germinability of stylosanthes spp. (fabaceae) accessions under water stress conditions”, genetic resources, 6(12), pp. 194–204. doi: 10.46265/genresj.kwvh7038. © copyright 2025 the authors. this is an open access article distributed under the terms of the creative commons attribution license (cc by 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. vitor oliveira dos santos*, aritana alves da silva, robson de jesus santos, uasley caldas de oliveira, marilza neves do nascimento, claudineia regina pelacani universidade estadual de feira de santana, feira de santana, bahia, brazil * corresponding author: vitor oliveira dos santos (vitor.agro.uefs@gmail.com) introduction the brazilian semi-arid region (sab) comprises 1,262 municipalities, most of which are located in the northeastern part of the country (ibge, 2023). this territory is notably characterized by a hot, dry climate and highly irregular rainfall throughout the year, which directly impacts the region’s socioeconomic development (simões et al, 2022), as water scarcity significantly influences local agricultural and livestock activity. with regard to livestock farming in the sab, the region is home to approximately 65% and 90% of the country’s sheep and goat herds, respectively, and about 14.8% of the national cattle herds (ibge, 2018). most of this livestock production is carried out under extensive systems that rely on lowyielding forages, especially during periods of reduced rainfall (souza et al, 2020). moreover, the climatic conditions of the sab limit water availability for the development of forage species, and animal feeding is often partially compromised (gusha et al, 2015). during the driest periods, producers are forced to seek alternative feed sources because pasture forage production becomes insufficient to meet the animals’ nutritional requirements, resulting in increased costs. in addition, few studies focused on improving forage plants adapted to the sab have been conducted to date. consequently, the search for alternatives to reduce livestock production costs in the region remains limited to a few studies. in light of these challenges, it is essential to explore plant https://doi.org/10.46265/genresj.kwvh7038 https://doi.org/10.46265/genresj.slta9371 https://www.genresj.org https://doi.org/10.46265/genresj.kwvh7038 mailto:vitor.agro.uefs%40gmail.com?subject= genetic resources (2025), 6(12), 194–204 germinability of stylosanthes spp. under water stress 195 genetic resources that are tolerant to the climatic conditions of the sab to mitigate the impacts of irregular rainfall in the region. among the forage species that demonstrate tolerance to water stress are elephant grass (cenchrus purpureus (schumach.) morrone.), lead tree (leucaena leucocephala (lam.) de wit), mexican lilac (gliricidia sepium jacq.), birdwood grass (cenchrus ciliaris l.), and species of the genus stylosanthes sw. the genus stylosanthes sw. (fabaceae lindl.) is widely distributed across the americas, with brazil harbouring the greatest diversity of species – 38 in total, 17 of which are endemic to the country (flora e funga do brasil, 2020). additionally, several species within the genus are considered plant genetic resources because of their suitability for animal feed (canzi et al, 2021) owing to their high forage potential, biomass production and protein content. furthermore, their adaptation to acidic soils and tolerance to water scarcity (gonzalez et al, 2000; liu et al, 2019; habermann et al, 2021) make them particularly suitable for cultivation in regions with edaphoclimatic conditions, such as the sab. notably, the semi-arid region is considered one of the centres of diversity for the stylosanthes genus, as evidenced by expeditions carried out in the semi-arid mesoregions of bahia between 2007 and 2019 (santos júnior et al, 2022). genotypes collected during these expeditions are conserved in the forage germplasm bank of the state university of feira de santana (bgf-uefs), located in the municipality of feira de santana, bahia, with approximately 370 accessions catalogued from these regions (santos júnior et al, 2022; silva et al, 2024). however, these materials still lack comprehensive studies on their genotypic, morphoagronomic and physiological traits, as well as their performance under different environmental conditions, especially under water stress, which is one of the main abiotic factors limiting productivity. water stress affects numerous physiological processes in plants, including the reduction in transpiration rates and degradation of photosynthetic pigments, ultimately impacting photosynthetic efficiency (lawlor and cornic, 2002; hussain et al, 2018). nevertheless, hussain et al (2018) highlighted that those plants in water-limited environments exhibit a range of adaptive responses that confer tolerance to water stress. during germination, the seed rehydrates its tissues through water absorption, triggering the embryo's metabolic processes and resuming its growth (bradford, 1990; bewley and black, 1994). in this context, water availability directly influences the success of this process, as germination may be inhibited or delayed under water deficits (bradford, 1990; roberts, 1973; bewley and black, 1994). consequently, when seeds are exposed to such environmental conditions in productive areas, germination may be interrupted or postponed, leading to field emergence irregularities, inefficient land use, and reduced forage production, negatively impacting the feed supply for livestock. thus, assessing seed germinability under water deficit or limiting moisture conditions provides valuable insights for identifying genotypes tolerant to semi-arid regions. this is especially relevant because germination is one of the most critical stages in the plant life cycle. polyethylene glycol 6000 (peg 6000) has been widely used to simulate water restriction during seed germination (braccini et al, 1996), as its high molecular weight prevents it from penetrating the plant cell membrane, thereby avoiding toxicity (marcos filho, 2002). in this context, understanding the germinative behaviour of genotypes subjected to water-limited conditions is essential for selecting materials suitable for cultivation in arid environments. therefore, this study aimed to evaluate and compare the germinability of seeds from stylosanthes spp. accessions held in bgf-uefs under water stress during germination. materials and methods the experiment was conducted in the seed germination laboratory (lager) at the state university of feira de santana (uefs), brazil. the genetic materials used in this study were conserved in the forage germplasm bank of uefs (bgf-uefs) and were obtained from accessions propagated between 2014 and 2020, with progenitors collected from the semi-arid region of bahia (table 1) and stored following the methodology of gómez-campo (2006), kept in properly labelled kraft paper envelopes, sealed with galvanized staples, and placed in airtight containers with silica gel as a moisture indicator, at room temperature (approximately 25.1ºc); with seed moisture content ranging approximately between 4.5% and 8%. moreover, the accessions were selected based on seed viability above 80%. table 1. passport data of stylosanthes spp. accessions stored in the forage germplasm bank of the state university of feira de santana (bgf-uefs) accession species year of seed regeneration municipality coordinates bgf 10-016 s. scabra 2020 queimadas 10°54’40”s/39°12’17”w bgf 12-014 s. humilis 2020 canarana 11º48’597”s/41º42’066”w bgf 014-p137-2 s. scabra 2020 seabra 12º27’311”s/42º11’452”w bgf 10-018 s. scabra 2014 candeal 11°49’49,8”s/ 39°07’08,5”w bgf 10-034 s. scabra 2014 feira de santana 12°09’719”s/38°57’696”w bgf 10-029 s. viscosa 2014 canudos 09°54’29,9”s/39°03’17,2”w genetic resources (2025), 6(12), 194–204196 oliveira dos santos et al to overcome seed coat dormancy, mechanical scarification was performed using sandpaper (sandpaper no. 150) (silva et al, 2024). additionally, to reduce contamination during the experiment, the seeds were disinfected in a 0.5% sodium hypochlorite solution for 10 min and then rinsed with distilled water. seeds from the accessions were subjected to treatment with peg 6000 solutions at different osmotic potentials (0.0mpa for pure distilled water, -0.2mpa, -0.4mpa, -0.6mpa and -0.8mpa), simulating water stress, as proposed by villela et al (1991), with adjustments made at a temperature of 30°c. germination tests were carried out in 60mm glass petri dishes lined with two sheets of sterilized germination paper (germitest type) moistened with 2ml of the respective preprepared peg 6000 solutions. every two days, the petri dishes and germination papers were replaced, and the osmotic solutions were replenished to maintain the target osmotic potentials for each treatment. the tests were conducted in biochemical oxygen demand (b.o.d.) germination chambers, under constant temperature (30°c) and in the dark. germination was defined as the emergence of the radicle (≥ 2mm), with daily counts recorded throughout the 5-day evaluation period. the choice of 5 days was based on preliminary tests (unpublished data), which showed that after this period, the germination of the remaining seeds was not significant. on the fifth day of evaluation, seeds that did not show radicle protrusion in -0,8mpa were removed from the peg solution, rinsed to eliminate residual peg 6000, and transferred to new petri dishes with fresh germitest paper moistened with 2ml of distilled water. this set of remaining seeds was returned to the b.o.d. chamber for an additional 5-day period, referred to as the ‘germination recovery’ phase. germination during this phase was recorded, and the results were expressed as the percentage of recovered seeds, based on a total of 25 sown seeds per replicate. the germination percentage (g%) was calculated according to laboriau and valadares (1976): g% = (n/a) × 100, where: g% = germination percentage, n = total number of germinated seeds, a = total number of seeds tested. mean germination time (mgt; days) was calculated following labouriau (1993): mgt = (σni·ti)/σni, where: ni = number of seeds germinated at time interval ti in days. germination speed index (gsi; seeds·day⁻¹) was calculated according to maguire (1962): gsi = g1/n1 + g2/n2 + ... + gn/nn, where: g1, g2, ..., gn = number of seeds germinated on each respective day, n1, n2, ..., nn = number of days from the start of the test to each respective counting day. the experiment followed a completely randomized design with four replicates, each consisting of one petri dish containing 25 seeds. the data were tested for the assumptions of residual normality using the shapiro–wilk test and for homogeneity of variances using bartlett’s test. for g%, because the assumptions required for analysis of variance (anova) were not initially met, the data were transformed using the arcsine square root function: arcsin√(x/100). once the assumptions were satisfied, anova was conducted. upon observing a significant interaction between the factors (p-value < 0.05), regression curves were constructed using the best-fit model (based on r² values). to compare accession performance within each osmotic potential, the means were grouped using the scott–knott test (scott & knott, 1974). all analyses and plots were conducted using r statistical software (version 2024.12.0+467) (r core team, 2024). results the analysis of variance (table 2) revealed a significant interaction between accessions and osmotic potentials for all variables analyzed: germination (g%), germination speed index (gsi) and mean germination time (mgt) indicating that the combination of osmotic potentials and genotypes had a differential effect on seed germination behaviour. table 2. summary of the analysis of variance (anova) for the variables evaluated during germination of six accessions of the genus stylosanthes spp. subjected to different water potentials of peg 6000. df, degrees of freedom; g%, germination; gsi, germination speed index; mgt, mean germination time; cv, coefficient of variation; **, significant (p-value < 0.01) and *, (p-value < 0.05) by the f-test, respectively. variation source df g% gsi mgt accession 5 12.41** 17.13* 4.62** osmotic potential 4 80.24** 499.60** 85.32** accession x osmotic potential 20 4.38** 6.23** 5.07** residual 90 cv(%) 13.19 12.19 21.58 figure 1 shows the influence of osmotic potential on the germination of seeds from different stylosanthes spp. accessions based on regression analysis. differences among accessions were evident, as indicated by the distinct slopes and intercepts of the regression curves. additionally, the r² values associated with each regression model indicated a good fit, ranging from 88.51% to 97.61%, demonstrating a strong correlation between osmotic potential and germination under the tested conditions. at osmotic potentials of 0.0, -0.2 and -0.4mpa, the accessions exhibited similar performance, with little variation in the germination percentage. however, at lower osmotic potentials (-0.6 and -0.8mpa), more pronounced differences in the performance of the tested genetic materials were observed than at higher potentials, highlighting that the genotypes exhibit divergent performances, especially under more severe water stress conditions. accessions bgf 10-029 (figure 1f), bgf 12-014 (figure 1b), and bgf 10-018 (figure 1d) exhibited less steep regression curves than the other materials, indicating lower sensitivity to water stress. moreover, under the most negative osmotic potential, these accessions maintained relatively high mean germination percentages, ranging from 63% to 66%. in contrast, accession bgf 014-p137-2 (figure 1c) showed intermediate performance under the same conditions, with a germination rate of 38%. accessions bgf 10-034 (figure 1e) and bgf 10-016 (figure 1a) showed the poorest performance under the imposed water stress conditions, with germination percentages at the most negative osmotic potential reaching only 19% and 6%, respectively, reflecting substantial reductions compared to the control treatment (0.0mpa), and indicating that under low water availability, these genetic materials are notably disadvantaged in terms of germination occurrence. figure 1. regression plots showing the effect of different concentrations of peg 6000 on the osmotic potential and seed germination of stylosanthes spp. accessions. peg 6000 concentrations were used to simulate water stress, ranging from 0.0 to -0.8mpa. each point represents the mean of four replications, and the lines represent the fit of the data using a quadratic regression model. genetic resources (2025), 6(12), 194–204 germinability of stylosanthes spp. under water stress 197 all analyses and plots were conducted using r statistical software (version 2024.12.0+467) (r core team, 2024). results the analysis of variance (table 2) revealed a significant interaction between accessions and osmotic potentials for all variables analyzed: germination (g%), germination speed index (gsi) and mean germination time (mgt) indicating that the combination of osmotic potentials and genotypes had a differential effect on seed germination behaviour. table 2. summary of the analysis of variance (anova) for the variables evaluated during germination of six accessions of the genus stylosanthes spp. subjected to different water potentials of peg 6000. df, degrees of freedom; g%, germination; gsi, germination speed index; mgt, mean germination time; cv, coefficient of variation; **, significant (p-value < 0.01) and *, (p-value < 0.05) by the f-test, respectively. variation source df g% gsi mgt accession 5 12.41** 17.13* 4.62** osmotic potential 4 80.24** 499.60** 85.32** accession x osmotic potential 20 4.38** 6.23** 5.07** residual 90 cv(%) 13.19 12.19 21.58 figure 1 shows the influence of osmotic potential on the germination of seeds from different stylosanthes spp. accessions based on regression analysis. differences among accessions were evident, as indicated by the distinct slopes and intercepts of the regression curves. additionally, the r² values associated with each regression model indicated a good fit, ranging from 88.51% to 97.61%, demonstrating a strong correlation between osmotic potential and germination under the tested conditions. at osmotic potentials of 0.0, -0.2 and -0.4mpa, the accessions exhibited similar performance, with little variation in the germination percentage. however, at lower osmotic potentials (-0.6 and -0.8mpa), more pronounced differences in the performance of the tested genetic materials were observed than at higher potentials, highlighting that the genotypes exhibit divergent performances, especially under more severe water stress conditions. accessions bgf 10-029 (figure 1f), bgf 12-014 (figure 1b), and bgf 10-018 (figure 1d) exhibited less steep regression curves than the other materials, indicating lower sensitivity to water stress. moreover, under the most negative osmotic potential, these accessions maintained relatively high mean germination percentages, ranging from 63% to 66%. in contrast, accession bgf 014-p137-2 (figure 1c) showed intermediate performance under the same conditions, with a germination rate of 38%. accessions bgf 10-034 (figure 1e) and bgf 10-016 (figure 1a) showed the poorest performance under the imposed water stress conditions, with germination percentages at the most negative osmotic potential reaching only 19% and 6%, respectively, reflecting substantial reductions compared to the control treatment (0.0mpa), and indicating that under low water availability, these genetic materials are notably disadvantaged in terms of germination occurrence. figure 1. regression plots showing the effect of different concentrations of peg 6000 on the osmotic potential and seed germination of stylosanthes spp. accessions. peg 6000 concentrations were used to simulate water stress, ranging from 0.0 to -0.8mpa. each point represents the mean of four replications, and the lines represent the fit of the data using a quadratic regression model. the breakdown of accession performance at each osmotic potential is presented in table 3. according to the scott–knott clustering test, under control conditions, the materials were divided into two groups, with accessions bgf 10-016, bgf 12-014, bgf 014-p137-2 and bgf 10-029 exhibiting higher germination rates. additionally, at -0.2mpa, all accessions were grouped together, except for bgf 10-036, whose mean value was significantly lower than that of the other genotypes. at osmotic potentials of -0.4 and -0.6mpa, no significant differences were detected among the accessions, as a single group was formed based on the genotype. furthermore, at the most negative osmotic potential, germination percentages among accessions once again showed significant differences, as this level of the factor yielded the greatest variation in the results. the accessions that demonstrated the best performance under these conditions were bgf 12-014, bgf 10-018 and bgf 10-029, with germination percentages of 63.0%, 66.0% and 64.0%, respectively. at the same osmotic potential, all other accessions were considered statistically different from the aforementioned group and from each other, as bgf 014-p137-2, bgf 10-034 and bgf 10-016 were each assigned to separate groups. genetic resources (2025), 6(12), 194–204198 oliveira dos santos et al table 3. germinability of seeds from stylosanthes spp. accessions subjected to different concentrations of peg 6000. means followed by the same letter in the columns do not differ from each other according to the scott–knott test at 5%. germination percentage (g%) osmotic potential (-mpa) accession 0.0 -0.2 -0.4 -0.6 -0.8 bgf 10-016 91.0 a 94.0 a 87.0 a 80.0 a 6.0 d bgf 12-014 96.0 a 97.0 a 94.0 a 92.0 a 63.0 a bgf 014-p137-2 100.0 a 96.0 a 90.0 a 77.0 a 38.0 b bgf 10-018 84.0 b 96.0 a 91.0 a 89.0 a 66.0 a bgf 10-034 83.0 b 78.0 b 82.0 a 75.0 a 19.0 c bgf 10-029 94.0 a 87.0 a 89.0 a 79.0 a 64.0 a the regression graphs showing the effect of peg 6000 on mgt of the tested materials are presented in figure 2. accession bgf 10-016 did not fit either the linear or quadratic regression models, preventing the construction of a curve to represent the data. most of the remaining accessions were best described by a quadratic regression model, with determination coefficients (r²) ranging from 93.75% to 99.16%, indicating a strong fit to observed trends. for accessions bgf 014-p137-2 (figure 2b), bgf 10-034 (figure 2d), and bgf 10-029 (figure 2e), significant increases in mgt began to occur at -0.4mpa, indicating that this osmotic potential was already sufficient to delay the germination process in these genotypes. in contrast, for accessions bgf 12-014 (figure 2a) and bgf 10-018 (figure 2c), this sharp increase was only observed starting at -0.6mpa, indicating that these materials are less sensitive to osmotic stress compared to the others, as changes in mgt were only triggered under lower water availability conditions. additionally, the scott–knott clustering test for mgt (table 4) showed that at osmotic potentials of 0.0, -0.2 and -0.6mpa, the accessions did not differ significantly from one another, indicating similar performances under these environmental conditions. however, at -0.4mpa, accessions bgf 12-014, bgf 10-018 and bgf 10-029 displayed superior mgt values, differing significantly from the other genetic materials, which exhibited higher mgts, highlighting the variation in sensitivity to water stress among the accessions. at the most negative potential (-0.8mpa), only bgf 10-016 showed a significantly different mgt compared to the others, with a lower mean germination time than the remaining accessions. table 4. mean germination time (mgt) of seeds from stylosanthes spp. accessions subjected to different concentrations of peg 6000. means followed by the same letter in the columns do not differ from each other according to the scott-knott test at 5%. mean germination time (mgt) (days) osmotic potential (-mpa) accession 0.0 -0.2 -0.4 -0.6 -0.8 bgf 10-016 1.10 a 1.10 a 1.72 a 3.00 a 1.00 a bgf 12-014 1.17 a 1.45 a 1.55 a 2.32 a 3.30 b bgf 014-p1372 1.05 a 1.37 a 2.25 b 2.40 a 3.20 b bgf 10-018 1.12 a 1.15 a 1.25 a 2.55 a 3.10 b bgf 10-034 1.22 a 1.35 a 2.02 b 2.72 a 3.42 b bgf 10-029 1.07 a 1.02 a 1.67 a 2.40 a 3.00 b the relationship between osmotic potential and gsi was modelled using quadratic regression equations (figure 3), with r² values ranging from 0.9110 to 0.9991, indicating a strong dependence of gsi on the osmotic conditions imposed by peg 6000 treatments. the accessions showed distinct gsi responses as the osmotic potential decreased. under the control treatment (0mpa), only accession bgf 10-034 exhibited a gsi below 20, while the other accessions showed similar values, approximately at the same level. the fitted equations suggest that the accessions respond differently to water availability, with greater or lesser reductions in gsi as water stress increases across the evaluated materials. figure 2. regression plots showing the effect of different concentrations of peg 6000 on the mean germination time (mgt) of stylosanthes spp. accessions. peg 6000 concentrations were used to simulate water stress, ranging from 0.0 to -0.8mpa. each point represents the mean of four replications, and the lines represent the fit of the data using a quadratic or linear regression mode. accession bgf 10-016 is not included in the figure because it did not fit either the linear or quadratic regression models, preventing the construction of a representative curve. genetic resources (2025), 6(12), 194–204 germinability of stylosanthes spp. under water stress 199 the control treatment (0mpa), only accession bgf 10-034 exhibited a gsi below 20, while the other accessions showed similar values, approximately at the same level. the fitted equations suggest that the accessions respond differently to water availability, with greater or lesser reductions in gsi as water stress increases across the evaluated materials. figure 2. regression plots showing the effect of different concentrations of peg 6000 on the mean germination time (mgt) of stylosanthes spp. accessions. peg 6000 concentrations were used to simulate water stress, ranging from 0.0 to -0.8mpa. each point represents the mean of four replications, and the lines represent the fit of the data using a quadratic or linear regression mode. accession bgf 10-016 is not included in the figure because it did not fit either the linear or quadratic regression models, preventing the construction of a representative curve. accession bgf 10-018 (figure 3d) maintained a high gsi up to -0.4mpa, with a sharp decline only observed at -0.6mpa, standing out as the material with the least variation in gsi as the osmotic potential decreased. accessions bgf 10-016 (figure 3a), bgf 10-034 (figure 3f) and bgf 10029 (figure 3e) maintained high gsi values at the two least concentrated peg levels (0.0 and -0.2mpa) but exhibited significant reductions at -0.4mpa and again at -0.8mpa. similarly, accessions bgf 12-014 (figure 3b) and bgf 014p137-2 (figure 3c) also showed marked decreases in gsi as osmotic potential declined; however, an osmotic potential of only -0.2mpa was already sufficient to significantly affect the gsi of these genotypes, as this condition caused noticeable differences compared to the control, highlighting genetic resources (2025), 6(12), 194–204200 oliveira dos santos et al that even a slight reduction in water availability is enough to exert a significant influence on this variable. according to the scott–knott test (table 5), variability in gsi among the accessions was evident at each osmotic potential. under the control condition (0.0mpa), accessions bgf 10-034 and bgf 10-018 had the lowest gsi values, whereas the other accessions had higher gsi means. figure 3. regression plots showing the effect of different osmotic potentials on the germination seed index (gsi) of stylosanthes spp. accessions. peg 6000 concentrations were used to simulate water stress, ranging from 0.0 to -0.8mpa. each point represents the mean of four replications, and the lines represent the fit of the data using a quadratic regression mode. moreover, variations in genotype responses were observed across the remaining osmotic potentials. under the most severe water stress (-0.8mpa), accessions bgf 12-014, bgf 10-018 and bgf 10-029 showed the highest gsi values, standing out under this condition of limited water availability. genetic resources (2025), 6(12), 194–204 germinability of stylosanthes spp. under water stress 201 table 5. germination speed index of seeds from stylosanthes spp. accessions subjected to different peg 6000 concentrations. means followed by the same letter in the columns do not differ from each other according to the scott-knott test at 5%. the analysis of variance (table 6) for the stage referred to as ‘germination recovery’ – which aimed to assess the proportional germination of the remaining seeds from the germination speed index (gsi) (seeds.days-1) osmotic potential (-mpa) accession 0.0 -0.2 -0.4 -0.6 -0.8 bgf 10-016 21.62 a 22.33 a 13.75 b 6.83 b 0.35 b bgf 12-014 22.25 a 19.52 b 18.64 a 12.25 a 4.87 a bgf 014-p137-2 24.37 a 19.87 b 10.80 c 8.50 b 2.99 b bgf 10-018 20.12 b 22.30 a 20.79 a 9.26 b 5.39 a bgf 10-034 19.05 b 16.89 c 12.60 c 8.03 b 1.45 b bgf 10-029 22.89 a 21.50 a 14.87 b 8.45 b 5.39 a accessions previously subjected to the most severe water stress treatment (-0.8mpa), now placed in distilled water (0.0mpa) – revealed significant differences among the accessions. table 6. summary of analysis of variance (anova) for germination recovery of stylosanthes spp. accessions after exposure to water stress at a potential of -0.8mpa. df, degrees of freedom; rec, germination recovery (%); cv, coefficient of variation; **, significant (p-value < 0.05). variation source df rec accession 5 4.78** residual 18 cv (%) 30.44 the scott–knott mean grouping test revealed the formation of two distinct groups for germination recovery. accessions bgf 10-016 and bgf 014-p137-2 exhibited the highest recovery percentages of 89.6% and 88.7%, respectively. all other accessions showed significantly lower recovery rates than these two, highlighting that water stress during the initial germination phase had a significant impact, leading to greater loss of seed viability in these genotypes (figure 4). figure 4. graph of germination recovery in distilled water of stylosanthes spp. accessions after water stress at a potential of -0.8mpa. different letters indicate significant differences in recovery rates according to the scott–knott test (5%). genetic resources (2025), 6(12), 194–204202 oliveira dos santos et al discussion the germination percentages at -0.8mpa (figure 1) confirmed that this concentration of peg 6000 reduced the number of germinated seeds. yamashita et al (2018) evaluated the effect of water stress induced by peg 6000 at different osmotic potentials (0.0; -0.2; -0.4; -0.6; -0.8; and -1mpa) on the germinability of the species stylosanthes capitata vogel, and found that the more negative the solution potential, the greater the reduction in seed germination percentage – results that are consistent with those observed in the present study. moreover, braccini et al (1996) explained the reduction effect caused by the use of peg 6000, who considered the high molecular weight of the substance, its high viscosity, and low o2 diffusion rate as factors that directly impair oxygen availability to seeds during germination. additionally, oliveira et al (2017) reiterated that the high viscosity of peg 6000 is also directly related to the seed’s ability to absorb water, because as the osmotic potential of the solution decreases, the availability of water falls below the level required for seeds to resume metabolic activities and, consequently, for the embryonic axis to grow. tolerance to water stress is an important characteristic to consider when recommending genotypes capable of withstanding different osmotic potentials and water scarcity, especially in ecologically challenging areas with low water availability and saline characteristics (rego et al, 2011). moreover, plants adapted to such conditions not only survive and establish successfully but also complete their reproductive cycle, ensuring population persistence in harsh environments (baskin & baskin, 2014; nicotra et al, 2010). the bgf-uefs accessions presented both intra and interspecific genetic variability, and combined with the fact that they were collected from different locations in the semi-arid region of bahia (oliveira and queiróz, 2016), this may explain the differing germination performances of the stylosanthes spp. genotypes under water stress induced by peg 6000, as observed in this study. the genetic materials bgf 12-014, bgf 10-018, and bgf 10-029 were superior to the other genotypes. the regression analysis for mgt of the accessions (figure 2) suggested that as peg 6000 concentration increased, seeds required more time to emit the radicle. duarte et al (2018), when observing the effect of water stress on the germination of white angico (anadenanthera colubrina var. cebil (vell.) brenan), also found results similar to those of this study, with an increased mgt under lower water availability. this can be explained by the fact that changes in water potential can affect the hydraulic properties of the seed coat, and from this perspective, the lower the potential, the lower the water diffusibility (antunes et al, 2011). this phenomenon delays water absorption by the seed and, consequently, germination (bradford 1990; ávila et al, 2007). the divergence in mgt results among the accessions may be attributed to genotypic variation, as they originated from different localities and may have different seed physiological qualities. the heterogeneity among them leads to differences in the average mgt (kolchinski et al, 2005). from an ecological perspective, delayed germination under low water potential can act as a selective filter, favouring genotypes capable of maintaining viability and responding only under favourable moisture conditions (bradford, 2002). in this regard, accession bgf 10-016 exhibited the most favourable germination pattern under the highest water stress level. however, the low number of germinated seeds in this accession suggests caution in interpreting this result, as the apparent performance may reflect a small number of highly vigorous seeds rather than a consistent tolerance across the seed lot. moreover, santos et al (2016), investigating water stress simulated by peg 6000 in seeds of caatinga species, catingueira (poincianella pyramidalis (tul.) l. p. queiroz) and white angico (anadenanthera colubrina (vell.) brenan), observed a higher mgt compared to all stylosanthes spp. accessions tested in this study at -0.8mpa, being 4.85 and 3.48 days, respectively. as pointed out by bewley and black (1994), when the available moisture is below the necessary level, enzymatic activity is significantly reduced, hindering seed hydration and compromising the ability to metabolize internal reserves. in this sense, the authors emphasized that this phenomenon results in slower and less efficient germination, as water stress limits the biochemical reactions essential for resuming seed metabolism. therefore, the results of this study are consistent with those reported in the literature, as higher peg 6000 concentrations led to a reduction in the gsi of the stylosanthes spp. accessions tested. simioni et al (2011) found similar results in sorghum seeds (sorghum bicolor (l.) moench) under water deficit conditions, with gsi decreasing from -0.4mpa. the same was observed by oliveira et al (2017), who analyzed the germination behaviour of cotton genotypes (gossypium hirsutum l.). azerêdo et al (2016) also found that water deficit simulated with peg 6000 at -0.2mpa was sufficient to reduce the gsi of white angico seeds, a species also used in animal feed. peg 6000, owing to its high molecular weight, cannot penetrate seed structures and thus only limits water availability (marcos filho, 2002). accordingly, marcos filho (2002) also noted that, since it does not exert toxic effects on the seeds, those that tolerate this temporary interruption in water supply during germination can survive and germinate when moisture becomes favourable. this explains the recovery of germination in some genotypes evaluated in this study. the differences observed among them may be attributed to genotypic variation, as some genotypes may not tolerate partial hydration. in such cases, metabolic activity may be initiated and then halted, where the continued lack of water becomes crucial for maintaining seed viability and successful germination. this study highlights the variability among bgf-uefs genotypes in terms of their performance under water stress conditions. the results enhance our understanding of germination dynamics under water-limited conditions and reveal significant variability among stylosanthes spp. accessions. this variation is particularly important for identifying promising genotypes with potential use as forage resources in semi-arid environments, where water scarcity poses a major challenge to seedling establishment and pasture productivity. while these findings provide valuable insights into early responses to water stress, further research is needed to assess the agronomic potential of these accessions under field conditions. field trials or common-garden experiments represent a logical next step to evaluate seedling establishment, early genetic resources (2025), 6(12), 194–204 germinability of stylosanthes spp. under water stress 203 plant development, and overall performance in more variable and realistic environments. such approaches are essential for selecting resilient accessions suitable for forage production systems facing increasingly unpredictable climatic conditions. conclusion the stylosanthes spp. accessions from bgf-uefs exhibited different germination performances under water stress conditions for the variables g%, mgt and gsi. the evaluated germination parameters of the stylosanthes spp. accessions were negatively affected by the reduction in osmotic potential. accessions bgf 012-014, bgf 10-018 and bgf 10029 were the most promising due to their performance in the evaluated germination parameters under water stress, simulated by peg 6000, during the initial germination phase. author contributions vos was responsible for conceptualization and methodology. vos, aas, and rjs handled data collection, while vos and rjs also performed formal analysis. vos and uco wrote the original draft. mnn and crp focused on methodology and visualization. vos, uco, mnn, and crp performed revision and editing. all authors read and approved the final manuscript. acknowledgements the authors acknowledge fapesb (foundation for research support of the state of bahia) for financial support during the execution of this work. conflict of interest statement the authors report no known financial or personal conflicts of interest that could have affected the research or findings presented in this article. references antunes, c. g. c., pelacani, c. r., ribeiro, r. c., souza c. l. m., castro r. d. 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(2020). water restriction, salinity and depth influence the germination and emergence of sourgrass. planta daninha. 38, 1-9. doi: https://doi. org/10.1590/s0100-83582020380100057 https://doi.org/10.1046/j.0016-8025.2001.00814.x https://doi.org/10.1046/j.0016-8025.2001.00814.x https://doi.org/10.1186/s12870-019-1822-y https://doi.org/10.1186/s12870-019-1822-y https://doi.org/10.2135/cropsci1962.0011183x000200020033x https://doi.org/10.2135/cropsci1962.0011183x000200020033x https://doi.org/10.1111/j.1442-9993.1985.tb00876.x https://doi.org/10.1111/j.1442-9993.1985.tb00876.x https://doi.org/10.1104/pp.51.5.914 https://doi.org/10.1016/j.tplants.2010.09.008 https://doi.org/10.1016/j.tplants.2010.09.008 https://doi.org/10.5555/19621604893 https://doi.org/10.5555/19621604893 https://www.r-project.org/ https://www.r-project.org/ https://doi.org/10.4336/2016.pfb.36.87.1017 https://doi.org/10.4336/2016.pfb.36.87.1017 https://doi.org/10.1590/0034-737x2024710012 https://doi.org/10.1590/0034-737x2024710012 https://doi.org/10.22256/pubvet.v8n13.1742 https://doi.org/10.22256/pubvet.v8n13.1742 https://doi.org/10.34117/bjdv8n3-053 https://doi.org/10.34117/bjdv8n3-053 https://doi.org/10.1590/s0100-83582020380100057 https://doi.org/10.1590/s0100-83582020380100057 _hlk208392673 _hlk211184148 genebank report genetic resources (2025), (s2), 185–202 doi: 10.46265/genresj.jwfv3378 https://www.genresj.org issn: 2708-3764 safeguarding, evaluating and valorizing fruit tree genetic resources in belgium: insights from nearly half a century of unsprayed orchard management baptiste dumont *,a, alain rondia a, laurent delpierre a, pascal dupont a, thibaut donis a, vincent ferrier a, julian reyser a, alexis jorion a, dominique mingeot a, patrick houben a, yves pennetreau a, rené stiévenard b, jean-baptiste rey b, guillaume bruneaux b, alain grifnée c, benjamin cerisierd, eva velghed, emilie mulot a, frédéric fauche a, christophe poirson a, françoise van roozendael a and marc lateur *,a a walloon agricultural research centre (cra-w), department of live sciences, biodiversity and plant & forest breeding unit, rue de liroux, 4, gembloux, b-5030, belgium b centre régional de ressources génétiques (crrg), chemin ferme lenglet, villeneuve d’ascq, 59650, france c collège des producteurs, av. comte de smet de nayer, b-5000, namur, belgium d fédération des parcs naturels de wallonie & diversifruits, rue de coppin, 20, b-5100, jambes, belgium abstract: in response to the rapid genetic erosion threatening belgium’s fruit tree cultivar heritage, the walloon agricultural research centre (cra-w, gembloux, belgium) initiated nationwide prospection campaigns in 1975 with support from citizens. these campaigns aimed to collect and conserve the country’s highly diverse fruit tree genetic resources (ftgr), including historically significant amateur-bred and landrace cultivars, for future breeding efforts. since then, the cra-w has maintained a diverse collection – primarily apples (1,629 accessions) and pears (1,198 accessions), but also cherries (355 accessions), plums (236 accessions), grapes (98 accessions), and peaches (29 accessions) – in ex situ unsprayed repository and experimental evaluation orchards. this approach makes it possible to assess these cultivars for multiple traits related to their tolerance and adaptability to biotic and abiotic stresses. this long-term evaluation method enables the identification of numerous quantitative traits and their impact on robustness and stress tolerance. moreover, cra-w has actively sought ways to promote the sustainable use of ftgr through partnerships with public institutions, private stakeholders and citizens. one key initiative was the gradual establishment of a nursery network governed by a participatory fruit tree quality charter, coupled with a traceability system for high-quality propagation material. this initiative led to the release of 33 well-performing heritage cultivars, notable for their sufficient robustness and disease tolerance, for use in both amateur and professional orchards. a decade later, a dedicated apple pre-breeding and breeding programme was launched to harness the extensive ftgr collection as a source of quantitative disease tolerance, robustness and quality traits. keywords: malus x domestica, pyrus communis, disease tolerance, robustness, untreated organic evaluation orchard, participatory breeding, collaborative breeding, low-input organic farming citation: dumont, b., rondia, a., delpierre, l., dupont, p., donis, t., ferrier, v., reyser, j., jorion, a., mingeot, d., houben, p., pennetreau, y., stiévenard, r., rey, j., bruneaux, g., grifnée, a., cerisier, b., velghe, e., mulot, e., fauche, f., poirson, c., van roozendael, f., lateur, m. (2025). safeguarding, evaluating and valorizing fruit tree genetic resources in belgium: insights from nearly half a century of unsprayed orchard management. genetic resources (s2), 185–202. doi: 10.46265/genresj.jwfv3378. © copyright 2025 the authors. this is an open access article distributed under the terms of the creative commons attribution license (cc by 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. received: 11.11.2024 accepted: 12.03.2025 published online: 30.04.2025 https://www.genresj.org https://www.doi.org/10.46265/genresj.jwfv3378 https://www.genresj.org https://www.doi.org/10.46265/genresj.jwfv3378 186 dumont et al genetic resources (2025), (s2), 185–202 the origins of the fruit tree genetic resources collections at gembloux the establishment of the fruit tree genetic resources collection and its evaluation was initiated in 1975 (populer, 1975) at the state plant pathology station of the former agricultural research centre of gembloux (cra, gembloux, belgium) under the leadership of plant pathologist charles populer. populer’s initiative (populer, 1979) stemmed from the observation that most cultivated apple (malus × domestica borkh.) and pear (pyrus communis l.) trees offered by nurseries to both amateur and professional growers were highly susceptible to diseases such as scab (venturia inaequalis on apples and venturia pirina on pears). additionally, the genetic diversity of these cultivars was quite limited, and most breeding programmes at the time focused on introducing monogenic resistance genes to improve apple tree resistance to apple scab. it therefore seemed wise to begin collecting cultivars adapted to local climatic conditions that had been cultivated before the advent of modern fungicides (pre-world war ii) and before the widespread use of bordeaux mixture (late 19th century). during the same period (between 1975 and 1980), several independent initiatives emerged across western europe, aiming to collect the remaining old fruit tree varieties, particularly apple trees. in 1975, corbaz and stoll began surveys in switzerland (corbaz, 1983). in france, similar projects started in 1979, including leterme’s work at the landes regional park (leterme, 1983), and in 1982, stievenard (1999) initiated a programme to conserve and develop local and heirloom fruit varieties in northern france at villeneuve d’ascq. in the netherlands, blommers (1983) and in spain, dapena (1996), also organized surveys in 1974 and 1987, respectively. at the walloon agricultural research centre (craw), the first intensive survey period occurred between 1975 and 1985. initially, efforts focused on visiting historical horticultural formal collections (1975–1980), where materials were collected based on criteria outlined in table 1. ten belgian collections – five in the flemish region, four in the walloon region and one in the brussels-capital region – were surveyed, resulting in the collection of 620 pear and 580 apple accessions. thirty-four accessions were recovered from foreign national collections (england, brogdale farm, kent, and france, inrae, angers), and 160 were collected from private citizens. this effort unexpectedly led to many historical collection managers dismantling their collections, arguing they were safeguarded at gembloux. ∗corresponding authors: baptiste dumont (b.dumont@cra.wallonie.be), marc lateur (m.lateur@cra.wallonie.be) the programme’s next phase was significantly boosted by widespread public interest, driven by media coverage in the press, radio and television highlighting efforts to preserve fruit tree heritage (populer et al, 1998). between 1980 and 1987, over 2,000 individuals contacted the institute, reporting hundreds of endangered old fruit trees in gardens and orchard meadows and requesting assistance in preserving them. in response, intensive prospection campaigns were organized across the walloon region. during this period, numerous landraces and previously unknown apple, pear, plum, cherry and peach varieties were collected. by 1987, the collection had grown to 2,181 accessions. each collecting mission involved engaging with tree owners to learn about the varieties’ qualities, traits and uses, and to gather valuable ethnobotanical knowledge. post-1987, the collection continued to expand through collaborations with institutions such as the proefstation voor de fruitteelt (wilhelminadorp, the netherlands), the centre régional de ressources génétiques de villeneuve d’ascq (france), the station d’amélioration des espèces fruitières et ornementales (inrae, angers, france), the long ashton research station (university of bristol, great britain), the university of illinois (usa), the institute of experimental botany (prague, czech republic), and the research and breeding institute of pomology (holovousy, czech republic), reaching 2,526 accessions by 1997. about one-third of these accessions came from partner collections, while two-thirds were sourced from the countryside with citizens’ assistance. this extraordinary public engagement attracted the attention of the european cooperative programme for plant genetic resources (ecpgr) and was presented at its second steering committee meeting in oeiras (portugal) in 1984 titled: ‘mobilization of public opinion (including practical involvement of the public) in the preservation of fruit tree genetic resources’ (ecpgr, 1984). regarding the selection of plant material (budwood) of cultivars to be introduced in a fruit tree collection (genebank), curators must prioritize based on objectives and available resources. table 1 outlines the main criteria used for introducing varieties into the walloon agricultural research centre (cra-w) collection. currently, collecting activities have slowed and are primarily driven by public requests for pomological consultations. each year, dozens of fruit identification requests are received through a standardized template that includes contact information, sampling location and details on tree characteristics, fruit traits, uses and history. annually, 300 to 900 fruit samples (mainly apple and pear) are submitted, though only a few are selected for inclusion in the collection. depending on selection outcomes and consultation context (e.g. local survey for developing a regional repository orchard), budwood may be requested for propagation to be introduced into the collection or planted in local repository orchards. upon receiving budwood, labels and passport data are mailto:b.dumont@cra.wallonie.be mailto:m.lateur@cra.wallonie.be genetic resources (2025), (s2), 185–202 187 table 1. criteria for selecting varieties introduced into the fruit genetic resources collection of cra-w (populer, 1980; lateur and populer, 1996) accessions collected from historical horticultural formal collections (1975-80) accessions collected with the help of the public from private gardens and orchards (ongoing process) varieties of belgian origin named varieties, of local origin and with a local history and use varieties from neighbouring countries with similar climates to belgium. varieties, even unnamed, that perform well against the main pests and diseases and/or abiotic stresses, which express good robustness. varieties dating back to before fungicide use (before 1850). varieties that significantly enhance existing diversity (hardiness, quality, storage ability, etc.). varieties noted in literature for good disease resistance/tolerance. varieties at risk of extinction and absent from other ecpgr collections. recorded, initiating a traceability process from storage and propagation to nursery monitoring, inventory, and eventual tree lifting and planting. a key principle of this programme has been to offer donors a young tree after successful propagation. this win-win approach acknowledges contributors by providing one or two young trees of the conserved variety and supports pragmatic on-site conservation by replanting original varieties in their native locations. definitions and categories of ‘old fruit varieties’ the main hypothesis of the cra-w biodiversity and plant & forest breeding research unit is that cultivars selected and propagated before the widespread use of fungicides underwent stronger selection pressures, leading to natural selection of more robust varieties that could thrive even without phytopharmaceutical treatments. this makes them more likely to exhibit greater tolerance to fungal diseases. similarly, cultivars selected and released prior to the significant shifts in agricultural practices following world war i and world war ii – and before the extensive use of mineral fertilizers – are presumed to be more resilient and better suited for low-input organic agriculture. to support this hypothesis, we propose a classification system for pome fruit cultivars based on the period when they were first documented (figure 1): • cultivars mentioned before 1760 are categorized as ‘ancient’ • cultivars mentioned between 1761 and 1850 are classified as ‘very old’ • cultivars first mentioned between 1851 and 1914 are designated as ‘old’ • cultivars mentioned between 1915 and 1945 are labelled as ‘pre-modern’ • cultivars mentioned after 1945 are categorized as ‘modern’. this classification framework helps to contextualize the historical development and adaptive traits of apple and pear cultivars across different agricultural eras. defining the concept of landraces for fruit trees the concept of landraces, introduced by von rümker (von rümker, 1908), originally referred to locally grown cultivars that were not consciously selected. camacho-villa et al (2005) further defined this concept highlighting the evolution of a genetically diverse and dynamic population. however, this definition primarily applies to seed-propagated crops. negri et al (2009) expanded the definition of landraces for seed-propagated crops as follows: “a landrace of a seed-propagated crop can be defined as a variable population, which is identifiable and usually has a local name. it lacks “formal” crop improvement, is characterized by a specific adaptation to the environmental conditions of the area of cultivation (tolerant to the biotic and abiotic stresses of that area) and is closely associated with the traditional uses, knowledge, habits, dialects, and celebrations of the people who developed and continue to grow it”. however, perennial plants like fruit trees are predominantly propagated vegetatively, resulting in clonal populations (e.g. groups of trees that all have the same genome because they have been vegetatively propagated e.g. by grafting). therefore, the concept of landraces must be adapted and redefined for these specific crops. historically, farmers propagated pome fruit from open-pollinated seedlings collected in their surroundings. through mass selection, some of these seedlings (known as ‘chance seedlings’) occasionally gave rise to new landraces. the most promising ones were propagated vegetatively within limited areas. the less interesting ones were used as rootstocks and grafted with landraces to make high-stem trees for orchard meadows. the landrace cultivars propagated by rural communities were usually well-adapted to local needs, uses and environmental conditions, including biotic and abiotic stresses. charles populer (lateur, 2001) provided a more nuanced definition using pear trees as an example. according to those authors, pear landraces differ from amateur-bred cultivars in several key aspects (table 2). these criteria are instrumental in distinguishing the safeguarding fruit tree genetic resources in belgium 188 dumont et al genetic resources (2025), (s2), 185–202 figure 1. classification of pome fruit (apple and pear) cultivars based on the period of their first documented mention. wwi, world war i; wwii, world war ii historical origin of pear cultivars within our collection (landraces or amateur-bred cultivars). nevertheless, since synonyms for cultivar names, mislabelling of material and errors are frequent in fruit tree genebanks (oger and lateur, 2004), it is essential to remain proactive in determining which material is true-to-type by cross-checking information, i.e. historical descriptions, accession evaluations and characterization data, expert knowledge and finally, genotypic data (e.g. molecular markers such as microsatellites and singlenucleotide polymorphisms). once materials have been carefully selected and ethnobotanical information has been gathered, a series of stages and activities follow over time, involving the active collaboration of multiple stakeholders. these steps and activities are illustrated in figure 2 and are described in detail below. vegetative fruit accession propagation: an experimental organic nursery after encountering challenges with local nurseries tasked with propagating our initial collected accessions, we established our own experimental nurseries in 1980. currently, the area dedicated to fruit tree propagation covers approximately 1.5ha per year. virus-free rootstocks are ordered from specialized professional nurseries. a decade ago, our nurseries transitioned to management under organic farming system regulations (eu, 2018), and for the past six years, they have been officially certified for organic production. each accession is grafted onto dwarfing or semidwarfing rootstocks. for apple trees, we primarily use ‘m9’ rootstocks, and more recently, the ‘geneva® g11’. due to frequent incompatibility or partial incompatibility between many pear accessions – particularly landraces – and quince (cydonia oblonga mill.) rootstocks, we traditionally used ‘quince a’ grafted with a ‘beurré hardy’ used as ‘interstock’ before grafting the desired accession. to simplify propagation procedures, over the past ten years, we have progressively transitioned to using the ’pyrodwarf’ pear rootstock. this semi-dwarfing rootstock (about 20% more vigorous than ‘quince a’) has a relatively short juvenile phase and, most importantly, is compatible with all pear varieties. for european plum and cherry trees, we use the semi-vigorous rootstocks ‘st. julien a’ and ‘gisela5’, respectively. recently, the ’rubira’ rootstock has shown promising results in propagating our peach accessions. nearly all grape accessions of our collection are propagated directly from cuttings. to ensure proper conservation and evaluation of the accessions in our collections, we aim for a minimum of two trees per accession in the ex situ repository orchard and one in the evaluation orchard. therefore, we routinely plan to graft at least five rootstocks per accession in order to get at least three trees per accession. organization of belgian fruit tree genetic resources conservation repository orchards used as ex situ collections our first repository orchards were established during the 1978–1979 period, primarily focusing on apple genetic resources (2025), (s2), 185–202 189 table 2. criteria that differentiate the historical origins of very old pear cultivars in two main classes: ‘landrace’ and ‘amateur-bred’, also known as ‘bourgeois’ cultivars (lateur, 2001). landrace cultivars amateur-bred or ‘bourgeois’ cultivars 1 no acquisition date date of the acquisition is very often known 2 name of the breeder and location are usually unknown. the name of the breeder is very often referenced. breeders were often belonging to higher social classes (aristocracy, bourgeoisie, artisans, clergy – never female breeders). 3 ‘chance seedlings’. discovered by anonymous peasants, propagated by rural non-profit users. often from deliberate crosses, clonal seedlings or named (re-named) ‘chance seedlings’, propagated and released by historical well-known professional nurseries 4 often distributed regionally or locally. distribution of the variety in larger areas, often international 5 rural/dialectic cultivar name (cvs. ‘poire de gros’, ‘poire de malades, ‘pwèr di fièr’, etc.) the name of the cultivar usually refers to the breeder and their entourage, historical figures, or the fruit itself, often using a high lexical style (‘souvenir de la reine des belges’, ‘hélène grégoire’, ‘napoléon savinien’, ‘nec plus meuris’). 6 almost never described in pomological historical literature nor in catalogues, information sharing nearly always linked with oral transmission. well described in the literature. can be found in old historical nursery catalogues. often found abroad in many collections. 7 fruits mostly selected for their long storage abilities or in order to enlarge the period of fruit consumption (from extremely early to extremely late). fruits usually selected for their taste or attractive visual appeal. 8 mainly ‘survival’ uses, coarse texture and/or astringent used as cooked and or processed fruit for local products (e.g. ‘sirop de liège’ or oven-baked pear). mainly dessert fruit, buttery and smelting flesh. 9 usually show better tolerance to pests and diseases and better robustness. not particularly selected for their robustness traits. 10 grafted on seedling vigorous rootstocks and grown traditionally on high-standard trees in orchard meadows. grafted and grown on dwarfing rootstocks such as quince (very often as espaliers, counter-espalier). 11 often graft incompatibility symptoms when grafted on quince. exceptionally rare graft incompatibilities on quince. figure 2. general overview of the steps and activities at the cra-w research programme aimed at safeguarding and promoting the sustainable use of biodiversity in old fruit varieties. safeguarding fruit tree genetic resources in belgium 190 dumont et al genetic resources (2025), (s2), 185–202 and pear accessions. due to limited land availability, a high-density planting system was employed, with 0.5m spacing between vertical cordons and 2.5m between rows, resulting in a density of 8,000 trees per hectare. each accession was grafted on two trees planted side by side. separate blocks were designated for apple and pear collections, planted adjacent to one another. these orchards were monitored but never sprayed against pests and diseases. to manage weeds, as dwarfing rootstocks were used, herbicides were periodically applied very locally in a narrow strip between grass and trunks at the base of the trees; however, we completely stopped using herbicides in 2000. the repository orchard for plum accessions was established with a lower-density planting system of 6m × 5m, with one tree per accession. thirty-five years later, the need to regenerate trees planted at such high densities prompted the development of a second generation of ex situ repository orchards. this new design aimed to reduce the risk of losses in unsprayed conditions and to accommodate mechanical weed control. key improvements in the second-generation repository orchards included: 1. separate locations for apple and pear collections: apple and pear repository orchards were planted in different locations to minimize the risk of disease epidemics, such as fire blight (erwinia amylovora), which often spreads from pear to apple. 2. dividing apple collections: the apple collection was split into two blocks located 1.5km apart, with a minimum of two copies but with one copy tree per accession planted in two different blocks, reducing the risk of total loss. 3. increased spacing: spacing between trees and rows was increased to 1m × 3.5m for both apple and pear orchards. this adjustment reduced the risk of pest and disease spread, improved light penetration and ventilation, and allowed for better adaptation to mechanical weed control machinery. 4. integrated hedgerows for pear orchards: in the new pear repository orchard, additional improvements included the introduction of multi-species hedgerows (excluding members of the rosaceae family) planted every seven rows of pear trees. these hedgerows act as natural wind barriers, reduce disease dispersal, and serve as banker plants by attracting beneficial insects and fauna. they provide alternative nectar and pollen sources as well as reproductive habitats. this innovative approach ensures the safe conservation and sustainable management of belgian fruit tree genetic resources while promoting ecological balance and minimizing chemical inputs. the in horto pear collection during the early 1990s, outbreaks of fire blight (erwinia amylovora) – one of the most devastating diseases affecting pear and apple trees – posed a significant threat in belgium. concerned about the potential loss of the pear collection, the most valuable cultivars were grafted onto ‘quince a’ rootstock and preserved in containers within an insect-proof greenhouse. remarkably, this pear collection has been maintained in horto as bonsai for over 30 years (figure 3). on-farm safe duplication orchard network since 1999, through collaboration with numerous partners, cra-w established the ‘walloon repository orchards network’ (wron) (villette et al, 2003). this initiative aimed to enhance the safe conservation of heritage diversity by dispersing it throughout the region. the network partners include farmers, local authorities, nature parks, regional administrations, associations, schools, universities and private owners dedicated to conserving and promoting local fruit tree heritage. the primary objectives of this network are to: 1. actively involve local stakeholders in safeguarding, conserving and developing their fruit tree heritage 2. coordinate the duplication of rare endangered local varieties identified in their areas of origin, as well as the true-to-type landraces from the ex situ cra-w collection in gembloux 3. reintroduce true-to-type old local varieties into their sub-regions of origin by increasing the number of genetic conservation sites. the spirit of this multi-partner conservatory orchard network is to reintroduce and duplicate the great diversity of true-to-type old varieties collected at craw, particularly the rarest and most threatened varieties, back to their places of origin. this approach counters decades of fruit tree diversity erosion while fostering a participatory dynamic in conserving fruit tree heritage. the involvement of local partners in the conservation orchards is vital for success, relying on integrating new local surveys of existing old orchards and trees, and maintaining a network of local partners to manage the primary conservation actions. we coordinate a collaborative and interactive network that enables enthusiasts of old fruit tree varieties to develop synergies and revive this fruit tree heritage. the network aims to coordinate actions, share expertise and develop strategies to enhance the value and uses of this diversity. expanding the range of species and varieties is crucial for expressing the best adaptive traits to climate change and countering biodiversity loss. bellon et al (2015) explored this concept and conducted an insightful study on the benefits and challenges of onfarm conservation. since 2019, a significant portion of on-farm repository orchards and their trees have been geo-referenced and monitored for health. technical support is provided to orchard owners to ensure the long-term viability of the trees. in the latest update (2024), wron includes 93 orchards covering 154ha for a total of 8,000 stangenetic resources (2025), (s2), 185–202 191 figure 3. view of the in horto pear cultivars repository collection. dard fruit trees representing approximately 3,000 accessions. according to our 2023 inventory, apple trees constitute 63% of the total, pears 21%, cherries 6%, plums 8%, and other species (peach, quince, walnuts, etc.) 2%. our ongoing goals are to continue planting new on-farm repository orchards, provide stakeholders with appropriate support, and involve local organizations in the inventory, conservation, utilization and public awareness efforts to maintain this heritage. current status of our ex situ fruit tree genetic resources collections cra-w ex situ field collections currently comprise 1,629 cultivated apple tree accessions, alongside 172 indigenous malus sylvestris (l.) miller unique genotypes forming a belgian ‘core collection’ (keulemans et al, 2007; jacques et al, 2009). the collections include also 1,198 cultivated pear tree accessions and 203 wild pyrus pyraster burgst. indigenous unique genotypes. additionally, we maintain 317 sweet cherry (prunus avium (l.)), 38 sour cherry (prunus cerasus (l.)), and 107 botanical/ornamental cherry tree (prunus spp.) accessions. other species in the collections include 236 european plum tree (prunus domestica (l.)) accessions, 98 table grape (vitis spp.) accessions, 29 peach (prunus persica (l.) batsch) accessions, and six local walnut (juglans regia (l.)) tree accessions (table 3). based on our latest data update, table 3 gives also a preliminary estimation of unique accessions and landraces per fruit species. management of unsprayed evaluation and repository orchards for each accession, one tree is planted in one of our evaluation orchards. the spacing is 2m × 4m for apple and pear, while plum trees are spaced 5m × 6m and trained as half-stem. additionally, two trees of each accession are systematically planted at distinct sites within our conservatory orchards (1m × 3.5m). similar conservation strategies are defined by other institutions, such as the german fruit genebank (gfg) (höfer et al, 2019; reed et al, 2004) and the usda-ars-npgs apple field collection managed by the plant genetic resources unit in geneva, ny (bramel and volk, 2019; volk et al, 2015). however, we do not employ alternative preservation methods such as in vitro or cryopreservation but – at least for pome fruits – we plan to implement the concept of storing dried open-pollinated seeds from diploid pome fruit accessions at low temperatures as a complementary safety conservation tool. safeguarding fruit tree genetic resources in belgium 192 dumont et al genetic resources (2025), (s2), 185–202 table 3. summary of the number of accessions and cultivars per crop/species currently preserved in the cra-w ex situ collections. 1, total number of entries present in the collection; 2,estimated number of different cultivars/genotypes among the accessions. as several accessions can be synonyms of the same cultivars or different origins of the same cultivars, we have fewer cultivars than accessions; 3, estimated number of cultivars that are classified as landraces among all the accessions; 4, total number of cultivars (all species combined) and percentage of accessions that represent different cultivars; 5, total number of landraces (all species combined) and percentage of cultivars that are classified as landraces. crops species no. of accessions1 estimated no. of cultivars/genotypes2 estimated no. of landraces3 apple malus × domestica borkh. 1,629 1,061 295 wild apple malus sylvestris (l.) miller 172 172 pear pyrus communis l. 1,198 730 121 wild pear pyrus pyraster burgst. 203 203 sweet cherry prunus avium l. 317 270 76 sour cherry prunus cerasus l. 38 28 5 botanical and ornamental cherry botanical & ornamental prunus spp. 107 107 european plum prunus domestica l. 236 135 77 peach prunus persica (l.) batsch 29 29 8 grapes vitis spp. 98 89 walnuts juglans regia l. 6 6 6 total 4,033 2,830 (70.2 %)4 588 (20.8% )5 the total land area dedicated to fruit tree genetic resources (ftgr) research and management spans approximately 20ha across seven sites, all located within a 7km perimeter. figure 4 depicts the principal apple, pear, grape and plum evaluation orchards situated near the main building. these orchards are managed in natural conditions without irrigation systems or crop protection measures such as hail netting. our philosophy emphasizes evaluating cultivars under the conditions they are expected to thrive in, particularly low-input, organic and regenerative agricultural systems. for this reason, our orchards, though certified for organic production, have never received plant protection treatments. in rare and exceptional cases, where pest damage threatens tree survival, we employ control methods that comply with organic production guidelines. to create a favourable micro-climate, protect trees from strong winds and enhance biodiversity, the orchards are surrounded by highly diverse hedges. the inter-rows are grassed, featuring a central flower strip, and include additional ecological enhancements such as nest boxes, bat boxes and insect boxes. since 2013, the oldest apple and pear evaluation orchard (established in 1978–79 and grafted on ‘m9’ and ‘quince a’ rootstocks, respectively) has been grazed by shropshire sheep at a density of four to five females per hectare (figure 5). this practice has proven effective in fostering synergies between livestock and fruit trees. sheep trample and compact vole galleries, fertilize the orchard, graze on grasses which also helps birds of prey to hunt voles, and manage weeds that compete with trees. they also consume fallen diseased leaves and fruits, while benefiting from abundant food, natural shelter from sun and wind, and a secure environment. consequently, the approach has been extended to a plum evaluation orchard (grafted onto ‘st julien a’ rootstock) and a 30-year-old apple repository orchard (grafted onto ‘m9’ rootstock). evaluation and characterization process of genetic resources fruit tree accessions the continuous adaptation of agriculture to ensure food security, through improvements in disease and pest resistance, tolerance to biotic and abiotic stresses induced by climate change, and other agronomic traits, relies directly on the genetic diversity of our genetic resources. these traits can only be effectively utilized if properly identified through evaluation activities. as stalker and chapman (1989) aptly noted: “a collection is of virtually no practical use until it has been properly evaluated and the data organised so that the content of the material collected can be known. otherwise, it could be compared to a library whose books are neither sorted nor catalogued”. evaluation data is therefore the most critical component, as it determines how and which parts of the collections can be utilized and improved. for this reason, our primary focus has always been on evaluation activities, with characterization being of secondary importance. table 4 delineates the differences between evaluation and characterization activities. given the significant size of genetic resource collections and the low probability of finding desirable traits in a single genotype, the evaluation process is typically conducted in stages. these stages are outlined in table 5. this structured approach is essential for broadening the selection base across numerous accessions. an important aspect of this process involves performing a preliminary evaluation before collecting a new genetic resources (2025), (s2), 185–202 193 figure 4. view of part of the evaluation orchards (apple, pear, grapes and plum) near the main building at the cra-w (gembloux, belgium). (courtesy of emilie mulot, 2024). figure 5. view of the oldest plum evaluation orchard (established in 1983–87) grafted on ‘st-julien a’ (left) and apple and pear evaluation orchard grafted on ‘m9’ and ‘quince a’ respectively. both orchards are grazed by shropshire sheep. photo: cra-w (gembloux, belgium). accession. this initial step allows for an informed selection based on priorities established by the collection manager. this general evaluation process also applies to assessing quantitative disease resistance traits, ensuring that the traits prioritized for future breeding programmes are thoroughly identified and understood. experimental growing conditions since the inception of the plant pathology station, the evaluation of tolerance to pathogens and pests has been the primary research focus. from the outset, both our evaluation orchards and our nursery have been managed according to organic farming practices. however, our approach goes far beyond standard organic requirements i.e. we never use fungicides, and the application of organic insecticides is exceptionally rare. this unique approach serves two critical objectives: 1. accurate evaluation of cultivar robustness: for an important part of the accessions we have more than 25 years of collected evaluation data which allows for a reliable and/or non-parametric assessment of each cultivar’s resilience in the absence of phytosanitary plant protection. 2. preservation of pathogenic and beneficial diversity: maintaining a diverse population of both pathogens and beneficial organisms in our longterm non-sprayed evaluation orchards ensures that new breeding cultivars are tested under high and varied selection pressures, providing a robust evaluation of their adaptability to biotic stresses prior to release (lateur et al, 2000). safeguarding fruit tree genetic resources in belgium 194 dumont et al genetic resources (2025), (s2), 185–202 table 4. description of evaluation and characterization activities applied to plant genetic resources collections (lateur, 2001). characterization evaluation definition description of the most stable traits with respect to interaction with the environment study of traits for which the degree of expression is influenced by environmental factors. main objective distinguish and identify genotypes improve knowledge of the potential of the accessions traits mostly qualitative mostly quantitative time required relatively short for trait stability validation relatively long to be able to define the extent of variability of traits depending on the interaction with the environment experimental protocols relatively simple, based on standardized descriptors experimental conditions need to be well defined and, for a given collection, should initially be relatively stable descriptors yes qualitative nominal variable scales or binary categories yes quantitative often ordinal variable scales – need of reference cultivars. examples specific descriptive fruit traits (fruit shape, presence of ribs, fruit crowning at apex, aperture of eye, length of stalk etc.) agronomic features, disease, pest or abiotic stress tolerance/resistance, flowering period etc. descriptors used aligned with ecpgr goals, considerable effort has been dedicated to developing harmonized and standardized protocols for evaluating and characterizing plant genetic resources. these efforts have been especially collaborative within the ecpgr prunus and malus/pyrus working groups, leading to the creation of comprehensive descriptor lists (lateur and populer, 1996; lateur et al, 1999; lateur, 1999a; lateur et al, 2002; lateur, 2010; kellerhals et al, 2012). most recently, this work culminated in the updated ecpgr characterization and evaluation descriptors for malus and pyrus genetic resources (lateur et al, 2022a,b) apple and pear accessions are systematically evaluated for a wide range of traits, including fruit and tree characteristics, agronomic performance, fruit quality attributes and tolerance to biotic and abiotic stresses. a notable recent development is the introduction of a global foliage quality descriptor. this integrated trait provides an overall assessment of tree health and tolerance to various stresses by combining multiple individual indicators. in the context of the european innobreed project, new descriptors are being developed to enhance the characterization and evaluation of fruit tree genetic resources’ tolerance to abiotic stresses associated with climate change (drought tolerance, sunburn and flower frost tolerance). direct valorization of best-performing cultivars through public–private partnerships a vital strategy for increasing public awareness of ftgr is to allow the public to visit the evaluation orchards. the orchards serve as open spaces where the public, professionals and policymakers can periodically visit to taste fruits, discover heirloom varieties and learn about their historical significance. although the programme initially focused on safeguarding ftgr and utilizing them as breeding material, several cultivars naturally exhibited desirable traits suittable 5. general stages in the evaluation process of genetic resource collections (dotlacil et al, 1994; horvath and szabo, 1997) stages description, objectives (o) and responsibilities (r) a preliminary evaluation (o) simple evaluation carried out before collecting the material in order to avoid accessions that are (1) virus-infected and/or (2) not adapted to the soil and climate conditions, (3) duplicates and (4) accessions that are too susceptible to pests and diseases or to abiotic stresses under in situ conditions. (r) fruit tree genetic resources managers. b basic primary evaluation (o) the first evaluation is carried out during a strict minimum of a 5-year period under experimental harmonized conditions and using standardized protocols, but the experimental setup is simple because it must be applied to a large number of accessions. (o) initial screening of accessions to highlight the most interesting material (best performing for the traits of interest). (r) fruit tree genetic resources managers and/or interested potential users. c secondary and specific evaluation (o) more accurate experimental design involving a sufficient number of replicates; multi-location trials; in the case of disease resistance, possible use of well-characterized pathogenic strains and artificial inoculation techniques. (o) a more detailed evaluation of the material that was pre-selected during the basic evaluation. (r) potential users such as breeders and multidisciplinary teams. genetic resources (2025), (s2), 185–202 195 able for direct propagation. these cultivars were propagated by a network of small family-run nurseries and offered to amateur gardeners and farmers for cultivation in unsprayed high-stem orchard meadows. since 1985, cra-w has actively promoted these outstanding old varieties, recognizing their excellent balance of disease tolerance, agronomic performance and unique qualities suitable for cultivation without plant protection products. this initiative led to the creation of a new range of varieties and a production chain through partnerships with belgian nurseries. these 33 exceptional heirloom varieties have been reintroduced to the market under the collective name ‘rgf-gblx varieties’, an abbreviation for ressources génétiques fruitières de gembloux. these are typically endangered, original varieties that align with the growing public interest in heritage and sustainable fruit cultivation. the varieties progressively released under the ‘rgfgblx’ label are primarily old, forgotten or neglected local varieties, often landraces or selections from former amateur breeders that had disappeared from the market. these varieties, once common in the belgian countryside, were rediscovered through survey campaigns and subsequently evaluated for a minimum of ten years in untreated orchards. this evaluation focused on their tolerance to diseases, pests and climatic stresses, their agronomic characteristics, quality and uses, and their adaptability to different rootstocks and regions. these varieties also stand out for their originality compared to the classic commercial range, offering a rich diversity of taste profiles, forgotten aromas and various uses – both for fresh consumption and processing. they feature staggered ripening periods, easy tree management, and a strong level of robustness, meaning a better overall ability to adapt to various stresses and efficient nitrogen use. more recently, new varieties resulting from the craw breeding programme have been introduced. at least one parent of these new cultivars is an old local variety known for its polygenic resistance to scab and robustness traits. these selections must demonstrate long-lasting and sufficient tolerance to major diseases, mainly scab-robust agronomic traits suited for amateur cultivation and untreated high-stem orchards, original qualities and diverse uses, and solid adaptation to different rootstocks and regions, all evaluated over a minimum 10-year period in untreated orchards. currently, the range of old fruit varieties released to nurseries under the ‘rgf-gblx’ label includes 18 apple varieties, 7 pear varieties, 4 european plum varieties, 3 cherry varieties, and 1 peach variety (figure 6). some of these are described in figure 7. the successful valorization of our ftgr is mainly due to the establishment of organized distribution channels through public-private partnerships. to support this, we outsourced the distribution of budwood and the associated phytosanitary monitoring activities to the figure 6. promotional poster for the certifruit ‘rgf-gblx’ cultivars available on certifruit nurseries and resellers. ormeignies nuclear stock managed by the regional centre d’essais horticole de wallonie (cehw). the certifruit quality charter and the associated nursery network to ensure better traceability and guarantee the true-totype identity of varieties for customers, the certifruit quality charter and label (figure 8) were developed for the ‘rgf-gblx’ old varieties of merit. this initiative was created through a participatory approach in collaboration with a group of volunteer nurserymen. the certifruit charter (available at www.certifruit .be) certifies: 1. a carefully selected assortment of more robust and disease-tolerant varieties 2. the guaranteed origin and identity of the propagation material, including cultivar, rootstock and any inter-stem 3. the superior quality of the nursery trees 4. local and artisanal production methods. additionally, the certifruit nursery network ensures high-quality advice and expertise from certified nurserymen. in 1991, a cehw nuclear stock (3.3ha) was established – initiated by nurserymen and the cehw, safeguarding fruit tree genetic resources in belgium 196 dumont et al genetic resources (2025), (s2), 185–202 fi gu re 7. sh or t de sc ri pt io n of a se le ct io n of ‘r g fg bl x’ ap pl e va ri et ie s. c r r g ,c en tr e ré gi on al de re ss ou rc es g én ét iq ue s de s h au ts de fr an ce genetic resources (2025), (s2), 185–202 197 with funding from the ministry of the walloon region – to distribute certified budwood to nurseries. currently, about half of the ‘rgf-gblx’ released cultivars are certified as ‘virus tested’, while the remaining cultivars are distributed under eu cac regulations (eu, 2008, 2014). figure 8. the certifruit logo promoting fruit tree heritage through the diversifruits association one of the key objectives of cra-w is to encourage the use of its extensive fruit tree collection gathered over the years. among several initiatives, the diversifruits association (www.diversifruits.be) was established in 2018, driven by cra-w and the fédération des parcs naturels de wallonie. the association, managed by volunteers and supported by two publicly funded project managers, brings together around 150 members. its mission is to unite the public and stakeholders in safeguarding and promoting this valuable fruit tree heritage. this is achieved through the planting of high-stem unsprayed orchard meadows and various agroforestry projects. diversifruits offers guidance in selecting the most suitable cultivars and support in orchard management. it is also involved in developing the economic sector related to both the direct sale of fruits and the production of processed goods (such as juice and cider) through its ‘wal4fruits’ project. over the past decade, more than 500ha of orchard meadows have been planted by farmers with the association’s support. each year, diversifruits organizes approximately 70 activities, including conferences, training sessions and awareness events for both the general public and professionals. to further promote and distinguish locally grown fruits – such as apples, pears, plums, cherries, walnuts and chestnuts – produced through this extensive and organic farming model (figure 9a), the association created the ‘vergers vivants’ label. this certification guarantees that fruits are cultivated in non-sprayed orchard meadows. officially recognized by the walloon region, the label also advocates for the fair remuneration of farmers. these extensive orchards (figure 9b, c, d) improve the ecosystemic services and provide fruits of superior quality. pre-breeding and breeding programme using fruit tree genetic resources the breeding programme at cra-w was initiated in 1988 with the primary objective of developing cultivars exhibiting polygenic resistance to apple scab (venturia inaequalis) and other biotic stresses, aiming for commercial production. this programme leverages both ancient and modern cultivars, using extensive phenotypic data collected over the years on our ftgr to select parent plants for crossing. the chosen parents possess complementary traits that help mitigate each other’s weaknesses (lateur, 1999b). the initial phase of seedling evaluation (figure 10) focuses on assessing tolerance to apple scab. at the 3-4 leaf stage, seedlings are sprayed with a mix of v. inaequalis strains with a defined concentration of spores using a pulverization bench. after a controlled incubation period, the seedlings are rated for apple scab tolerance using a simplified scale based on the percentage of leaf surface affected by lesions. our selection is not limited to fully resistant seedlings; those with up to 25% – and occasionally up to 50% – leaf damage are also retained for further evaluation. once transplanted to our nursery and evaluation orchard, these young trees undergo comprehensive assessments for various traits. this includes tolerance to apple scab, powdery mildew, european canker, anthracnose (elsinoë piri), and apple rosy aphid. elite cultivars that perform well are subsequently grafted and further evaluated for fruit production and quality traits. this comprehensive, multi-stage selection process ensures the development of robust cultivars that combine resilience to biotic stresses with desirable agronomic and fruit quality traits, supporting sustainable and low-input fruit production systems. novafruits: a transborder participatory breeding programme since 2014, our breeding activities have primarily focused on participatory breeding within the framework of a public–private partnership. this programme involves two distinct growers’ associations, with the novafruits association serving as a transborder collaboration between partners from northern france and southern belgium (wallonia). novafruits brings together 31 organic fruit tree growers, two regional public institutes – the espaces naturels régionaux (enrx) and crrg – and cra-w, along with both the gawi and the chambre d’agriculture de normandie organic fruit extension services. through this partnership, elite cultivars selected by cra-w and crrg are planted by organic growers under professional cultivation conditions. each year, novafruits members convene to evaluate the traits of the fruits and corresponding cultivars. since many growers sell their products directly to consumers, they gather and share public feedback on fruit quality and performance. safeguarding fruit tree genetic resources in belgium 198 dumont et al genetic resources (2025), (s2), 185–202 figure 9. a, fruit harvest of one conservatory orchard; b, high-stem orchard meadow at the harvesting period; c, visit to a young high-stem orchard meadow; d, an old high-stem orchard meadow. figure 10. illustration of the different steps of our breeding programme selection process. genetic resources (2025), (s2), 185–202 199 additionally, since 2012, cra-w and crrg have been officially linked by a collaboration agreement through which both institutes are: (1) developing shared database facilities, (2) pooling expertise in the identification of local fruit cultivars, (3) rationalizing and sharing responsibilities for cross-border fruit tree genetic resources, (4) mutualizing high-quality plant propagation material, (5) co-steering participatory apple and pear breeding activities (e.g. through the cross-border novafruits association, including planning common breeding objectives and sharing breeding material and offspring), and finally (6) jointly managing two organic variety and elite testing experimental apple and pear orchards. this collaborative approach has led to the release to cross-border organic fruit growers of several cultivars, the most recent being ‘ducasse’ (ocvv/cpvo), a cross between ‘reinette libotte’ and ‘rubinola.’ perspectives the emergence of new pathogens, such as apple blotch disease (diplocarpon coronariae) and anthracnose (elsinoë piri), along with the increasing frequency of abiotic stresses like sunburn, prolonged drought, and to a lesser extent, partial lack of chilling requirement, have become evident through the monitoring of our collections. this situation compels us to develop new descriptors to better assess the individual tolerance of our cultivars to these emerging threats and to enhance parent selection in our organic breeding programme. another ambition of the cra-w collection is to broaden the diversity of cultivars for certain species, including peaches and table grapes, and to introduce new fruit species such as persimmons and fig trees. these species may prove suitable for cultivation in belgium, offering organic farmers both increased resilience to climatic variability and opportunities for income diversification. we firmly believe that the success of robust organic fruit farming relies on a systems-based approach. this approach integrates the use and selection of more robust, more resilient and well-adapted cultivars such as recently described by serrie et al (2024) with agricultural practices that promote biodiversity, soil health, and the regeneration of agroecosystems. consequently, we are also exploring the impact of several practices – such as fruit hedges, grazed orchard meadows, agroforestry and successional agroforestry – alongside measures designed to enhance functional biodiversity on fruit production and crop health. managing and monitoring our collection is both time-consuming and complex. to address this, we are investing in digital tools that streamline data acquisition and analysis, making data collection more efficient. additionally, we are adopting sequencing technologies to deepen our understanding of the genetics within our collections, thereby guiding our breeding strategies. as emphasized in the ecpgr report dedicated to strengthening the ‘aegis’ european strategy (engels et al, 2019), we believe that it is essential for collection managers to use a standardized and common tool for genotyping their germplasm, such as the set of apple 16 ssr markers (linked to the malus unique genotype codes, munq) developed for apple (muranty et al, 2020) and similarly on pear (durel et al, 2023), or the recently proposed snp-based munq system based on a set of 96 snps (muranty et al, 2024). these valuable tools help eliminate duplicates from collections, verify that accessions are true-totype, and facilitate comparisons between collections at national and international levels. this enables the identification of common and unique accessions across collections, thereby supporting the development of a robust conservation strategy for the most valuable genotypes. the challenges ahead for low-input organic farming and integrated fruit production are significant. addressing these challenges will require strong collaboration and synergy between genetic resource collection curators and research institutes. to this end, we are actively working toward and advocating for the establishment of participatory organic breeding programmes, supported by european research initiatives such as innobreed (h ttps://innobreed.eu/, grant agreement no. 101061028) and fruitdiv (https://fruitdiv.eu/, grant agreement no. 101133964). conclusions low-input organic fruit production and integrated fruit production face numerous challenges: (1) the emergence or the increased impact of new pathogens on crops, (2) the rise of abiotic stresses linked to climate change, (3) evolving restrictions and standards requiring the development of innovative, environmentally friendly control techniques, and (4) the accelerating erosion of genetic diversity in cultivated plants. in this context, the conservation and valorization of ftgr have become increasingly important. the collection and conservation efforts initiated nearly 50 years ago at cra-w underscore the enduring importance of the preservation and valorization of plant genetic resources. it is essential to continue expanding their collection, not only by increasing the number of accessions but also by integrating new species that may demonstrate promising adaptation to changing environmental conditions. additionally, there is a critical need to improve the characterization and evaluation of existing genetic resources, focusing on their tolerance to emerging biotic and abiotic stresses and deepening our genetic understanding of these resources. this article presented our diverse approaches and experiences in managing and promoting the use of ftgr collections. a pivotal aspect of this work is the systematic, long-term evaluation of varieties under unsprayed conditions. this process identifies superior-performing cultivars with valuable traits such as enhanced disease tolerance and greater overall robustness – qualities that are increasingly vital in the safeguarding fruit tree genetic resources in belgium 200 dumont et al genetic resources (2025), (s2), 185–202 face of climate change. evaluation data are crucial for releasing robust old varieties directly for use through public–private partnerships, enabling their marketing to individuals and farmers via the certifruit and diversifruits associations. moreover, these evaluations support the development of participatory breeding programmes, exemplified by the novafruits association, which aim to introduce new fruit varieties with broader genetic diversity, improved robustness and better adaptation to low-input organic and integrated fruit production systems. our overarching goal is to sustain more durable production systems with fruit tree cultivars that exhibit greater resistance and adaptability. the conservation, deeper understanding and promotion of agrobiodiversity and fruit genetic resources depend on collaborative efforts among research institutes, farmers, small familyrun nurseries, ngos and the general public (lateur, 2003). this work would not have been possible without the support and interest of the public. therefore, it is crucial to return to citizens the best cultivars we have safeguarded, along with the new varieties developed from crosses using these valuable genetic resources. authors’ contributions marc lateur and baptiste dumont wrote this manuscript. the other authors contributed to the review of this manuscript. conflict of interest the authors declare no conflict of interest. acknowledgements this work was funded by the horizon europe framework programme of the european union under grant agreement no. 101061028 (innobreed project). we extend our sincere gratitude to our dedicated team of passionate individuals committed to the preservation and promotion of belgian fruit genetic resources. this work, along with the maintenance of our collections, would not have been possible without their efforts. we also 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(1999). bilan d’une expérimentation de relance de variétés fruitières locales dans le nord de la france. in le patrimoine fruitier hier, aujourd’hui, demain, ed. chauvet, m., (paris: afcev, brg, inra), 231-243. villette, i., lateur, m., and delpierre, l. (2003). création d’un réseau wallon de conservation in situ de ressources génétiques fruitières. bulletin de l’institut royal des sciences naturelles de belgique biologie 73, 97–101. volk, g. m., chao, c. t., norelli, j. l., brown, s., fazio, g., peace, c., mcferson, j., zhong, g. y., and bretting, p. (2015). the vulnerability of u.s. apple (malus) genetic resources. genet. resources crop evol 62, 765– 794. doi: https://doi.org/10.1007/s10722-014-01942 von rümker, k. (1908). die systematische einteilung und benennung der getreidesorten für praktische zwecke. jahrbuch der deutschen landwirtschaftsgesellschaft 23, 137–167. https://doi.org/10.1186/s12870-019-2171-6 https://doi.org/10.1186/s12870-019-2171-6 https://doi.org/10.17660/actahortic.2024.1412.4 https://doi.org/10.17660/actahortic.2024.1412.4 https://doi.org/10.17660/actahortic.2004.663.104 https://doi.org/10.17660/actahortic.2004.663.104 https://www.upov.int/edocs/pubdocs/en/upov_pub_438_23.pdf https://www.upov.int/edocs/pubdocs/en/upov_pub_438_23.pdf https://hdl.handle.net/10568/105045 https://hdl.handle.net/10568/105045 https://doi.org/10.1093/jxb/erae150 https://doi.org/10.1093/jxb/erae150 https://doi.org/10.1007/s10722-014-0194-2 https://doi.org/10.1007/s10722-014-0194-2 the origins of the fruit tree genetic resources collections at gembloux definitions and categories of `old fruit varieties' defining the concept of landraces for fruit trees vegetative fruit accession propagation: an experimental organic nursery organization of belgian fruit tree genetic resources conservation repository orchards used as ex situ collections the in horto pear collection on-farm safe duplication orchard network current status of our ex situ fruit tree genetic resources collections management of unsprayed evaluation and repository orchards evaluation and characterization process of genetic resources fruit tree accessions experimental growing conditions descriptors used direct valorization of best-performing cultivars through public–private partnerships the certifruit quality charter and the associated nursery network promoting fruit tree heritage through the diversifruits association pre-breeding and breeding programme using fruit tree genetic resources novafruits: a transborder participatory breeding programme perspectives conclusions authors' contributions conflict of interest acknowledgements genebank report genetic resources (2025), (s2), 91–105 doi: 10.46265/genresj.gydy5145 https://www.genresj.org issn: 2708-3764 the german federal ex situ genebank for agricultural and horticultural crops – conservation, exploitation and steps towards a bio-digital resource centre stephan weise a, frank r blattner a, andreas börner a, klaus j dehmer b, marion grübe a, dörte harpke a, ulrike lohwasser a, markus oppermann a, nils stein a,c, evelin willnerd and manuela nagel *,a a genebank department, leibniz institute of plant genetics and crop plant research (ipk), ot gatersleben, corrensstr. 3, 06466 seeland, germany b genebank department, leibniz institute of plant genetics and crop plant research (ipk), parkweg 3a, 18190 sanitz, germany c martin-luther-university halle-wittenberg, universitätsplatz 10, 06108 halle, germany d genebank department, leibniz institute of plant genetics and crop plant research (ipk), inselstr. 9, 23999 malchow/poel, germany abstract: over more than 80 years, the collections of the german federal ex situ genebank for agricultural and horticultural crops have grown to around 152,000 accessions of 3,000 species preserved at three locations: gatersleben, groß lüsewitz and malchow/poel. more than 96% of the material is stored as desiccation-tolerant orthodox seeds according to the active–base–safety (a-b-s) replicate approach at -18◦c. almost 70,000 freshly regenerated safety replicates are stored in the svalbard global seed vault. however, 4% of the material (2,000 field, 3,000 in vitro and 2,500 cryopreserved accessions) can only be maintained vegetatively, as no or few seeds or no true-breeding seeds are available. most of the accessions are provided via the standard material transfer agreement (smta) and more than 1.2 million samples have been distributed since the genebank was founded. to guarantee the identity of the living plant material, reference samples comprising about 450,000 voucher specimens, 110,000 seed and fruit samples and 57,000 cereal spikes are used for comparisons. genebank workflows are supported by the genebank information system (gbis), which also manages workflow-independent data to describe the genebank accessions by passport, phenotypic and taxonomic data, thus allowing users to make targeted selections of material. the genebank-related processes, including acquisition, preservation, regeneration, documentation and material distribution, are certified for quality management in accordance with iso 9001. nowadays, the genebank is undergoing a transformation process to become a bio-digital resource centre to improve utilization of the genetic resources in research and breeding to address future challenges. keywords: german crop genebank, plant genetic resources, conservation, ex situ, in vitro, cryo, documentation, exploitation citation: weise, s., blattner, f. r., börner, a., dehmer, k. j., grübe, m., harpke, d., lohwasser, u., oppermann, m., stein, n., willner, e., nagel, m. (2025). the german federal ex situ genebank for agricultural and horticultural crops – conservation, exploitation and steps towards a bio-digital resource centre. genetic resources (s2), 91–105. doi: 10.46265/genresj.gydy5145. © copyright 2025 the authors. this is an open access article distributed under the terms of the creative commons attribution license (cc by 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. received: 16.10.2024 accepted: 11.02.2025 published online: 10.03.2025 https://www.genresj.org https://www.doi.org/10.46265/genresj.gydy5145 https://www.genresj.org https://www.doi.org/10.46265/genresj.gydy5145 92 weise et al genetic resources (2025), (s2), 91–105 introduction and historical background the availability, accessibility and diversity of plant genetic resources (pgr) are the basis for the adaptation of our crops to environmental challenges and human needs. pgr are pivotal for breeding towards increased biotic and abiotic stress tolerance, optimizing human and animal nutrition and efficient use of renewable resources, including for the energy, chemical and pharmaceutical industries (grusak and dellapenna, 1999; hoisington et al, 1999; metzger and bornscheuer, 2006; tilman et al, 2006; qian et al, 2018). however, since the beginning of industrialization and the introduction of the targeted selection of advantageous local plant varieties – so-called landraces – pgr have steadily disappeared (tanksley and mccouch, 1997). this effect was already recognized by various researchers at the turn of the 20th century and led to the first collecting missions, e.g. those organized by nikolai ivanovich vavilov and frank nicholas meyer (hammer and diederichsen, 2009; baranski, 2013). against this background, the seed and plant introduction office (beltsville, usa) and the office for agricultural crops (st. petersburg, russia) were established in 1893 and 1894, respectively (hammer, 2020), and are considered the two most important forerunners of today’s genebanks. a first organized seedbank was established in the predecessor institution of today’s n.i. vavilov institute of plant genetic resources (vir) in st. petersburg (then petrograd) and stimulated the worldwide movement to preserve the diversity of agricultural and horticultural plants as a basis for food security. vavilov’s postulation of geographical centres of origin, defining assumed regions where the domestication of cultivated plants began, played an important role in the guidance of early collecting trips (vavilov, 1926). these narrowly defined geographical areas were characterized by a great diversity of cultivated and wild forms of domesticated species. although only some of vavilov’s centres of origin turned out to be areas for crop domestication, the high genetic diversity in these regions is still present today. in germany, the latest findings on genetic mechanisms stimulated researchers such as fritz von wettstein and erwin baur to argue for the preservation and exploitation of the diversity of crops. at a seed breeding conference organized in berlin in february 1914, erwin baur stated: “it is very urgent now to become active to save and maintain the quickly disappearing old and primitive varieties of our cultivated crops” (baur, 1914). since that time, efforts were initiated to establish an institute for research on crops which was finally founded in 1943 on the tuttenhof estate near vienna as kaiser wilhelm institute for crop plant research (kaiserwilhelm-institut für kulturpflanzenforschung). the first collections included mainly materials from expeditions carried out before the institute was founded. after the ∗corresponding author: manuela nagel (nagel@ipk-gatersleben.de) second world war, the first director, the geneticist hans stubbe, successfully re-established the institute in gatersleben and initiated a period of systematically planned collecting trips all over the world (müntz and wobus, 2013). larger collecting trips were made to southern italy, afghanistan, china and mongolia, among others (supplemental table 1). from 1948 onwards, there was also an intensive exchange of seeds with botanical gardens, agricultural and horticultural institutes and breeders. while the collections comprised approximately 3,500 accessions at the time of the transfer to gatersleben, by 1962 they had already grown to 23,000 (lehmann, 1963). for the first years, the seeds of genebank accessions could only be stored at ambient conditions and thus had to be regenerated every 3–5 years (lehmann and mansfeld, 1957). the construction of a seed cold-storage facility, completed in 1976 (anon, 1978), led to a drastic change in conservation management. the increased storage capacity and storage temperatures of -15 to -18◦c extended the storage periods of the seeds, resulting in fewer regeneration cycles and lower costs (figure 1). however, systematic large-scale screening on various crops for raw protein content and the essential amino acid lysine began in the late 1960s, see e.g. lehmann et al (1978) and grebenščikov (1985), and led to a sharp increase in seed regeneration in some years. the gatersleben genebank collections had grown to more than 65,000 accessions by the end of the 1980s. however, with the german reunification in 1990 and the desire to consolidate the pgr for agriculture and horticulture in one institute, the collections in pillnitz (fruit genetic resources), gülzow (rye and triticale), malchow (oil and forage crops) and groß lüsewitz (potato) were integrated. the total collection size thus increased to almost 96,000 accessions by 1992. the institute was now renamed the institute of plant genetics and crop plant research (ipk). between 2001 and 2003, around 50,000 accessions from the former west german genebank were transferred to the ipk genebank. originally, the west german genebank was established at the research centre for agriculture (forschungsanstalt für landwirtschaft, fal) in braunschweig in 1970 (hammer, 1998). it was later assigned to the federal centre for breeding research (bundesanstalt für züchtungsforschung, baz) now part of the julius kühn institute (jki). in this context, the collection of fruit genetic resources in pillnitz was transferred to the baz by the end of 2002 and the ipk genebank was renamed the ‘german federal ex situ genebank for agricultural and horticultural crops’. the composition and conservation of the genebank collections composition the ipk genebank collections today comprise almost 152,000 accessions of 3,000 species from 750 genera (table 1). they are actively managed by eight curator mailto:nagel@ipk-gatersleben.de genetic resources (2025), (s2), 91–105 the german federal ex situ genebank for pgrfa 93 figure 1. development of the number of accessions and the regeneration percentage of the ipk genebank collections since the relocation to gatersleben in 1945/1946. selected events that can be seen from the curves are: 1) the regeneration rate of almost 100% due to the transfer of 3,500 accessions to gatersleben, 2) the increase in the number of accessions due to the establishment of a lively exchange of seeds between botanical gardens, research institutes and breeders from 1948, 3) the renewed increase in the regeneration rate due to the incorporation of material from the first major collecting trips, 4) the start of large-scale screening of the raw protein content and the essential amino acid lysine in various cultivated plants, 5) the introduction of seed cold storage, which led to longer storage times and thus to a reduction in regeneration cycles, 6) the integration of the collections from pillnitz, gülzow, malchow and groß lüsewitz, 7) the integration of the west german genebank collections and transfer of the fruit genetic resources in pillnitz to baz. supplemental table 1 provides an overview of collecting missions of genetic resources worldwide that have been at least partially incorporated into the ipk genebank and have contributed to the continuous increase in the number of accessions. four of these are mentioned here as examples: i) integration of material from the fao collecting missions to iran under h. kuckuck (1952–1954) from 1956, ii) integration of e. mayr’s alpine landrace collection (1922–1932) from 1964, iii) start of various landrace collections in slovakia and moravia and integration into the genebank from 1974, iv) various collecting missions to italy and continuous integration into the genebank (1980–1992). groups – cereals, vegetables, tomatoes and beans, legumes, medicinal plants, potatoes, oil and forage crops, and in vitro and cryopreservation – organized in three research groups at three different locations. all groups collaborate intensively and contribute to the reference collection (figures 2 and 3). about 86% of the material is maintained at the main site in gatersleben (deu146), the remainder at two satellite stations in groß lüsewitz (deu159, 4%) and malchow/poel (deu271, 10%). overall, the largest collections comprise accessions of wheat (18%), barley (15%), phaseolus bean (6%) and potato (4%), which are among the largest global genebank collections. for example, ipk holds 6% of the total accessions of barley, 5% of phaseolus bean and 11% of the potato held in the global genebanks (wiews, 2025). about 37% of the accessions are classified as traditional cultivars/landraces, 28% as advanced or improved cultivars, 15% are wild or weedy and 10% are breeding/research material. the remainder is not specified. the country of provenance is known for almost 125,000 accessions in the collections. most accessions originated in europe (66,400 accessions), followed by asia (32,200), the americas (13,800), africa (12,000) and oceania (500) (figure 4). seedbank about 96% of the material is preserved as orthodox, desiccation-tolerant seed and maintained according to the genebank standards for plant genetic resources for food and agriculture (fao, 2014). every year, about 8,000 to 10,000 accessions are regenerated or multiplied in the fields, following best agricultural practices concerning fertilizer supply, pest/weed control and crop rotation. self-pollinating species are grown side by side on areas of 10–15 hectares. most accessions are separated by a different crop, e.g. wheat accessions by barley or forage grasses by rye. cross-pollinators such as rye are grown in separation strips with larger distances or in more than 170 isolation greenhouses of 5–10m2. the latter are mainly used for insect-pollinated accessions and are equipped with solitary bees, bumblebees or flies. biennial accessions are often grown in the open field and transferred to isolation greenhouses or cages after evaluation in the second year. during the growing season, crop-specific descriptors based on ipgri/bioversity descriptor lists (bioversity, 2024) are used for characterization. extended morphological and physiological information about adaptation and resistances towards environmental stresses and diseases are often obtained during targeted projects, e.g. on 94 weise et al genetic resources (2025), (s2), 91–105 figure 2. overview of genebank management for conservation of seed and clonal accessions including safety storage of seeds at the svalbard global seed vault (sgsv) and of cryosamples at the german collection of microorganisms and cell cultures (dsmz). arrows indicate the direction of the main workflows. the genebank information system (gbis) provides process support including management and curation of data, allowing users to specifically select and order material, and is linked to international information systems, bioinformatics hubs and research projects. 1-9, numbers in circles indicate steps visualized in figure 3. legumes, forage grasses (supplemental table 2) and support data complementation and breeders to select and utilize pgr. maturity of seeds is crucial for the development of optimal desiccation tolerance and seed longevity (leprince et al, 2017). when full seed maturity is reached, the plants are cut manually. most of the material is placed in drying cabinets at 20% relative humidity (rh) and a temperature of 20◦c. depending on the workload, the material is further threshed and cleaned. clean seeds or separately harvested spikes are compared with reference material and then transferred to drying cabinets at 15% rh and 20◦c to reach a final seed moisture content of 5–7% depending on the species. in parallel, the initial germination capacity and moisture content of the seeds are tested. if germination of cultivated species reaches more than 80%, the material is further processed and separated into active–base–safety replicates. active replicates are mainly kept in sealed glass jars with silica gel tops and stored at -18◦c, in some cases also at -8◦c or 4◦c. base and safety replicates are vacuum sealed and stored at -18◦c. once per year, safety replicates are transferred to the svalbard global seed vault, spitsber-gen. within the last 16 years, freshly reproduced seeds of almost 70,000 ipk accessions have been deposited at the global backup storage, providing an important level of security against the loss of seeds due to human-caused or natural disasters. highly vigorous seed material is the basis for the longterm availability of genetic resources (ellis and roberts, genetic resources (2025), (s2), 91–105 the german federal ex situ genebank for pgrfa 95 figure 3. various steps during conservation of seed and clonal genebank accessions. 1, regeneration of cereal accessions in the gatersleben fields (photo: michael grau, 2008); 2, allium field genebank in gatersleben (photo: manuela nagel, 2020); 3, in vitro slow-growth storage of potato (solanum tuberosum l.) in groß lüsewitz (photo: manuela nagel, 2019); 4, regeneration of red clover (trifolium pratense l.) accessions in malchow/poel (photo: daniela impe, 2019); 5, active storage of runner beans (phaseolus coccineus l.) in gatersleben (photo: heike müller, 2014); 6, long-term cryostorage of clonal accessions (photo: lynne main, 2016); 7, spike reference collection (photo: sam rey, 2012); 8, voucher specimen (ipk herbarium); 9, seed reference collection in gatersleben (photo: sam rey, 2012). 96 weise et al genetic resources (2025), (s2), 91–105 table 1. composition of the ipk genebank collections shown by species groups by june 2024. species groups accessions species groups accessions cereals and grasses 66,434 vegetables 17,861 wheat 28,307 tomatoes 3,910 barley 23,839 pepper 1,533 oat 4,863 eggplants 113 rye 2,582 beta beets 2,376 triticale 1,619 raphanus 766 aegilops 1,513 carrots 505 millets 841 chicory 673 maize 1,532 allium 1,974 others 1,338 brassica 2,178 lettuce 1,145 legumes 27,862 spinach 215 phaseolus 9,013 celery 254 field beans 3,038 quinoa 953 soybeans 1,491 others 1,296 other beans 615 pea 5,392 medicinal and spice plants 8,244 chickpea 527 poppy 1,135 vetchling 514 tobacco 590 vetches 1,845 others 6,519 lupines 2,712 lentils 473 mutants 1,684 clover 1,970 tomato mutants 743 others 272 soybean mutants 527 antirrhinum mutants 414 cucurbitaceae 2,668 pumpkins 1,054 potatoes 6,357 melons 728 cucumbers 738 small-grained oil and forage crops 15,157 others 148 oilseed rape and forage kale 2,645 grasses 11,157 larger-grain oil, fibre and dye plants 5,470 red clover and alfalfa 1,344 flax 2,324 sunflower 677 dye plants 458 fibre plants 191 oil plants 548 others 1,272 total 151,737 1980). a lower number of regeneration cycles increases the cost-efficiency of the genebank and lowers the risk of loss of genetic integrity. therefore, all accessions stored at -18◦c are regularly checked for seed germination after 8–20 years and are considered for regeneration when seed germination has dropped to less than 70% of the initial germination. regeneration is also considered when the number of actively stored seeds is reduced due to the distribution of seed samples. depending on the species, most seeds have been regenerated after 20 to 40 years. however, seed storability depends on the genetic background, the environmental conditions during growth and the storage conditions (nagel et al, 2015). in future, advances in sensor technology may allow the individual control of e.g. seed moisture content and temperature in storage to optimize survival periods. field genebank maintaining clonal plants in the field is the most traditional conservation method. it allows characterization and evaluation on site and immediate distribution of material (engels and visser, 2003; panis et al, 2020). at ipk, about 4% of the accessions are preserved vegetatively, because no or little seeds or no true breeding seeds are available. of these, about 2,000 accessions genetic resources (2025), (s2), 91–105 the german federal ex situ genebank for pgrfa 97 figure 4. overview of main genera presented by continent. approximately 750 genera have been summarized as ‘others’. of various allium species, changing accessions of potato landraces, as well as mint and other species are grown in a field genebank. the management of the field genebank varies in terms of growth requirements, propagation cycle and field design depending on the species. for example, the present allium collection, merged from the taxonomic reference collection and the allium crop collection (keller and kik, 2018), comprises 1,080 accessions of 189 species and has been located at the main site for 5–6 years. although best field management practices are applied, allium accessions lose vigour over time due to soil exhaustion and need to be transplanted to another site to minimize the risk of infections and diseases. in the case of 392 garlic (allium sativum l.) accessions, for example, cloves and bulbils are harvested in july after full senescence of leaves and stems. the material is then cleaned, prepared for planting and kept at 7◦c. in autumn, the cloves are planted in plots of 1.5×1.5m and develop adventitious roots and flat leaves before winter. some accessions bolt and develop inflorescences with flower buds and bulbils in may. other materials, i.e. 82 shallot accessions and approximately 200 potato accessions are grown annually. at the ipk site in groß lüsewitz, the potato collections (glks) comprise 2,800 accessions from europe and north america and approx. 650 native landraces from the andes that are maintained clonally, most of them in vitro. for field reproduction and characterization, 10 tubers are pregerminated and planted in the field between march and april. over the vegetation period, various phenotypic traits are recorded following huaman et al (1977), and tubers are harvested after 4–5 months before or at maturity. on average, about 400 field accessions have been distributed annually to 122 users, mainly private individuals, since 2017. the accessions are available for distribution but require phytosanitary certificates for shipment abroad and systematic evaluation of quarantinable diseases. recent projects, i.e. ‘ecpgr garli-ccs’ and ‘obivonknobi’ (see supplemental table 2) intensively evaluate the morphology, composition and genetic architecture to provide more comprehensive data to breeders and support identification of unique and duplicate genotypes for further decision-making processes. major challenges for field collections depend on the year’s climate and are the potential exposure of accessions to unfavourable conditions or threats such as pests and diseases. the allium collection, for example, was exposed to an infestation of larval stage click beetles (elateridae family, known as wireworms) in 2013. as a consequence, 52 allium accessions were lost while 73 could be rescued by replanting (panis et al, 2020). in addition, material that is maintained permanently in the field accumulates viruses, bacteria, fungi and mutations (mckey et al, 2010). this increases the necessity for careful evaluation and selection, besides frequent weeding, and seed or propagule harvest to avoid mixing of different accessions. due to this high workload in field collections, in vitro slowgrowth storage and cryopreservation were established at the gatersleben genebank in the 1980s and 1990s, respectively. in vitro slow-growth storage in vitro slow-growth storage is an essential tool for the conservation of accessions that are permanently propagated clonally, as they fail to produce seeds due to sub-optimal field/greenhouse conditions. the 98 weise et al genetic resources (2025), (s2), 91–105 storage of in vitro cultures allows the preservation of disinfected, pathogen-free material under precisely controlled environmental conditions, which is available for distribution. if plant physiology permits, lower temperatures and light intensities are used to reduce the metabolic activity, which extends the storage period and reduces the workload (panis et al, 2020). at ipk, 2,900 potato, 150 mint, 30 dioscorea and 50 accessions from other species are preserved in in vitro slow-growth storage. here, 967 samples of in vitro potato accessions have been distributed to breeders and researchers since 2017. similar to the field genebank, the conservation practices vary between species, specifically regarding media composition and growth conditions. in the case of potato, apices from sprouting potato tubers are excised, surface-sterilized and grown on a murashige and skoog (1962) medium (ms). accessions are tested for the common virus strains, such as potato virus a (pva), potato leaf roll virus (plrv), potato viruses m, s, x, y (pvm, pvs, pvx, pvy), and quarantine pests, e.g. bacterial ring rot (brr), among others (nagel et al, 2022). if plants test positive for six common potato viruses, they are subjected to chemoor thermotherapy (depending on the virus) followed by meristem isolation. the procedure is repeated until the viruses are eliminated. the meristems are then grown on ms media supplemented with 6% sucrose and exposed to a combination of warm (20◦c for 1–2 months) and cold phases (10◦c for 2–4 months, low light intensity). under cold conditions, in vitro potato plants develop microtubers, which can be kept in a dormant state at 4◦c and low light intensity for 12–15 months. when microtubers begin to sprout, either these or the nodal segments are transferred to fresh media and the cycle is initiated again. for other in vitro cultures, nodal segments of young plants grown in the field or greenhouse are surface-sterilized and grown on ms media supplemented with 3% sucrose and speciesspecific phytohormone compositions (senula and nagel, 2021). most mint accessions, but also 18 antirrhinum and 17 brassica accessions, are kept for 12–20 months under two different cold regimes at 2◦c and 6◦c, and 16h light before they need to be sub-cultured. warm-adapted mint, dioscorea, but also eight artemisia, three salvia, three sechium, three orthosiphon and two plectranthus accessions are kept at 25/20◦c and 16/8h light/dark and need to be sub-cultured after 2–5 months. although in vitro slow-growth storage has been established for a number of species, some plant species fail to grow and develop (benson, 2000). this phenomenon, also called in vitro recalcitrance, was observed in allium species. in 1995, ipk maintained 645 allium accessions in vitro. after some sub-cultures, the plants failed to grow and were contaminated indicating that growth conditions were not optimal and favoured growth of endophytic microorganisms. unfortunately, efforts to adapt the media and conditions failed, and hence, plants rejuvenated in the greenhouse were used for immediate cryopreservation. for the remaining accessions, field material, i.e. bulbs, cloves and bulbils, was collected and used to introduce allium species directly into cryopreservation. for potato and mint, in vitro propagation is an essential step to achieve yearround cryopreservation and long-term preservation of clonal plants with minimal workload and costs. cryobank cryopreservation is the storage of biological material at ultralow temperatures, usually below -130◦c. this is realized in liquid nitrogen (ln, -196◦c), in its vapour phase (between -165◦c and -190◦c) or in electric freezers (-150◦c). under these conditions, molecular movements cease, which increases the possibility of storing biological material indefinitely. however, the cryopreservation of plants was only established in the 1980s, when particular challenges, such as uncontrolled ice crystallization due to the presence of stiff plant cell walls and vacuoles, had to be overcome (panis et al, 2020; nagel et al, 2024). at ipk, international progress in cryopreservation triggered the start of safety duplication of the clonal potato collection stored in groß lüsewitz and led to the cryopreservation of the first potato accessions in 1997. later, as a part of the restructuring of the german federal ex situ genebank, 578 accessions were transferred from baz braunschweig to gatersleben, resulting in a collection of 900 potato accessions in 2002 (keller and dreiling, 2003). over the next two decades, a range of methods, i.e. dmso droplet freezing, pvs2 and pvs3 vitrification, were tested and adapted (keller et al, 2014) and form the basis for about 2,100 potato, 250 allium and 160 mentha accessions cryopreserved by 2024 (nagel et al, 2024). nowadays, ipk routine cryopreservation of potato, allium and mentha is based on a vitrification approach using the cryoprotectant pvs3. this method has been applied to a range of clonal species preserved at the ipk genebank and proven the most convenient, successful, rapid and reproducible for these accessions. in brief, 1–2mm shoot tips are excised, precultured on ms media with 3% sucrose and exposed first to a loading solution with 13.7% sucrose and 18.4% glycerol and then to pvs3 solution containing 50% sucrose and 50% glycerol (senula and nagel, 2021). the increased sucrose concentrations facilitate osmotic dehydration and stabilize proteins and membranes (lerbret et al, 2011). glycerol permeates quickly into cells, replaces hydrogen bonds and prevents ice formation by separating water molecules (towey et al, 2012). shoot tips treated with a combined solution are transferred to vials or aluminium foil strips containing fresh pvs3 droplets and submerged to ln. the rapid temperature drop of ~130 k/s results in vitrification of the cytoplasm which reduces the potential to develop lethal intracellular ice. based on statistics of dussert et al (2003) and availability of propagules, 300 shoot tips for potato and mentha, and 150 for allium species are cryopreserved, of which 90 and 50, genetic resources (2025), (s2), 91–105 the german federal ex situ genebank for pgrfa 99 respectively, are thawed to evaluate the cryopreservation success. if more than 30 shoot tips regrow, they are considered safely cryopreserved. on average, however, potato, allium and mentha regrew at higher percentages of 47%, 38%, 64%, respectively, which is a promising basis to increase the threshold to 35%, as suggested by an international team of cryoexperts (volk et al, 2017). after successful cryopreservation, the number of shoot tips is divided into triplicates; two replicates are stored in separate tanks at ipk and one in tanks at a backup storage facility at the german collection of microorganisms and cell cultures (dsmz) in braunschweig, germany. the cryopreserved material is occasionally requested for activation, comparisons and distribution, which provides information about their status of viability. however, activating the material is time-consuming and costly. therefore, permanent conservation in cryo is only considered at ipk if the accessions are not actively used, such as duplicates or non-requested accessions, or material which does not survive in the field or in vitro. for the allium collection, 60 accessions exist only in cryo due to unfavourable field conditions. reference collection the ipk genebank has been operating reference collections of preserved plants and plant parts since 1946 (anon, 1953). some reference materials even date back to the early 19th century (e.g. allium angulosum, gat0011009, from 1809). today, the reference collections comprise more than 450,000 herbarium voucher specimens, 110,000 reference seed and fruit samples and 57,000 cereal spikes, which serve as important sources to guarantee the identity of the reproduced genebank material. besides the genebank reference collection, the herbarium contains a representative specimen collection of cultivated plants and their wild relatives which provided the basis for mansfeld’s encyclopedia of agricultural and horticultural crops (hanelt, 2001). moreover, the herbarium stores important types, i.e. the specimens of organisms to which newly described taxonomical units such as species or subspecies refer, and also functions as a repository for physical references of plants used in molecular systematics studies. to prepare herbarium vouchers, entire plants or plant parts important for determination and differentiation are collected during the vegetation period, pressed, dried and mounted as voucher herbarium specimens. a label including taxonomic and collection information is attached to the voucher, which is then stored in the ipk herbarium. plant parts that cannot be prepared such as tubers or fruit clusters were preserved dry or wet (in alcohol) (anon, 1953). however, due to the high workload, the latter activity had not been continued and only the available reference material is refreshed occasionally. to ensure long-term preservation of the reference collections, they are protected by separate quarantine areas where the vouchers are prepared and frozen at -20 ◦c for one week to kill parasites before they are introduced into the collection. insects are prevented by mosquito meshing at the windows and annual fumigations with phosphine (ph3) help to keep museum beetles (anthrenus museorum l.) in particular out of the collections. the herbarium collection is continuously processed and digitized in high resolution and currently provides about 53,000 digital images of the vouchers that can be accessed online in the joint herbarium management system jacq (https://www.jacq.org/, herbarium code: gat) and, hence, via the global biodiversity information facility (gbif). further scans are currently being processed. this reduces shipping of the valuable specimens among herbaria, thus minimizing the danger of losing materials. moreover, researchers who work on taxonomic revisions of specific plant groups have fast access to digital collections, which very much speeds up taxonomic procedures, as high-resolution scans provide the most important details. the availability of digitized vouchers will support emerging machine learning approaches for species determination, help in understanding the geographic distribution and ecological settings of certain species, and allow easier search for and compilation of datasets of developmental and anatomical features of the taxa. documentation documentation plays an important role in both conservation and exploitation, and thus, utilization of pgr. the more information is available about a resource, the more precise statements can be made about its value for breeding and research. furthermore, a genebank collection can only be developed further in a meaningful way if its composition is well documented. this makes it possible, for example, to identify species or geographic regions that are underrepresented in the collection via gap analysis. moreover, the management of information is essential both for the physical management of the collection and for the fulfilment of legal obligations (weise et al, 2020). there are three categories of data: 1) pure management data, 2) data of legal significance and 3) data that allows the assessment of pgr value. the first category includes data like germination percentage, age of samples, storage quantities and locations, results of health tests and responsibilities for conservation. this data needs to be stored in a structured way. the second category comprises the documentation of collecting permits, correspondence with other institutions or documentation of receipt. the third category can be further subdivided into different kinds of data. passport data comprise the basic information on pgr, in particular they facilitate the identification of the material. stable and unique identifiers, such as digital object identifiers (dois), are of great importance in this context (garrity et al, 2009; alercia et al, 2018). in addition, passport data contains, among others, the scientific name, information on origin and acquisition as well as the type of material and 100 weise et al genetic resources (2025), (s2), 91–105 is based on the multi-crop passport descriptors (mcpd) data standard (alercia et al, 2001, 2015). other important data that help to assess the potential value of an accession for research and breeding are phenotypic characterizations, including morphological and agronomic traits. at ipk, this information is initially collected during the first cultivation of each accession and checked during each subsequent regeneration. the first system for the management of the ipk genebank data was established in the 1980s and has been continuously developed thereafter. as part of the fusion of the former eastern and western german genebank collections (see above), resources were also made available to develop an integrated information and management system, the genebank information system (gbis) (oppermann et al, 2015). in 2006, gbis started to operate and has been managing the above-mentioned data. in parallel, gbis supports the processes for maintaining genebank accessions. for this purpose, it is made up of three components. the gbis/m management module is primarily used to support the daily work processes in the genebank and enables the management and curation of data on the preserved material. the gbis/b evaluation module is used for the electronic recording of phenotypic data with mobile devices during the regeneration, and the gbis/i internet module provides potential users of genebank material with relevant information via a public web interface, thus allowing them to specifically select and order material (figure 5). gbis also documents genebank-related processes including acquisition, preservation, regeneration, documentation and material provision under the regulations of the standard material transfer agreement (smta) for quality management. furthermore, it supports the fulfilment of reporting obligations at national and international levels, e.g. with regard to the international treaty on plant genetic resources for food and agriculture (itpgrfa). as a result, all of the genebanks’ accessions are also listed in international aggregator systems such as eurisco, genesys and fao-wiews. the active curation of data on pgr is becoming increasingly important (shaw et al, 2023). the genebank’s information pool is therefore continuously updated. this includes comparing and supplementing existing data sets with those from external sources, e.g. from information systems of other collections. this significantly increases the quality and quantity of information on genebank accessions. furthermore, historical data has also been explored and stepwise added. even unbalanced data, i.e. phenotypic data recorded during reproduction in different years, can provide added value, for example by being used to predict the phenotypic performance of genebank accessions (philipp et al, 2018; berkner et al, 2024). quality management the ipk genebank aims to efficiently use the available economic, human and technical resources to ensure figure 5. development of the number of samples distributed. in 2007, the genebank information system (1, gbis) was introduced allowing scientists, breeders and private persons to order accessions free of charge. this led to a continuous increase in the number of samples reaching over 50,000 samples in 2016 (2). due to the financial burden and workload, a processing fee was introduced in mid-2016 (3), which has limited the annual distributions to a manageable level of 25.000 samples on average (yellow line). overall, the ipk genebank has distributed more than 1.2 million samples over the last 80 years. the permanent availability of collection material and to offer the users a high-quality service. therefore, the ipk genebank introduced a quality management (qm) system according to iso 9001 in 2007. quality management is a tool for monitoring all activities, tasks and processes required to maintain a desired level of quality in products and/or services. an effective qm system involves clear organizational strategies and goals, efficient and transparent processes, measurable results and continuous process improvements. the establishment of a qm system and certification according to iso 9001 is a measure to increase the satisfaction of the stakeholders (service quality) and to improve the internal genebank management. moreover, the documentation of the individual processes is a key issue to perpetuate the longstanding experience of the employees and their knowledge for a sustainable continuation of pgr conservation. finally, the transparency of the genebank processes ensures that they are aligned with agreed genebank quality standards. since 2007, all relevant key processes have been visualized in 51 procedure instructions and described in detail in 72 working instructions. a quality management handbook and an operational genebank manual, available on the ecpgr website (https://www.ecpgr.org/a egis/aquas/genebank-manuals/), describe the qm system. internal and external quality audits are planned and carried out annually, and a certification company recertifies the genebank every three years. the continuous improvement is pursued through the development and implementation of state-of-the-art knowledge and research conducted at ipk. these collaborative activities guarantee high-quality services and progress in the field of preservation, propagation, conservation, taxonomic classification as well as information technologies. genetic resources (2025), (s2), 91–105 the german federal ex situ genebank for pgrfa 101 development of a bio-digital resource centre progress in life sciences is increasingly centred around data availability, quality and management. in line with this, the genebank is undergoing a transformation process to become a bio-digital resource centre (mascher et al, 2019). this means describing pgr on an ever-better scale in order to optimize their use for research and breeding. the aims are to successively raise existing data to a higher level through curation and complementation, and to obtain additional data. the latter pursues two goals: on the one hand, additional data from domains that are already being used will be tapped. this includes, among other things, further phenotypic data integrated from additional sources, e.g. from high-throughput phenotyping. on the other hand, data from domains that have not yet been used in the past will be harnessed, in particular genetic characterizations. genomic data can help to decipher genetic diversity and provide insights into geographical origin, row type, growth habit or domestication status, for example. it can also help with the identification of duplicates and enables applications such as genome-wide association studies. entire sub-collections are increasingly being genotyped, for example barley (milner et al, 2019) and wheat (schulthess et al, 2022), and their data are made available via crop portals. the above-mentioned processing of historical data from the last 80 years, particularly from seed regenerations, also plays an important role for the bio-digital resource centre as it helps to assess the value of pgr accessions for breeding and research purposes. this data is extensively curated and published in accordance with the findability-accessibility-interoperability-reusability principles (fair; (wilkinson et al, 2016)). in addition, this data is also analyzed together with genotyping data. a cooperation with the dsmz in braunschweig has been established with regard to a safety backup for cryomaterial (see above). to store valuable resources together with their most important data, a pilot project together with the norwegian company genever was initiated and special cryoboxes developed. the boxes combine cryovials with data on a roll of film (piql film). this technology is extremely robust and promises to last for centuries. until the end of 2024, all cryo backup samples stored at the dsmz will be supplemented with data on film strips. in recent years, a great deal of energy has been invested in establishing efficient data management at ipk, and previously isolated information systems have been and are being successively interlinked. in addition, ipk is also involved in the establishment and further development of data standards such as ‘minimum information about a plant phenotyping experiment’ (miappe; krajewski et al (2015); papoutsoglou et al (2020)) and is embedded in national and international networks for pgr. for example, the european search catalogue for plant genetic resources (eurisco) has been operated and further developed by an ipk genebank working group on behalf of the european cooperative programme for plant genetic resources (ecpgr) since 2014 (weise et al, 2017; kotni et al, 2023). challenges and future plans as in any genebank, there are a number of challenges associated with the various activities; maintenance and regeneration in particular are labour-intensive and costly. in order to utilize the available resources as efficiently as possible, one of the options currently being discussed is to rely on a higher degree of automation and digitization. furthermore, cryopreservation of heterozygous, short-lived and hybrid seeds might also be a backup solution for material which cannot be maintained adequately by conventional long-term storage. the identification of duplicates also plays an important role in the more efficient use of resources. in large collections comprising hundreds or even thousands of accessions of a species (such as wheat and barley in the german genebank), duplicates within the collection are unavoidable. in addition, there is a large percentage of duplicates between genebanks (van hintum and visser, 1995). unfortunately, the identification of duplicates is not a trivial task; reliable statements can only be made by jointly analyzing passport data, phenotypic data and genotyping data in combination with comparative cultivations. in addition, the definition of threshold values is useful here. such approaches have been tested as examples, but have not yet been carried out on a larger scale. however, duplicates, both within and between collections, open up possibilities for normalizing data, especially historical phenotypic data. this is an approach that is currently being pursued in the agent project (https: //www.agent-project.eu/). despite progress, at least in the large sub-collections (see e.g. gonzález et al (2018); philipp et al (2018)), there is still a great need for the digitization and curation of historical data. however, consistent recording, storage and curation of data also require continuous maintenance and further development of the genebank information system. this includes the regular porting of both data and software components. to facilitate the recording of phenotypic data, a new client for mobile devices was recently finalized. it is based on the phenoapp (röckel et al, 2022) and has been specially extended to meet the needs of the genebank. a particular challenge is the integration of phenotypic data that was not collected as part of the regeneration of material by the genebank staff themselves, but in the context of research projects. there are still no widely accepted standards regarding the collection of phenotypic data using standardized traits and methods (krajewski et al, 2015). however, approaches such as miappe facilitate description and reproducibility, at least for future data. not all biodiversity is secured in the world’s genebanks. especially against the backdrop of the 102 weise et al genetic resources (2025), (s2), 91–105 climate crisis, this represents a race against time. it is therefore necessary to specifically analyze existing subcollections and to identify priority species and regions for collecting. such an analysis has already been carried out using oilseed rape as an example (weise et al, 2023). this allows the targeted acquisition of material from other collections and, if possible, the organization of collecting trips. the ipk genebank is involved in various infrastructure projects and research programmes, and has genotyped entire sub-collections. however, the participation in the exploitation and utilization of (neglected) crops and crop wild relatives (e.g. legume generation (https:// www.legumegeneration.eu/) and cousin (https://cou sinproject.eu/) projects) as well as the participation in the establishment of a european research infrastructure for pgr (pro-grace project, https://www.grace-ri.eu /) will continue to conserve and utilize our european pgr as efficiently as possible. supplemental data supplemental table 1: collecting trips by germanspeaking researchers supplemental table 2: recent third party-funded projects under participation of the ipk genebank author contributions sw and mn drafted the manuscript. all authors revised and edited the manuscript, and approved the final version. conflict of interest statement the authors declare that they have no competing interests. acknowledgements we 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(2017). eurisco: the european search catalogue for plant genetic resources. nucleic acids research 45, d1003–d1008. doi: https://doi. org/10.1093/nar/gkw755 wiews (2025). world information and early warning system on plant genetic resources for food and agriculture (wiews). url: http://www.fao.org/ wiews/en/. wilkinson, m. d., dumontier, m., aalbersberg, i. j., appleton, g., axton, m., baak, a., blomberg, n., boiten, j. w., santos, l. b. d. s., bourne, p. e., bouwman, j., brookes, a. j., clark, t., crosas, m., dillo, i., dumon, o., edmunds, s., evelo, c. t., finkers, r., gonzalez-beltran, a., gray, a. j. g., groth, p., goble, c., grethe, j. s., heringa, j., ’t hoen, p. a. c., hooft, r., kuhn, t., kok, r., kok, j., lusher, s. j., martone, m. e., mons, a., packer, a. l., persson, b., rocca-serra, p., roos, m., van schaik, r., sansone, s. a., schultes, e., sengstag, t., slater, t., strawn, g., swertz, m. a., thompson, m., van der lei, j., van mulligen, e., velterop, j., waagmeester, a., wittenburg, p., wolstencroft, k., zhao, j., and mons, b. (2016). the fair guiding principles for scientific data management and stewardship. scientific data 3, 160018. doi: https://doi.org/10.1038/sdata.2016.18 https://doi.org/10.1021/jp3093034 https://doi.org/10.1007/bf02539517 https://doi.org/10.1007/bf02539517 https://doi.org/10.1007/s10722-016-0460-6 https://doi.org/10.1007/s10722-016-0460-6 https://doi.org/10.3389/fpls.2023.1244467 https://doi.org/10.3389/fpls.2023.1244467 https://doi.org/10.3390/plants9081050 https://doi.org/10.3390/plants9081050 https://doi.org/10.1093/nar/gkw755 https://doi.org/10.1093/nar/gkw755 http://www.fao.org/wiews/en/ http://www.fao.org/wiews/en/ https://doi.org/10.1038/sdata.2016.18 introduction and historical background the composition and conservation of the genebank collections composition seedbank field genebank in vitro slow-growth storage cryobank reference collection documentation quality management development of a bio-digital resource centre challenges and future plans supplemental data author contributions conflict of interest statement acknowledgements original article genetic resources (2025), 6 (12), 57–72 doi: 10.46265/genresj.fqlf1923 https://www.genresj.org issn: 2708-3764 received: 02.05.2025 | accepted: 01.08.2025 | published online: 17.09.2025 ethnography of traditional healers and their indigenous medicinal plants in southern philippines: implications for conservation and sustainable use abstract: this paper investigates indigenous medicinal plants, the threats they face and the healing knowledge and profiles of traditional healers in the sarangani uplands, southern philippines. during field and community floral inventories, 39 medicinal plant species were documented, belonging to 18 orders, 20 families, and 31 genera. while this study unveiled diverse utilization of medicinal plants, interviewed healers unfortunately revealed local losses which they attributed to (1) climate change, (2) overharvesting, (3) forest denudation, and (4) the shift to over-the-counter medicines. additionally, the gradual erosion of healing knowledge was ascribed to (1) christianization suppressing traditional healing practice, (2) local losses of medicinal plants, (3) shift in culture and lifestyle brought by increasing market integration, (4) reluctance of tribal healers to share healing knowledge, (5) devaluation of indigenous knowledge by the younger generation, (6) advanced ages of knowledge keepers, and (7) the oral nature of mentoring. moreover, this paper reports that conservation was accomplished mainly through continuous utilization/cultivation and the judicious collection of medicinal plants. these efforts are, however, grossly insufficient and without complementary in situ and ex situ conservation initiatives, these invaluable genetic treasures will face local extinction. in addition to sarangani’s medicinal plant losses, the valuable cache of associated traditional knowledge will likewise be lost, resulting in a culturally impoverished and less resilient community. finally, to foster inclusivity, promote knowledge pluralism, and aid in the preservation of traditional healing knowledge, the involvement of healers in the crafting of a comprehensive healthcare strategy for southern philippines is recommended. keywords: traditional healers, indigenous medicinal plants, conservation, utilization, sarangani province citation: buay, b. m. g., aguilar, c. h. m., banaticla-hilario, m. c. n., rodriguez, c. r. m. and zapico, f. l. (2025) “ethnography of traditional healers and their indigenous medicinal plants in southern philippines: implications for conservation and sustainable use”, genetic resources, 6(12), pp. 57–72. doi: 10.46265/genresj.fqlf1923. © copyright 2025 the authors. this is an open access article distributed under the terms of the creative commons attribution license (cc by 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. bhegie may g. buaya,d, catherine hazel m. aguilarb, maria celeste n. banaticla-hilarioc, chenny rose m. rodriguezd and florence l. zapicod,* a department of biology, institute of mathematics, arts, and sciences, davao del sur state college, digos city, the philippines b global crop diversity trust, platz der vereinten nationen, 7 53113 bonn, germany c plant biology division, institute of biological sciences, university of the philippines-los banos, college, laguna, the philippines d department of biology, college of natural sciences and mathematics, mindanao state university general santos, the philippines * corresponding author: florence l. zapico (florence.zapico@gmail.com) introduction in biodiversity-rich, low to middle-income countries like the philippines, traditional healing knowledge and medicines remain the primary refuge of ethnic peoples for their healing and therapeutic needs. in rural communities, medicinal plants are abundant, freely available and reputed to be efficacious based on millennia of traditional use (mahmoud and gairola, 2013; bankole et al, 2015; barata et al, 2016). indigenous groups have been utilizing indigenous medicinal plants (imps) since time immemorial, leading to a wealth of accumulated traditional healing knowledge (thk). even up to this time, developed and developing countries alike rely on imps as direct sources of medicine or as raw materials for the processing of therapeutic drugs (miano et al, 2011; ambasta et al, 2016), thereby resulting in an increasing demand for these priceless natural resources. studies from nepal (aryal et al, 2016) and nigeria (stoffersen et al, 2011; borokini et al, 2013) report on agehttp://doi.org/10.46265/genresj.fqlf1923 mailto:https://doi.org/10.46265/genresj.apnr6909%0d?subject= https://www.genresj.org https://doi.org/10.46265/genresj.fqlf1923 mailto:florence.zapico%40gmail.com?subject= genetic resources (2025), 6(12), 57–7258 buay et al medicinal plants, (4) carry out initiatives for evidence-based ex situ and in situ conservation of sarangani medicinal plants and (5) recommend for the inclusion of traditional healers in the crafting of a comprehensive health strategy. it is envisioned that meticulous documentation of imp utilization and conservation status will contribute significantly to the preservation of sarangani traditional healing knowledge for future generations. materials and methods study locations figure 1 shows a location map of sarangani province with all study sites indicated. this study was conducted in seven upland villages (sitios) in four towns (viz. kinam and banlas in malapatan, datal anggas and ihan in alabel, miasong and ligaya in glan and gasie in maasim) in sarangani province. in malapatan and glan, b’laans are the predominant tribal group. t’bolis inhabit maasim while b’laans and tagakaulos reside in the upland villages of alabel. the study sites and their geophysical coordinates are shown in table 1. preliminary preparations and ethical considerations letters seeking permission for the study were sent to the provincial governor, the mayors, heads of agriculture offices, and barangay (village) captains before field visits. subsequently, both oral and written consents were secured from healers before the study commenced. elderly healers who were illiterate appended their thumbprints to the consent form. the participants were informed about the study, its objectives and the information to be solicited from them. they were assured that their identities would be maintained with the utmost confidentiality. additionally, they were apprised of their right to decline to respond to inquiries or to terminate the key informant interview (kii) at any moment for any personal reason. research design, respondent selection, inclusion and exclusion criteria this study is a qualitative research that employed ethnographic methods, such as direct participant observation, interviews with key informants and a semi-structured questionnaire to chronicle the traditional knowledge of local healers based on their lived experiences. purposive sampling based on information from sitio officials was utilized to identify potential study respondents. subsequently, the researchers scoured the remote upland areas of alabel, glan, malapatan and maasim to locate identified healers. while the municipalities of maitum, malungon and kiamba were initially considered for inclusion in the study, several factors rendered this difficult, if not outright impossible. in maitum, traditional healers visited by the team declined requests for an interview, believing that divulging secret information would diminish their healing powers. as for the municipalities of malungon and kiamba, locals reported shifting to modern medicines and that no more healers were found in the sitios. moreover, the remoteness and isolation of some villages and safety concerns prevented the researchers from penetrating these far-flung areas. old thk and utilization of imps by local communities as well as their economic potentials (batugal et al, 2004). known as ethnomedicine, this field of anthropology deals with medicinal plants and the wealth of healing knowledge of traditional healers (cotton, 1996; cheikhyoussef et al, 2011). thk is, therefore, an indispensable source of socioculturally coherent information about imps, their indigenous uses, and their natural habitats. nonetheless, thk is vulnerable to sociocultural and ecological transformations that define traditional communities in contemporary times. considered as one of the 18 mega-biodiverse countries in the world, the philippines ranks fifth in terms of plant species richness (cbd, 2025). recently, meniza et al (2024) documented 1,500 species of medicinal plants in the philippines, with over one-third (or 530 species) found in mindanao. recent studies (dapar et al, 2020; paraguison et al, 2020; alinsug et al, 2022; cabugatan et al, 2022; ilagan et al, 2022) revealed the prevalent use of medicinal plants in rural mindanao communities. however, these priceless genetic resources and associated indigenous knowledge are threatened by emerging social-ecological realities in these areas. among the identified major pressures to medicinal plant species in the philippines are agricultural expansion, deforestation, mining, environmental degradation, unregulated resource extraction and climate change (mendoza et al, 2016; cordero et al, 2022; agduma et al, 2023; belgica et al, 2024). moreover, alinsug et al (2022), dapar et al (2020), fiscal (2017), and ong and kim (2014) reported about the propensity of the younger generation to embrace modernization, resulting in knowledge erosion. other identified causes of knowledge erosion are acculturation, outmigration and increasing access to over-the-counter medicine (dapar et al, 2020; cordero et al, 2022). at the national level, the philippine government has been promoting the shift to imps, given the exorbitant prices of modern medicines. after thorough evaluation, the department of health (doh) endorsed ten medicinal plants, viz. senna alata (l.) roxb., momordica charantia l., allium sativum l., psidium guajava l., vitex negundo l., combretum indicum (l.) de filipps, blumea balsamifera (l.) dc., ehretia microphylla lam., peperomia pellucida (l.) kunth and clinopodium douglasii (benth.) kuntze for widescale use (dapar et al, 2020). another initiative of doh was the promotion of herbal medicine gardens in rural communities and the integration of traditional healing into mainstream healthcare (maramba-lazarte, 2020). unfortunately, there is a paucity of information about traditional healthcare practitioners, especially those residing in far-flung areas. sarangani province in southern mindanao is home to lumad (tribes of non-muslim ethnicity) groups such as b’laans, tagakaulos, and t’bolis. these groups inhabit remote and inaccessible upland areas and consequently receive no (or very little) basic social services from the local government. moreover, the lack of government presence in these areas and an official census of the tribal population have resulted in a dearth of information about their exact numbers. these lumads are, therefore, the most disadvantaged sector of philippine society, whose dependence on local resources was highlighted when the covid-19 pandemic isolated their remote communities. this study was thus designed to (1) profile traditional healers, (2), document sarangani medicinal plants and how they are used in healing rituals, (3) investigate threats to sarangani genetic resources (2025), 6(12), 57–72 indigenous medicinal plants in the philippines 59 medicinal plants, (4) carry out initiatives for evidence-based ex situ and in situ conservation of sarangani medicinal plants and (5) recommend for the inclusion of traditional healers in the crafting of a comprehensive health strategy. it is envisioned that meticulous documentation of imp utilization and conservation status will contribute significantly to the preservation of sarangani traditional healing knowledge for future generations. materials and methods study locations figure 1 shows a location map of sarangani province with all study sites indicated. this study was conducted in seven upland villages (sitios) in four towns (viz. kinam and banlas in malapatan, datal anggas and ihan in alabel, miasong and ligaya in glan and gasie in maasim) in sarangani province. in malapatan and glan, b’laans are the predominant tribal group. t’bolis inhabit maasim while b’laans and tagakaulos reside in the upland villages of alabel. the study sites and their geophysical coordinates are shown in table 1. preliminary preparations and ethical considerations letters seeking permission for the study were sent to the provincial governor, the mayors, heads of agriculture offices, and barangay (village) captains before field visits. subsequently, both oral and written consents were secured from healers before the study commenced. elderly healers who were illiterate appended their thumbprints to the consent form. the participants were informed about the study, its objectives and the information to be solicited from them. they were assured that their identities would be maintained with the utmost confidentiality. additionally, they were apprised of their right to decline to respond to inquiries or to terminate the key informant interview (kii) at any moment for any personal reason. research design, respondent selection, inclusion and exclusion criteria this study is a qualitative research that employed ethnographic methods, such as direct participant observation, interviews with key informants and a semi-structured questionnaire to chronicle the traditional knowledge of local healers based on their lived experiences. purposive sampling based on information from sitio officials was utilized to identify potential study respondents. subsequently, the researchers scoured the remote upland areas of alabel, glan, malapatan and maasim to locate identified healers. while the municipalities of maitum, malungon and kiamba were initially considered for inclusion in the study, several factors rendered this difficult, if not outright impossible. in maitum, traditional healers visited by the team declined requests for an interview, believing that divulging secret information would diminish their healing powers. as for the municipalities of malungon and kiamba, locals reported shifting to modern medicines and that no more healers were found in the sitios. moreover, the remoteness and isolation of some villages and safety concerns prevented the researchers from penetrating these far-flung areas. table 1. study sites, their geophysical coordinates and dominant ethnic group/s. ip, indigenous people; masl, metres above sea level. figure 1. map of sarangani province showing the municipalities where the study was conducted the inclusion criteria encompassed traditional healers of any gender, aged 25 years or older, who utilized imps for healing and provided explicit consent to participate in the study. a prospective study participant had to be a recognized traditional healer within the community. conversely, individuals who shifted to modern medicine and those who declined invitations for participation were excluded from the study. data collection methodologies and analyses ethnobotanical assessment the semi-structured questionnaire used consisted of open-ended questions, which enabled respondents to articulate comprehensive views regarding traditional sitio municipality gps coordinates altitude (masl) dominant ip group datal anggas alabel n 06°11.995’ e 125°27.158’ 900 b’laan, tagakaulo sitio ihan alabel n 06°12’21.7” e 125°22’21.7” 905 b’laan, tagakaulo sitio ligaya glan n 05°46’17.5” e 125°19’50.5” 426 b’laan sitio miasong glan n 05°45.638’ e 125°20.954’ 428 b’laan sitio banlas malapatan n 06°06.508’ e 125°28.065’ 342 b’laan kinam malapatan n 06°06.125’ e 125°25.0158’ 406 b’laan sitio gasie maasim n 05°57.495’ e 124°54.083’ 1,016 t’boli healing knowledge in their own words. this questionnaire contained four main sections: (1) demographic profiles of healers and knowledge acquisition, (2) medicinal plants and their modes of utilization, (3) medicinal plant sources, processing and storage, and (4) threats to medicinal plants and conservation awareness. after pre-testing, the validated questionnaire (see supplemental material 1) was used as the main data-gathering tool during face-to-face interviews. the questionnaire, written in english, was administered using the predominant dialect of sarangani (cebuano) or the tribal dialect through a local intermediary for respondents who could only converse in their native language. to validate the results of the questionnaire and delve deeper into the healing knowledge of the sarangani ethnic groups, key informant interviews were carried out. as a qualitative ingenetic resources (2025), 6(12), 57–7260 buay et al depth interview method, a key informant interview is flexible and extracts knowledge from key respondents who have particularly informed perspectives and first-hand knowledge about the topic at hand. being a descriptive study, answers to the questionnaire and key informant interviews were analyzed thematically, classified a posteriori, and discussed narratively to minimize bias in the conclusions. subsequently, relevant information was presented in tables for easier reference and interpretation. inventory, collection, identification and conservation of indigenous medicinal plants field observation and community walks were conducted to triangulate information and corroborate data from the questionnaires and key informant interviews, thereby lending credence to the results. with the assistance of the healers, medicinal plants were identified and gathered (with consent) from backyards, home gardens, roadsides and adjacent woodlands. two distinct collection methods were utilized, contingent on the biological characteristics of the plants. for sexually reproducing plants, viable seeds were collected and placed in labelled coin envelopes. on the other hand, vegetatively propagated herbs and seedlings were immediately planted in black polypropylene seedling bags filled with garden soil. both coin envelopes and seedling bags were labelled with passport information such as the plant’s local name, collection date and site, the name of the household head (if collected in home gardens), and its geophysical coordinates. during community walks, the healers initially recognized medicinal plants by their ethnic names, which were subsequently verified through appropriate references. collected medicinal plants were classified according to their growth habits and their habitats, among other information. taxonomic identification was done up to the species level whenever possible using suitable references and consultation with a botanist. no scientific identification was conducted for some imps growing deep in the forests, and the researchers relied solely on the healers’ memory and knowledge. subsequently, collected seeds from sarangani medicinal plants were sent to the national plant genetic resources laboratory of the university of the philippines in los banos, laguna for conservation purposes. results profile of the traditional healers the demographic profiles of the traditional healers are presented in table 2. of the 12 traditional healers who consented to participate in the study, 5 were from malapatan, 3 were from alabel, and glan and maasim had 2 healers each. female healers and those with b’laan ancestry predominated at 11 and 8, respectively, with 8 healers having no formal education and the rest having 1–6 years of primary education because of the distance of schools. moreover, healers’ ages varied across a broad spectrum (27 to 110 years old), with a mean age of 63.9 years. in terms of healing experience, traditional practitioners ranged from being neophytes (1 year) to veteran healers (90 years), with most of them falling within the range of less than 20 years of experience. in terms of religion, the healers professed to be christians, a fact borne out by small chapels established by protestant missionaries from the lowlands. when asked about their sources of healing knowledge, all respondents received information through oral transmission, with ten healers learning from their parents and grandparents. the remaining healers availed of instruction from government-sponsored seminars or the radio. unfortunately, a traditional healer refused to participate in an interview and disclose healing knowledge, as doing so could jeopardize her healing abilities. table 2. demographic profiles of the traditional healers. demographic profiles details frequency gender male 8% female 92% ethnicity b’laan 67% t’boli 17% aklanon 8% lowland tribes 8% education primary 36% secondary 0% tertiary 0% none 64% mean age 63.9 number of years practising healing ≤ 20 years 75% ≥ 20 years 25% sources of healing knowledge oral transmission 83% government-sponsored seminars/radio 17% perceived health status healthy sickly 100% 0% religion christian 100% genetic resources (2025), 6(12), 57–72 indigenous medicinal plants in the philippines 61 moreover, the interviewed tribal healers disclosed that the ability to heal is a gift, and that it stays in the family. while most healers use plants alone to treat sick individuals, a few invoke the spirits through incantations. biodiversity assessment of indigenous medicinal plants a total of 39 imp species were documented based on the accounts of the traditional healers (table 3). however, only 32 species, belonging to 31 genera, 18 orders and 20 families, were collected during community walks. seven taxa were identified up to the genus level only as they lack reproductive structures needed for species level identification. the comparable number of families and orders reflect the phylogenetic diversity of the medicinal plant species in the sarangani upland communities. families asteraceae and lamiaceae were the most represented with five species each. these were followed by families euphorbiaceae, moraceae, poaceae and zingiberaceae with two species each. of the collected plants, 26 were foraged within the community (backyards, roadsides and transition zones between forests and villages), while 6 species were collected in the forests by the healers. moreover, of the 39 plant species identified by healers as having medicinal properties, 21% were trees, 56% were herbs, 5% were climbers or vines, and the remaining 18% were classified as shrubs or small trees. indigenous medicinal plants and traditional healing knowledge from the semi-structured questionnaires and key informant interviews, information about the preparation and utilization of imps was collated. most herbal remedies were prepared using primarily leaves (82%), while others used stems (or tree bark), roots, and plant sap. moreover, traditional healers favoured decoction (85%) over fresh material use in their herbal concoctions. for some medicinal preparations, tree bark (or other tougher parts of the plant) was mixed with coconut oil and used as a liniment by spreading liberally on the skin. as for fresh leaves, some were directly applied to the skin as a poultice. moreover, except for t’kulu (tibouchina sp.), t’kas (elytropappus sp.), kataas (colocasia esculenta (l.) schott), k’lol (tinospora crispa (l.) hook. f. & thomson), langka (artocarpus heterophyllus lam.) and luy-a (zingiber officinale roscoe), which were combined with other plants in coconut oil during the preparation of the herbal remedies, the remainder of the medicinal plants were given as single preparations. incidentally, the traditional healers did not have any concept of dosage and were not mindful of the proportions of plant parts to water or oil that they used in their preparations. traditional healers likewise disclosed that most of the imps (56%) were readily available in the neighbourhood, did not need to be planted, and could be foraged anytime. conversely, imps (predominantly trees and shrubs) found in the forests were less accessible, and their harvesting posed significant risks. shown in table 3 are the imps of the ethnic groups along with their scientific and local names, their habit, utilization and their availability in the upland areas of sarangani province. traditional healers in sarangani province classified human ailments into nine categories: (1) gastro-intestinal diseases, (2) head, ear, nose, and throat diseases, (3) cuts or wounds, (4) diseases of the bone and muscles, (5) urinary ailments, (6) cough/colds, (7) fever, (8) mouth/tongue/tooth problems and (9) other ailments (table 3). in the upland areas, the more prevalent disorders were cough, loose bowel movement (lbm), kabuhi, stomachache and headache. a diagnosis of kabuhi is made when the patient experiences dizziness, nausea, chills and severe abdominal pain. this disorder is akin to heartburn, hyperacidity, or gastroesophageal reflux disease, depending on the nature and severity of symptoms. furthermore, the healers revealed their capability to treat buyag, a culturally recognized ailment that can be loosely defined as a malady resulting from a curse imposed by someone with malevolent intent, an 'evil eye', or nature spirits. this supposed ailment necessitates incantations in addition to the imps. figure 2 shows some indigenous medicinal plants used by the traditional healers. as for their healing practices, traditional healers disclosed that decoctions of combined langka leaves/ ginger and kataas/k’lol are effective against kabuhi. among the t’bolis in maasim, stems and leaves of t’kulu and t’kas are chopped into small pieces and put in a receptacle containing coconut oil. the resulting liniment or salve (haplas) is used to relieve shoulder pain and stomachache. for cough and colds, leaf decoctions of gabon (blumea balsamifera), lagundi (vitex negundo), mayana (coleus scutellarioides), tawa-tawa (euphorbia hirta), kalabo/ balbas pusa (orthosiphon aristatus) and mertaan (ficus septica) are used by healers. for persons with lbm, root decoction of banlo-banlo (galinsoga sp.) and leaf decoctions of star apple (chrysophyllum cainito) and white flower (andrographis paniculata) are given. in contrast, a tawa-tawa leaf decoction is given to a person suffering from headaches. on the other hand, patients with dengue are given papaya (carica papaya) and tawa-tawa leaf decoctions, while fresh samples of mertaan, root decoctions of bulong baltang (heliotropium indicum) and leaf decoctions of blibid (eleusine indica) are given for bughat. bughat, which loosely translates to relapse, is used to describe a condition when a person who is recovering from illness or who has shortly recovered from it becomes sick again. in addition, sili (capsicum sp.) and tawal leaf decoctions are used to treat snake bites while gabon and galong are used for urinary tract infections by the traditional healers. to treat fever, healers use alingatong or dendrocnide sp. (despite its itchiness), angelika/skaan bulan (kalanchoe pinnata) and native sibuyas (allium cepa). regarding their utilization, tawa-tawa, kyama (stevia rebaudiana) and mertaan have a wide range of medicinal uses. for instance, traditional healers use tawa-tawa for the treatment of malaria, cough, swelling, stomachache and headache, whereas kyama is utilized to address stomachache, toothache, dysmenorrhea, flatulence, and to facilitate wound cleansing. in addition, mertaan serves as a remedy for toothache, cough and bughat, while coleus amboinicus, commonly referred to as oregano, is utilized for cough treatment and is widely utilized by the tribes. moreover, another remarkable observation was related to plants having redundant utilities. for instance, tribal healers utilized ten, six, and five imps for common disorders like cough, lbm, and stomachache, respectively. genetic resources (2025), 6(12), 57–7262 buay et al ta bl e 3. m ed ic in al p la nt s us ed in s ar an ga ni u pl an d co m m un iti es . l bm , l oo se b ow el m ov em en t; u ti , u ri na ry tr ac t i nf ec tio n; iu cn c at eg or ie s: l c, le as t c on ce rn ; e n , e nd an ge re d; d d , d at a de fic ie nt . lo ca l n am e en gl is h/ co m m on na m e sc ie nt ifi c na m e fa m ily o rd er u se d fo r pl an t ha bi t pl an t pa rt us ed m od e of pr ep ar at io n m od e of ad m in is tr at io n w he re ha rv es te d so ur ce m et ho d of u se av ai la bi lit y iu cn a lin ga to ng st in gi ng ne tt le d en dr oc ni de sp . u rt ic ac ea e ro sa le s fe ve r tr ee le af d ec oc tio n d ru nk co lle ct ed fr om th e w ild w ild ha rv es te d si ng ly le ss av ai la bl e a m pa la ya bi tt er go ur d m im or di ca ch ar an tia cu cu rb ita ce ae cu cu rb ita le s co ug h cl im be r le af d ec oc tio n d ru nk h om e ga rd en cu lti va te d si ng ly re ad ily av ai la bl e a ng el ik a / sk aa n bu la n ka ta ka ta ka / a ir pl an t / ca th ed ra l be lls ka la nc ho e pi nn at a (l am .) pe rs . cr as su la ce ae sa xi fr ag al es fe ve r; co ug h h er b le af d ec oc tio n; fr es h d ru nk ; e xt er na l/ ru bb in g h om e ga rd en cu lti va te d si ng ly le ss av ai la bl e av oc ad o av oc ad o pe rs ea am er ic an a m ill . la ur ac ea e la ur al es lb m tr ee le af d ec oc tio n d ru nk h om e ga rd en cu lti va te d si ng ly re ad ily av ai la bl e lc ba nl oba nl o g al la nt so ld ie r g al in so ga s p. a st er ac ea e a st er al es lb m h er b ro ot d ec oc tio n d ru nk h om e ga rd en cu lti va te d si ng ly re ad ily av ai la bl e bl ili d g oo se g ra ss el eu si ne in di ca (l .) g ae rt n. po ac ea e po al es bu gh at (r el ap se ) h er b le af d ec oc tio n d ru nk u bi qu ito us w ild ha rv es te d si ng ly le ss av ai la bl e lc bu lo ng ba lta ng in di an he lio tr op e h el io tr op iu m in di cu m l . bo ra gi na ce ae bo ra gi na le s bu gh at (r el ap se ) h er b ro ot d ec oc tio n d ru nk h om e ga rd en cu lti va te d si ng ly re ad ily av ai la bl e g ab on sa m bo ng bl um ea ba ls am ife ra (l .) d c. a st er ac ea e a st er al es co ug h; u ti sh ru b le af d ec oc tio n d ru nk co lle ct ed fr om th e w ild ; h om e ga rd en w ild ha rv es te d; cu lti va te d si ng ly re ad ily av ai la bl e lc g al on g tr ee le af d ec oc tio n d ru nk co lle ct ed fr om th e w ild w ild ha rv es te d si ng ly le ss av ai la bl e ka la bo / ba lb as p us a ca t’s w hi sk er s o rt ho si ph on ar is ta tu s (b lu m e) m iq . la m ia ce ae la m ia le s co ug h; co ld h er b le af d ec oc tio n d ru nk h om e ga rd en cu lti va te d si ng ly le ss av ai la bl e ka pe co ffe e co ffe a ar ab ic a l. ru bi ac ea e ru bi al es lb m sh ru b le af d ec oc tio n d ru nk h om e ga rd en cu lti va te d si ng ly re ad ily av ai la bl e en ka ta as ta ro co lo ca si a es cu le nt a (l .) sc ho tt a ra ce ae a lis m at al es ka bu hi h er b le af fr es h ex te rn al / ru bb in g co lle ct ed fr om th e w ild w ild ha rv es te d w ith o th er pl an t le ss av ai la bl e lc genetic resources (2025), 6(12), 57–72 indigenous medicinal plants in the philippines 63 ta bl e 3 co nt in ue d lo ca l n am e en gl is h/ co m m on na m e sc ie nt ifi c na m e fa m ily o rd er u se d fo r pl an t ha bi t pl an t pa rt us ed m od e of pr ep ar at io n m od e of ad m in is tr at io n w he re ha rv es te d so ur ce m et ho d of u se av ai la bi lit y iu cn k’ lo l ti no sp or a cr is pa ( l. ) h oo k. f. & th om so n r an un cu la le s ka bu hi cl im be r ro ot fr es h ex te rn al / ru bb in g co lle ct ed fr om th e w ild w ild ha rv es te d w ith o th er pl an t le ss a va ila bl e ku so l a ro m at ic gi ng er ka em pf er ia ga la ng a (l .) zi ng ib er ac ea e zi ng ib er al es ea ra ch e h er b le af fr es h ea r d ro ps h om e ga rd en cu lti va te d si ng ly le ss a va ila bl e d d ky am a ca nd y le af st ev ia re ba ud ia na (b er do ni ) be rd on i a st er ac ea e a st er al es h er b le af d ec oc tio n d ru nk co lle ct ed fr om th e w ild w ild ha rv es te d si ng ly le ss a va ila bl e la ga no o re ga no co le us am bo in ic us lo ur . la m ia ce ae la m ia le s co ug h h er b le af d ec oc tio n d ru nk h om e ga rd en cu lti va te d si ng ly re ad ily av ai la bl e la gu nd i la gu nd i vi te x ne gu nd o l. la m ia ce ae la m ia le s co ug h sh ru b le af d ec oc tio n d ru nk h om e ga rd en cu lti va te d si ng ly re ad ily av ai la bl e lc la ng ka ja ck fr ui t ar to ca rp us he te ro ph yl lu s la m . m or ac ea e ro sa le s ka bu hi tr ee le af d ec oc tio n d ru nk co lle ct ed fr om th e w ild w ild ha rv es te d w ith o th er pl an t re ad ily av ai la bl e la ye t l an ga s h ea da ch e; lb m h er b le af d ec oc tio n d ru nk u bi qu ito us w ild ha rv es te d; cu lti va te d si ng ly le ss a va ila bl e lu ya g in ge r zi ng ib er offi ci na le ro sc oe zi ng ib er ac ea e zi ng ib er al es ka bu hi h er b st em d ec oc tio n d ru nk co lle ct ed fr om th e w ild ; h om e ga rd en w ild ha rv es te d; cu lti va te d w ith o th er pl an t re ad ily av ai la bl e d d m ay an a co le us co le us sc ut el la ri oi de s (l .) b en th . la m ia ce ae la m ia le s co ug h h er b le af d ec oc tio n d ru nk h om e ga rd en cu lti va te d si ng ly re ad ily av ai la bl e m er ta an fi g tr ee fi cu s se pt ic a bu rm . f . m or ac ea e ro sa le s to ot ha ch e; co ug h; re la ps e sh ru b le af fr es h po ul tic e co lle ct ed fr om th e w ild w ild ha rv es te d si ng ly le ss a va ila bl e lc n ab ol to ng ue so re tr ee sa p fr es h h om e ga rd en cu lti va te d si ng ly re ad ily av ai la bl e n at iv e si bu ya s o ni on al liu m c ep a l. a m ar yl lid ac ea e a sp ar ag al es fe ve r h er b le af d ec oc tio n d ru nk h om e ga rd en cu lti va te d si ng ly re ad ily av ai la bl e pa pa ya pa pa ya ca ri ca p ap ay a l. ca ri ca ce ae br as si ca le s d en gu e tr ee le af d ec oc tio n d ru nk h om e ga rd en cu lti va te d si ng ly re ad ily av ai la bl e d d genetic resources (2025), 6(12), 57–7264 buay et al ta bl e 3 co nt in ue d lo ca l n am e en gl is h/ co m m on na m e sc ie nt ifi c na m e fa m ily o rd er u se d fo r pl an t ha bi t pl an t pa rt us ed m od e of pr ep ar at io n m od e of ad m in is tr at io n w he re ha rv es te d so ur ce m et ho d of u se av ai la bi lit y iu cn si li si li ca ps ic um s p. so la na ce ae so la na le s sn ak e bi te h er b le af d ec oc tio n d ru nk h om e ga rd en cu lti va te d si ng ly re ad ily av ai la bl e si sl ok w ou nd cl ea ni ng h er b le af fr es h po ul tic e u bi qu ito us w ild ha rv es te d; cu lti va te d si ng ly le ss a va ila bl e s’ lo t ca ra ba o gr as s pa sp al um co nj ug at um p. j. be rg iu s po ac ea e po al es vo m iti ng h er b le af d ec oc tio n d ru nk u bi qu ito us w ild ha rv es te d; cu lti va te d si ng ly le ss a va ila bl e lc st ar a pp le st ar a pp le ch ry so ph yl lu m ca in ito l . sa po ta ce ae er ic al es lb m tr ee le af d ec oc tio n d ru nk h om e ga rd en cu lti va te d si ng ly re ad ily av ai la bl e lc ta lil vo m iti ng h er b st em d ec oc tio n d ru nk co lle ct ed fr om th e w ild w ild ha rv es te d si ng ly le ss a va ila bl e ta m bi sa n m ug w or t ar te m is ia s p. a st er ac ea e a st er al es co ug h h er b le af d ec oc tio n d ru nk h om e ga rd en cu lti va te d si ng ly re ad ily av ai la bl e ta w al sn ak e bi te tr ee le af d ec oc tio n d ru nk co lle ct ed fr om th e w ild w ild ha rv es te d si ng ly re ad ily av ai la bl e ta w ata w a a st hm a pl an t eu ph or bi a hi rt a l. eu ph or bi ac ea e m al pi gh ia le s m al ar ia ; co ug h; sw el lin g; h er b le af ; ro ot d ec oc tio n d ru nk co lle ct ed fr om th e w ild ; h om e ga rd en w ild ha rv es te d; cu lti va te d si ng ly le ss a va ila bl e t’ ka s el yt ro pa pp us sp . a st er ac ea e a st er al es sh ou ld er pa in sh ru b st em d ec oc tio n d ru nk co lle ct ed fr om th e w ild w ild ha rv es te d w ith o th er pl an t le ss a va ila bl e t’ ku lu ti bo uc hi na s p. m yr ta le s sh ou ld er pa in sh ru b le af d ec oc tio n d ru nk co lle ct ed fr om th e w ild w ild ha rv es te d w ith o th er pl an t le ss a va ila bl e tu ba -t ub a tu ba ng ba ko d ja tr op ha cu rc as l . eu ph or bi ac ea e m al pi gh ia le s m ou th so re ; h ea da ch e sh ru b le af d ec oc tio n d ru nk h om e ga rd en cu lti va te d si ng ly re ad ily av ai la bl e lc vi ck s si bu m in t m en th a sp . la m ia ce ae la m ia le s fe ve r; co ug h h er b le af d ec oc tio n d ru nk h om e ga rd en cu lti va te d si ng ly re ad ily av ai la bl e w hi te flo w er g re en ch ir et ta an dr og ra ph is pa ni cu la ta (b ur m . f .) w al l. ex n ee s a ca nt ha ce ae la m ia le s lb m h er b le af d ec oc tio n d ru nk h om e ga rd en cu lti va te d si ng ly re ad ily av ai la bl e w uh to ot ha ch e h er b le af d ec oc tio n d ru nk h om e ga rd en cu lti va te d si ng ly re ad ily av ai la bl e genetic resources (2025), 6(12), 57–72 indigenous medicinal plants in the philippines 65 indigenous medicinal plants: threats and conservation initiatives questionnaire administration and in-depth conversations with key informants were carried out to ascertain threats to their medicinal plant resource base. figure 3 illustrates the challenges traditional healers encounter in the sarangani uplands about their thk and the imps they utilize. the cultural practice of swiddening or alnigo (figure 3a) before upland rice cultivation led to significant deforestation and considerable biodiversity losses (figure 3c). another legitimate concern raised by the locals during field observations was the considerable deforestation caused by unregulated resource extraction in the mountainous regions. figure 3b, conversely, depicts an elderly tribal healer who has surpassed the remarkable age of 100 years but who was still active on the farm. regrettably, these elderly healers expressed their sorrow over their failure to pass on thk to the younger generation, attributing this to a notable disinterest from the latter. upon the demise of these knowledge keepers, the invaluable information they possess will be irretrievably lost to humanity. finally, the waning interest of the younger generation in traditional healing and their preference for modern medicines (figure 3d) were also identified as significant threats to the perpetuation of traditional healing practices and the conservation of imps. conversations with tribal healers indicated that although certain medicinal plants could be harvested year-round from home gardens and forest fringes, other ipms were diminishing due to various stresses. from 2016 to 2017, the prolonged effects of el niño resulted in wide-scale losses of flora in the sarangani uplands. other threats identified by the healers were climate change-related devastation (such as heavy rains and flash floods), pest infestation, unregulated harvesting, forest denudation, and the shift to modern medicines. the healers also disclosed that they had to walk long distances to harvest rarely occurring medicinal plants previously abundant in the figure 2. images of medicinal plants collected in sarangani upland communities. (a) blilid (eleusine indica); (b) mertaan (ficus septica); (c) white flower (andrographis paniculata); (d) alingatong (dendrocnide sp.); and (e) balbas pusa (orthosiphon aristatus). communities. moreover, residents from relatively accessible settlements such as lamlifew (malungon) and sitio malaya (kiamba) disclosed that they forsook traditional remedies in favour of modern medicines several years back, owing to the perceived superiority of the latter. furthermore, the healers acknowledged the potential extinction threats of medicinal plants if conservation efforts are not implemented. through continuous cultivation of imps in their backyards and home gardens, the healers inadvertently conserved these invaluable genetic treasures. furthermore, the healers disclosed that to avert the extinction of imps in their natural habitats, they exclusively harvest what is necessary. discussion sarangani traditional healers: a dying breed? one important feature of the sarangani traditional healthcare system is the preponderance of female healers – a fact that emphasizes their vital roles in the traditional healing system. this association of women with traditional healing practices transcends cultures due to their nurturing nature and profound knowledge of plants that thrive in their communities. struthers (2003) reported that aboriginal women healers in the united states and canada generally practised holistic healing using age-old methods passed down by their ancestors. however, the idea that women are the predominant healers is not a universal truth, as there are cultures where male healers serve as the primary practitioners (gessler et al, 1995; semenya and potgieter, 2014). in morocco, bakker (1992) documented the notable rise of berber women as traditional healers when their male counterparts lost influence due to political upheavals in the region. french colonization and the consequent succession of the moroccan makhzen in the political sphere stripped male healers of prestige, leading to the remarkable emergence of women healers to fill this healthcare vacuum. this is one case genetic resources (2025), 6(12), 57–7266 buay et al wherein politics, and not modernization, was the identified cause of shifting gender roles in the traditional healthcare system. another study about busoga healthcare practitioners in uganda (isiko, 2018) revealed that while men and women are equally accepted in traditional medicine, the power and influence that they wield depend on societal expectations relating to gender roles. it must likewise be emphasized that traditional healers possess a critical understanding of medicinal plants, owing to their long years of experimentation and use. acquiring and mastering utilization of such material requires significant time and effort. this knowledge, transmitted to them through generations, enables them to provide healing and therapy for poverty-stricken communities beyond the reach of basic social services. however, one factor that does not bode well for the perpetuation of traditional knowledge was the advanced ages of the sarangani knowledge keepers. similarly, teves et al (2023) reported that eskaya healers in bohol, philippines, are dwindling in numbers because of old age-related deaths. these are elderly people who become weak and decrepit with advancing years, lack of proper nutrition and inadequate health care due to the remoteness of their villages. moreover, traditional healing knowledge, being passed on to chosen members of the family, limits its transmission, especially when the appointed successor does not show any interest in learning these skills. consequently, when these elderly figure 3. threats to medicinal plants and traditional healing knowledge in the sarangani uplands: a, ongoing alnigo (or swiddening); b, advanced ages of knowledge keepers and non-transmission of healing knowledge to succeeding generations; c, denudation of mountains; d, proliferation of modern medicines. healers pass on, the priceless healing knowledge that they possess will forever be lost to humanity. sarangani indigenous medicinal plants: current state and major pressures this study revealed a significant number of imps and a diverse repertoire of healing rituals and knowledge among the visited ethnic communities in the sarangani uplands. this prevalent use of medicinal plants for healing in other areas of mindanao has been reported in the mount matutum protected landscape (alinsug et al, 2022), agusan del sur (dapar et al, 2020), davao occidental (paraguison et al, 2020; cabugatan et al, 2022) and surigao del sur (ilagan et al, 2022). community inventory also revealed that sarangani medicinal plants with high utilization were more frequently found closer to home. this fact underscores the direct correlation between the availability and cultural importance of medicinal plants to the sarangani tribes. with greater accessibility, healers have more opportunities to test by trial and error the efficacy of potentially medicinal plants (vandebroek et al, 2008). furthermore, in sarangani upland tribal communities, a direct relationship between the redundancy of medicinal plant use and the more prevalent physical ailments was established. this relationship was likewise borne out by studies done by kunwar et al (2015), genetic resources (2025), 6(12), 57–72 indigenous medicinal plants in the philippines 67 kumar and bussman (2011), and vandebroek et al (2008) in nepal, india, and bolivian andes, respectively. however, during recent years, the multi-fold effects of rapidly evolving social-ecological scenarios have severely impacted the sarangani traditional agroecosystem, resulting in wide-scale natural resource losses. moreover, species introductions from the lowlands resulted in a mosaic of indigenous and non-indigenous medicinal plants, giving the healers a more expansive repertoire on which to experiment. study results revealed, however, that imps (especially those thriving in the wild) are declining in terms of numbers in their natural habitats. among the identified pressures leading to these losses are climate change-related devastation (such as heavy rains and flash floods), pest infestation, unregulated harvesting and forest denudation. in addition, imps thriving in transition areas and roadsides are more often subjected to anthropogenic pressures, resulting in their diminished numbers. while some imps are still available, several could no longer be found in the vicinity, and tribal healers reported travelling long distances to harvest them. in limpopo province, south africa, locals identified over-harvesting and indiscriminate collection of imps as factors leading to large-scale losses (mathibela et al, 2015). moreover, several studies in sri lanka and northern ethiopia ascribe losses of imps to ecological devastation, climate change-associated devastation, agricultural expansion and human habitation, among other factors (de silva and wettasinghe, 2004; mesfin et al, 2013). owing to the burgeoning global population, the increased demand for modern medicines as well as the international trade for medicines by pharmaceutical companies, imps are disappearing in the wild because of overharvesting (soetan and aiyelaagbe, 2009; oladele et al. 2011; otieno and analo, 2012; ganie et al. 2015). another study by birhanu et al (2015) identified the shift to modern medicines as a factor leading to the extinction of imps. in the sarangani uplands, conversations with locals revealed the use of modern medicines, such as paracetamol (for headaches), loperamide (for lbm), phenylephrine hcl or neozep (for colds), bioflu (colds and flu) and mefenamic acid (toothache). to prevent infection, tribal people open an amoxicillin capsule and sprinkle its contents on an open wound. accessibility of the tribal communities was also identified as a factor contributing to medicinal plant losses in the sarangani uplands. villages like lamlifew (malungon) and sitio malaya (kiamba) are relatively accessible and can be reached by 4x4 vehicles and single motorcycles. the former community is dominated by b’laans and is frequently frequented by tourists and researchers for its school of living tradition for abaca weaving. local inhabitants of lamlifew enjoy the creature comforts of modernization (i.e. running water, electricity, cellphone service, internet service and cable television, among others). sitio malaya, on the other hand, is inhabited by t’bolis who manage a thriving abaca industry for local and international markets. all household needs (food and medicines) are purchased from the lowland markets. in both these places, traditional healing had been wholly abandoned for modern medicine. in a study done in the himalayas, tali et al (2014) identified agricultural expansion, road improvement, overgrazing and deforestation as threats leading to the extinction of medicinal plants. furthermore, increased accessibility and foreign introductions resulted in a mosaic of indigenous and non-indigenous medicinal plants in the sarangani ethnic communities, resulting in a more diversified pharmacopoeia. needless to say, medicinal plants remain the sole refuge of remote tribal communities in sarangani province, where the provision of basic social services is scanty at best and non-existent at worst. the need for self-sufficiency was highlighted during the covid-19 pandemic (2019–2021) when these communities were isolated, travel was prohibited, food/resource supply chains were disrupted, and prices of commodities soared beyond the reach of many. furthermore, the practice of traditional healing was more prevalent in rural communities with very little/no access to modern medicines owing to geographical isolation and economic reasons (maramba-lazarte, 2020). in another study done in six middle-income countries (china, ghana, india, mexico, russia and south africa), oyebode et al (2016) identified factors leading to the decline in the use of medicinal plants in these countries. these were the significant shifts in social trends and cultural beliefs, as well as the political support and provision of resources for training, practising and increasing public awareness of modern medicines. traditional healing knowledge: headed for oblivion in the remote sarangani uplands, conversion to christianity primarily resulted in the abandonment of belief in nature spirits. for some healers, however, some vestiges of spiritism persisted. in fact, this belief that human activities intersect with the goings-on in the spiritual world persists even with the incursion of modernization and christianization. similarly, local inhabitants in namibia, south africa and bangladesh believe that nature spirits influence their health and that the efficacy of imps hinges on a complete understanding of their physical and spiritual purposes (motaleb et al, 2010; cheikhyoussef et al, 2011; stofferson et al, 2011). this persistence of spiritism in healing practices, which has been labelled as pagan and backwards, has caused a significant number of sarangani locals to forsake traditional medicine for modern medicine. in the sarangani uplands, losses of thk can be ascribed to additional pressures such as acculturation, education, increasing accessibility of the communities, the lure of modernization/technology, aging knowledge keepers (healers) and the oral nature of knowledge transmission that predisposes it to loss (posey, 1996). mahwasane et al (2013) concurred that the thk is gradually becoming extinct in the absence of a writing system and because healers do not keep written records. in south africa, thk has been largely ignored because, being verbally transmitted, it could not be included in school curricula or policy documents (mathibela et al, 2015). consequently, huge volumes of thk worldwide remain undocumented, thereby underscoring now more than ever the need to record thk and conserve imps, before they completely disappear. in addition, the unwillingness of some healers (particularly in kiamba and maitum) to divulge healing knowledge that they consider secret information likewise exacerbates thk losses. this unwillingness to share thk was also reported by kala et al (2006) and giday et al (2003). others are wary about researchers coming to their communities and extracting traditional knowledge that had been passed on to them by their ancestors. the lack of written genetic resources (2025), 6(12), 57–7268 buay et al records due to the oral nature of knowledge transmission compounds these losses, while the secretive nature of some healers dooms knowledge transmission to permanent loss. further exacerbating these losses is the diminished intergenerational knowledge transfer of thk. consequently, there is no mentoring for the next generation and, if the elderly healers pass on, their thk will be lost forever. in the sarangani uplands, thk is losing its appeal among the younger generation, who consider these as backward, primitive, unchristian and inferior to modern medicine. in rural malaysia, the oral nature of mentoring has also caused failure in thk transmission to the next generation (batugal et al, 2004). consequently, the older generation of healers will have no pool of recruits to inherit their knowledge and practices. further contributing to the decline in the practice of thk is the proliferation of private and state-sponsored health facilities all over the province, especially in more accessible areas. future directions sarangani traditional healthcare system: ways forward in sarangani province, healers’ knowledge results from learning and social interactions between knowledge keepers and those who seek it. however, the majority of thk practitioners still lack scientific justifications for their methodologies. although they believe in the outcomes of their practices, they lack understanding of the mechanisms that produce these effects. interviewed healers were unable to elucidate the mechanisms and efficacy of the remedies. bannerman (1977) emphasized the necessity of assessing thk through the lens of contemporary science, thereby enhancing beneficial practices and safeguarding against detrimental ones. rather than depending on trial and error resulting from arbitrary screening methods, well-documented thk could assist scientists in identifying plants with potential medical characteristics. scientific validation of imps through phytochemical, toxicological and pharmacological studies is thus warranted to eliminate quackery associated with thk, warn against inappropriate usage, and identify potential sources of medicines. however, one crucial matter that needs to be considered is the lack of coherence among healers about modes of preparation and proper dosages of imps (wilcox and bodeker, 2004). once the effectiveness of thk is properly assessed, the next logical step is its integration into a nation’s healthcare delivery system.  integrating thk into national healthcare will preserve the indigenous peoples’ cultural heritage for future generations (mahwasane et al, 2013). one way to do this is by including traditional healers in the crafting of a comprehensive healthcare strategy that is grounded in inclusivity, knowledge pluralism and sharing of information. in 1997, the philippine government passed the traditional and alternative medicine act, which affirmed government support for thk (marambalazarte, 2020). kaido (1997) proposed that traditional healers could be significantly used if they were organized and trained. empowering traditional healers to participate in the crafting of local healthcare strategies for disadvantaged people whose views are oftentimes ignored during deliberations is also suggested. moreover, policy positions relating to thk promotion should be more nuanced, and the use of imps must not be encouraged when there are reasons to doubt their effectiveness in comparison to modern medicine. ultimately, collaborative initiatives by the scientific community and governments are essential to generate the impetus for such actions. dissemination of pertinent research findings and facilitating further education and training programmes for traditional healers will empower them to document and share their significant indigenous knowledge independently. the use of audiovisual media, espoused by bidwell et al (2011), can serve as an effective means for knowledge transfer. biocultural conservation a comprehensive inventory of imps must be undertaken before any conservation initiative commences. soetan and aiyelaagbe (2009) stated that imps, when not recorded or classified, can hamper conservation efforts. the importance of documenting imps as a prelude to their conservation was also espoused by hamilton (2003). in the sarangani upland communities, emerging sociocultural scenarios underscore the need for complementary ex situ and in situ conservation of imps. through continuous utilization and cultivation of imps in their backyards and home gardens, the healers are unknowingly conserving these priceless genetic resources. the healers also practice judicious collection of imps and only harvest when these are needed. in a rural nigerian community, oladele et al (2011) reported that the cultivation of imps contributed to their conservation while batugal et al (2014) espoused continuous cultivation as an efficient conservation strategy. in africa, okigbo et al (2008) maintained that only through sustainable conservation practices can a constant supply of imps be ensured for prolonged periods. in southern italy, regional and national parks (considered protected areas) that feature medicinal plants conserved ex situ attract ethnobotanists from all over the country because of the joint preservation of imps and associated thk of the rural people (menale et al, 2016). along these lines, communityinitiated ex situ conservation of sarangani imps and thk must be undertaken in collaboration with academia, the local government and other stakeholders to preclude further losses of imps. in doing this, priority should be given to imps having redundant utilities since this will help offset pressure on imps that are considerably threatened in the wild. as for the imps collected in the sarangani uplands, these were sent to the philippine national genebank for ex situ conservation. this backup collection will serve as an ‘insurance policy’ against the extinction of medicinal plant species amidst widespread environmental devastation in their natural habitats in the sarangani uplands. additionally, these imps are potential sources of new drugs for present and future health needs, not just of the locals but also of the communities at large. the need to preserve thk via knowledge transfer is crucial for the perpetuation of tribal culture and the promotion of local communities' rights over their plant genetic resources. moreover, thk, when harmonized with frontier knowledge, may prove useful in addressing problems that may arise in the future. recognizing the significance of the knowledge held by these healers and meticulously recording essential elements with sensitivity and respect for intellectual property rights are paramount in assisting the healers (mathibela et al, 2015). therefore, it is important to have a governance framework to effectively protect the thk of the tribes for posterity. genetic resources (2025), 6(12), 57–72 indigenous medicinal plants in the philippines 69 limitations of the study this study covered a limited number of study sites and gleaned valuable healing knowledge from a handful of traditional healers. some of the sitios were inaccessible due to geographic isolation and difficult terrain, while others, unfortunately, still experience peace and order issues brought by insurgent groups, specifically in the towns of banlas, malapatan, datal anggas and alabel. in addition, the danger posed by armed jihadist muslim groups also prevented the research team from exploring areas in maasim and maitum. as for the healers themselves, some were unwilling to share healing knowledge in the belief that doing so would diminish their powers, while others were wary about sharing traditional knowledge with outsiders who they believe are merely out to extract information from them. moreover, in some sitios, the locals had completely shifted to modern medicine and traditional healers were nowhere to be found. furthermore, on the part of the researchers, the expensive and lengthy process of securing government permits for every community visited often discourages them from visiting more communities. consequently, as some of the healers did not impart their thk, it is possible that some of the imps were not documented in this study. conclusion this study unveiled the healing knowledge of local healers and the diversity of medicinal plants in the sarangani uplands. results, however, revealed that these imps are declining in numbers because of environmental degradation and the shift to modern medicines, among other pressures. also revealed by this study are thk losses due to the advanced ages of knowledge keepers and the shifting preferences of the sarangani locals towards modern medicine. to preclude further losses of imps, the following are recommended: (1) complementary in situ and ex situ conservation, (2) rehabilitation of the natural habitats of imps, (3) documentation of the thk of elderly healers before they pass on, (4) further scientific studies to validate thk, (5) establishment of a school of living traditions for the preservation of thk and its transmission to the younger generation, (6) protection of imps from exploitation and biopiracy through appropriate policy and legislation and (7) promotion of value-adding, utilization and widescale cultivation of imps. having a continuous supply of imps (along with their associated traditional knowledge) to promote and support the health of the locals will bring the community (and eventually the philippines) closer to the attainment of its sustainable development goals (sdgs), such as improved health (sdg3), empowerment of the tribal women (sdg5), and self-sustainability (sdg11) in the face of disasters and isolation. furthermore, when validated by scientific knowledge and promoted, these imps can be a potential livelihood source for tribal households, resulting in improved household income. through these interventions, it is envisaged that the sarangani imps and their associated thk will be preserved for future generations of tribes. finally, the conservation of thk will likewise make tribal households more resilient against the ravages of climate change, pandemics, and other future problems and perturbations. traditional healers’ organization, empowerment and participation are essential for developing suitable and culturally sensitive healthcare interventions for the local community. supplemental data supplemental material 1. questionnaire about sarangani indigenous medicinal plants acknowledgments the authors extend their most profound thanks to the tribal healers who took part in the study. authors’ contributions flz conceptualized the study, supervised its implementation, revised the first draft, and co-wrote the final paper with mcbh, who also carried out confirmation of taxonomic identification of medicinal plants and finalization of the article. bmgb carried out the majority of field work, wrote the first draft and did data analysis. crmr provided good quality pictures for the paper, helped in field collection and in the processing of the paper for publication while chma assisted in field preparations, collection and ex situ conservation of collected medicinal plants at the philippine national genebank. conflict of interest statement the authors have declared that no competing interests exist. ethics statement a free and prior informed consent for this study was 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genetic resources (2025), 6 (11), 24–40 doi: 10.46265/genresj.bwnf7962 https://www.genresj.org issn: 2708-3764 overview of germplasm collecting activities for plant genetic resources for food and agriculture in sudan from 2002 to 2022 ibrahim mohamed el tahir *, ali zakaria babiker, elsadig ahmed abdalla, awadelkarim alam elhuda ahmed, mashaer obaed yousif goda and magdi ahmed mohamed elgabri agricultural plant genetic resources conservation and research centre, agricultural research corporation, sudan abstract: from 2002 to 2022, the agricultural plant genetic resources conservation and research centre (apgrc) in sudan conducted 56 collecting missions for plant genetic resources for food and agriculture (pgrfa) in sudan. these missions aimed to conserve the country’s crop genetic diversity and covered different states and almost all ecological zones within the country, from the desert in the north to the high-rainfall savannah in the far south. different farming systems were included, such as rain-fed, irrigated and flood-irrigated systems. the most covered states were west darfur in the far west followed by south kordofan in the western-central region. a total of 7,720 pgrfa accessions were collected encompassing diverse crops and plant species within different plant groups. cultivated varieties made up 90% of the whole collection, while crop wild relatives accounted for 8%, and range plants represented the remaining 2%. cereals were the most collected group (48%), followed by vegetables (17%). the least represented groups were range plants, medicinal plants and fibre crops. sorghum was the most represented crop in the collection with 2,481 accessions, followed by pearl millet with 1,022 accessions. hundreds of accessions of cowpea, okra, sesame and other crops were also collected. a total of 181 accessions of natural range plants were collected from selected states. the materials collected during these germplasm collecting missions will be conserved at the apgrc genebank, characterized and evaluated for different traits. further germplasm collection activities may be carried out in the future to address any identified gaps. keywords: pgrfa, collecting missions, conservation, cultivated varieties, crop wild relatives, range plants, south kordofan citation: el tahir, i. m., babiker, a. z., abdalla, e. a., ahmed, a. a. e., goda, m. o. y., elgabri, m. a. m. (2025). overview of germplasm collecting activities for plant genetic resources for food and agriculture in sudan from 2002 to 2022. genetic resources 6 (11), 24–40. doi: 10.46265/genresj.bwnf7962. © copyright 2025 the authors. this is an open access article distributed under the terms of the creative commons attribution license (cc by 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. introduction sudan is the third largest country in africa, after algeria and the democratic republic of congo, with an area of 1.88 million km2 (unep and hcenr, 2020). it is located between latitudes 10◦ and 22◦ north, and longitudes 22◦ and 38◦ east. sudan is ecologically divided into five vegetation zones according to rainfall patterns from north to south (figure 1). these are: 1) desert (0–75mm), 2) semi-desert (75–300mm), 3) low-rainfall savannah on clay or sand (300–800mm), ∗corresponding author: ibrahim mohamed el tahir (eltahir81@yahoo.com) 4) high-rainfall savannah (800–1500mm) and 5) mountain vegetation (300–1000mm) (hcenr, 2015). according to the ministry of agriculture and forestry (2015), sudan’s cultivable arable land is estimated at 86 million hectares. however, less than 20% of the potential area is utilized in three major farming subsectors (ministry of agriculture and forestry, 2015): • the irrigated system subsector, estimated at 5 million hectares, where 100% of wheat and 25% of sorghum are produced, in addition to others including vegetables and fruit trees, oil crops such as groundnut, and cotton as a cash crop; • the semi-mechanized rain-fed subsector, estimated at 6 million hectares, where the two main received: 28.10.2024 accepted: 28.01.2025 published online: 07.03.2025 https://www.genresj.org https://www.doi.org/10.46265/genresj.bwnf7962 https://www.genresj.org https://www.doi.org/10.46265/genresj.bwnf7962 mailto:eltahir81@yahoo.com genetic resources (2025), 6 (11), 24–40 collecting pgrfa in sudan from 2002–2022 25 crops are sorghum and sesame, in addition to sunflower; • the agro-pastoral traditional rain-fed subsector, of about nine million hectares, where main crops produced are sorghum, pearl millet, sesame and groundnut, as well as seed watermelon and roselle. sudan is endowed with different ecosystems making it rich in biodiversity, including plant genetic resources for food and agriculture (pgrfa). sudan is part of the east african primary region of crop genetic diversity, with high diversity for coffee, cotton, cowpeas, melons, millets, olives, peas, sesame and sorghum (khoury et al, 2016). wild relatives of crops such as sorghum, pearl millet, okra, watermelon and melon are known in the country (mahmoud et al, 1995). old introduced cultivars for crops such as maize, faba bean and tomato are still existing and utilized by some farmers. in addition, rangelands encompass different ecological zones extending from desert and semi-desert in the north to low and high rainfall savannah to the southern border and include a wealth of natural range plant species (hcenr, 2013). factors threatening the genetic diversity of pgrfa in sudan include several natural and man-made factors. the most important ones are drought spells and seasonal rain fluctuations, expansion in modern agriculture and use of advanced improved cultivars, expansion in new constructions and activities other than crop cultivation, and disturbance of normal traditional systems of life, including agricultural activities due to civil strives and wars in several regions within the country (hcenr, 2015). in order to safeguard these important genetic resources, the agricultural research corporation (arc) of sudan established the plant genetic resources programme (pgr programme). historically, the pgr programme dates back to the early 1980s, when activities for collecting and conserving local genetic resources of horticultural crops were conducted by the horticultural research section of the arc in collaboration with the international board for plant genetic resources (hassan et al, 1983). in 1995, the programme was mandated for collecting, conserving and enhancing the use of the genetic resources for different agricultural crops under what was by then called the plant genetic resources unit. following expansion in the total germplasm holdings and the human and physical capacities of the programme, this unit was upgraded in 2014 into a centre now known as the agricultural plant genetic resources conservation and research centre (apgrc), which is currently managing the pgr programme. the apgrc headquarters are located in the city of wad medani in central sudan. during the 1990s and 2000s, the coverage of pgrfa acquired by the pgr programme expanded to different groups such as cereals, oil crops, grain legumes, in addition to horticultural crops including vegetables, fruit-producing plants and medicinal and aromatic plants. significant progress in the efforts to collect and acquire pgrfa from different sources occurred from 2003, thanks to the availability of necessary financial resources to conduct germplasm collecting missions from different regions, as well as the improvement in the physical and human capacities of the pgr unit. funding for the pgr unit came from the eastern africa plant genetic resources network (eapgren) through a project funded by the swedish international development cooperation agency (sida), which extended up to 2010, and other funding sources such as the global crop diversity trust (crop trust). moreover, the budgetary payments to the pgr programme were significantly increased by the national government on an annual basis since 2014. this paper is intended to provide a general overview of the collecting activities conducted under apgrc, and summarize the results obtained over 20 years from 2002 to 2022 for collecting indigenous pgrfa within sudan for conservation in the apgrc genebank facilities. those genebank facilities consist of seedbanks, and field genebanks for vegetatively propagated crops. information on germplasm collecting activities has been documented within the apgrc genebank documentation system, as well as in the global pgr data platform genesys (http://www.genesys-pgr.org/wiews/sd n002). this review provides information on the methodologies used to collect pgrfa, the locations visited, the germplasm materials collected as well as the phenotypic variations observed on the collected materials. the results are thoroughly discussed to explain the extent and importance of the geographical coverage and the pgrfa materials collected. the conclusion highlights the major achievements from the germplasm collecting activities and identifies major gaps to be addressed in the future. methodology the germplasm collecting activities followed a structured process, starting with planning and ending with germplasm sampling and data recording on the collected pgrfa from the targeted geographical locations before their conservation at the apgrc genebanks. planning planning for germplasm collection was conducted through consultative processes involving all apgrc scientific staff during regular programme planning activities within the arc national research programme planning process. geographical locations and taxa for collecting were identified and prioritized based on the following: a. geographical prioritization ecological zones and geographical areas were selected and prioritized based on the following criteria: http://www.genesys-pgr.org/wiews/sdn002 26 el tahir et al genetic resources (2025), 6 (11), 24–40 figure 1. map of sudan vegetation zones (eltoum et al, 2023) 1. geographical areas known for their richness in pgrfa diversity across different ecological zones in the country. this was the case for kordofan and darfur regions in western sudan, blue nile state in central to eastern sudan, river nile and northern states in northern sudan. 2. major geographical gaps that were identified after 2014 in the darfur region in far western sudan. b. taxonomical prioritization different crops and plant taxa for collection were identified and prioritized according to the following criteria: 1. specific taxonomical gaps within the existing germplasm collections for specific crops already held by apgrc, as identified in consultation with different crop specialists, such as for crops like sorghum and pearl millet. 2. overall taxonomical gaps in terms of crops and plant groups almost or fully absent within the apgrc collection, as was the case for taxa and plant groups such as banana, date palm, crop wild relatives and natural range plants. detailed collection work plans were developed annually by the apgrc researchers in consultation with the apgrc technical staff and/or directors of the arc stations in the targeted regions, as well as with technicians and local communities there. the whole processes of planning at different stages were coordinated, guided, supervised and approved by the apgrc director, who afterwards closely followed the implementation processes. targeted pgrfa regular germplasm collecting missions were organized annually across the different cultivation seasons starting from season 2002–2003 to season 2013–2014. they targeted different pgrfa, including cereals such as sorghum and pearl millet; oil crops such as sesame and groundnut; legumes such as cowpea and faba bean; vegetables and cucurbits such as okra, tomato, melons and watermelon; medicinal and aromatic plants such as roselle and fenugreek, and fruit producing plants such as banana and date palm. starting from 2014, the focus was on filling geographical and taxonomical gaps, of which the latter included date palm, natural range plants and crop wild relatives. targeted locations germplasm collecting activities targeted various farming systems in the different ecological zones during the appropriate seasons, including rain-fed, irrigated and river-flooded systems. the administrative states of the country were the broad targeted geographical locations for the different collecting missions. the ecological zones covered across the different states included (supplemental table 1): • the desert and semi-desert zones in northern, river nile, khartoum, kassala and red sea states; • the low-rainfall savannah on clay and sand in the states of gezira, sennar, blue nile, gedarif, and in the states within kordofan and darfur regions; • high-rainfall savannah in southern parts of the blue nile state, and kordofan and darfur regions. the major geographical gaps targeted since 2014 were the different states within the darfur region of far western sudan, which was difficult to cover earlier because of the civil war since 2003. means of transportation to these locations was usually a 4 wd vehicle facilitating reaching to different collection points that were usually away from asphalt roads. however, they were accessible under the guidance of the representatives of the local authorities and communities within the collecting teams. collecting approaches the collecting missions were implemented following either multi-species or single-species collecting approaches. the cultivation season in sudan usually starts in summer from may to october, and extends to winter from november to april. therefore, the germplasm collecting activities from in situ habitats were usually carried out during the harvesting times, which were from october to december or beyond for summeradapted crops such as sorghum, sesame, cowpea and okra; and from february to april for winter-adapted crops such as wheat, faba bean and tomato. multi-species collecting missions a total of 39 multi-species collecting missions were organized across 17 years during the period extending genetic resources (2025), 6 (11), 24–40 collecting pgrfa in sudan from 2002–2022 27 table 1. approaches and total numbers of collecting missions conducted from 2002 to 2022. more details are available in supplemental tables 1 and 2. approach targeted plants total multi-species different crop groups 27 vegetables 1 crop wild relatives 8 range plants 3 sub-total 39 single-species banana 11 date palm 4 sorghum 1 watermelon 1 sub-total 17 grand total 56 between the cultivation seasons of 2002–2003 and 2021–2022, with the exception of three seasons (2009–2010, 2011–2012 and 2012–2013), with at least one mission per year (table 1 and supplemental table 1). those missions covered different areas of the country and targeted different crop and plant species, including in some years vegetables, crop wild relatives or natural range plants (supplemental tables 1 and 2). single-species collecting missions seventeen single species collecting missions were organized during the specified period. they targeted sorghum, watermelon, banana and date palm from different states (supplemental table 1, table 1). collecting teams each team carrying out a collecting mission was usually headed by an apgrc researcher and might include a collaborating or assisting scientist and/or a technician from apgrc or arc (supplemental table 1). missions conducted since 2014 in the states of the darfur region were led by directors of the agricultural research stations in such states. each team also included a representative from the state ministry of agriculture, and a representative from the local community in the targeted area. germplasm sampling germplasm samples were collected from farmers’ fields, wild habitats or rangelands. each population at each specific site was sampled separately with a basic approach targeting around 50 plants per population. however, for populations with fewer than 50 plants, only the available plants were sampled. materials collected included seeds, panicles, fully ripe or dry fruits in the form of soft berries, pods or capsules, corms for banana, and off-shoots for date palm (figure 2). seeds were then extracted and processed for long-term conservation in the seedbank, while banana corms and date palm off-shoots were prepared and planted in the field genebanks. data recorded a germplasm collection form was used to record passport data on each sample collected (supplemental material 1). the form was derived from the fao-ipgri multi-crop passport descriptors (fao and ipgri, 2001). it consisted of major information such as taxonomic data, georeferenced data on the site of collection, date of germplasm collection, and local names. however, a specific germplasm collection form including more descriptors was used for collecting crop wild relatives during 2016 and 2017 (eastwood et al, 2022). gps was used to determine the coordinates and altitudes of the collection sites. results results obtained through the collecting activities across the cultivation seasons from 2002–2003 to 2021–2022 are summarized in terms of geographical locations covered, germplasm materials collected and phenotypic variations observed on the collected samples. missions conducted and sites covered a total of 56 germplasm collecting missions were conducted, including 39 for multiple species and 17 for single species (table 1). among the single-species missions, 11 and 4 targeted banana and date palm genetic resources respectively, while sorghum and watermelon were targeted each by only one mission in delta elgash in kassala state, and north and west kordofan states, respectively. germplasm samples were collected from 1,155 sites within the 18 states of sudan (supplemental table 2). they were located within latitudes ranging between 09.52◦ n in south kordofan state and 20.93◦ n in northern state; and longitudes ranged between 21.85◦ e in west darfur state and 38.43◦ e in red sea state (figure 3). in terms of collection sites, the most covered states were west darfur and south kordofan, where germplasm entries were sampled from 142 and 135 sites, respectively. the least covered states were sennar and khartoum, where germplasm entries were sampled from five and nine sites, respectively. the number of states contributing to the total accessions of each crop varied considerably between the different crops, reflecting the diverse growth habitats and requirements. while okra (abelmoschus spp.) accessions were sampled from 17 states, and sorghum (sorghum spp.) and roselle (hibiscus sabdariffa) were sampled from 16 states each, genetic resources of 20 crops were sampled from only five states or less, including the date palm accessions, which were sampled from only two states (supplemental figure 1). germplasm materials collected a total of 7,720 accessions were collected from more than 40 cultivated crops and other pgrfa species (supplemental table 3). they were composed of different crop groups including cereals such as sorghum 28 el tahir et al genetic resources (2025), 6 (11), 24–40 figure 2. banana and date palm materials collected (sorghum spp.), pearl millet (pennisetum spp.) and maize (zea mays); grain legumes such as cowpea (vigna spp.), faba bean (vicia faba) and chickpea (cicer arietinum); oil crops such as sesame (sesamum spp.) and groundnut (arachis hypogea); vegetables such as okra (abelmoschus spp.), tomato (solanum lycopersicum), and pumpkins and squashes (cucurbita spp.); medicinal and aromatic plants such as roselle (hibiscus spp.) and fenugreek (trigonella foenum-graecum); fruit-producing plants such as banana (musa spp.) and date palm (phoenix dactylifera), in addition to natural range plants. cereal genetic resources were the most represented in the collected materials, accounting for 49%, followed by vegetables at 17%, while medicinal and aromatic plants, range plants and fibre crops were the least represented, comprising 5%, 2% and less than 1%, respectively (figure 4). the total number of collected accessions varied greatly between cultivated crops ranging from only one to a few thousands. out of 47 cultivated crops covered, 12 crops were represented by more than 100 accessions of each, collected from different numbers of states that varied between different crops (table 2), adding up to 6,656 accessions and representing 86% of the total collection. the rest (1,064 accessions) were from other cultivated crops, wild plants or natural range plants, including 38 genera of other cultivated crops and wild plants, in addition to 39 genera of range plants. the most collected crops were sorghum and pearl millet, of which 2,481 accessions and 1,022 accessions were collected, respectively (table 2). hundreds of accessions ranging from 179 to 449, were collected from date palm, melons, maize, watermelon, roselle, groundnut, banana, okra, sesame and cowpea. all 18 states of sudan were represented in this top collection, with the total number of accessions varying from 14 in khartoum and sennar states to 1,550 from south kordofan state (table 2). almost 90% of the accessions collected were from cultivated varieties, while only 8% were wild, including crop wild relatives and other wild plants apart from range plants. the rest of the accessions were from natural range plants, representing 2% of the total materials collected. taxa of crop wild relatives fully identified ranged between five for sorghum (sorghum virgatum, s. purpureosericeum, s. halepense, s. bicolor verticilliflorum and s. aethiopicum), three for eggplant (solanum incanum, s. dubium and s. cerasiforme), and two for pearl millet (pennisetum stenostachyum and p. glaucum monodii) and melons (cucumis melo agrestis and c. metulikerus). others including watermelon, sesame, cowpea, and rice had only one fully identified wild species, which were citrullus colocynthis, sesamum alatum, vigna vaxillata and oryza barthii, respectively (supplemental figure 2). the total accessions collected of natural range plants were 181. they included 151 accessions, fully or partially identified taxonomically, belonging to 50 species across 37 genera. however, 30 accessions were taxonomically unidentified and documented only using their local names (supplemental figure 3). genetic resources (2025), 6 (11), 24–40 collecting pgrfa in sudan from 2002–2022 29 figure 3. sites where most germplasm accessions were collected from 2002–2022 (map a) across different states of sudan, with state names shown on map b. figure 4. distribution of percentages of total accessions collected from different plant groups 30 eltahir et al g enetic resources (2025),6 (11),24–40 table 2. distribution of number of accessions of the top 12 crops collected among different states in sudan between 2002 and 2022. state sorghum pearl millet cowpea okra sesame banana groundnut roselle watermelon maize melon date palm total south kordofan 706 93 158 124 122 0 69 62 9 132 75 0 1,550 south darfur 237 168 58 40 38 22 46 23 14 11 21 0 678 west darfur 147 147 42 52 45 0 69 94 44 1 25 0 666 blue nile 172 94 57 33 68 84 27 18 0 36 10 0 599 north kordofan 134 167 31 26 27 0 4 40 19 10 13 0 471 kassala 273 3 2 9 2 151 2 2 10 2 12 0 468 east darfur 135 49 7 16 13 0 79 54 11 0 6 0 370 north darfur 144 141 11 7 21 0 13 17 7 0 0 0 361 gedarif 142 35 52 13 51 0 21 6 4 9 16 0 349 northern 9 0 6 16 0 21 0 2 5 11 4 139 213 central darfur 69 53 7 12 6 0 15 14 2 4 2 0 184 white nile 169 3 4 1 1 0 1 2 0 0 0 0 181 red sea 89 48 0 26 0 0 0 4 0 1 13 0 181 west kordofan 18 19 5 5 2 0 2 1 120 0 7 0 179 river nile 28 0 7 5 0 12 0 4 3 3 0 40 102 gezira 9 2 2 3 2 49 1 0 4 1 3 0 76 sennar 0 0 0 0 0 14 0 0 0 0 0 0 14 khartoum 0 0 0 6 0 4 0 1 0 0 3 0 14 total accessions 2,481 1,022 449 394 398 357 349 344 252 221 210 179 6,656 total states 16 14 15 17 13 8 13 16 13 12 14 2 genetic resources (2025), 6 (11), 24–40 collecting pgrfa in sudan from 2002–2022 31 as a result of these collecting efforts and other germplasm acquisition activities, the total pgrfa accessions conserved by apgrc reached more than 16,000 accessions by 2022 compared to around 6,000 accessions in 2002, as shown in the apgrc genebank documentation system, with a total increase of around 10,000 accessions (figure 5). the collecting missions reported here contributed significantly to this increase with 7,720 accessions, while the rest came from other germplasm acquisition activities during the same period, including repatriation of sudanese germplasm from outside the country, and donations of germplasm samples by others such as crop breeders. phenotypic variations observed remarkable phenotypic variations on some morphological traits were visually observed on the plant organs collected such as the panicles, fruits and seeds. such observations will be further studied and documented through detailed characterization using standard descriptor lists. in cereals, variations in shapes, sizes and colours were observable on panicles and seeds of sorghum, pearl millet, maize and rice (figure 6). seed colours of sesame and groundnut, both oil crops, were varied among the accessions collected from different sites (figure 7). remarkable variations were also observed in colours and sizes of seeds of some leguminous crops such as cowpea, faba bean, hyacinth bean and bambara groundnut (figure 8). variations were also observed on a number of horticultural crops, including malvaceous and solanaceous vegetables and medicinal plants (figure 9). capsule shapes were variable among the okra accessions, as well as the colours of dry enlarged calyces of roselle accessions. fruit sizes and shapes also varied among tomato, hot pepper and eggplant accessions. prominent variations were also observed in shapes, sizes and colours of fruits and seeds of different cucurbits such as watermelons, melons, pumpkins and squashes, and bottle gourds (figure 10). discussion the total number of pgrfa accessions conserved by apgrc before 2002 was approximately 6,000. a significant increase in the apgrc holdings occurred during the following 20 years resulting in more than 16,000 accessions by 2022. this increase of around 10,000 accessions, was primarily due to the intensive germplasm collecting activities undertaken during the cultivation seasons from 2002 to 2022, which accounted for 77% of the germplasm acquired, in addition to repatriation of sudanese germplasm from outside the country, and donations of samples by others. on the other hand, direct collection by apgrc from in situ habitats contributed by only 21% before 2002, during the 20 years since the start of formal germplasm collecting activities in 1982. in fact, the horticultural germplasm materials collected in 1982, 1983 and 1985, as reported by hassan et al (1983), hassan et al (1984) and geneif et al (1985), were the first accessions deposited in the genebank unit that later evolved to become apgrc. the increased role of apgrc as a germplasm collecting institute after 2002 was mainly due to the availability of necessary resources from different streams, including funding for germplasm collecting. for example, the period between 2002 and 2010 witnessed a substantial increase in the resources available for apgrc from the capacity-building project for pgr under the eapgren network, of which sudan was a founding member (marandu and kamau, 2008). this project, financed by the swedish international development cooperation agency, aimed to implement national and regional activities, including germplasm collecting activities. also, apgrc obtained funds from a project financed through the crop trust from 2015 to 2017 for collecting crop wild relatives, which contributed significantly to increasing the apgrc holdings of crop wild relatives (eastwood et al, 2022). additional financial support was obtained from the crop trust through the cgiar genebank management platform and the international center for agricultural research in the dry areas (icarda) in 2021 for collecting pgrfa to fill gaps in the collections held by apgrc and other international research centres. the number of sites sampled was considerably large, which was made possible by reliable transportation means that enabled access to various locations, even those far from asphalt roads. membership in the collecting teams of representatives from local authorities and communities was invaluable in guiding those teams through local internal roads connecting villages and leading to wild habitats. almost half (48%) of the accessions collected from 2002 to 2022 were cereal crops, of which sorghum was represented by 66%, and pearl millet by 27%. this highlights the importance of cereals, especially sorghum and pearl millet, in the country in terms of the extent and distribution of cultivated areas, as well as the diversity they encompass, including farmers’ cultivars and wild relatives. both crops are major staples in sudan, and are annually cultivated in areas of more than 10 million hectares for sorghum, and 4 million hectares for pearl millet (ministry of agriculture and forestry, 2015). moreover, sudan is part of the primary diversity regions for these crops in east and west africa, as indicated by khoury et al (2016). it is located within sub-saharan and northeast africa, which is the primary centre of origin and diversity of sorghum as mentioned by barmel et al (2022b). pearl millet was domesticated in west africa and then diffused into eastern africa, southern africa and south asia (barmel et al, 2022a). therefore, eastern africa, including sudan, contains remarkable variability in pearl millet as was observed in the number of pearl millet accessions collected by apgrc and the variations observed. this was proved by bashir et al (2014), who identified clear genetic 32 el tahir et al genetic resources (2025), 6 (11), 24–40 figure 5. comparisons between total accessions acquired by the agricultural plant genetic resources conservation and research centre (apgrc) from different sources during the periods before and after 2002 figure 6. variations among panicles and seeds of different cereal crops accessions (sorghum, pearl millet, maize and rice) collected from different sudan states during different seasons diversity within a sample of 214 accessions from sudan, using marker-based analysis. vegetables, including malvaceous, solanaceous and cucurbit crops were the second largest group among the materials collected, representing 17%. various vegetables are consumed in sudan; among them are wild species, indigenous vegetables and introduced types, as mentioned by ahmed and mohamed (1995). among the collected vegetables, true indigenous forms of okra, melons and watermelons were present showing remarkable morphological variation. one piece of evidence of the geographical origin of okra is partially based on the presence of a putative ancestor (a. ficulneus) in east africa (kumar et al, 2011). schippers (2002) reported that a. ficulneus was found in sudan and other regions of the sahel and east africa, but more so in south and south east asia. okra is one of the most important traditional vegetables in sudan that is used almost all over the country in a number of ways of cooking either after being dehydrated or as fresh pods (mohamed, 1991). this explains the widest geographical range from which okra genetic resources were collected making it the leading crop in terms of the number of states (17) from which samples were obtained. variations observed among okra fruits collected were further shown on vegetative, inflorescence and fruit traits through morphological characterization results as recently reported by el-tahir (2023). the collections made over 20 years contained a considerable number of melon accessions totalling 210, including cultivated and wild melons with observed genetic resources (2025), 6 (11), 24–40 collecting pgrfa in sudan from 2002–2022 33 figure 7. variations among seeds of different oil crops accessions, including sesame and groundnut, collected during different seasons from various sudan states variations among them. mohamed and yousif (2004) described sudan as unique, in terms of the presence of different melon subspecies, containing both wild and cultivated genotypes. cultivated melons in sudan are composed of sweet melon, known locally as shammam; snake melon, known locally as ajjour; a local vegetable melon named ‘tibish’; and a local type of melon of which seeds are eaten, known locally as seinat. wild melon plants (c. melo agrestis) are also found in sudan and are known as humaid. among them are types of melon that are truly indigenous to the country, either of typical wild type such as c.melo agrestis, or grown only in sudan such as tibish (mohamed and pitrat, 1999). in contrast, cultivars of sweet melons and snake melons are introductions from outside the country. according to pitrat (2013) the tibish melon is considered a primitive cultivated melon that evolved and domesticated independently in a domestication event separate from other cultivated melons in africa and belonging to the subspecies agrestis. the latter subspecies agrestis was considerably represented in its wild form in the collections made from 2002 to 2022 by 153 accessions, providing supportive proof of the probability that tibish melon might have been domesticated in sudan through evolution from such wild forms. this collection is uniquely important since the wild and cultivated agrestis melons tibish and humaid have been proved to be sources of resistance to a number of viral and fungal diseases, as well as insect pests, as reviewed by mohamed and yousif (2004). the collecting efforts resulted in 252 accessions of watermelon, of which more than 50% were from kordofan region. one of the reasons for the importance of this collection is the recently reported information by renner et al (2021), indicating that the closest relative to the domesticated watermelons was the kordofan melon (citrullus lanatus subsp. cordophanus) from sudan. an earlier study by goda (2007) on morpho34 el tahir et al genetic resources (2025), 6 (11), 24–40 figure 8. variations among seeds of different accessions of leguminous crops, including cowpea, faba bean, hyacinth bean and bambara groundnut, collected during different seasons from different sudan states. figure 9. variations among accessions of malvaceous crops, including capsules of okra and dry calyces of roselle, as well as among fruits of the solanaceous crops tomato, hot pepper and wild relatives of eggplant, collected from some sudan states during different seasons genetic resources (2025), 6 (11), 24–40 collecting pgrfa in sudan from 2002–2022 35 figure 10. variations among fruits and seeds of some accessions of cucurbit crops, including watermelon, melon, pumpkin and bottle gourd, collected during different seasons from different sudan states logical characterization of 28 citrullus accessions from sudan, revealed remarkable variations shown by clustering analysis distinguishing between four main distinct groups. the first group was characterized by 88% similarity within the group members and consisted of cultivated accessions collected mainly from kordofan and darfur regions in the west. this group was further divided into three subgroups indicating the variation of the genetic resources of this group within a region, from where watermelon is believed to have originated (goda, 2007). among the solanaceous vegetables collected were tomatoes, hot peppers and eggplants. all of them originated in regions far away from sudan but have been introduced into the country some time ago. an earlier study on tomato accessions collected from the western and northern regions of sudan showed distinctive features of each compared to the other (eltahir, 1993). tomatoes from western sudan were mostly small or very small in size and were mostly used for making dried tomato paste from fruit slices. on the other hand, tomatoes from the northern region of sudan were medium or relatively big and mainly used as salad tomatoes. such wide variations in tomato fruits were proved to be still there as shown within the collections obtained from 11 states, including from western and northern sudan. capsicum species originated in south america from where they dispersed into different regions of the world forming secondary centres of diversity, among which eastern and southern africa, where sudan lies. west and central africa represent a significant secondary region of diversity for c. chinense, while east africa is likely to be an important secondary region of diversity for c. frutescens (barchenger and khoury, 2022); both hot pepper species were present in sudan as suggested by el-tahir (2001), and, therefore, are likely to have been collected during the last 20 years. the popularity of this crop and its production in smallholdings, in diverse environmental conditions of different regions substantially contributed to the observable vast variability of this crop in sudan (geneif, 1984). this variability was further confirmed by el-tahir (1994), who indicated the availability of high variation between and within 116 accessions from sudan in plant and fruit characters. accordingly, it was recommended to have further collections of hot peppers to cover new areas. the collections made after 2002 resulted in dozens of accessions from nine states in response to this earlier recommendation. among the food grain legumes, the total collected accessions of cowpea ranked third after sorghum and pearl millet with 426 accessions, originating from different states in the west and south-east with clear phenotypic variations of the collected seeds (figure 8). in a study on cowpea accessions from sudan, collected in the last 20 years, sudanese germplasm showed high similarity to west african cowpeas (ali et al, 2015). this was interpreted by an earlier suggestion that the cowpea crop was introduced to sudan from west african countries to the western part of sudan (kordofan and darfur), from where it spread to the rest of the country (ali et al, 2015). this is further supported by 36 el tahir et al genetic resources (2025), 6 (11), 24–40 the relatively large collection obtained from kordofan and darfur regions in sudan. a considerable number of accessions of oil crops sesame and groundnut were collected especially from the states of south kordofan, south darfur, east darfur and west darfur in the west of the country, and blue nile and gedarif states in the southeast and east of the country, the traditional production areas for such crops. the sesame collection was relatively abundant, ranking fourth among the top collected crops from 13 states, encompassing mostly farmers’ cultivars with about 10% of wild sesame species. such a relatively big collection of sesame from a wide geographical distribution with observable variation in seed colours is indicative of the broad diversity of sesame genetic resources in sudan, similar to that of cowpea and okra. this could be attributed to the fact reported by mahmoud et al (1995) that the selection by subsistence farmers resulted in many landraces of sesame adapted to different ecological areas, varying mainly in rainfall and soil, and to the needs of the farmers. sesame was suggested to have been domesticated in africa (bedigian, 2004), as several wild relatives are still growing there. sudan is one of the countries in sub-saharan africa where a variety of cultivated and wild sesame is found, as proved by the germplasm collecting activities reported here. this indicates the importance of the country as a place of diversity for this crop, and suggests the possibility that it may be one of the regions where sesame was domesticated. as discussed by williams (2022), the groundnut is believed to have originated in south america in the area of southern bolivia and northwestern argentina. it was introduced to west africa by portuguese traders in the 16th century, and in the following centuries it became fully integrated into subsistence farming systems and various ethnic cuisines, causing the crop to diversify into dozens of distinct african landraces. west african immigrants are believed to have brought the crop to sudan about 200 years ago, and they grew it in parts of western sudan and along the blue nile (mahmoud et al, 1995). this is supported by the germplasm collecting activities resulting in a relatively large collection of locally cultivated varieties of groundnut, the absolute majority of which was from darfur and kordofan regions in the west, and the blue nile state in the southeast of the country. according to what was mentioned by mcclintock and el-tahir (2004), roselle (hibiscus sabdariffa) probably originated from africa, where it may have been domesticated in sudan about 6,000 years ago. it is locally known as karkade and is grown in various parts of sudan, particularly kordofan and darfur, where it is one of the cash crops cultivated by traditional farmers under rain-fed conditions, for local consumption and export (mohamed et al, 2012). it is therefore understandable that it ranked among the top crops, with a relatively large number of accessions collected, totalling 344. the absolute majority of these were from darfur and kordofan states, where the crop is traditionally grown. the variation observed during collection, especially on enlarged calyces, which are the main parts of the plant used to make cold or hot beverages, is indicative of high diversity to be detected through morpho-agronomic and genetic characterization studies. the vegetatively propagated fruit-producing crops represented in this collection were banana and date palm. banana is a plant that is grown in sudan for its sweet-flavoured fruits that are used as dessert by sudanese people. it is produced commercially in small and medium scattered orchards along the river nile and its tributaries banks, and in large plantations at kassala (elsadig, 2014). the dwarf cavendish has been the only old cultivar grown in sudan for about 100 years (mahmoud et al, 1995). however, new banana cultivars were introduced, tested and released in the early 2000s, and might replace the traditional cultivar for their superior yield potential and quality. the collection of local banana germplasm was planned to safeguard it against any possible losses due to the use of the new improved varieties or any other factors, including the recently frequent seasonal flooding of the rivers over the banks where banana plantations are established. therefore, the collecting activities since 2003 have reasonably covered the different areas where bananas used to be grown. the highest number of accessions (151) came from kassala state, where banana production is believed to have started during the late 18th century and is considered historically one of the most important banana-producing centres in sudan (elsadig, 2014). date palm has been grown in sudan for a long time in the northern region using local cultivars producing different types of dates, including dry, semi-soft and soft dates. a study to describe the phenotypic vegetative and fruit characters of a number of date palm cultivars at on-farm level in northern sudan has revealed high variability between these cultivars, where 14 out of 16 qualitative and quantitative traits used showed a strong discriminating factor (elsafy et al, 2015). genetic variation has also been detected within and between 18 cultivar groups obtained from northern sudan as reported by elsafy et al (2016). findings of a study reported by ezebilo et al (2013) revealed that the date palm cultivars in northern sudan are diverse, implying that date palm farms in sudan can also serve as sites for conserving genetic resources. however, having an ex situ collection of such diverse date palms in sudan has been an apgrc strategy to back up collections as a means to safeguard such materials against hazards at on farm level. following that, the collection of date palm accessions from northern and river nile states began in 2015 resulting in materials from both states for conservation in a field genebank. rangelands in sudan are estimated at 68.6 million hectares or 36% of the total country area (ministry of agriculture and forestry, 2015). surveys conducted genetic resources (2025), 6 (11), 24–40 collecting pgrfa in sudan from 2002–2022 37 from 2000 to 2014 revealed that certain important range plant species were becoming scarce or extinct and some areas were invaded by unpalatable plant species. thirteen valuable herbaceous species were reported as decreasing in semi-desert and low-rainfall savannah zones. among these, were species belonging to genera such as andropogon, aristida and blepharis (hcenr, 2013). the apgrc’s collection of genetic resources of range plants started relatively late, in 2015. the areas surveyed for collection were limited to only three states in the semi-desert and low-rainfall savannah zones, indicating the pressing need for intensive collecting efforts in the future for range plants as an important component of the pgrfa in the country. it is interesting to note that south kordofan state was significantly represented in the collection of the top twelve crops, with a total of 1,550 accessions, a number much higher than that of the secondranking state, south darfur, which contributed 683 accessions. this can be attributed to the fact that south kordofan was one of the states most intensively covered during germplasm collection, with 135 sites visited. additionally, south kordofan is known for its high diversity of cultivated crops grown in different farming systems including what is known as the ‘jubraka’, the local name of kitchen home gardens, as well as in small lands held by smallholder farmers locally known as bildat. moreover, materials were collected from sites located at a wide range of altitudes from 117 to 976 masl. on the other end, sennar state, which is part of the central clay plains in sudan, was the least covered in terms of crops collected, as only banana clones were collected from the farms along the river banks of the blue nile. this makes sennar state among the top geographical gaps for germplasm collection in the future. planning for multi-species collecting from sennar should consider areas around and within the dinder national park, which is one of the largest protected areas in the country covering about 890,000 hectares (unep and hcenr, 2020). it stretches in areas within sennar, gedarif and blue nile states, with the biggest area within sennar state. this will require close collaboration with the park authorities for effective targeted germplasm collection within and around the park. around 65% of the accessions collected (4,982 accessions) were from crops and species covered by the multilateral system (mls) for access and benefit sharing established by the international treaty on plant genetic resources for food and agriculture (itpgrfa) (fao, 2009). the total crops belonging to this system that were sampled were 22 accounting for 43% of the total crop genera covered. such a relatively big number of accessions collected from the mls crops and species indicates the importance of this collection from a global perspective, as sudan is a party to the itpgrfa and obliged to provide facilitated access to such accessions based on the mls terms. moreover, the crops and plant species belonging to the mls covered by these collections were almost more than 30% of the total plant genera covered by the mls. they included crops under genera such as sorghum, pennisetum, zea, vigna, musa, vicia, phaseolus, solanum, and a range plant under the genus andropogon. this shows how sudan could be an important contributing party to the effective functioning and success of this system at the global level. in fact, sudan has officially notified the itpgrfa secretary on 23 september 2010 that 6,351 samples of sorghum, pearl millet, banana and other pgrfa listed in annex i and maintained in the plant genetic resources unit of the agricultural research corporation in wad medani, sudan, have been included in the multilateral system (https://www.fao.org/plant-treaty/areas-o f-work/the-multilateral-system/collections). since then, apgrc has been engaged in exchanging pgrfa with others using the standard material transfer agreement, which has been set out by the itpgrfa to regulate the germplasm exchanges within the mls. conservation of collected pgrfa is challenging in sudan. arrangements have been taken through an evolutionary process that started in the early 1980s with the establishment of genebanks at central and subnational levels. the seed samples of different accessions have been kept under long-term seed storage conditions in the central seedbank at wad medani (14.3931◦ n, 33.5392◦ e), with copies of some of them in a subnational seedbank unit in elobeid agricultural research station at elobeid (13.1782◦ n, 30.2167◦ e) in north kordofan state. however, the war that erupted in the country in mid-april 2023 and extended to wad medani in mid-december 2023 has posed serious threats to seed collections at the apgrc headquarters there. hence, there is a pressing need to accelerate the duplication of all the materials in the svalbard global seed vault (sgsv), where only around 3,000 accessions have already been deposited by 2022. if the situation continues to deteriorate due to the ongoing conflict, temporary relocation of the collection from wad medani to other safer place(s) should be an option, as experienced by other genebanks in conflict areas. a recent example is the icarda genebank, which was originally located in aleppo, syria. as a consequence of the syrian civil war and severe combat operations in aleppo starting in 2012, the genebank had to be relocated in 2016 to lebanon and morocco. part of the germplasm collection could be restored, with safety duplicates preserved at international genebanks and sgsv, as mentioned by herbold and engels (2023) in their study on genebanks risks. overall, some african and asian countries were identified as having high political instability, as is currently the situation in sudan, necessitating the implementation of effective mitigation measures including safety duplication outside the country, which has occurred at low rates so far, and relocation to safer places if the war continues. 38 el tahir et al genetic resources (2025), 6 (11), 24–40 conclusion the activities for collecting pgrfa from in situ habitats in sudan during the reported period have reasonably succeeded in covering almost all agroecosystems, as well as different farming systems within almost all the sudan states resulting in a substantial number of accessions from different cultivated crop and plant groups. among the areas covered were regions that had been affected by armed conflicts in previous years, such as south and west kordofan states before 2004, and the darfur region in the far western parts of the country before 2014. the germplasm collecting activities have successfully gathered a considerable number of accessions from crops for which sudan is part of the region of diversity and/or origin. among those were crops such as sorghum, pearl millet, sesame, watermelon, melon, okra and roselle, with each crop having between 200 and more than 2,000 accessions collected. the collection of local genetic resources of vegetatively propagated crops such as banana and date palm, was initiated for the first time during this period. however, further collecting activities are needed for these crops as well as other vegetatively propagated fruitproducing plants such as mango, guava and citrus. the genetic resources of indigenous range plants remain a major taxonomic gap, which needs to be filled through extensive germplasm collecting activities. this should include a wide coverage of rangelands across the different ecological zones, including the desert, semidesert, low-rainfall savannahs on both sandy and clayey soils, as well as high-rainfall savannahs towards the borders with the south sudan republic, and the unique mountain vegetation such as in jebel marra in the far west. moreover, crop wild relatives, although relatively covered, still remain poorly represented, and continue to be another major taxonomic gap, including relatives to mls crops such as sorghum, pearl millet and eggplant, which were somehow sampled during the last years, as well as relatives to non-mls crops of local importance in sudan such as melons, watermelon, okra and sesame. it was interesting to note that accessions were collected in relatively large numbers from about 30% of the genera covered by the mls of the itpgrfa, positioninig sudan, as a party to this treaty, to play a significant role in conserving and providing facilitated access to such pgrfa for use under the mls terms. acknowledgements authors are very grateful to the arc administration, under which apgrc operates, for assigning and facilitating access to necessary resources for implementing the different germplasm collecting plans, including financial resources and logistical facilities. a major part of the germplasm collecting activities conducted during the specified period would not have been possible without the substantial financial support from the swedish international development cooperation agency through eapgren, to both of them we are very thankful. the global crop diversity trust and other supporters such as icarda are greatly appreciated for providing and facilitating the obtaining of the necessary financial resources to conduct some of the collection missions. the effective roles of all collaborators, including scientists, technicians and directors of agricultural research stations in different regions, during planning and conducting the collecting missions, are very much appreciated, and we are very grateful to them. special thanks and appreciation are extended to the directors of the research stations within darfur region for their effective collaboration in planning and conducting the collecting activities in the region. thanks and appreciation are also extended to all representatives of state ministries of agriculture, and local communities for their cooperation and effective contributions during the collecting processes. supplemental data • supplemental table 1. germplasm collecting missions across the cultivation seasons from 2002–2003 to 2021–2022. • supplemental material 1. germplasm collection form. • supplemental table 2. total of collection sites visited by the germplasm collection missions in the different states during the cultivation seasons between 2002 and 2022 and the ranges of coordinates within which they were geographically located. • supplemental table 3. total accessions collected from each crop or plant and the total number of states from where they were collected. • supplemental figure 1. total number of states from where total accessions of the genetic resources of different crops collected. • supplemental figure 2. total number of accessions collected from different crop wild relatives. • supplemental figure 3. total number of accessions collected from each range plant species. author contributions i.m. el tahir: leading germplasm collection planning and coordination, proposing the paper, writing the first draft, revising and finalizing the manuscript. a.z. babiker, e.a. abdalla, a.a.e. ahmed: participation in planning, leading some collection missions and revising manuscript drafts. m.o.y. goda: participation in planning, leading documentation process for all collection data and revising manuscript drafts. m.a.m. elgabri: participation in planning, assisting in leading some collection missions, and revising manuscript drafts conflict of interest statement the authors declare that there are no conflicts of interest. https://www.genresj.org/index.php/grj/article/view/genresj.bwnf7962/suppdata235 https://www.genresj.org/index.php/grj/article/view/genresj.bwnf7962/suppdata235 https://www.genresj.org/index.php/grj/article/view/genresj.bwnf7962/suppdata235 https://www.genresj.org/index.php/grj/article/view/genresj.bwnf7962/suppdata235 https://www.genresj.org/index.php/grj/article/view/genresj.bwnf7962/suppdata235 https://www.genresj.org/index.php/grj/article/view/genresj.bwnf7962/suppdata235 https://www.genresj.org/index.php/grj/article/view/genresj.bwnf7962/suppdata235 https://www.genresj.org/index.php/grj/article/view/genresj.bwnf7962/suppdata235 genetic resources (2025), 6 (11), 24–40 collecting pgrfa in sudan from 2002–2022 39 references ahmed, m. k. and mohamed, e. i. 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(2022). global strategy for the conservation and use of peanut genetic resources (bonn, germany: global crop diversity trust). doi: https://doi.org/10.5281/zenodo.7545106 https://doi.org/10.3390/plants12152874 https://doi.org/10.1098/rspb.2016.0792 http://www.academicjournals.org/ajar https://hdl.handle.net/10568/105066 https://hdl.handle.net/10568/105066 https://doi.org/10.5511/plantbiotechnology.13.0813a https://doi.org/10.5511/plantbiotechnology.13.0813a https://doi.org/10.1073/pnas.2101486118 https://doi.org/10.5281/zenodo.7545106 introduction methodology planning targeted pgrfa targeted locations collecting approaches multi-species collecting missions single-species collecting missions collecting teams germplasm sampling data recorded results missions conducted and sites covered germplasm materials collected phenotypic variations observed discussion conclusion acknowledgements supplemental data author contributions conflict of interest statement review and position paper genetic resources (2025), (s2), 203–223 doi: 10.46265/genresj.unvv5571 https://www.genresj.org issn: 2708-3764 a significantly enhanced role for plant genetic resource centres in linking in situ and ex situ conservation to aid user germplasm access nigel maxted a, anne-françoise adam-blondon b, catherine hazel aguilar c, ana maria baratad, béla bartha e, riccardo bocci f, domenica de paola g, heli susanna fitzgeraldh, louis john fresta i, pietro fusani j, giovanni giuliano k, filippo guzzon l, philipp holzherr e, vojtech holubecm, josé maŕıa iriondo alegŕıan, juozas labokas o, lorenzo maggioni l, joana magos brehm a,d, anna palmé p, jade phillips a, jaime prohens q, lorenzo raggi r, parthenopi ralli s, dainis ruņģis t, karuine sarikyanu, jelka šuštar vozlič v, imke thormannw and goran zdunić x a school of biosciences, university of birmingham, b15 2tt, birmingham, united kingdom b institut national de recherche pour l’agriculture, l’alimentation et l’environnement, 78026 versailles, france c leibniz institute of plant genetics and crop plant research (ipk) gatersleben, 06466 seeland, germany d banco português de germoplasma vegetal, instituto nacional de investigação agrária e veterinária i.p. (bpgv-iniav), braga, 4700-859, portugal e prospecierara, unter brüglingen 6, basel, ch-4052, switzerland f rete semi rurali, via di casignano 25, scandicci (fi), 50018, italy g cnr-ibbr institute of biosciences and bioresources, national research council of italy, bari, italy h finnish museum of natural history, university of helsinki, helsinki, fi-00014, finland i plant protection directorate, rural affairs department, ministry for agriculture, fisheries, food and animal rights, lja1910 lija, malta j council for research in agriculture and economics, research centre for forestry and wood (crea-fl), trento, 38100, italy k enea, centro ricerche casaccia, via anguillarese 301, rome, 00123, italy l ecpgr secretariat, alliance of bioversity international and the international center for tropical agriculture (ciat), via di san domenico, 1, rome, 00153, italy m czech agrifood research centre, drnovska 507, 16100, praha 6 -ruzyně, czech republic n instituto de investigación en cambio global (iicg-urjc), universidad rey juan carlos, c/ tulipan s/n, móstoles, madrid, 28933, spain o state scientific research institute nature research centre, akademijos g. 2, 08412, vilnius, lithuania p nordic genetic resource center (nordgen), box 162, 234 23, lomma, sweden q universitat politècnica de valència, camino de vera 14, es46022, valencia, spain r dipartimento di scienze agrarie, alimentari e ambientali (dsa3), università degli studi di perugia, borgo xx giugno 74, 06121, perugia, italy s hellenic agricultural organization dimitra (elgo dimitra), institute of plant breeding & genetic resources, po box 60458, 570 01 thermi – thessaloniki, greece t genetic resource centre, latvian state forest research institute (lsfri) silava, lv-2169 salaspils, latvia u scientific center of vegetable and industrial crops, ministry of economy, ararat marz, darakert community, 808 ararat region, armenia v agricultural institute of slovenia, hacquetova ulica 17, 1000 ljubljana, slovenia w federal office for agriculture and food, information and coordination centre for biological diversity (ibv), 53179 bonn, germany x institute for adriatic crops and karst reclamation, 21000 split, croatia citation: maxted, n., adam-blondon, a., aguilar, c. h., barata, a. m., bartha, b., bocci, r., de paola, d., fitzgerald, h. s., fresta, l. j., fusani, p., giuliano, g., guzzon, f., holzherr, p., holubec, v., iriondo alegŕıa, j. m., labokas, j., maggioni, l., magos brehm, j., palmé, a., phillips, j., prohens, j., raggi, l., ralli, p., ruņģis, d., sarikyan, k., šuštar vozlič, j., thormann, i., zdunić, g. (2025). a significantly enhanced role for plant genetic resource centres in linking in situ and ex situ conservation to aid user germplasm access. genetic resources (s2), 204–223. doi: 10.46265/genresj.unvv5571. © copyright 2025 the authors. this is an open access article distributed under the terms of the creative commons attribution license (cc by 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. received: 03.04.2025 accepted: 27.04.2025 published online: 09.05.2025 https://www.genresj.org https://www.doi.org/10.46265/genresj.unvv5571 https://www.genresj.org https://www.doi.org/10.46265/genresj.unvv5571 abstract: plant genetic resources (pgr) serve as the cornerstone for global varietal enhancement and food security. however, these resources face significant threats, including diversity erosion and extinction, are often inadequately conserved, and frequently remain inaccessible for practical use. traditionally, pgr have been primarily conserved through population seed samples stored ex situ in genebanks. in contrast, complementary in situ techniques – whether involving crop wild relatives (cwr) in genetic reserves or crop landraces (lr) on-farm – have largely remained experimental. the demand from breeders for a broader diversity is driving a more integrated approach that combines ex situ and in situ methods. this paper posits that such an integrated strategy would be mutually advantageous for pgr, biodiversity, and farmer-based conservation communities. as a foundation for future pgr science, we propose the three ‘principles of pgr conservation and use congruence’ and outline the practical processes involved in in situ and on-farm conservation. we also review the challenges associated with integrating ex situ and in situ conservation, specifically addressing how collaborative resource management can be established, how potential resource users can access in situ and on-farm conserved pgr, how to promote user access to in situ conserved populations, and the progress made thus far in integrating in situ and ex situ efforts. while it is acknowledged that full integration may be unrealistic without adequate resources for genetic resource centres and the rectification of skill gaps, the potential to significantly enhance the long-term, sustainable conservation of pgr diversity holds profound existential benefits for humanity in the 21st century. keywords: crop wild relatives, ex situ, genebank, genetic reserves, in situ, landraces, on-farm conservation, integrated conservation citation: maxted, n., adam-blondon, a., aguilar, c. h., barata, a. m., bartha, b., bocci, r., de paola, d., fitzgerald, h. s., fresta, l. j., fusani, p., giuliano, g., guzzon, f., holzherr, p., holubec, v., iriondo alegría, j. m., labokas, j., maggioni, l., magos brehm, j., palmé, a., phillips, j., prohens, j., raggi, l., ralli, p., rungis, d., sarikyan, k., šuštar vozlič, j., thormann, i., zdunić, g. (2025). a significantly enhanced role for plant genetic resource centres in linking in situ and ex situ conservation to aid user germplasm access. genetic resources (s2), 203–222. doi: 10.46265/genresj.unvv5571. © copyright 2025 the authors. this is an open access article distributed under the terms of the creative commons attribution license (cc by 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 204 maxted et al genetic resources (2025), (s2), 203–223 genetic resources (2025), (s2), 203–223 plant grc roles in linking in situ and ex situ conservation 205 introduction plant genetic resources (pgr) conservation is unique among conservation methods as it aims to preserve biodiversity while also utilizing conserved resources (maxted et al, 1997a). this process involves several steps: identifying genetic diversity across plant species, prioritizing target taxa, planning and implementing conservation actions, and characterizing, evaluating and utilizing resources by farmers, breeders or researchers. clarity and expediency in this model’s application are essential for global, regional, national and local initiatives focused on food security, poverty reduction, and enhancing human well-being, thereby supporting many un sustainable development goals (un, 2015). pgr conservation employs two main strategies: in situ, where resources are conserved in their natural habitats, and ex situ, where resources are relocated to safer environments for conservation and accessibility (see definitions in supplemental table 1). it is widely accepted that in situ and ex situ actions should complement each other, enhancing overall conservation effectiveness (fao, 1996). historically, formal pgr conservation and germplasm application for orthodox-seeded species have relied heavily on ex situ seed storage in genebanks and, latterly, cryogenic preservation, with field genebanks and tissue culture techniques primarily used for recalcitrant-seeded species and clonally propagated crops. genebanks can secure long-term viability at low cost and have successfully made this diversity available to plant breeders and researchers (fao, 1998, 2011). however, ex situ approaches alone do not fully address the growing demand for broader diversity in a rapidly changing environment. the science of in situ and on-farm pgr conservation has advanced significantly, with refined techniques and a solid evidence base (maxted et al, 1997c, 2002, 2020; brush, 2000; eyzaguirre and linares, 2004; heywood and dulloo, 2005; jarvis et al, 2007, 2016; iriondo et al, 2008, 2021; veteläinen et al, 2009; hunter and heywood, 2011; hunter et al, 2017). initially, in situ and ex situ techniques were viewed as independent, even competitive (ford-lloyd and maxted, 1993), but the case for their complementarity is now widely accepted, though their practical integration remains incomplete (maxted et al, 1997a, 2020; van hintum et al, 2021). lack of integration limits conservation effectiveness, resulting in unconserved resources being unavailable to users and preventing their potential utilization. the challenge of increasing food production to feed a growing human population while mitigating climate change impacts on agriculture is escalating for the pgr and breeding communities fao (2010, 2012). lack of breadth and access to conserved genetic diversity is now a barrier to crop improvement (mccouch et al, 2013; ipcc, 2014; dempewolf et al, 2017; zhang et al, 2017). there is an opportunity to better serve farmers and breeders by integrating in situ conservation, genebanks, and germplasm use into a cohesive continuum that could significantly enhance breadth and access to diversity for users (maxted and brehm, 2023). failure to integrate these activities reduces the potential role of genebanks in leading pgr conservation and meeting user demands. maxted et al (2016) suggested that expanding the role of genebanks to include both ex situ and in situ conservation was logical and required change to the pgr paradigm and would warrant their renaming as genetic resource centres (grc), as the term ‘genebank’ implies a more restrictive focus. to explore this enhanced grc role, a questionnaire was prepared in 2024 on european genebank activities for the horizon europe project ‘promoting a plant genetic resource community for europe’ (pro-grace https://www.grace-ri.eu/pro-grace). the results indicated that 76% of genebanks (13 of 17 respondents) were interested in adopting complementary in situ/on-farm roles alongside traditional ex situ activities. genebanks have historically succeeded in supporting breeders and farmers while maintaining the pgr foundation for diverse crop varieties, but human population increase and climate change’s impact on crop production and food security are forcing a change of practice. although some grc may face limitations in skills and resources, with appropriate support, their roles could evolve to become even more critical for humanity’s future. this discussion focuses on how to better integrate in situ, ex situ, and user access in pgr conservation to provide greater diversity. we highlight current opportunities to: (1) clarify pgr conservation aims through proposed principles of pgr conservation and use congruence; (2) summarize practical processes for in situ and on-farm conservation; (3) promote resource management collaboration; (4) enhance user access to in situ and on-farm conserved pgr populations; (5) facilitate access to in situ conserved populations via the european search catalogue for plant genetic resources (eurisco, http://eurisco.ecpgr.org); and (6) identify future ways to better integrate in situ and ex situ conservation. for pgr actors and germplasm users, the clear advantage lies in addressing current challenges and ensuring greater diversity availability, with an enhanced role for genebanks or grc at the core, ultimately leading to increased sustainable food production and long-term food security. the principles of pgr conservation and use congruence the aim of pgr conservation may be summarized in three fundamental principles, to ensure: (1) longterm, sustainable maintenance of pgr1 diversity, (2) 1 the scope of pgr found outside of grc, or breeding collections is commonly focused on crop wild relative (cwr) and landrace (lr) diversity both of which are highly threatened. 206 maxted et al active2 conservation and characterization of crop, varietal and related wild taxon diversity using complementary3 techniques and (3) conserved resource documentation and availability for utilization within the applicable legislative context. the use of complementary techniques provides additional security by employing multiple, diverse approaches to conserve these resources, ensuring greater security as each technique backs up and supplements the others. there could as well be other subordinate objectives, such as maintaining seed viability, phenotypic and genotypic characterization and evaluation of conserved resources, and ensuring standard material transfer agreement (smta) enforcement, but the three fundamental objectives should hold true for whatever form of conservation strategy is applied. together, these objectives may be referred to as the principles of pgr conservation and use congruence; overall, conservation should, in the long-term, maintain the full breadth of genetic diversity, employ multiple conservation techniques, and make the conserved resources available to actual or potential users. these three objectives are met for most ex situ holdings (except for the requirement to link to complementary in situ conservation). ex situ pgr conservation and use is well tested, and we know it already ‘works’, but there is now an urgent need to further develop in situ conservation approaches. hawkes (1991) commented in the early 1990s that in situ techniques were in their “infancy”, and although advances in this area have been made (maxted et al, 2020), in situ and on-farm conservation is still largely experimental and not based on more than 60 years of practice and the associated extensive evidence-base available for ex situ conservation. additionally, effective standardization of in situ conservation techniques is itself challenging, as their application occurs in natural or semi-natural environments, or in on-farm locations, where diverse environmental, socioeconomic and cultural factors impact the target taxa, and effective pgr population managers (e.g. farmers, foresters, estate managers, etc.), may not be professional conservationists or have the necessary skills to maintain intrinsic genetic diversity. this is not to devalue the efforts of farmers or other landrace (lr) maintainers, or landscape managers, who have retained crop wild relative (cwr) populations on the estates they manage for extended periods of time. however, if in situ pgr conservation is to function as intended and be appropriately resourced, it must meet all three principles and objectives, as do ex situ approaches. populations and diversity of in situ resources must be maintained in the long term via the application of complementary techniques, and the conserved resources must be 2 active conservation implies targeted management and monitoring of conserved cwr or lr populations, as opposed to passive maintenance of cwr or lr populations, where there may be a conservation ethos but no targeted management and monitoring. 3 complementary conservation implies the use of both ex situ and in situ techniques to conserve cwr or lr populations. available to users. if in situ pgr conservation does not ensure availability of the conserved resource, it will not meet the principles of pgr conservation and use congruence and it is unlikely ever to be seen as truly complementary to ex situ conservation. it should also be noted that the third principle, which conserved resources are available for use, may not always be achievable, for example, when the in situ conserved populations are rare or threatened, and few, or an ex situ conserved accession has limited seed numbers and low viability. in both cases, the sample may need to be multiplied or regenerated before it can be made available to users. the principle remains that resource availability is paramount, and any periods of unavailability should be temporary until germplasm can be offered. practical processes of in situ and on-farm conservation to identify potential opportunities for integration, we need first to summarize and understand how in situ and on-farm conservation operate. the conservation–utilization continuum for in situ conservation is divided into four component steps and summarized in figure 1 (adapted from maxted et al (2020)): 1. conservation planning. this involves: (i) selection of target conservation units, either cwr or wild food plant (wfp) taxa or crop lr (maxted et al, 1997c; brehm et al, 2017); (ii) prioritization, usually based on potential use value, relative crop value and threat, identifying an easily implementable inventory of highest priority cwr, wfp or lr (brehm et al, 2017; nilsen et al, 2017; fao, 2019b); (iii) ecogeographic and gap analyses to identify concentrations of the conservation units and predict which sites with target populations (maxted and kell, 2008; maxted et al, 2012b; fao, 2019b); and (iv) field exploration to check the validity of the previous prediction and establish where the target diversity will be conserved in genetic reserves, other effective area-based conservation measures (oecm), on-farm, or in home garden. 2. conservation technique implementation. conservation targets are actively managed either in nature for cwr or wfp or cultivated on-farm or in-garden for lr diversity. this involves: (i) selection of sites with targeted resource diversity (hawkes et al, 2000; maxted et al, 2002; dulloo et al, 2008; veteläinen et al, 2009; iriondo et al, 2021); (ii) formulation of the management plan, a detailed plan for how the population(s) of the target taxa/crop are to be maintained and enhanced (maxted et al, 2002, 2008; dulloo et al, 2008; veteläinen et al, 2009; iriondo et al, 2021); (iii) implementation of the management plan, including the site interventions, implementation of which is likely to be experimental initially until targenetic resources (2025), (s2), 203–223 207 get population retention is sustainable (veteläinen et al, 2009; iriondo et al, 2021); (iv) resource monitoring at set time intervals to check the success the management regime (veteläinen et al, 2009; iriondo et al, 2021); and (v) formation and upkeep of partnerships essential for in situ and on-farm conservation of the genetic resources to occur. 3. conserved resource description. the preutilization stage will involve characterization and evaluation (maxted et al, 2020). these data may be uploaded alongside passport data in eurisco to facilitate germplasm selection. 4. conserved resource utilization. the in situ conserved resource should be available for use by breeders, farmers, researchers and other potential bona fide users. forms of traditional utilization should be encouraged, provided it is not detrimental to the target taxon or taxa, thus fostering local support for conservation actions. proposed resource management collaboration it is important to clarify not only how the target populations are managed, but also (1) who should provide oversight of the networks of in situ or on-farm sites and populations, and (2) who should practically implement the management interventions of individual in situ or on-farm sites and populations. there are several potential communities that might fulfil these roles: existing population managers, national grc staff and other diverse pgr stakeholders (including allied nongovernmental organizations (ngos), research centres and universities). as noted above, given that often the conserved in situ or on-farm genetic resources have been managed by the reserve/protected area (pa) manager, landowner, farmer or gardener for extended periods, one might assume they are the most appropriate to play both roles. while existing in situ and on-farm site managers should continue their successful management of individual in situ or on-farm sites and populations, the question is: do they have the necessary skills, tools and resources to provide oversight of the network(s) of in situ or onfarm sites and populations established? it can be argued that it would be impractical for individual in situ and on-farm site managers to provide oversight of the network(s) of in situ or on-farm sites and populations given they: (1) are unlikely themselves to use trait diversity from the conserved cwr or wfp populations; (2) lack skills and expertise in international and national policy and legislation; (3) lack skills and expertise in field trials or genomic analysis; (4) lack access to a pgr information system to aid in situ population management and transfer of germplasm to the end user; and (5) already have an existing heavy core load of activities in managing biodiversity populations or producing food and their scope to adding a significant additional activity is limited. therefore, it would seem appropriate that national grc staff (or other appropriate national pgr agency or pgr-focused ngos) would be better placed with the necessary skills, tools, resources and long-term experience from ex situ pgr applications, to provide multi-site pgr governance and overall oversight of the networks of in situ or on-farm sites and populations, including overall monitoring of natural reserves, other effective areabased conservation measures (oecm) sites or on-farm systems to prevent population losses (maxted et al, 2016). however, it is important to stress that national grc staff cannot work in isolation. the pgr conservation goal of maximum pgr diversity conservation and availability can only be achieved by the three communities working in integrated collaboration, with national grc staff providing national pgr leadership and oversight, individual pgr field population maintainers (i.e. reserve/pa manager, landowner, farmer or gardener) managing the genetic resources under their responsibility, and other pgr stakeholders (allied ngos, research centres and universities) providing the necessary additional support. furthermore, as the in situ or on-farm resource is maintained outside of a controlled unit, like a grc, the local community within the vicinity of the in situ/on-farm resource site should also be involved in the conservation project management and associated decisions. individual roles will vary depending on multiple factors (e.g. taxa included, whether wild or cultivated, resources available, value of resource conserved, etc.), therefore stakeholder discussions and negotiations will form part and parcel of the process of defining the roles of each actor, however it can be safely stated that the key expertise and areas of responsibility are likely to include those presented in table 1 . to aid clarity, figure 2 highlights those components managed by grc staff, and in situ site maintainers alone, and which may be managed jointly. collaboration between the three communities would be critical and involve periodic meetings of a pgr in situ population management committee. such an integrated approach to in situ and ex situ collaboration would extend each communities roles and responsibilities. however, for those maintaining pgr populations (pa, oecm or on-farm field maintainers) and given the target populations were selected because of their ‘health’, the additional workload is not foreseen as being significant, at least initially, as it would primarily involve monitoring target populations, while the provision of additional ecosystem and food services from the site would underpin the public good value of maintaining pgr populations. furthermore, in some countries, additional targeted pgr conservation could generate additional subsidies or added income for the site maintainers/owners through government funding (such as payments for ecosystem services, subsidies for farmers who cultivate and conserve landraces that suffer from genetic erosion), so the benefit to pgr field population maintainer could be substantial. the proposed changes outlined for the national grc would also be significant, possibly requiring additional staff with in situ expertise and genetic resources (2025), (s2), 203–223 plant grc roles in linking in situ and ex situ conservation 208 maxted et al figure 1. schematic description of key elements of in situ conservation to utilization pathway. green, in situ; brown, ex situ; red, threatened populations; gold, utilized pgr; blue, conservation steps; cwr, crop wild relatives; lr, landraces; oecm, other effective area-based conservation measures; wfp, wild food plants. additional resources, but the additional role would fall within the existing genebank’s remit – genebank managers network (https://www.ecpgr.org/about/gen ebank-managers-network) and aegis initiative (https: //www.ecpgr.org/aegis) of the european cooperative programme for plant genetic resources (ecpgr) – and would substantially boost the genebank’s role in national biodiversity conservation. for all three collaborating communities, increased collaboration will involve additional time and resource commitments, incurring additional costs. therefore, it is crucial to identify sustainable funding mechanisms to cover these costs, even if they are anticipated to be minor. however, any additional costs incurred due to collaboration and changes in roles would be far outweighed by the potential benefits of increased diversity available for breeders and other stakeholder’s use (maxted and brehm, 2023). access to and conservation of additional germplasm significantly enhances the diversity of collections, a core grc and genebank objective, thereby better fulfilling their professional mandate. as a final point, the collaboration as outlined in this document, involves the transfer of in situ or onfarm samples from their original locality to a nominated ex situ grc for backup and to facilitate access for germplasm users. this means that the provisions emanating from the international treaty on plant genetic resources for food and agriculture (itpgrfa) (fao, 2001) and the convention of biological diversity (cbd) nagoya protocol (cbd, 1992, 2011) are triggered and there is the need for an smta or internationally recognized certificate of compliance (ircc) respectively, between the in situ maintainer and the recipient nominated grc. this would need enacting even if the grc had no intention to utilize the germplasm itself, but simply to conserve the in situ or on-farm sample and make it in turn available to more active users. by virtue of the relationship between the in situ/on-farm source, the grc and the end user, the involved actors would be required to address the requirements to ensure fair and equitable sharing of benefits arising from the sample‘s potential final utilization, depending on the terms established under national regulations. the actual scope of the three-way (source, grc and end user) relationship would require expert deconstruction and is therefore beyond the scope of this document but must be resolved before any germplasm transfer occurs. genetic resources (2025), (s2), 203–223 209 ta bl e 1. c ol la bo ra ti ve ac ti vi ti es of na ti on al g en et ic r es ou rc e c en tr e (g r c ) st af f, pl an tg en et ic re so ur ce (p g r ) po pu la ti on m ai nt ai ne rs an d ot he r st ak eh ol de rs .c w r ,c ro p w ild re la ti ve s; lr ,l an dr ac es ;w fp ,w ild fo od pl an ts c w r ,w fp or lr in si tu po pu la ti on co n se rv at io n n at io n al g r c st af f’ s ro le pg r po pu la ti on m ai n ta in er ’s ro le o th er st ak eh ol de r’ s ro le le ad pr ep ar at io n an d pe ri od ic re vi si on of n at io na lp g r st ra te gy an d a ct io n pl an ,i nc lu di ng in si tu si te se le ct io n an d m an ag em en t pl an pr od uc ti on . c on tr ib ut e to pr ep ar at io n an d pe ri od ic re vi si on of n at io na lp g r st ra te gy an d a ct io n pl an ,l ea d in si tu si te se le ct io n an d m an ag em en t pl an pr od uc ti on . c on tr ib ut e to pr ep ar at io n an d pe ri od ic re vi si on of n at io na lp g r st ra te gy an d a ct io n pl an ,a nd in si tu si te se le ct io n an d m an ag em en t pl an pr od uc ti on . le ad na ti on al pg r in si tu co ns er va ti on si te ne tw or k m an ag em en t. c on tr ib ut e to pg r in si tu co ns er va ti on si te ne tw or k m an ag em en t. c on tr ib ut e to pg r in si tu co ns er va ti on si te ne tw or k m an ag em en t. a ss is t w it h im pl em en ti ng th e si te ’s in di vi du al m an ag em en t pl an . le ad im pl em en ta ti on of in di vi du al si te m an ag em en t pl an . a ss is t w it h im pl em en ti ng of in di vi du al si te m an ag em en t pl an . a ss is t w it h pe ri od ic m on it or in g of ta rg et po pu la ti on s an d an al ys is of de m og ra ph ic an d ge ne ti c tr en ds . le ad pe ri od ic m on it or in g of ta rg et po pu la ti on s an d an al ys is of de m og ra ph ic an d ge ne ti c tr en ds . a ss is t w it h pe ri od ic m on it or in g of ta rg et po pu la ti on s an d an al ys is of de m og ra ph ic an d ge ne ti c tr en ds . a ss is t w it h pe ri od ic re vi si on of in di vi du al si te m an ag em en t pl an an d bu ild in g ev id en ce ba se . le ad pe ri od ic re vi si on of in di vi du al si te m an ag em en t pl an an d bu ild in g ev id en ce ba se . a ss is t w it h pe ri od ic re vi si on of in di vi du al si te m an ag em en t pl an an d bu ild in g ev id en ce ba se . ta rg et po pu la ti on ch ar ac te ri za ti on an d ev al ua ti on .e ns ur in g us er ac ce ss to in si tu co ns er ve d re so ur ce s (v ia ex si tu ba ck up sa m pl es ). pe ri od ic co lle ct io n of ta rg et po pu la ti on s fo r ex si tu re pr es en ta ti ve ba ck up sa m pl es . d iv er se re se ar ch pr oj ec ts fo cu se d on ai di ng ef fe ct iv e pg r di ve rs it y co ns er va ti on an d us e. en su re in te gr at io n of in si tu an d ex si tu co ns er va ti on ac ti vi ti es an d su pp or t to ol s an d ap pl ic at io ns to ai d in si tu co ns er va ti on si te ne tw or k m an ag em en t, e. g. na ti on al in ve nt or ie s, m an ag em en t an d m on it or in g, ge rm pl as m ac ce ss , ch ar ac te ri za ti on an d ev al ua ti on da ta ba se s an d in fo rm at io n m an ag em en t. c ol la ti on of si te an d pg r po pu la ti on da ta ,a nd in te gr at io n w it h na ti on al pg r da ta ba se s, ac ti vi ti es as si st ed by ne tw or k to ol s an d ap pl ic at io ns . d iv er se re se ar ch pr oj ec ts su pp or ti ng na ti on al pg r po pu la ti on m an ag em en t an d us e, an d as so ci at ed to ol an d ap pl ic at io n de ve lo pm en t. pr om ot io n of na ti on al in te gr at io n in to in te rn at io na lp g r co m m un it y. pr om ot io n of pg r in te gr at io n in to th e br oa de r bi od iv er si ty co m m un it y. pa rt ic ip at io n in na ti on al an d in te rn at io na lr es ea rc h ac ti on s. le ad an d pa rt ic ip at e in th e n at io na lp g r in si tu po pu la ti on m an ag em en t c om m it te e. pa rt ic ip at e in th e n at io na lp g r in si tu po pu la ti on m an ag em en t c om m it te e. pa rt ic ip at e in th e n at io na lp g r in si tu po pu la ti on m an ag em en t c om m it te e. genetic resources (2025), (s2), 203–223 plant grc roles in linking in situ and ex situ conservation 210 maxted et al figure 2. schematic description of key elements of in situ conservation, highlighting genetic resource centre (grc) staff (dark red), in situ populations manager (green) and joint (orange) responsibilities. cwr, crop wild relatives; lr, landraces; oecm, other effective area-based conservation measures; wfp, wild food plants. user access to in situ and on-farm conserved pgr populations the endpoint of pgr conservation is not conservation itself but ensuring that conserved germplasm is available for present or potential future utilization (maxted et al, 1997a). the pathway of use for ex situ conserved pgr is tried and tested, but, apart from the positive activities of farmers and farming ngos focusing on pgr diversity and farming systems, the in situ pathway to utilization has yet to be established. without effective in situ conservation-to-use linkage, it is doubtful whether in situ conservation sites and site networks will ever be established (maxted, 2019). therefore, establishing links between in situ resources and use is fundamental to ensure additional germplasm access and the promotion of in situ conservation itself (maxted and brehm, 2023). maxted and kell (2008); maxted and palmé (2016) and maxted (2019) each reviewed potential models for how in situ conserved resources might be linked to user access, either accessed for use directly from the in situ population or indirectly via an ex situ conservation facility (figure 3). five potential options have thus far been proposed for promoting user access to in situ and on-farm conserved pgr and are elaborated in table 2. except for option 3, users request an in situ pgr population sample and ≈(20-) 40–50 viable seeds are dispatched to the end user, fulfilling the in situ to-use prerequisite outlined in the principles of pgr conservation and use congruence. the chosen option may vary based on grc facilities, available resources, conservation practices, and constraints from pgr maintainers or national authorities. however, assuming resources are adequate and constraints do not limit distribution, option 5 achieves the principles of pgr conservation and use congruence, making the in situ resource-to-user link via the grc, while placing the minimum additional burden on the grc staff and their resources. however, such an approach has not been practically implemented in any country. the reason is not thought to be that option 5 or the other options are not conceptually sound, but due to funding limitations, risk aversion, lack of formal incentives, or the necessary skills and tools to promote in situ utilization. it could also simply be that active cwr, wfp in situ or lr on-farm conservation itself is only now being tentatively initiated, in situ conserved resources are uncharacterized and evaluated, the potential of in situ or on-farm germplasm access is unflagged so potential users are unaware such resources are accessible or how to access them. genetic resources (2025), (s2), 203–223 211 fi gu re 3. fi ve op ti on s to lin kp la nt ge ne ti c re so ur ce (p g r ) in si tu co ns er ve d re so ur ce s to us er ac ce ss :1 .d ir ec t in si tu su pp ly ,2 .s ta nd ar d ex si tu co ns er va ti on ,3 .b la ck bo x in si tu sa fe ty ba ck up ,4 .i n si tu de m an d an d su pp ly ,a nd 5. in si tu ba ck up & su pp ly .o ec m ,o th er ef fe ct iv e ar ea -b as ed co ns er va ti on m ea su re s. genetic resources (2025), (s2), 203–223 plant grc roles in linking in situ and ex situ conservation 212 m axted et al g enetic resources (2025), (s2), 203–223 table 2. options proposed for promoting user access to in situ and on-farm conserved plant genetic resources (pgr). the addition of an asterisk to option number means the option meets the principles of pgr conservation and use congruence. abs, access and benefit sharing; cbd, convention on biological diversity; cwr, crop wild relatives; grc, genetic resource centre; itpgrfa, international treaty on plant genetic resources for food and agriculture; lr, landraces; wfp, wild food plants. option option description advantages disadvantages 1 direct in situ supply: involves the user being made aware of the availability of particular in situ pgr populations and their characteristics, the user contacts the pgr in situ maintainer and the maintainer sends a sample directly to end user. a simple procedure agreed and organized by the in situ or on-farm maintainer and the user, which would not necessarily imply grc involvement. in some cases, users may be granted permission to autonomously collect by the appropriate national authority. (a) in general, in situ population maintainers (protected area managers, farmers, land agents, gardeners, etc.) do not see germplasm supply as one of their core activities, have no experience with such activities and are unable to engage in direct user supply. further, they rarely have legislative knowledge of cbd (2011) and itpgrfa-related legislation or its national application and/or international abs statutes (fao (2001); art. 12.3(h) and art. 15.1(b)), therefore cannot enact the legislation. (b) germplasm supply outside of the country of origin requires phytosanitary certification and testing to ensure seeds are free from specific pests/pathogens and the in situ population maintainers would not have the required processing skills. while it might be feasible to supply such knowledge to some maintainers, such as protected areas managers, extending it to all potential farmers, land agents and gardeners, is unrealistic. (c) training in situ population maintainers in germplasm supplier skills would be almost meaningless as the chances of each individual supplier supplying conserved germplasm would be limited given their large number and the limited number of seed requests. (d) in situ population maintainers could only supply germplasm during the pgr fruiting season, so there would be significant delays between request and user supply. 2* standard ex situ conservation: describes the typical route by which germplasm enters the grc: populations are sampled from the wild or on-farm location, transferred to the grc, registered and documented, processed following the standard guidelines (fao, 2014) and supplied to users. a tried and tested route applied widely for ex situ conservation that effectively meets users’ needs, but here is applied to an in situ conserved population. it meets the principles of pgr conservation and use congruence. (a) if each country maintains a substantial number of in situ conservation sites for cwr, wfp or lr population conservation and these all ex situ backup accessions in the grc, the processing of additional in situ samples and making them available to users would require significant additional resources. continued on next page 213 table 2 continued option option description advantages disadvantages 3 blackbox in situ safety back-up: a sample is either collected by the in situ maintainer or collected by grc staff and stored in the nominated ex situ facility and is only available to the donor for their use, or in situ population reinforcement or reintroduction. a simple, inexpensive procedure agreed and organized by the in situ or on-farm maintainer and the grc. (a) this option does not meet one of the imperatives of the principles of pgr conservation and use congruence which mandates that conserved pgr should be available for utilization, therefore this cannot be considered effective as a primary pgr conservation measure. (b) if the in situ population is rare, highly threatened or has known unique, adaptive allelic diversity, then it should be conserved in situ and backed up ex situ4. 4* in situ demand and supply: proposed by van hintum et al (2021) to minimize the grc additional workload. it involves users identifying the in situ population they wish to obtain, requesting a sample from the appropriate grc, and a staff member travelling to the site, collecting and processing a sample and distributing it to the end user. this option does minimize the additional grc workload and ensures the in situ or on-farm maintained population is provided to the user. (a) this option would involve additional work for the grc staff in sampling and processing in situ samples, though grc sampling costs could potentially be shared with the user. costs could be reduced by providing guidance to in situ maintainer so that they collect and forward the sample either directly to the user or via the grc. however, any additional costs of in situ supply might act as a disincentive to potential users, especially if no such cost is associated with ex situ grc holdings. (b) user supply would involve one-off population sampling and would not be as cost-effective as expedient sampling while undertaking a routine grc collection mission. (c) seasonality would mean seed, cuttings or tissue samples would not be available year-round and this might significantly delay in situ sample supply to the user, which would add a further disincentive to potential users (maxted, 2019), while ex situ conserved grc samples are available for distribution year-round. (d) for cwr and wfp taxa natural seed dispersal mechanisms make it difficult for collectors to gather the required target number of seed at the optimal time for conservation and supply during a brief one-off visit to a natural population. (e) also, in situ populations are less likely to be characterized and evaluated for adaptive traits, although users could apply predictive characterization techniques to aid in situ population selection (thormann et al, 2014). continued on next page 4 the assumption is that availability would be granted by the in situ maintainer in the future when target population levels have risen, and black box in situ back-up would not be a long-term preferred option. g enetic resources (2025), (s2), 203–223 plant g rc roles in linking in situ and ex situ conservation 214 m axted et al g enetic resources (2025), (s2), 203–223 table 2 continued option option description advantages disadvantages 5* in situ backup and supply: iriondo et al (2012) proposed as a standard that in situ conserved populations should be backed up in nominated ex situ facilities. it involves users identifying the in situ population they wish to obtain, requesting a sample from the appropriate grc, and a staff member supplying a sample from the in situ backup material to the end user. each in situ population should be backed up ex situ to facilitate reintroduction of the original material, if necessary. the sample could be collected by the in situ maintainer and sent by them to the nominated grc. the backup sample could be maintained using a partial ex situ protocol and used for characterization and evaluation to promote user application. (a) backing up each in situ conserved population in the grc would be costly, especially if all samples were collected and processed using standard ex situ models (fao, 2014). to minimize the grc costs of in situ sample processing: (i) the sample and associated data could be collected by the in situ population manager and sent to the grc, rather than collected by grc staff; (ii) on arrival in the grc, the in situ sample would be processed using ex situ protocols, except regeneration5 and germination monitoring would be omitted (maxted, 2019), regeneration being replaced by regular in situ population resampling6, which would also reduce the requirement for periodic germination testing; (iii) periodically resampling will also ensure that the genetic diversity captured in the ex situ backup sample accurately reflects the ongoing evolutionary trajectory of the in situ population. (b) the in situ backup sample needs to sufficiently large for the grc to supply the end user. 5 note for cwr samples, it may be difficult to collect recommended standard sample sizes quantities (fao, 2014) and therefore, initial sample seed bulking may be required before formal seed storage, especially if the sample is to be used subsequently for characterization and evaluation, and user provision. 6 although germination testing as a relatively inexpensive task might be retained to confirm the initial quality of the sampled seeds and as an indicator to trigger in situ population resampling. 215 it is also true that there has been some initial resistance to changes in roles and responsibilities from both current in situ population maintainers and grc staff; true in situ and ex situ grc integration will add additional roles and responsibilities, especially when many staff are already over-committed and additional resources are limited. although in situ and ex situ grc integration will add additional roles and responsibilities for both grc staff and in situ population maintainer communities, it is likely to be mutually beneficial. for grc staff it would extend the range of diversity they are able to provide to users, whereas for in situ maintainers, it presents a good example of applied additional ecosystem services from the pgr resources they manage, graphically demonstrating the fundamental value of area-based conservation and diversity-based farming systems to the public. by providing access to in situ population samples, grc extend their expertise in user seed supply — an area in which in situ population managers lack experience and have no institutional mandate. this aligns with the grc’s existing key role in effectively addressing user demand for genetic diversity. furthermore, adoption of this option could be expanded if the additional commitment remained minimal for site managers and grc staff, and if it were adequately resourced. such integration would also likely facilitate more coherent pgr policy development and implementation, rather than pgr policy being the responsibility of each discrete site managers and grc communities, plus those from the third research community. it is appropriate that the grc takes a lead role in pgr conservation and user provision because it: (1) has experience in pgr long-, mediumand short-term genetic conservation, collection management and meeting user requests for germplasm effectively, as well as promoting a supportive policy environment over the past 60 years globally; (2) possesses practical expertise in national and international germplasm transfer, as well as meeting associated phytosanitary and legislative requirements; (3) is already known as the germplasm source for diverse users and are accustomed to germplasm access procedures; and (4) has the potential to extend their role to supply samples from in situ conservation sites. it should be stressed that even if the grc provides the overall pgr national lead they must ensure collective decision-making and implementation among the three communities involved, site managers and grc communities, plus those from the third research community, site manager, pgr researcher and grc communities, potentially plus more peripheral communities (e.g. biodiversity, informatics, systematics, etc.). how such managerial cooperation is achieved is likely to vary from country to country based on local contexts, species biology, resource constraints and broader socio-political factors, but it is likely to involve the establishment of a pgr conservation committee to promote collection management, user access promotion research direction and policy development discussion, chaired by grc staff. it should be noted that the partnership between the in situ population maintainer and the ex situ component of the grc is critical to facilitating in situ germplasm user access. to ensure this relationship is effective, it is preferable that each in situ population be partnered with a nominated ex situ grc, this will be the national or a national grc. however, in countries with a decentralized grc network, matching specific crop group cwr, wfp or lr with their corresponding specialist grc would be appropriate and beneficial. the preceding discussion has focused on professional roles in pgr conservation and use, but locally, community biodiversity management is increasingly shown to be effective in facilitating local conservation management of pgr; a role that seems particularly pertinent in the in situ context linking local pgr conservation effort to local pgr use. it seems unlikely many local communities would be interested in cwr use because of the potential need for advanced techniques to overcome interspecific breeding barriers and problems associated with linkage drag of unwanted additional traits, though even here local communities have shown interest in cwr population surveying. however, wfp and lr could be conserved and used more directly via community seedbanks initiated by local communities. local community seedbanks could also function as a conduit to the more formal grc community (bocci et al, 2025), aiding in situ characterization, adaptive trait recognition, in situ population sampling for ex situ duplication and backup, and even cwr prebredvarietal introductions, as well as provision of associated datasets. this could encourage greater recognition of the informal conservation sector, provision of resources and skills training, and inclusion of community seedbank holdings in national pgr inventories and eurisco. community seedbanks could take the role of lr population maintainers working in collaboration with grc staff to maximize diversity maintenance. improving integration between the pgr formal and informal systems will surely prove mutually beneficial and help secure existentially important food security resources. aiding user selection of in situ conserved populations via eurisco significant progress has recently been achieved in advancing in situ pgr conservation documentation through the incorporation of information on active in situ population conservation into eurisco (https://w ww.ecpgr.org/working-groups/crop-wild-relatives/cwr -in-eurisco). this was accomplished through a project funded by the german federal ministry of food and agriculture (referred to as eurisco project below), commenced in november 2021 and focused on countrybased case study incorporation of cwr in situ population data in eurisco. although this initiative was developed for cwr populations, a similar approach genetic resources (2025), (s2), 203–223 plant grc roles in linking in situ and ex situ conservation 216 maxted et al could, in the future, be implemented for wfp and lr population data, marking a significant step forward in pgr science. the extension of eurisco is endowing the european region with a centralized, public and web-searchable inventory of priority in situ cwr populations’ passport data, along with a fine-tuned data flow mechanism that uses an internationally agreed data exchange standard (van hintum and iriondo, 2022). the new in situ module of the eurisco catalogue was built in compliance with the ‘fair principles’: findable, accessible, interoperable and reusable data (wilkinson et al, 2016). the online central catalogue of in situ cwr population data has been available since the beginning of 2024, and more european countries are being trained and encouraged to add their country data. this provides easy-to-access information to potential users seeking novel sources of diversity for breeding and pre-breeding programmes and other uses. the implementation of these international commitments prioritized by the cbd, global plan of action (gpa) and itpgrfa, as well as by the european plant genetic resources strategy (ecpgr, 2021), will prove beneficial to pgr conservationists and users alike, ultimately promoting food security and wellbeing. a proposal, including principles and requirements for data inclusion, the definition of a data flow mechanism and the proposed data exchange standard (cwr passport descriptors), was developed and published on the ecpgr website (van hintum and iriondo, 2022). it includes recommendations for identifying the most relevant cwr populations to be recorded in eurisco. it also outlines a set of descriptors for in situ conserved populations, including their current location, precise coordinates, and where samples are being actively conserved to guarantee their long-term persistence. it addresses how samples from these populations can be accessed, potentially based on the terms and conditions of the itpgrfa multilateral system. furthermore, it describes the structure of information shared between the cwr-national inventory (cwr-ni) and eurisco, the necessary steps to upload cwrni elements into eurisco and the modifications to eurisco to accommodate such type of data. two annexes containing ’descriptors recommended for the generation of a national inventory of in situ crop wild relatives’ and ’descriptors for uploading passport data of in situ cwr to eurisco’ complete the document. as of january 2025, eleven countries (albania, bulgaria, cyprus, germany, italy, lithuania, the netherlands, poland, romania, spain and the united kingdom) have provided in situ cwr data to eurisco, with data from a total of 5,764 populations. incorporating in situ data into eurisco is a key step toward addressing some of the accessibility issues related to in situ material that have been discussed in this review. the eurisco project has already played a key role in establishing in situ pgr conservation and documentation as being truly complementary to ex situ efforts in europe and in helping ensure that in situ conservation meets the principles of pgr conservation and use congruence. without this initial step, the establishment of in situ genetic reserves would have progressed more slowly. further initiatives are likely to be agreed between the pgr in situ site and population maintainers, researchers and grc to ensure a future fully integrated and effective complementary in situ– ex situ conservation–use continuum. some first thoughts include: • while a periodic update of in situ data to eurisco, such as every five years, may be suitable long-term, more frequent updates might be necessary during the initial establishment of in situ genetic reserves. • recently, eurisco has begun to support the linkage of characterization and evaluation data with the germplasm passport data held in eurisco as a means of aiding user selection of germplasm and promoting further utilization of conserved resources. there is significant opportunity for further extending utilization by building tools to aid germplasm selection (tags) and links to additional data sets. one obvious tags would be a predicted characterization tool, where the crop and the desired trait required are selected and the tool suggests a subset of cwr and lr accessions that might have the trait for the crop. another tool is a lr repatriation tool that allows the user to choose lr from certain localities to aid repatriation of lr lost from those locations. • just as cwr and wfp diversity is actively conserved in other non-pgr contexts, e.g. as a rare or threatened taxa by biodiversity specialists or as wild species by botanic gardens, so biodiversity specialists and botanic gardens are interested in cwr and wfp diversity, and organic crop producers and diversity-based farmer specialists, for example, are interested in pgr germplasm for their own non-pgr based utilization. to this end, eurisco could be better designed to meet additional user communities. • it is widely agreed that national in situ and onfarm conservation should be managed in a network structure rather than each site being managed independently. the likely benefits include systematic conservation coordination and reporting, stronger partnerships and mutual support, integration of global, regional, national and local actions, truly in situ– ex situ conservation integration with improved data interoperability and coordinated policy development, facilitation of abs for protected areas and farmers/farming communities, and tools and methodologies to aid safeguarding in situ pgr populations. with so many potential benefits and many different potential governance structures possible, it would be wise to start planning now to maximize national pgr in situ genetic resources (2025), (s2), 203–223 217 networking that links in situ, ex situ, user access and impact. • it would also be useful to define what data will be included and excluded from eurisco. what data might be better maintained at individual cwr, wfp or lr population site level and or at national network level, and where there is no benefit in collating at the regional level. one example that could be considered such data is currently provided by genebank holding curatorial data (e.g. size of seed collection, germination percentages, location of sample in genebank). similar curatorial data exists for in situ populations (e.g. monitoring data for in situ populations over time, levels and timings of management interventions, or age of lr maintainer cultivating a lr). some such data might appropriately be recorded in national inventories and some at site level, but boundaries need to be established to maximize overall efficiency. future challenges and opportunities for in situ– ex situ integration with agrobiodiversity conservation budgets limited and becoming tighter, it is imperative to maximize the efficiency of conservation expenditure. horizon scanning, a participatory approach to the establishment of future priorities, is getting increasingly recognized as a useful tool to help prioritize and plan conservation action, inform resource allocation and provide an evidence base for conservation implementation (for its pgr application see maxted et al (2012a)). this exercise is carried out here in the context of in situ– ex situ integration for cwr and lr conservation over the next ten years and the results are summarized in supplemental tables 2 and 3, respectively. those involved in the 2025 assessment were partners in the eu-funded project pro-grace7 (23 experts from 11 countries + ecpgr secretariat), members of the ecpgr on-farm conservation and management (85 experts from 43 countries) and cwr working groups (87 experts from 38 countries). these experts were also asked to identify emerging pgr-related issues with implications for ex situ and in situ conservation that they felt were of european importance to cwr, wfp and lr diversity in europe, and required resolution by 2035. the experts identified a set of 23 issues related to cwr and wfp, and 24 issues related to lr. it is anticipated that the issues detailed in supplemental tables 2 and 3 will be used in three primary ways. firstly, that policymakers will critically examine the issues identified, assessing their potential impact on policy development and considering appropriate implementation timelines. secondly, it is expected that this exercise will help the integrated ex situ and in situ pgr community better target their activities for 7 https://www.grace-ri.eu/pro-grace, grant n. 101094738 the immediate and longer-term future, considering the relative success of the previous pgr horizon scanning initiative. it is hoped that researchers, funders, and those working on pgr policy and regulation will use the outcome of this exercise when considering the future direction of strategic cwr, wfp and lr research. finally, this exercise may encourage further consideration and debate about the issues that are on the horizon and the ways in which scientists and decisionmakers can best communicate about them. discussion this paper discusses the largely unexplored challenges and opportunities associated with integrating ex situ and in situ plant genetic resources (pgr) communities. historically, these communities have worked semiindependently, but there are now significant mutual benefits for humanity in their integration, transforming and enhancing the paradigm of pgr conservation and use. traditionally, formal pgr conservation has relied almost exclusively on ex situ storage of seed samples in genebanks, providing users with easy access to meet evolving needs. conversely, in situ and on-farm applications for pgr conservation have been extensively discussed (jain, 1975; maxted et al, 1997b, 2002, 2020; safriel et al, 1997; brush, 2000; eyzaguirre and linares, 2004; heywood and dulloo, 2005; jarvis et al, 2007, 2016; iriondo et al, 2008, 2021; veteläinen et al, 2009; hunter and heywood, 2011; fao, 2013; hunter et al, 2017), particularly post-cbd established prioritized in situ techniques (cbd, 1992), but rarely practically applied except for farmer-based maintenance of lr. the strength of integrating both conservation strategies, ex situ and in situ, lies in maximizing the long-term and sustainable maintenance of a more comprehensive representation of pgr diversity. historically, commercial plant breeding has been hesitant to utilize non-domesticated or highly heterogeneous cwr and lr germplasm, likely due to a lack of economic incentive for broader diversity, limited availability of non-domesticated cwr or diverse lr germplasm, challenges in identifying germplasm with known adaptive and desirable traits, and the economic, time and complexity costs associated with pre-breeding and elimination of unwanted traits inadvertently introduce via linkage drag. however, the status quo is shifting: climate change and ecosystem instability necessitate a greater breadth of pgr diversity to sustain agricultural production, while precision techniques facilitate the identification of valuable adaptive traits and enhance the precision of trait introgression (prohens et al, 2017). this knowledge highlights that ex situ approaches alone cannot satisfy users’ demands for a comprehensive range of diversity, prompting a renewed focus on in situ conservation. despite recent progress in experimental in situ applications, experience over the past 30 years indicates that implementing in situ methods independently of ex situ approaches is both ineffective and counterproductive. genetic resources (2025), (s2), 203–223 plant grc roles in linking in situ and ex situ conservation 218 maxted et al in situ conservation should be complemented by ex situ strategies to: (1) provide long-term backup for security and potential population reinforcement or reinstatement; (2) assist in characterization and evaluation; and (3) ensure ease of access for end users. likewise, ex situ conservation should be complemented by in situ approaches to: (1) maximize the preservation of taxonomic and genetic diversity; (2) allow for the evolution of adaptive traits in changing environments; and (3) address the evolving demands of end users. thus, both ex situ and in situ conservation methods are interdependent and should function in a mutually supportive manner. however, unlike ex situ conservation, which can be largely managed within controlled environments, in situ conservation necessitates the active participation of diverse actors with various skill sets (ecology, wild plant biology, field genoand phenotyping, remote monitoring, climate change management, invasive species and pest management) to enact conservation actions, adding layers of complexity and associated challenges. integration of these diverse actors in a distributed research infrastructure on plant genetic resources is likely to unite these additional actors. moreover, a critical question arises: who will take primary responsibility for coordinating in situ conservation efforts? the experiences of the ecpgr cwr and on-farm conservation and management working groups have demonstrated that neither protected areas nor farming communities can effectively coordinate in situ pgr diversity conservation activities, and many are reluctant to engage in formal in situ pgr conservation. protected area managers focus on biodiversity rather than crop diversity conservation, while farming communities are primarily engaged in commercial agricultural production rather than systematic diversity conservation. therefore, there is a pressing need for additional training for grc staff and/or extending collaboration with actors possessing the necessary skills and experience in ecology, pest management and field conservation to complement the existing grc staff’s expertise in genotypic, phenotypic and agronomic evaluation, sampling, viability and phytosanitary testing, documentation, data upload to eurisco, and distribution to users, including knowledge of national and international legislative implementation. this collaboration is fundamental to enhancing the conservation of in situ diversity, its description and its availability to end users. additionally, the existential problem of user supply is often underestimated by the in situ pgr community. for pgr conservation to be effective, meaningful, and serve a utilitarian purpose beyond its intrinsic value in nature preservation, a link must exist between conservation and utilization. however, neither protected areas nor farming communities possess experience in germplasm supply within the context of access and benefit-sharing legislation. consequently, it can be argued that without the involvement of the ex situ community in these roles, in situ implementation risks becoming limited to ’academia,’ ’hobbyists’ or shortterm project support without long-term sustainability. therefore, it is evident that the application of ex situ and in situ strategies is mutually dependent, and their complementary integration should be led by grc. leadership from grc would entail adjusting their perspective to encompass both ex situ and in situ aspects, along with appropriately increased resources to fund the necessary structural and skill provisions for practical implementation. conversely, if the in situ or on-farm community was able to take such a leadership role, would the genebanks welcome the competition? there is also an economic argument for grc to adopt a more proactive role in in situ conservation. as outlined, one justification for pgr conservation is to enhance user access and benefits, which may encompass various industries, with the most prominent being those related to economic and food security, medicinal products and material uses. the most recent estimate of the use value for cwr closely related to 29 globally important crops is us$42 billion, with a potential future value of $120 billion. the annual gross added value was $581 billion in 2010, indicating that cwr are already valued at about 7% of the annual production value of these 29 crops (pwc, 2013). this valuation is conservative, as it does not account for the potential expansion of cwr use in breeding these or other crops, nor the value of utilizing lr diversity. therefore, the overall annual gross added value of using pgr diversity in crop improvement could approach a trillion us dollars. this significant valuation raises the question: does not the potential revenue stream justify the modest investment required now in pgr conservation to secure future substantial benefits? the rationale for integrating ex situ and in situ conservation lies in the fact that ex situ collections typically capture only a snapshot of the genetic diversity present in natural populations at the time of collection. it also should be acknowledged that over time, genetic drift or selection during storage and regeneration can lead to the loss of some of this genetic variation. in contrast, in situ conservation allows the remaining spectrum of genetic diversity to persist and evolve naturally in response to environmental changes. without leveraging both approaches, a significant portion of the genetic diversity available in natural populations remains untapped, limiting its potential contribution to crop improvement and other industries. while the practical establishment of cwr genetic reserves or lr on-farm diversity maintenance sites has progressed more slowly than anticipated, this may be partly attributed to the pgr community’s longstanding focus on the established ’in-nature and on-farm sampling to genebank to user’ paradigm (guarino et al, 1995, 2012; hawkes et al, 2000; smith et al, 2003; fao, 2014). this paradigm has proven resilient and successful over the last century, consistently meeting the needs of breeders and consumers. however, the very success of this established paradigm poses a significant challenge to the adoption of in situ conservation approaches. to gain wider acceptance, these approaches genetic resources (2025), (s2), 203–223 219 must articulate an equally robust and straightforward model that demonstrates long-term effectiveness – the pgr germplasm user is indifferent to the conservation source if it meets their trait needs. promoting in situ application includes clearly communicating the value of the proposed in situ to ex situ to use paradigm and its mutual advantage in diversity breadth. although the clarification of the principles of pgr conservation and use congruence and the derived proposals presented provide an initial foundation for a proposed in situ to ex situ to use paradigm led by the national grc, further development will be necessary based on a growing evidence base. another related topic that has progressed more slowly than anticipated is the systematic ex situ and in situ conservation of wfp. these include fruits, leafy vegetables, woody foliage, bulbs and tubers, cereals and grains, nuts and kernels, saps and gums, mushroomsand seaweeds (wunder, 2014). wfp have historically served as a coping strategy for many rural households, particularly during the ’hungry season’ before the next season’s crops ripen and as part of subsistence farming systems (hunter et al, 2015; kennedy et al, 2017). fao (2019a) estimates that around one billion people globally incorporate wild foods into their diets regularly, and forests alone provide livelihoods and food for approximately 300 million people through non-timber forest products. however, wfp are rarely included in pgr conservation initiatives and are unlikely to be targeted for biodiversity conservation only if they are threatened or rare. fao (2019a) calls for (1) active ex situ and in situ conservation and sustainable use, (2) breeding of improved varieties, and (3) raising awareness of the importance of wfp, particularly local and traditional foods that are vital for nutritionally balanced, healthy diets and food security. wfp, like cwr, are simply wild species with specific food value, although the former is associated with direct consumption rather than trait provision. therefore, wfp planning and conservation implementation are unlikely to differ significantly from cwr-based actions, making it timely to test this assumption. implementing wfp conservation falls within the remit of national grc activities and should be integrated with other pgr activities. most importantly, wfp can provide material for future domestication efforts, thereby expanding the foundation of our food production systems. here much has been made of expanding in situ/ex situ integration, but there is also significant leverage in in situ/on-farm working more closely with biodiversity communities. cwr and lr could be used as ‘cultural ambassadors’ to help promote pa-based conservation or traditional cultivation practices. the collaboration offers opportunities to marry biodiversity conservation management with food security or traditional foods associated with healthier lifestyles. while such collaboration would also help conserve the critical pgr resource more extensively and effectively – demonstrating the mutual relevance of each community contribution – pa don’t only maintain birds, mammals and reptiles, they conserve the founding resource for our food. traditional farming is not just picturesque, it sustains cultural benefits such as recreation, education, spiritual and creative enrichment, and improved mental health and wellbeing. whilst pa management may recognize the importance of these ecosystem services, their consideration and usefulness in site management decision-making is worth closer understanding. there exists an opportunity and a central role for the proposed grace research infrastructure (see https:// www.grace-ri.eu/pro-grace), which builds on 55 years of ecpgr collaborative networking aimed at ensuring long-term conservation and facilitating utilization of pgr to implement the necessary transition from genebanks to grc and enact more effective in situ pgr conservation. this role may prove existentially important for humanity in the future. without appropriate financing, skills and capacity provision, and cooperation with the broader biodiversity community, establishing and maintaining in situ and on-farm networks would be unsustainable in the medium to long term, even under grc direction. the core mission of the pgr community remains unchanged, as summarized in the principles of pgr conservation and use congruence, and it is essential to reassess and reconfigure this mission to ensure it is fit for purpose today and in the future. conclusions the dual challenges of human population growth and climate change’s negative impact on crop production have resulted in increased demand from germplasm users and consumers for greater breadth of diversity. ex situ genebanking alone is unable to secure such breadth of diversity, as are in situ or on-farm conservation activities; the urgency of the situation is such that the muchdiscussed but rarely applied implementation of complementary pgr conservation offers the only practical and expedient solution. the principles of pgr conservation and use congruence describe the fundamental principles of pgr conservation (long-term, sustainable conservation, application of complementary conservation techniques, and documentation and availability of the conserved resource for utilization) and provide a framework for indicating success. evidence and experience have shown that neither ex situ, in situ nor on-farm conservation functions adequately in isolation, but further that systematic in situ and on-farm genetic conservation is not a priority for practitioners of either biodiversityfocused conservationists or production-based farmers. the comprehensive integration of ex situ, in situ and on-farm conservation communities and their activities, with the local communities where the bulk of the genetic resources exist, led by national grc and cgiar institutes, is now critical for global, regional, national and local food security; failure to address this issue could have devastating consequences for humankind in the 21st century. specific recommendations are outlined for collaborative resource management, user access to in genetic resources (2025), (s2), 203–223 plant grc roles in linking in situ and ex situ conservation 220 maxted et al situ and on-farm conserved pgr, improving user selection of in situ conserved populations and what the future challenges and opportunities there might be for future in situ– ex situ integration. other recommendations will undoubtedly come from further steps toward pgr community integration. although realistically this initiative is doomed to failure unless national grc step up to take the lead, skill gaps are filled, and they are adequately resourced. supplemental data supplemental table 1. genetic conservation strategies and techniques (maxted et al, 2020). supplemental table 2. horizon scanning issues associated with cwr in situ– ex situ conservation in 2025 that require resolution by 2035. supplemental table 3. horizon scanning issues associated with lr in situ– ex situ conservation in 2025 that require resolution by 2035. acronyms used • abs − access and benefit sharing • aegis − a european genebank integrated system • c & e − characterization and evaluation • cbd − convention on biological diversity • cwr − crop wild relatives • cwr-ni − cwr-national inventory • ecpgr − european cooperative programme for plant genetic resources • eurisco − european search catalogue for plant genetic resources • fao − food and agriculture organization of the united nations • gpa − global plan of action • gr – genetic reserve • grc − genetic resource centre • ircc − internationally recognized certificate of compliance • itpgrfa − international treaty on plant genetic resources for food and agriculture • lr − crop landrace • ngo − non-governmental organization • oecm − other effective area-based conservation measures • pa − protected area • pgr − plant genetic resources • smta − standard material transfer agreement • tags − tools to aid germplasm selection • wfp − wild food plant acknowledgements we gratefully acknowledge the support of the horizon europe programme, project ‘promoting a plant genetic resource community for europe (pro-grace)’, n. 101094738 in preparation for this review. part of this paper derives from the results of deliverable 1.3 of prograce. conflict of interest statement the authors declare no conflict of interest. author contributions nm drafted the first iteration of the text and coordinated production of the final text, all authors contributed to the conception, discussion, and text of the paper. references bocci, r., bartha, b., maierhofer, h., arndorfer, m., and salvan, m. 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(2025). strategies to balance productivity and genetic diversity for the sustainable use of indigenous livestock breeds: a case study of ethiopia. genetic resources 6 (11), 82–98. doi: 10.46265/genresj.nnfe5064. © copyright 2025 the authors. this is an open access article distributed under the terms of the creative commons attribution license (cc by 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. introduction in many developing countries, livestock (cattle, sheep, goats, poultry, camels, horses and donkeys) play a vital role in rural economies (csa, 2021). livestock provide meat, milk, eggs and other products essential for human nutrition. livestock also serve as a source of income, draught power and manure for crop production. moreover, livestock is deeply intertwined with cultural practices and traditions, making it an integral part of the social fabric (adane and girma, 2008; gizaw, 2009; csa, 2022). the sector is dominated by indigenous ∗corresponding author: amine mustefa (amine.mustefa@ebi.gov.et) animals that have evolved over centuries and are managed in diverse production environments, including lowlands, highlands, arid and semi-arid areas (ebi, 2016; assefa and hailu, 2018). however, the sustainable use of livestock in most developing countries has been significantly affected by two major challenges: climate change and the poor productivity of indigenous breeds. climate change is one of the most pressing challenges of our time, with far-reaching impacts on agriculture and food security (el-bilali et al, 2020). developing countries, which are often more vulnerable to climate variability, face significant risks to their livestock production systems. climate change exacerbates existing challenges in livestock production, including water scarcity, feed received: 06.02.2025 accepted: 12.05.2025 published online: 13.06.2025 https://www.genresj.org https://www.doi.org/10.46265/genresj.nnfe5064 https://www.genresj.org https://www.doi.org/10.46265/genresj.nnfe5064 mailto:amine.mustefa@ebi.gov.et genetic resources (2025), 6 (11), 82–98 balancing livestock productivity and diversity in ethiopia 83 shortages and disease outbreaks (degefu and milkias, 2024). rising temperatures and changing precipitation patterns can reduce the availability of pasture and water, leading to decreased productivity and increased mortality rates (woldeyohannes et al, 2023). at the same time, the poor productivity of indigenous breeds poses a significant challenge to the livestock sector in many developing countries, limiting the sector’s potential benefits. this issue stems from a combination of low genetic potential and environmental constraints, including inadequate feed, veterinary care and management practices (gizaw, 2009; mustefa, 2022). therefore, to ensure the sustainable use of livestock, addressing these two major challenges is essential. in this context, two primary strategies have emerged: genetic improvement and the conservation of indigenous breeds. livestock production and productivity can be enhanced through the implementation of various genetic improvement programmes. selective breeding, crossbreeding and breed substitution are viable options for improving the genetic potential of indigenous breeds (philipsson et al, 2006). on the other hand, the application of in situ and ex situ conservation, or a combination of both can help maintain the diversity of indigenous livestock breeds, enabling them to cope with upcoming climate-driven changes. indigenous livestock breeds are known for their ability to adapt, produce and reproduce under harsh environmental conditions, such as scarce feed and water, extreme temperatures, disease challenges and prolonged drought periods (ebi, 2016; assefa and hailu, 2018; endris et al, 2022). in addition to their adaptability, indigenous breeds are valued for their desirable products, such as eggs, meat and milk. the market value of products from indigenous breeds is often higher than those from exotic breeds. however, enhancing productivity and diversity simultaneously is challenging because the concepts of genetic improvement are often associated with decreasing diversity (ebi, 2016). moreover, according to article 2 of the convention on biological diversity (cbd), sustainable use is defined as “the use of components of biological diversity in a way and at a rate that does not lead to the long-term decline of biological diversity, thereby maintaining its potential to meet the needs and aspirations of present and future generations” (cbd, 2004). therefore, balancing genetic improvement with the conservation of indigenous livestock breeds is crucial. to achieve this, it is necessary to review the principles and on-the-ground impacts of these approaches. thus, the current study aims to recap the advantages and limitations of both options (conservation and genetic improvement) for the sustainable utilization of indigenous livestock breeds with a focus on cattle, sheep, goats and chicken, using ethiopia as a case study, and to recommend a win-win solution. a systematic review was conducted, following five steps as stated in khan et al (2003): • step 1: framing review questions. the review question focused on the advantages and limitations of livestock genetic improvement and conservation programmes related to the sustainable utilization of the livestock production sector. • step 2: identification of relevant work. relevant published articles as well as unpublished msc and phd thesis works addressing the framed review questions were extensively searched. • step 3: quality assessment. articles published in reputable journals were selected alongside the msc and phd thesis works. • step 4: summarization of evidence. information related to the genetic improvement programmes using within-breed selection, crossbreeding and breed substitution approaches, as well as in situ and ex situ conservation programmes, was compiled. • step 5: interpretation of findings. the main findings from step 4 were interpreted by comparing the achievements and limitations of genetic improvement and conservation approaches, as well as examining them against scientific justifications. genetic improvement livestock genetic improvement refers to the enhancement of the genotype of live animal breeding populations to increase their productivity, efficiency and resilience (mueller and van eenennaam, 2022; tesfa et al, 2024). the primary goals of livestock genetic improvement activities in most developing countries are to increase meat, milk and egg production, as well as improve feed efficiency. this can be achieved through selective breeding, crossbreeding, breed substitution and the use of advanced biotechnologies such as genomic selection and gene editing (belew et al, 2016; haile et al, 2020; woldeyohannes et al, 2023). within-breed improvement involves selecting superior animals from the same population to serve as parents for the next generation while culling lowperforming animals from the flock (haile et al, 2020). crossbreeding improves the genotype of indigenous animals by crossing them with high-performing exotic breeds. breed substitution, on the other hand, involves replacing low-performing indigenous animals with highperforming exotic breeds (vaccaro and steane, 1990; solomon et al, 2014). ethiopia has successfully implemented genetic improvement programmes of cattle (beneberu et al, 2021), sheep (getachew et al, 2020), goats (solomon et al, 2014), and chicken (yigzaw et al, 2024). alongside within-breed selective breeding initiatives, numerous crossbreeding and breed substitution programmes have been implemented, introducing several exotic cattle, chicken and small ruminant breeds (table 1). 84 mustefa genetic resources (2025), 6 (11), 82–98 table 1. exotic livestock breeds that were introduced into ethiopia in the past decades livestock species/breeds introduction year references cattle angus 1950s tucho et al (2021) brahman 1950s chebo and alemayehu (2012) brown swiss 1947 hunde (2018) hereford 1950s tucho et al (2021) holstein-friesian 1950s albero (1983) jersey 1987 beneberu et al (2021) simmental 1950s mwenya (1992) sheep awassi 1980 getachew et al (2020) corriedale 1967 getachew et al (2016) dorper 2007 habtegiorgis et al (2025) hampshire 1967 sheriff and alemayehu (2018) merino 1944 getachew et al (2016) rambouillet 1967 tibbo et al (2006) romney 1967 sheriff and alemayehu (2018) goats anglo-nubian 1970s workneh (2000) boer 2007 mustefa et al (2019b) saanen 1940s awgichew et al (1989) toggenburg 1975 girma et al (2000) chicken arbor acre 2000s alemneh and getabalew (2019) australorp 1953 gage and suntebo (2023) bovans brown 1950s melkamu et al (2017) brown leghorn 1950s chebo et al (2022) cobb-500 2000s sidrak et al (2021) dominant brown d102 2000s guteta (2021) dominant sussex 2000s yigzaw et al (2024) fayoumi 1996 geleta et al (2013) hubbard classic 2015 fekadu et al (2022) hubbard jv 2015 tolasa (2021) isa brown 1950s ebi (2016) koekoek 1950s abadi et al (2020) lohman brown 1950s kidie et al (2024) lohmann silver 2022 fekadu et al (2022) new hampshire 1953 gage and suntebo (2023) novo brown 1950s yigzaw et al (2024) rhode island red (rir) 1953 hussen and anja (2017) sassot44 2014 chebo et al (2022) sussex 1950s chebo et al (2022) white leghorn 1953 chebo et al (2022) genetic resources (2025), 6 (11), 82–98 balancing livestock productivity and diversity in ethiopia 85 advantages of livestock genetic improvement programmes increased productivity the primary significance of genetic improvement methods lies in enhancing productivity (mueller and van eenennaam, 2022). the objectives of genetic improvement programmes can differ with focuses on aspects like growth, production and reproductive performance. some of the reported results for each species are presented below. cattle most of the cattle genetic improvement programmes carried out in ethiopia so far were aimed at increasing milk yield (getahun et al, 2020). the lactation milk yield results of both the indigenous and crossbred cows are presented in table 2. notable differences were observed between the indigenous and crossbred cows where the latter performed more than threefold in most cases. the holstein friesian crosses were observed to produce better milk yield than the jersey crosses. furthermore, the on-farm results were lower than the on-station reports. this might be due to the suboptimal management practices of the farmers as well as limited adaptability of the crossbred animals to the local environment. small ruminants thus far, most of the small ruminant genetic improvement programmes carried out in ethiopia have targeted growth traits (tesema et al, 2020). body weight results from birth to yearling age of both the indigenous and crossbreds are presented in table 3. differences were observed between the indigenous and crossbreds in most cases. most of the crossbreds had better growth performances than the indigenous breeds except for afar sheep and central highland goat crosses where the indigenous performed better than the crosses. this might be due to the lower adaptability of the crossbreds which were managed on station with intensive and semi-intensive management systems. on the other hand, communitybased breeding programmes (cbbp) were observed to bring outstanding results in bonga sheep while their effect was small in menz sheep and abergelle goats. chicken chicken genetic improvement programmes primarily aimed to improve egg production, while growth and reproduction traits were also given due consideration (dana et al, 2010; esatu, 2015; chebo et al, 2022). according to alemneh and getabalew (2019), the overall egg production of the ethiopian indigenous chicken breeds was reported to be 30–60 per hen per year. compared to this value, notable productivity gain were obtained through the implementation of genetic improvement programmes under intensive and extensive management systems (table 4). extremely lower egg production was also observed for sasso (133) and bovans brown (124), which might be due to adaptation problems under certain production conditions (assefa et al, 2019; litigebew et al, 2021). similarly, notable successes have also been documented using the within-breed selection approach where a 21% egg number increment at 24 weeks was reported for the indigenous horro chicken selective breeding programme (esatu, 2015). moreover, improved horro chicken showed a 124% egg increment by week 45 (wondmeneh et al, 2016), and were also reported to produce 150 eggs/hen/year, which is significantly higher than the egg production per year of the unimproved horro chicken (moges et al, 2010). climate change mitigation climate change, driven by rising temperatures, is among the factors limiting the sustainable use of animals and their products. the emission of greenhouse gases, including methane, carbon dioxide, nitrous oxide and halocarbons, is regarded as the primary driver of temperature increases. while livestock production is often seen as a victim of climate change, it is also identified as a major contributor to the process (cassandro, 2020). therefore, minimizing the contribution of livestock production to climate change is imperative. genetic improvement is recognized as an important tool for mitigating climate change by reducing greenhouse gas emissions (cassandro, 2020; stranden et al, 2022). the intensification approach to genetic improvement, which reduces the total number of animals while improving their efficiency, has been reported to decrease emissions (cassandro, 2020). according to jardine et al (2012), reducing the number of animals could result in an estimated 8% drop in greenhouse gas emissions. alongside decreasing animal numbers, increasing feed efficiency has been shown to significantly reduce greenhouse gas emissions in dairy production (edwards-jones et al, 2009; bell et al, 2011). hence, genetic improvement in addition to extensive pasture-based farming systems is regarded as a cost-effective approach to climate change mitigation (cassandro, 2020; stranden et al, 2022; marchegiani et al, 2025). reducing aggressiveness animal temperament, or docility, is an important trait in cattle production, influencing not only human safety but also animal welfare and productivity (norris et al, 2014). according to norris et al (2014), poor animal temperament is associated with reduced performance, carcass quality and animal health. thus, temperament affects the sustainable use of a given breed, with docile animals often preferred over aggressive ones (dickson et al, 1969). most indigenous breeds are reported to be more aggressive than exotic breeds. therefore, crossing indigenous breeds with or replacing them with exotic breeds may reduce their aggressive temperament. one widely introduced cattle breed in most developing countries is the holstein friesian. according to dickson et al (1969), holstein friesians, known for their high 86 mustefa genetic resources (2025), 6 (11), 82–98 table 2. lactation milk yield (kg) of representative indigenous and crossbred cows in ethiopia. hf, holstein-friesian; je, jersey. breeds/genotypes lactation milk yield breed type management references arsi (ar) 809 indigenous on station niraj et al (2014) 50% hf x 50% ar 2,247 crossbreds on station million et al (2004) 75% hf x 25% ar 2,497 crossbreds on station million et al (2004) 50% je x 50% ar 1,741 crossbreds on station niraj et al (2014) begait (be) 672 indigenous on station tadesse and dessie (2003) 50% hf x 50% be 2,312 crossbreds on station tadesse and dessie (2003) 75% hf x 25% be 2,373 crossbreds on station tadesse and dessie (2003) 50% hf x 50% be 1,488 crossbreds on farm bekele et al (2011) 50% je x 50% be 970 crossbreds on farm bekele et al (2011) borana (bo) 771 indigenous on station demeke et al (2000) 50% hf x 50% bo 2,203 crossbreds on station getahun et al (2020) 75% hf x 25% bo 2,959 crossbreds on station getahun et al (2020) 50% je x 50% bo 1,684 crossbreds on station gebregziabher et al (2014) 75% je x 25% bo 1,832 crossbreds on station gebregziabher et al (2013) horro (ho) 559 indigenous on station gizaw et al (2011) 50% hf x 50% ho 1,836 crossbreds on station gebregziabher et al (2013) 75% hf x 25% ho 2,184 crossbreds on station gebregziabher et al (2013) 50% je x 50% ho 1,621 crossbreds on station gebregziabher et al (2013) 75% je x 25% ho 1,724 crossbreds on station gebregziabher et al (2013) table 3. growth performance of some indigenous and crossbred small ruminants in ethiopia. bhs, black head somali; tu, tumelie; do, dorper; aw, awassi; chg, central highland goats; wg, woyto guji; bw, birth weight; ww, weaning weight; smw, six months’ weight; yw, yearling weight; in, indigenous; cr, crossbred; os, on station; of, on farm; cbbp, community-based breeding programme. species breed/genotype bw ww smw yw breed type management references sheep afar (af) 2.7 11.5 26.6 in os yibrah (2008) 50% do x 50% af 2.6 9.5 13.2 25.0 cr os abebe et al (2016) bhs 2.5 11.3 23.1 in os yibrah (2008) 50% do x 50% bhs 3.0 15.1 cr os teklebrhan et al (2014) menz (me) 2.1 9.1 17.3 in os markos (2006) 50% do x 50% me 2.8 12.3 17.3 31.3 cr os abebe et al (2016) menz 2.6 9.0 13.3 19.9 in cbbp abebe et al (2020) tumele (tu) 2.4 8.5 11.9 22.4 in os lakew et al (2014b) 50% do x 50% tu 3.2 15.0 20.4 31.4 cr os lakew et al (2014b) wollo (wo) 1.9 10.8 15.7 21.6 in of amare et al (2018) 50% aw x 50% wo 2.4 13.8 22.7 30.4 cr of amare et al (2018) bonga 16.7 in of moa (2018) bonga 3.9 16.3 27.8 in cbbp arega et al (2024) goats abergelle (ab) 2.2 6.9 9.5 14.2 in of hagos et al (2018) 50% bo x 50% ab 2.9 15.3 19.6 27.9 cr os belay et al (2014) abergelle (ab) 2.0 7.2 10.1 15.9 in cbbp gobeze et al (2017) chg 2.0 9.0 13.8 20.6 in of deribe and taye (2013) 50% bo x 50% chg 2.6 8.8 11.2 16.7 cr os mustefa et al (2019b) wg 2.0 9.0 11.5 in of zergaw et al (2016) 50% bo x 50% wg 2.8 11.6 16.2 29.2 cr os dea et al (2019) genetic resources (2025), 6 (11), 82–98 balancing livestock productivity and diversity in ethiopia 87 table 4. egg production per hen per yearof exotic chicken breeds in ethiopia under intensive and extensive production management. intensive management system breed eggs references bovans brown 292 melkamu et al (2017) lohman brown 275 kidie et al (2024) faoumi 160 geleta et al (2013) extensive management system breed eggs references bovans brown 218 melkamu et al (2017) koekoek 176 abadi et al (2020) sasso 133 assefa et al (2019) bovans brown 124 litigebew et al (2021) milk yield and good temperament, have been selected for docility over generations. moreover, within-breed selection for more docile animals may reduce the aggressiveness of indigenous breeds; however, this type of selection is not commonly practised in developing countries, including ethiopia. the sheko cattle breed of ethiopia, known for its trypano-tolerant ability, is also noted for its aggressiveness (desta et al, 2011; aleme and mengistu, 2023). for this reason, farmers often choose to cross it with other relatively docile cattle breeds (desta et al, 2011; aleme and mengistu, 2023). therefore, reducing aggressiveness is another advantage of genetic improvement approaches. limitations of livestock genetic improvement programmes genetic erosion the concept of genetic improvement in indigenous livestock breeds is often associated with a reduction in withinand among-breed genetic variation. both the less destructive within-breed selection approach and the more destructive indiscriminate crossbreeding and breed substitution approaches contribute to decreasing genetic diversity in indigenous breeds (belew et al, 2016; woldeyohannes et al, 2023). even though its effect is less severe than other methods, continued within-breed selection can lead to genetic erosion due to random genetic drift. this occurs when a few genes responsible for economically important traits are favoured, while a large proportion of genes responsible for survival and adaptation traits are lost. the more severe options, such as crossbreeding and breed substitution, are even more destructive, with their contribution to genetic erosion observable within a short period (rahman et al, 2013). genetic erosion, caused by genetic drift, reduces adaptive genetic variation, limiting evolutionary responses (köhler-rollefson and mundy, 2010). therefore, genetic erosion negatively affects the long-term sustainable utilization of indigenous livestock breeds. thus, genetic improvement approaches contribute negatively to the future sustainable use of indigenous livestock breeds due to their role in the erosion of adaptive genotypes (rahman et al, 2013). maladaptation maladaptation is a significant limitation of genetic improvement approaches using exotic livestock breeds. crossbreeding and breed replacement are not always effective potentially due to the poor adaptation of exotic breeds to local environments (köhler-rollefson and mundy, 2010). morbidity and mortality rates of crossbred livestock breeds in different locations of ethiopia are presented in table 5. accordingly, a significantly higher mortality rate was observed for the crossbreds of boer goats with central highland and woyto guji goats indicating their suboptimal adaptability to local conditions. similarly, higher morbidity and mortality rates were also observed for the crossbreds of holstein friesian cattle. moreover, the lower egg production per hen per year presented in table 4 for sasso (133) and bovans brown (124) might be due to their maladaptation to the local environment (assefa et al, 2019; litigebew et al, 2021). exotic breeds, developed and selected for specific environmental conditions, often perform poorly when introduced to new environments due to adaptation problems (köhler-rollefson and mundy, 2010). inbreeding genetic improvement is associated with the selection and use of a few high-performing sires as parents for the next generation (mueller and van eenennaam, 2022). the use of elite sires, increased selection pressure and reproductive technologies like artificial insemination (ai) increase the likelihood of offspring being half-siblings, leading to inbreeding in successive generations (de-roos et al, 2011). one of the negative effects of inbreeding is inbreeding depression, where the increased likelihood of offspring inheriting two copies of harmful recessive genes leads to reduced fertility, vigour and overall fitness (tongsiri et al, 2019; lozada-soto et al, 2021). reduced genetic diversity is another negative effect of inbreeding. inbreeding decreases genetic diversity within a population, making it more vulnerable to diseases, parasites and environmental changes. these factors decrease productivity and increase mortality rates, making livestock production less sustainable (tongsiri et al, 2019; lozada-soto et al, 2021). moreover, ethical concerns arise from inbreeding, as it can lead to increased suffering and reduced welfare for animals due to genetic defects and health problems (frankham, 2005; skotarczak et al, 2020). on the other hand, selecting traits that enhance animal welfare, such as reduced aggression, can lead to more humane and sustainable use of animal populations. cost and time implementing genetic improvement programmes can be expensive, requiring investments in infrastructure, 88 mustefa genetic resources (2025), 6 (11), 82–98 table 5. morbidity and mortality rates of crossbred livestock breeds in different locations of ethiopia. exotic breeds (hf, holsteinfriesian cattle; do, dorper sheep; bo, boer goats; bovans brown; sasso); indigenous breeds (ghc, gojjam highland cattle; am, ambo cattle; go, gofa cattle; tu, tumelie sheep; wl, wolaita sheep; ad, adilo sheep; chg, central highland goats); os, on station; of, on farm. breed/genotype morbidity (%) mortality (%) management location references cattle 50% hf x 50% ghc 56.5 28.1 of bahir dar zuria ferede et al (2014) 50% hf x 50% ghc 65.0 37.0 of gozamen ferede et al (2014) 50% hf x 50% am 62 22.0 of ada’a liben wudu et al (2008) 50% hf x 50% go 66.7 20.0 of wolaita soddo assefa and ashenafi (2016) 50% hf x 50% ghc 47.3 17.9 of bahir dar yeshwas (2015) sheep 50% do x 50 tu 7.0 os tumelie lakew et al (2014a) 50% do x 50 wl 28.4 of mente dubo habtegiorgis et al (2025) 50% do x 50 ad 9.8 os boloso gemiyo et al (2017) goats 50% bo x 50 chg 56.1 os ataye mustefa et al (2019a) 75% bo x 25 chg 64.0 os ataye mustefa et al (2019a) 50% bo x 50 wg 48.0 os jinka molla (2016) 50% bo x 50 wg 41.0 os konso dea et al (2019) chicken bovans brown 3.2 os mekelle melkamu et al (2017) bovans brown 20.3 of mekelle melkamu et al (2017) sasso 16.1 12.7 os sidama hailegebreal et al (2022) technology and skilled personnel (wojtkowski, 2008; biscarini et al, 2015). moreover, genetically improved animals often require better management because the genetic modifications that enhance certain productivity traits can also create new vulnerabilities or amplify existing issues, making them more susceptible to environmental stressors and requiring more precise care to maintain their optimal health and productivity (wojtkowski, 2008; biscarini et al, 2015). however, smallholder farmers in developing countries often have limited access to the technology and resources needed to implement effective genetic improvement programmes. therefore, the high cost of genetic improvement programmes can be a significant barrier for smallholder farmers in developing countries. in addition to higher costs, genetic improvement approaches also require considerable time. genetic improvement is a long-term process, often taking many generations to achieve significant results. this can be a challenge for farmers who need immediate solutions to improve their livelihoods (biscarini et al, 2015). conservation conservation of farm animal genetic resources refers to various human interventions aimed at maintaining the diversity of farm animal genetic resources, without genetic change as far as possible, to contribute to current and future food and agricultural needs (henson, 1992). conservation of indigenous breeds not only preserves their genotypes but also allows farmers and breeders to select and develop new breeds that can adapt and produce under changing environmental conditions, making this approach critically important for sustainable utilization (gicquel et al, 2020). animals can be conserved using in situ and ex situ conservation methods. in situ conservation maintains live animal breeding populations in their production environments (henson, 1992). under this approach, the animals continue to contribute to the food and agriculture of their breeding areas. on the other hand, ex situ conservation maintains genetic resources outside their production systems. there are two ways of conserving genetic resources using the ex situ approach: ex situ in vivo and ex situ in vitro. ex situ in vivo involves maintaining live animal breeding populations outside their production environments, while ex situ in vitro involves the cryopreservation of semen, oocytes, embryos, cells and/or tissues in genebanks (fao, 2012a). in recent decades, several in situ and ex situ conservation programmes have been implemented in ethiopia (table 6). the primary objective and progress of the in situ conservation programmes have been to create exotic-free breeding tracts for the mentioned breeds. similarly, the ex situ in vivo approach conserves live animal populations at ranches or research centres to produce pure parental lines for genetic improvement programmes, but has been applied to two indigenous cattle breeds so far: the sheko and fogera cattle breeds (tibbo et al, 2004). moreover, the ex situ in vitro conservation programmes aim to preserve the semen of genetic resources (2025), 6 (11), 82–98 balancing livestock productivity and diversity in ethiopia 89 the cattle breeds for future restoration purposes, but has been applied to five indigenous cattle breeds so far: the sheko, fogera, borana, begait, and irob cattle breeds out of the country’s registered 28 cattle breeds (assefa et al, 2021). the success of these programmes has been directly linked to the restoration of dwindling population sizes. restoration of endangered breeds requires more budget and time than other conservation programmes, which are typically carried out through successive awareness-raising campaigns. below are some of the advantages of conservation approaches for the sustainable utilization of indigenous livestock genetic resources. advantages of conservation genetic diversity one of the main advantages of conservation programmes is the maintenance of genetic diversity (köhler-rollefson and mundy, 2010; gicquel et al, 2020). maintaining genetic diversity is crucial because once genes are lost, they cannot be replaced except through cumulative selection or mutation (smith, 1984). maintaining withinand among-breed variability supports current and future research and development activities. it also enhances the effectiveness of within-breed selection-based genetic improvement programmes. genetic improvement is more attainable in highly variable populations than in populations with low variability. highly variable populations provide opportunities for the development of specialized breeds. the creation of synthetic breeds for specific purposes through crossbreeding requires the conservation of pure parent stocks. maintaining the variability of indigenous breeds is also essential for their ability to adapt, produce and reproduce under future environmental changes (silva et al, 2019; gicquel et al, 2020). moreover, ensuring a diverse genetic pool helps secure a more reliable and resilient food supply, which is particularly important in the face of climate change and other unpredictable challenges that can impact food production (köhler-rollefson and mundy, 2010). according to smith (1984), the conservation of indigenous breeds provides alternative breeding stock for future changes in market demands, husbandry practices and climatedriven environmental changes. therefore, the conservation of indigenous breeds maintains genetic diversity, which in turn supports the sustainability of livestock production. adaptability adaptation to local environments can be defined in various ways, including disease resistance and tolerance to harsh conditions. many indigenous breeds have natural resistance to local diseases and parasites (köhlerrollefson and mundy, 2010). for example, the sheko cattle breed is known for its trypano-tolerance (desta et al, 2011; aleme and mengistu, 2023). such genetic resilience is invaluable in most developing countries, where access to veterinary care and modern disease control methods is limited. on the other hand, indigenous breeds are often well-suited to challenging environments, such as arid and mountainous regions. they can thrive on limited resources and withstand harsh weather conditions, making them valuable assets for food security in marginal areas. the overall adaptability of indigenous breeds is due to their long-term evolution in specific local environments. in addition, in the face of future climate change, indigenous breeds often offer several advantages over exotic breeds (silva et al, 2019). thus, the conservation of indigenous breeds makes them more efficient and sustainable under current local climatic and management conditions, as well as future climate change (köhler-rollefson and mundy, 2010; gicquel et al, 2020). unique traits the conservation of indigenous breeds is significantly associated with maintaining their unique traits. for example, the conservation of sheko cattle in southwest ethiopia is directly linked to preserving their trypanotolerant ability (desta et al, 2011; aleme and mengistu, 2023). similarly, other special traits of indigenous breeds have been reported, such as the screw horns of racka sheep in hungary (bodo, 1994) and the seaweed-eating sheep (balasse et al, 2019). moreover, indigenous breeds are known to produce items with special qualities, such as coloured wool, super-fine fibre, and tasty products like milk, meat and eggs (köhlerrollefson and mundy, 2010). therefore, for local communities that have adapted to these traits, the sustainable approach is to conserve them rather than crossbreed or replace them with high-yielding exotic breeds. cultural heritage indigenous breeds are deeply woven into the fabric of many cultures, playing significant roles in various aspects of life, from sustenance and livelihoods to social customs, traditions and religious practices (smith, 1984; köhler-rollefson and mundy, 2010; marsoner et al, 2018). indigenous breeds are often associated with specific cultural identities and traditions. they may be used in ceremonial events, festivals and traditional practices, symbolizing heritage and cultural continuity. similarly, in many cultures, indigenous breeds are used as sacrificial offerings in religious ceremonies, symbolizing devotion and gratitude (marsoner et al, 2018; silva et al, 2019). therefore, the conservation of indigenous breeds helps preserve cultural identity and history, which in turn supports the sustainable use of these breeds. market demand in most developing countries, consumers highly prefer products from indigenous livestock breeds over those from exotic breeds (sharif and farooq, 2004; silva et al, 2019). several traditional beliefs and scientific reasons 90 mustefa genetic resources (2025), 6 (11), 82–98 table 6. ethiopian indigenous livestock breeds under conservation. livestock breeds conservation type references cattle sheko in situ & ex situ in vivo aleme and mengistu (2023) ex situ in vitro assefa et al (2021) fogera in situ & ex situ in vivo tesfa et al (2024) ex situ in vitro assefa et al (2021) borana in situ tessema et al (2022) ex situ in vitro assefa et al (2021) begait ex situ in vivo mekuriaw and kebede (2015) ex situ in vitro assefa et al (2021) begaria in situ aseged et al (2023) raya in situ assefa et al (2021) irob ex situ in vitro assefa et al (2021) sheep washera in situ amane et al (2010) menz in situ & ex situ in vivo gizaw et al (2013) bonga in situ & ex situ in vivo mustefa (2023) wollo in situ assefa et al (2021) horro in situ & ex situ in vivo molla (2020) gedeo in situ assefa et al (2021) goats highland in situ assefa et al (2021) arsi-bale in situ assefa et al (2021) chicken horro in situ taye (2024) metekel in situ assefa et al (2021) jarso in situ assefa et al (2021) kundudo ex situ in vivo sufiyan (2022) can explain this preference. in sri lanka, for example, it is traditionally believed that milk from indigenous cows has medicinal and therapeutic properties due to its low likelihood of causing milk allergies in humans (rajapakshe et al, 2015). according to silva et al (2019), milk from indigenous cows is preferred in the southern province of sri lanka due to its high-fat content, which produces a firm curd structure and good flavour. scientifically, meat from indigenous chickens has been reported to have better physicochemical and sensory parameters than meat from commercial broilers (rajapaksha et al, 2014). senarathne et al (2016) reported high mineral and fat contents in eggs from indigenous chickens. physical and chemical analyses by lordelo et al (2020) indicated higher quality in eggs from indigenous chicken breeds in portugal compared to commercial breeds in many characteristics. therefore, due to consumer preferences, eggs, meat and milk from indigenous breeds have become highly priced products in most developing countries (silva et al, 2019). thus, maintaining indigenous breeds helps ensure the availability of these preferred products in the market which also improves the income of farmers. limitations of conservation maintaining indigenous livestock breeds is significant for securing the sustainable utilization of these genetic resources; however, it comes with its own set of challenges. below some of the limitations are presented. lower productivity compared to modern, high-yielding exotic breeds, indigenous cattle breeds often have lower milk yields, slower growth rates and lower feed conversion efficiency. for example, the average milk yield of ethiopian indigenous cattle breeds (1.32–2.19 litres/cow/day) (ayalew et al, 2018) and the average egg production of most indigenous chicken breeds (45–75 eggs/hen/year (tolasa, 2021) and 30–60 eggs/hen/year (alemneh and getabalew, 2019)) are significantly lower than those of their exotic counterparts. similarly, the slower growth rates of indigenous livestock breeds mean it takes longer for them to reach market weight, leading to increased feeding costs and delayed returns on investment for farmers. moreover, the lower feed conversion efficiency of indigenous breeds means they require more feed to produce the same amount of meat or milk compared to modern breeds under uniform management and controlled envigenetic resources (2025), 6 (11), 82–98 balancing livestock productivity and diversity in ethiopia 91 ronment. this increases production costs and reduces profitability. these factors can result in lower profits for farmers, making indigenous breeds less attractive to those who need to maximize their outputs to remain profitable. thus, solely maintaining indigenous breeds can affect sustainable food security and income generation goals, which can further influence their sustainable use. discussion in ethiopia, livestock genetic improvement programmes have been implemented through within-breed selection, crossbreeding and breed-substitution programmes. the within-breed selection programmes were mainly implemented in small ruminants and chicken through community-based breeding programmes (cbbps) and on-station selection programmes. similarly, several exotic breeds of cattle, sheep, goats and chicken were also imported to conduct crossbreeding and breedsubstitution programmes. accordingly, notable achievements were reported in cattle milk yield, chicken egg production and growth performances of small ruminants. alongside increasing livestock productivity, livestock genetic improvement programmes were also reported to minimize the aggression of indigenous livestock breeds. these programmes were also reported to contribute to climate change mitigation. however, despite these achievements, livestock genetic improvement programmes were reported to have some limitations. these include the facilitation of genetic erosion and inbreeding, maladaptation of the exotic and crossbred to the local production environment, the need for a long implementation time, and high costs for both importing and managing the high-producing exotic breeds. similarly, conservation programmes were also reported to have advantages and limitations regarding sustainable utilization of the livestock production sector. the advantages of conservation programmes include the preservation of genetic diversity of indigenous adaptable breeds, the maintenance of unique traits and cultural heritage, and the availability of products from indigenous breeds that meet market demands. however, maintaining indigenous breeds without genetic improvement is often associated with keeping low-productivity animals, which hinders food security and income generation for farmers. therefore, designing a balanced approach is recommended to achieve optimal productivity while preserving the genetic diversity of indigenous breeds. the way forward although genetic improvement and conservation approaches are inherently opposite and cannot be applied simultaneously to the same livestock population, it is essential to find a win-win solution for the sustainable utilization of indigenous livestock genetic resources to optimize outcomes in the livestock production sector. to achieve this, some recommendations are proposed below. identification of indigenous breeds characterization of indigenous cattle breeds is a foundational step prior to any breeding programme (fao, 2012b). several variables need to be considered at this stage, including the assessment of morphometric and morphological traits, identification of their production environments (origin/breeding tract and distribution areas), identification of unique traits, cultural values, adaptability to harsh environments (e.g. extreme weather conditions and climate change), adaptability to limited resources (e.g. feed, water and veterinary care), and assessment of indigenous knowledge associated with these breeds (fao, 2012b). alongside phenotypic and environmental variables, assessing withinbreed genetic diversity is necessary to identify a breed. similarly, assessing the degree of genetic relationship with other indigenous breeds (population structure) is required to determine the number of breeds in the country. therefore, phenotypic, genomic and historic characterization is recommended. breed-level population size census after identification, conducting a breed-level population size census is essential to assess the endangered status of each breed. therefore, data on the number of animals by breed, sex and age are required to determine whether conservation or genetic improvement programmes should be implemented. conservation programmes can be recommended for breeds with small populations to help maintain their genotype. based on their current population size, appropriate conservation methods can be selected. in situ and ex situ conservation methods can be applied to critically endangered breeds. an indigenous breed with a large population size and a wide distribution area may be considered for a controlled crossbreeding programme to enhance targeted traits. breed evaluation the assessment of on-station and on-farm phenotypic performances – such as growth, production, reproduction and survival traits – is necessary to understand the potential of each breed. a genomic evaluation of a breed for specific traits is also essential to assess its genetic potential. this evaluation is crucial for selecting a breed, trait, method and location for genetic improvement. furthermore, withinbreed selection-based genetic improvement programmes are recommended for populations with high genetic diversity. in contrast, populations with low genetic diversity may require a controlled crossbreeding programme. before implementing any livestock breeding programme, evaluating the complementarity of each parent breed is essential. therefore, breed evaluation is mandatory. 92 mustefa genetic resources (2025), 6 (11), 82–98 breed and area delineation for breeding programmes the results of characterization, breed-level census and breed evaluation activities need to be used to identify suitable breeding programmes for each livestock breed and production environment. accordingly, breeds and areas can be delineated either for conservation or genetic improvement programmes. based on this, a conservation programme can be applied to the economically important and endangered livestock breeds. it is also advisable not to implement crossbreeding and breedsubstitution programmes in the origin and breeding tract of the indigenous livestock breeds. within-breed selection approaches can be considered in these areas to bring the desired genetic improvement. areas out of the indigenous livestock breed origin can be considered for either crossbreeding or breed-substitution programmes based on the complementarity of these livestock breeds with exotic ones. additionally, exotic livestock breeds can be recommended in commercial farms with intensive management systems and controlled environments. in situ and ex situ conservation in situ and ex situ conservation options can be applied simultaneously or separately for economically important endangered indigenous breeds according to the situation (fao, 2012a). in situ conservation, the maintenance of livestock breeds in their natural production environment, can be carried out for livestock breeds with relatively higher population sizes. establishing a community and designing an incentive-based approach can be considered during the in situ conservation programmes. similarly, ex situ conservation can be applied to livestock breeds with alarming population size or as a complementary method to in situ conservation. in vivo and/or in vitro can be considered simultaneously or separately to the livestock breeds with dwindling population size. ex situ in vitro/cryoconservation is an advanced method of preserving genetic material at extremely low temperatures, typically using liquid nitrogen (-196◦c). this technique is widely used in the conservation of livestock breeds, wildlife, plant species and in human medicine (e.g. preserving sperm, eggs and embryos). for indigenous livestock breeds, cryoconservation is a powerful tool to protect genetic diversity and ensure the survival of rare or endangered breeds. the implementation of these methods is expensive and needs a more skilled workforce than the in situ method fao (2012a). husbandry practices alongside genetic improvement, improving husbandry practices enhances livestock productivity and diversity by optimizing animal health, nutrition, breeding selection and environmental management (dristan, 2025). these improvements lead to increased yields of meat, milk and eggs, and the development of better genetic traits, while also promoting a broader range of livestock breeds suited to various ecological conditions and market demands (dristan, 2025). therefore, improving husbandry practices is essential to ensure the sustainable use of indigenous livestock genetic resources. acknowledgements the author is grateful to the authors of the published 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(2016). growth performance of woyto-guji and central highland goat breeds under traditional management system in ethiopia. liv res rural dev 28(1). url: http://www. lrrd.org/lrrd28/1/nets28008.htm. https://doi.org/10.3923/asb.2024.763.770 https://doi.org/10.3923/asb.2024.763.770 https://doi.org/10.1007/s11250-013-0513-1 https://www.researchgate.net/publication/349324158_current_status_of_livestock_crossbreeding_in_ethiopia_implications_for_research_and_extension https://www.researchgate.net/publication/349324158_current_status_of_livestock_crossbreeding_in_ethiopia_implications_for_research_and_extension https://www.researchgate.net/publication/349324158_current_status_of_livestock_crossbreeding_in_ethiopia_implications_for_research_and_extension https://www.researchgate.net/publication/349324158_current_status_of_livestock_crossbreeding_in_ethiopia_implications_for_research_and_extension https://doi.org/10.46265/genresj.lyoq7265 https://doi.org/10.46265/genresj.lyoq7265 https://doi.org/10.5897/ijlp2021.0787 https://doi.org/10.5897/ijlp2021.0787 https://doi.org/10.1017/s1014233900001814 https://doi.org/10.1155/2021/8848388 https://doi.org/10.1155/2021/8848388 http://dx.doi.org/10.5713/ajas.18.0690 http://dx.doi.org/10.5713/ajas.18.0690 https://doi.org/10.17352/2455-815x.000100 https://doi.org/10.1016/b978-012374117-2.50005-4 https://doi.org/10.1016/b978-012374117-2.50005-4 https://doi.org/10.36648/2577-0594.7.2.38 https://doi.org/10.5897/ajar2015.10493 https://doi.org/10.1007/s11250-007-9104-3 https://doi.org/10.1007/s11250-007-9104-3 https://doi.org/10.35248/0970-1907.24.40 http://www.lrrd.org/lrrd28/1/nets28008.htm http://www.lrrd.org/lrrd28/1/nets28008.htm introduction genetic improvement advantages of livestock genetic improvement programmes increased productivity cattle small ruminants chicken climate change mitigation reducing aggressiveness limitations of livestock genetic improvement programmes genetic erosion maladaptation inbreeding cost and time conservation advantages of conservation genetic diversity adaptability unique traits cultural heritage market demand limitations of conservation lower productivity discussion the way forward identification of indigenous breeds breed-level population size census breed evaluation breed and area delineation for breeding programmes in situ and ex situ conservation husbandry practices acknowledgements conflicts of interest review genetic resources (2021), 2 (4), 21–43 doi: 10.46265/genresj.wjeu8358 https://www.genresj.org issn: 2708-3764 history and impact of a bean (phaseolus spp., leguminosae, phaseoleae) collection daniel g debouck *, marcela santaella and lúıs guillermo santos genetic resources program, alliance of bioversity international and international center for tropical agriculture (ciat), km 17 recta cali-palmira, cali, aa 6713, colombia abstract: this work explains the reasons why a bean collection was established in 1973 at the international center of tropical agriculture (ciat) near palmira in colombia. it shows the impact of the collection on plant breeding and in agricultural development through the distribution of germplasm to the center’s bean breeding program, to successively find resistances to pests and diseases, adaptation to low phosphorus and drought, and more recently higher content of iron and zinc in seeds. the collection was also used to progress knowledge in biological sciences, as shown by a dozen of examples. a reason behind these successes was foresight and focus on diversity per se in the collection. the paper ends with a number of suggestions for the way ahead for the genetic resources conservation and management of these bean crops, and possible take-home lessons for curators in charge of other similar collections. keywords: distribution, yield, biotic stress, abiotic stress, breeding, phaseolus citation: debouck, d. g., santaella, m., santos, l. g. (2021). history and impact of a bean (phaseolus spp., leguminosae, phaseoleae) collection. genetic resources 2 (4), 21–42. doi: 10.46265/genresj.wjeu8358. © copyright 2021 the authors. this is an open access article distributed under the terms of the creative commons attribution license (cc by 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. the purpose for establishing a collection of genetic resources at ciat created in 1967, the international center of tropical agriculture (ciat for its spanish acronym) was the third international center of agricultural research whose mandate was to increase the agricultural productivity in the tropics. because of population growth a sure food availability crisis was anticipated and yields of key staple crops had to increase (kastner et al, 2012) and, fortunately, this actually happened, namely in asia (evenson and gollin, 2003). although ciat was originally designed to improve agricultural systems in the lowland tropics (the center had a couple of animal production programs), it became clear that the level of human talents, physical and financial resources required for such a task was beyond the donors’ capacity and time frame, and these limits indicated to re-focus instead (lynam and byerlee, 2017). after the successful experience of the international rice research institute ∗corresponding author: daniel g debouck (d.debouck@cgiar.org) (irri) in los baños, philippines, on rice and that of the centro internacional para el mejoramiento del máız y trigo (cimmyt) in el batán, mexico, on wheat, and as an outcome of international conferences (hernándezbravo, 1973; rachie, 1973; voysest-voysest, 1983), ciat moved from a food legumes production systems program into a program focused on common bean, phaseolus vulgaris l. (hidalgo, 1991); the bean program initiated in january 1974 (voysest-voysest, 2000). given the production problems faced for this crop (hernández-bravo, 1973; singh, 1992), very often managed by small-holder farmers with limited access to inputs (broughton et al, 2003), the next strategic decision was to increase productivity by transferring resistance to diseases and pests into target varieties. by then, the most severe diseases, out of more than one hundred affecting the crop (zaumeyer and thomas, 1957; singh, 1999), often caused a 70-100% loss in yield (sanders and schwartz, 1980; singh, 1999). the first cycle of breeding (which took about 8-10 years in beans in the 1960s) aimed at securing the potential yield of the landraces (rachie, 1973). thus, farmers would have a secure food stock at home and a surplus for received: 25.06.2021 accepted: 06.10.2021 published online: 08.11.2021 https://www.genresj.org https://www.dx.doi.org/10.46265/genresj.wjeu8358 https://www.genresj.org https://www.dx.doi.org/10.46265/genresj.wjeu8358 mailto:d.debouck@cgiar.org 22 debouck et al genetic resources (2021), 2 (4), 21–43 figure 1. evolution of the breeding strategy in bush common bean in the tropics over the last decades. sale in local markets. this strategy was likely to work because in subsistence agriculture beans were planted in mixtures to where resistant genotypes would lower the disease pressure for the total crop (clawson, 1985), and in market-oriented agriculture with fewer genotypes under cultivation a higher part of the production would be saved for sale. it was envisioned to tackle the next most limiting factor to productivity in a cumulative way, through the production and distribution of elite varieties, with the active participation of the national partners, who then would work with extension services for the diffusion of such a technological package. that participation was critical for impact, given the high number and regional variation of commercial seed types (voysest-voysest, 1983; voysest and dessert, 1991). figure 1 shows how this strategic approach has worked for common bean over five decades. with each breeding cycle tackling a new challenge, the strategy had to be cumulative, because the entire production chain and the final users would hardly accept to go backwards. in this regard, it is worth noting that after fifty years, diseases and pests continue to be among the highest priorities of bean breeding, especially in africa (assefa et al, 2019). while the first breeding cycle was under way, improvements were introduced to agronomical practices, taking into account planting density, planting date versus water availability versus solar radiation/photoperiod, minimum of nitrogen-phosphoruspotassium fertilization, or ph correction by lime application (thung, 1991). once these improved agronomical practices were implemented, it was clear that progress in yield had to come from plant breeding (borlaug, 1983), but there was a critically important assumption for the whole strategy to work: the immediate availability of well characterized and evaluated genetic resources, which would be the ultimate source of all desirable genes. practically, because there were none at the center, this meant assembling large collections of genetic resources of beans and evaluating such collections by multidisciplinary teams. in the early years of ciat, that assumption had to quickly become reality for the efficiency and impact of the breeding efforts. the need for multiple sources for disease and pest resistance and abiotic stress tolerance was also related to the wide diversity of conditions of deployment in the many countries benefiting from that technology. note that apart from assembling collections, it was also the time of setting the founding principles of genebank management (allard, 1970; frankel and hawkes, 1975). how the phaseolus collection was assembled with the establishment of the ciat bean program, the first introductions of bean collections from other institutes (e.g. united states department of agriculture [usda], pullman, usa; instituto nacional de investigación agŕıcola, chapingo, mexico; instituto de ciencias y tecnoloǵıa agŕıcola, chimaltenango, guatemala; centro agronómico tropical de investigación y enseñanza [catie], turrialba, costa rica) (hernández-xolocotzi, 1973; vieira, 1973) were made thanks to the constant cooperation of the instituto colombiano agropecuario, palmira, colombia on plant quarantine matters (figure 2). accessions were registered as germplasm numbers (e.g. g4017 for ‘carioca’, perhaps the most planted bean variety in the world; voysest-voysest (2000). thanks to the support of the international board for plant genetic resources (ibpgr), rome, italy (established in 1974), several collecting missions were organized for landraces and wild species. before the entry into force of the convention on biological diversity (cbd), these crop genetic resources were considered as common heritage of humankind, and there was free exchange of genetic resources for breeding and agricultural research purposes. after december 1993, acquisition by introduction of copies of germplasm collections and explorations came to a halt (as experienced in other genebanks of the consultative group on international agricultural research (cgiar); halewood et al (2020). but in recent years, target explorations were carried out under the legal framework set by the international treaty on plant genetic resources for food and agriculture (fao, 2002), for example in costa rica. because beans as a staple are often associated with maize, collections of bean germplasm have been established across the tropics and subtropics, particularly in latin america (e.g. in chapingo in mexico, medelĺın in figure 2. increase in size of the bean collection introduced into the ciat genebank. genetic resources (2021), 2 (4), 21–43 impact of a bean collection 23 colombia or campinas in brazil) (vieira, 1973), which could be introduced into ciat. it was not rare for a maize breeder to pick up seeds of local bean landraces when visiting a farmer or a local market and give them to colleagues in charge of bean breeding. as a general practice in latin america the maintenance of bean germplasm collections was a side activity of the bean breeders. given this kind of attention, it is no surprise that the landraces of the market classes worked on by the breeders were well represented in the respective collections. retrospectively, this is positive because some of these collections were made in the 1940-1960s, just before massive rural transportation blurred the origins of many local landraces. by that time, the varieties selected or bred over the last decade were starting to replace many landraces. this replacement was seen in small black and red-seeded bean varieties of central america. consequently, the most original genetic variation in landraces that existed in the 1940s is by now either in genebanks or lost. collecting today will only result in duplicates or in samples of bred materials. the focus on common bean and the need to find resistances for several market classes of beans as final targets influenced the makeup of the collection (table 1). as the bean program was working with central american countries and brazil (voysest-voysest, 1983), many small-seeded collections were introduced, but because ciat also worked together with andean countries, large-seeded collections were included as well (both collections but particularly the last ones were important for africa: martin and adams (1987). cultivated p. vulgaris makes up the biggest part of the collection, the other cultivated species follow, with a total of 32,183 landraces and 2,797 improved varieties. the wild forms of the cultivated species and the wild species are represented by over 2,000 accessions (table 1). the number of country depositors is 110. the top five countries that have contributed most are: mexico (6,237 accessions), colombia (3,927 accessions), peru (3,798 accessions), guatemala (2,853 accessions) and the united states (1,863 accessions), followed by brazil, ecuador, turkey, malawi and costa rica (with around 1,000 accessions each). restoration of national bean diversity has been done for bhutan, chile, costa rica, india, iran, and mexico, based on institutional agreements. after partial safety duplications at catie and centro nacional de pesquisa de recursos genéticos e biotecnologia, brasilia, it was decided in 1996 to make a complete backup at cimmyt because extra space was kindly offered by bent skovmand then in charge of the wheat collection. this included a check for viability and absence of diseases of quarantine importance. by 2019, 92% of the bean collection had been safely duplicated at cimmyt. when the global svalbard seed vault (gssv, longyearbyen, norway) was opened in february 2008 (fowler, 2016), a second safety backup was implemented there. by 2019, 94% of the bean table 1. accessions of the in-trust phaseolus collection kept at ciat palmira (information also available in genesys: https:// www.genesys-pgr.org/a/overview/v2zw8lqwlep) species biological status no. accessions p. vulgaris, common bean cultivated (landraces, commercial varieties) 30,571 wild and weedy forms 1,804 p. lunatus, lima bean cultivated (landraces, commercial varieties) 3,031 wild and weedy forms 274 p. coccineus, scarlet runner cultivated (landraces, commercial varieties) 760 wild and weedy forms 198 p. dumosus, year-bean cultivated (landraces) 475 wild and weedy forms 15 p. acutifolius, tepary cultivated (landraces, commercial varieties) 161 wild and weedy forms 165 other species (411) wild forms only 484 1 this figure represents half of the number of species of the genus (debouck, 2021). collection was safely duplicated at gssv. in addition, ciat safeguards in its vault the seed backups of the food legume collection of the international institute of tropical agriculture (iita), ibadan, nigeria, and the tropical forage legumes of the international livestock research institute (ilri), addis ababa, ethiopia. the diversity in the bean collection and its use given the breeding priorities (figure 1), the bean collections were evaluated by multidisciplinary teams in multiple locations in colombia, first in palmira, in quilichao since 1977 and in popayán since 1978 (cuellar, 2003). resistances to several highly damaging diseases (e.g. anthracnose, angular leaf spot (als), bean common mosaic virus (bcmv), rust) were found (table 2) and transferred as their mode of inheritance was progressively defined (singh, 1992; beebe et al, 1997; beebe, 2012). however, bean breeders soon realized that further genetic progress would be obtained only by screening the diversity outside the respective market classes, and that for many traits (e.g. ascochyta blight, bean golden mosaic virus (bgmv), bean golden yellow mosaic virus (bgymv), common bacterial blight, halo blight, web blight, white mold, bruchids and leafhoppers) there were very few or no sources of workable resistance (miklas et al, 2006; singh and schwartz, 2010; beebe, 2012). in a copy of the usda collection there were a few accessions of wild common bean from mexico collected in the 1960s by howard scott gentry in which, later on, césar cardona and his team found bruchid resistance associated with a particular seed protein (osborn et al, 1988). the screening for the right variants of that protein by electrophoresis https://www.genesys-pgr.org/a/overview/v2zw8lqwlep https://www.genesys-pgr.org/a/overview/v2zw8lqwlep 24 debouck et al genetic resources (2021), 2 (4), 21–43 opened the way for marker assisted selection (kelly and miklas, 1999), widely applied in breeding for bgymv resistance (broughton et al, 2003), anthracnose resistance (kelly, 2004) and other traits. the wide secondary gene pool (e.g. p. coccineus, p. costaricensis, p. dumosus) has just started to be evaluated, and has already shown promise against ascochyta blight (schmit and baudoin, 1992), angular leaf spot (mahuku et al, 2003) and white mold (singh et al, 2013). such resistances were expected because the species of the section phaseoli thrive in montane humid forests (debouck, 2000) where these fungi diseases are frequent selection pressures (cattan-toupance et al, 1998) and have likely been present over hundreds of thousands of years, given the age of these species (approximately 1 million years) (delgado-salinas et al, 2006; rendón-anaya et al, 2017). once disease resistances were transferred to the preferred varieties in the different market classes, yield came as the next challenge, in order to keep bean as a competitive (mono-)crop as compared to soybean, cowpea or sorghum. an early approach, in line with the spirit of the green revolution in wheat (donald, 1968), was to optimize the ideotype under favorable environments (adams, 1973). that breeding effort on plant architecture continued (kelly, 2001), although with limited success in the tropics (beebe, 2012). an important outcome, however, has been the rise of growth habit 2 or bush erect indeterminate (race 3 of evans (1973) for mechanical harvesting in bean improved germplasm, little present in traditional landraces of latin america. this also offered opportunities to enrich the collection (hidalgo et al, 1992). another breeding goal was to combine the productivity of the smallseeded varieties with the grain size of the large-seeded ones, many of them demonstrated to be of mesoamerican and andean origin, respectively (evans, 1976). thus came the works evidencing the two major gene pools (gepts et al, 1986; singh et al, 1991b; kwak and gepts, 2009; bitocchi et al, 2013), and the races within them (singh et al, 1991a; beebe et al, 2000b; blair et al, 2007, 2012). the presence of races was a bit unexpected in an autogamous crop but could be explained by the role of outcrossing during early domestication (chacón-sánchez et al, 2021). some genetic isolation and poor recombination have been shown to exist between the two major genepools (singh and gutiérrez, 1984) and since the wild state (koinange and gepts, 1992). but significant heterosis was demonstrated to exist (nienhuis and singh, 1986; bannerot, 1989), especially between races (singh et al, 1993; singh and urrea, 1995), while genetic disorders between races were sometimes observed (singh and molina, 1996). the significant interactions with the environment, however, have resulted in a narrow commercial applicability of this approach (gutiérrez and singh, 1985; nienhuis and singh, 1986). another strategy inspired from the quantitative developments in tomato breeding (tanksley et al, 1996) was the advanced backcross qtl analysis using a wild form. the accessions of wild p. vulgaris g12947 (acosta-gallegos et al, 2007), g19892 (buend́ıa et al, 2003), g24404 (blair et al, 2006) and g24423 (kelly, 2004) were found to contribute a significant qtl for yield (a 27% increase as compared to the recurrent parent in the last example). in some cases, the use of weedy types would help reduce the number of backcrosses needed to recover the appropriate seed size (acosta-gallegos et al, 2007). another innovative approach has been the use of lines coming from crosses with the year-bean (for transfer of high iron in the grain) or with tepary (for transfer of bacterial blight resistance) in order to bring more monocarpism into common bean (klaedtke et al, 2012; mej́ıa-jiménez et al, 1994). the bean crop with exceptions in growth habits 1 and 2 still has the ancestral trait of continuing shoot production and lateral flowering, while the first pods already enter into maturity. in the wild it makes all sense, but not in a crop aimed at mechanical harvesting. the desert ephemerals of the genus such as p. acutifolius a. gray, p. filiformis benth, or p. microcarpus mart. (freytag and debouck, 2002) invest much less in profuse branching but soon move all photosynthesis products into their seeds. thus, under heat or drought stress, it makes sense to quickly redirect such products to the only part that will be harvested (rao et al, 2013; suárez et al, 2020). one outcome of the increase in size of the collection and the first phase of germplasm evaluations at ciat was the establishment of core collections (along the concept introduced by frankel and brown (1984). the ciat common bean core collection was established by use of geographic information systems maximizing the environmental diversity of landraces, and a few morpho-agronomic descriptors (tohme et al, 1995a). the core collection, consisting of 1,556 accessions, has been used for the identification of germplasm tolerant to low phosphorus (beebe, 1997) or containing high levels of micronutrients (islam et al, 2002). for both traits less than 10% of the total collection had been evaluated at that time (beebe et al, 2000b,a), explaining the recourse to the core collection. using core collections was, in part, the consequence of internal duplication or redundancy in general collections, which for cultivated common bean has been estimated at 50% across the major genebanks (lyman, 1984). this figure is perhaps on the high side but reflects the amount of commercial and breeding materials kept in genebanks as compared to primary sources of variation (landraces, wild species). as mentioned, the management of germplasm collections was often a side activity of bean breeders who would hesitate to eliminate all variants close to the target market class. given the cost of keeping accessions versus the cost of tracking down internal copies (this was then achieved by multi-site characterization, in addition to passport data) (koo et al, 2004), the problem was not given high priority in the past. with the development of snp genotyping technology, this issue should be revisited, genetic resources (2021), 2 (4), 21–43 impact of a bean collection 25 table 2. bean accessions from the ciat collection used as sources of resistance to diseases and pests. trait material used references diseases angular leaf spot g10613 from guatemala pastor-corrales et al (1998) interspecific hybrids with p. coccineus; g4691 pastor-corrales et al (1998); islam et al (2002); mahuku et al (2003) angular leaf spot and anthracnose g3991 from costa rica schwartz et al (1982) anthracnose aliya g02333 young and kelly (1996) kaboon g1588; cornell 49-242 g5694 melotto and kelly (2000) interspecific hybrids with p. coccineus g35252 mahuku et al (2002) ascochyta blight p. dumosus g35369 from costa rica schmit and baudoin (1992) p. dumosus g35182 from guatemala garzón et al (2011) bacterial wilt wild p. vulgaris g12883 from mexico urrea and harveson (2014) bean golden yellow mosaic virus (bgymv) p. coccineus g35172 from rwanda beaver et al (2005) bean common mosaic virus (bcmv) porillo sintético g04495, royal red g04450 singh et al (2000) beet curly top virus california pink g06222, red mexican g05507 larsen and miklas (2004) porillo sintético g04495, burtner, tio canela 75 singh and schwartz (2010) common bacterial blight interspecific hybrids with acutifolius vax4, mbe7 zapata et al (1985); singh and muñoz (1999); michaels et al (2006); navabi et al (2012) montana no. 5; pi 207262 miklas et al (2003, 2006) halo blight montcalm g06416, ica tundama g14016 beaver (1999) palomo g12669 schwartz (1989) pinto us 14 g18105 singh and schwartz (2010) wis hbr 72 g03954 taylor et al (1996) fusarium root rot porillo sintético g04495; wild p. vulgaris g12947 beebe et al (1981); acosta-gallegos et al (2007) pythium root rot pi 311987 g02323 beebe et al (1981) rhizoctonia solani rot n203 g00881 beebe et al (1981) rust compuesto negro chimaltenango g05711 stavely (1984) ecuador 299 g05653 stavely and pastor-corrales (1989) redlands pioneer g05747 liebenberg et al (2006) pi 260418 singh and schwartz (2010) web blight bat 93; flor de mayo g14241 beaver et al (2002) white mold p. coccineus pi 175829 from turkey abawi et al (1978) p. dumosus pi 417603 from mexico hunter et al (1982) interspecific hybrids with p. coccineus g35172 singh et al (2009) interspecific hybrids with p. costaricensis g40604 singh et al (2013) pests acanthoscelides weevil wild p. vulgaris from western mexico g12952; ques van schoonhoven et al (1983); zaugg et al (2013) apion godmani pod weevil amarillo 154 g03982; g03578 beebe et al (1993); garza et al (2001) empoasca leafhoppers turrialba 1 g03712 galwey (1983) california dark red kidney, from usa g17638 schaafsma et al (1998) ophiomyia bean fly p. coccineus g35023 and g35075, and interspecific hybrids kornegay and cardona (1991) whiteflies aleyrodidae dor 303 blair and beaver (1992) zabrotes weevil wild p. vulgaris from chiapas, mexico g24582 acosta-gallegos et al (1998) 26 debouck et al genetic resources (2021), 2 (4), 21–43 with the merging of near identical accessions and the review of the core collection (as already suggested for the usda core collection by kuzay et al (2020). the most significant costs ahead are likely to be about regeneration and evaluation, not the chasing of internal copies helped by robotics! distribution of the bean collection since being established in august 1977 as an internal service unit (hidalgo, 1991), the genebank has distributed to the bean program and the biotechnology research unit of ciat a total of 318,148 samples (or 69.4 % of the total distributed) (figure 3). externally, the genebank has distributed 140,109 samples (or 30.6% of the total) to users in 105 countries. the total distributed was 458,257 samples of 37,657 accessions, or 99% of the bean collection. these figures indicate that: i) the collection has been studied and used initially by the scientists of ciat, ii) the number of countries benefiting from the conservation work through distribution almost matches with the number of country depositors, and iii) the collection has been distributed almost entirely (this percentage could be even higher since some accessions have not been distributed due to lack of seeds). one should note that apart from ciat programs, the most important users were national agricultural research services, universities and research institutes. the shares of farmers, commercial companies and non-governmental organizations in the distribution were low in comparison. assuming farmers are aware of the existence of germplasm collections, reasons for the low number of requests might be related to the farmers’ access to on-line request processes (mail requests were honored), as well as the capacity to deal with phytosanitary regulations in the respective countries. as dry bean breeding has been mostly carried out by public institutions, requests of genetic diversity by the private sector were few, often related to specific sources of variation for snap bean breeding (e.g. sources of resistance to anthracnose). the purposes of distribution have generally followed the breeding priorities shown in figure 1: interest in resistances to diseases and pests, nutritional quality and more recently tolerance to abiotic stresses such as drought and high temperature. as discussed below and shown in figure 4, a significant part of the distribution has been for the purpose of advancing knowledge. in figure 4, breeding activities (38.9%) and applied research (e.g. in pathology or entomology: 37.1%) were the top purposes for seed requests, followed by agronomy (11.7%) and basic research (e.g. in genetics or evolutionary studies: 9.6%). the variation in number of distributed samples from one year to another can be significant, namely if the core collection with over 1,500 accessions was requested and sent. the peak in distribution in the period 19781996 practically matches with that of the activities of ciat bean program (voysest-voysest, 2000). for the period 1973-2019, the top five recipient countries were: usa (26,093 samples), colombia (18,444 samples), brazil (9,198 samples), guatemala (7,430 samples) and mexico (6,787 samples). the term ‘samples’ is preferred over ‘accessions’ as a country recipient could ask for a specific accession more than once. apart from germplasm, the genebank also distributed information related to the in-trust collections. an indicator of this service is given by the number of consultations of the genebank website (https://ciat.cgia r.org/what-we-do/crop-conservation-and-use/) to have access to data (figure 5). statistics about access to genebank information and services in recent years show that most of the visitors (81%) reach the genebank website directly, indicating a user knowledge and confidence that relevant information can be found there, while 19% of visitors find the website through a browser search or are referred to it through another link. users also benefit from specialized technical information currently consisting of 658 documents (including articles, book chapters, conference proceedings, germplasm exploration reports, posters and presentations). these documents can be accessed through the genebank website or the institutional document repository cgspace (https://cgspace.c giar.org/handle/10568/35697), that registered 27,278 downloads in 2017-2019. impact of the bean genetic resources collection the 225 varieties released in 17 countries of latin america and the 88 varieties released in 14 countries of africa in 1974-1999 by the bean program (voysestvoysest, 2000), the check of bgymv in central america (beebe, 2012), as well as the yield gain from 688 kg/ha to 782 kg/ha in eastern africa (lynam and byerlee, 2017) eventually have their origin in the ciat genebank. once the interesting traits were identified (table 2); (hidalgo and beebe, 1997), through different breeding schemes, elite varieties were produced, tested and released via international nurseries such as the international bean yield and adaptation nursery (voysest-voysest, 1983; beebe, 2012), generating significant economic and social benefits (johnson et al, 2003). in this last work, over the period of analysis, and because of the varieties involved, some countries of latin america and the caribbean were net beneficiaries (argentina, brazil), while others were net contributors (mexico, el salvador). overall, and over the duration, however, it seems that all countries benefit from conservation and unrestricted international exchange of germplasm (johnson et al, 2003). as an example of changing context over time, the gene gy. originating from peru and conferring an intense and sustained yellow color (bassett et al, 2002) was used since 1978 in northwestern mexico (lépiz-ildefonso and navarro-sandoval, 1983), because it gave a premium price as compared to the fading color in yellowseeded traditional landraces. it ended up in the variety ‘azufrado peruano 87’ (voysest-voysest, 2000) and also https://ciat.cgiar.org/what-we-do/crop-conservation-and-use/ https://ciat.cgiar.org/what-we-do/crop-conservation-and-use/ https://cgspace.cgiar.org/handle/10568/35697 https://cgspace.cgiar.org/handle/10568/35697 genetic resources (2021), 2 (4), 21–43 impact of a bean collection 27 figure 3. number of samples distributed in the period 1973-2019. important recipients were the bean and biotechnology programs of ciat, the national agricultural research services (nars), universities and research institutes. an undue patent granting (genetically dissected in great detail by pallottini et al (2004). it is because the in-trust collection was rich in yellow-seeded accessions from mexico and peru, and because the ciat genebank kept past records such as old catalogs of varieties (hedrick, 1931), it was possible to demonstrate ample prior art, and the patent was turned down in 2008. legumes have been called the ’meat of the poor’ (heiser, 1990) and in many parts of latin america (e.g. brazil, mexico, cuba) people with low income eat beans daily. similarly, the highest consumption rates per capita are currently registered in eastern africa (oecd, 2015). given this, it was becoming evident that improved varieties should also fight the ‘hidden hunger’ or the deficiency in minor minerals such as iron and zinc where the diet is not sufficiently diverse. using the core collection, the evaluation to find accessions with high iron and zinc was expanded (islam et al, 2002), and good sources were identified (g21242, g23818, g23834) (blair et al, 2011), primarily from the andean region. nothing on the seed aspect indicates high values in these micronutrients. likewise, g14519, an old landrace named ‘hickman pole bean’ from the united states and belonging to the mesoamerican genepool, also has shown potential (blair et al, 2010). from the start, the genebank had interest in assembling variation for any future need, and this is precisely the wide scope that made biofortification possible thirty years later and with a lasting impact where it is today most needed, e.g. in east africa (sellitti et al, 2020). with changing context of bean production over the last forty years, for example, common bean being pushed towards the west in the plains of the usa or in the canadian prairie, the northwest in mexico and the northeast in brazil (singh, 2001), new challenges like drought, cold, heat or low phosphorus are set for breeding. some drought tolerance can be found in the ‘durango’ race (singh, 2007; beebe et al, 2013), in other landraces such as g21212 (beebe et al, 2008) and in wild forms (cortés and blair, 2018). root architectural and physiological traits identified in an andean landrace, g19833, may contribute to phosphorus acquisition (beebe et al, 2006). 28 debouck et al genetic resources (2021), 2 (4), 21–43 figure 4. number of samples distributed annually to users in the period 1973-2019, according to the purposes of requests. figure 5. number of consultations of the ciat genebank website (https://ciat.cgiar.org/what-we-do/crop-conservation -and-use/) in the period 2009-2019. the impact of the bean germplasm collection has also been through the direct adoption of genebank accessions by farmers after the screening of international nurseries. no less than thirty-four accessions have been registered in national catalogs of varieties in thirty-eight countries in 1974-1999 (voysest-voysest, 2000), or 13% of the total of improved genetic materials distributed by ciat. this figure may not look impressive, but bean varieties produced by breeding have been released in the same geographic areas since the 1940s. that said, it is anticipated that apart from the use of landraces for specific niche markets (see popping beans below), for specialized studies for example in pathology (e.g. disease differential sets) or for servicing gardeners, a significant coming impact of genetic resources will materialize through libraries of genetic stocks (van https://ciat.cgiar.org/what-we-do/crop-conservation-and-use/ https://ciat.cgiar.org/what-we-do/crop-conservation-and-use/ genetic resources (2021), 2 (4), 21–43 impact of a bean collection 29 treuren and van hintum, 2014), targeted diversity panels (cichy et al, 2015; moghaddam et al, 2016), and sequence tagged traits (lobaton et al, 2018). but in order to allow the ‘molecular’ breeder to do advanced searches throughout the collection substantial changes must be brought to databases (mccouch et al, 2012; byrne et al, 2018). learning from experience, the design should be for use by non-database experts, modular and scalable, moving from passport data into accession traits and ending into annotated genes. somehow, this focus re-emphasizes the role of genebanks as keepers of the primary genetic variation, and perhaps less of all allelic combinations of that variation (i.e. the sister lines of simple crosses), since tools now exist to recombine that variation to better meet human needs or agricultural contexts (towards precision agriculture for instance). appraising that variation by curators might be difficult (what should be kept in the genebank remains a cornerstone and recurrent question), although they will be helped by sequence information. from a pragmatic perspective genebanks may keep interest in old landraces since these have been tested over long durations in farmers’ fields. for similar efficiencies in breeding, genebanks might be interested in keeping recombinants between gene pools, for example from southern europe (gioia et al, 2013), part of the guarani area in brazil (burle et al, 2010) or the northern andes (chacón-sánchez et al, 2021), especially if they represent novelties in agronomic or nutritional attributes. the other impact: the contribution to knowledge the in-trust collections, because of the open access set forth by fao in the early years and then the facilitated access approved by the parties to the international treaty (fao, 2002), have helped increase knowledge in many fields of biological sciences (dudnik et al, 2001). conversely, the increased knowledge contributed tremendously to the efficiency of the breeding and varietal deployment efforts. this was particularly applicable to the mandate crops of ciat, since with the exception of rice, not much basic biology and genetics was known in the late 1960s when crop improvement efforts were launched. for instance, the ancestry of common bean became firmly established at a time not far away from the founding of ciat (burkart and brücher, 1953; gentry, 1969). the double domestication of common bean became obvious only in 1986 (gepts et al, 1986), and that of lima bean in 1989 (debouck et al, 1989), and the existence of a fifth case of domestication in the genus was clarified as late as 1991 (schmit and debouck, 1991)! such increased knowledge also helped to better define what should be conserved in genebanks; for example, studies on the founder effect due to bean domestications have stressed the importance of wild forms for accessing the total genetic diversity of three bean crops (sonnante et al, 1994; mart́ınez-castillo et al, 2015; mina-vargas et al, 2016). as expected, that founder effect was less marked in the scarlet runner (guerra-garćıa et al, 2017). that knowledge also helped in the development of disease indexing methods for the safe movement of germplasm (kumar et al, 2021). some examples are provided in table 3 (by tracking accessions distributed in 1973-2019). the breadth of disciplines, evidenced by the diversity of peer-reviewed journal titles and linked to genebank accessions, is striking but just reflecting the diversity of the collection. no less important are the opportunities of scientific collaborations around ‘problems’ set up by the bean crops and materials provided by the genebank, as reflected by the institutions and countries of authorships. knowledge often overlooked, although associated with crop germplasm for millennia because germplasm collections were assembled at ciat primarily in relation to breeding, traditional knowledge associated with specific accessions was rarely documented. as the first phase of breeding was focused on resistance to diseases and pests, with systematic inoculation of known strains, there were often no incentives for a time-consuming effort to document vernacular names, culinary and other folk practices. one such example is that of popping beans consumed toasted (national research council, 1989). that group of landraces still exists in the andes, from cajamarca in peru down to chuquisaca in bolivia (tohme et al, 1995b). elder farmers in the countryside will tell which variety can pop, while migrants to urban areas one generation after will simply process them all in water cooking, even with a slight increase in digestibility (van beem et al, 1992). documenting this property by the genebank is doubly important. first, consumer preferences change over time (voysest-voysest, 2000) and, in contrast to the 1960s, there is a renewed interest nowadays in local gastronomy that can provide a better income to mountain farmers (zimmerer, 1992). second, water and fossil energy might become expensive inputs to food processing or transportation, as it is still the case in many parts of rural eastern africa. in pre-ceramic times in the andes, these two inputs (excepting fire) were either difficult to carry or to access. producing a hot surface with the help of solar energy might not be an excessively difficult or expensive technology to implement in the andes or in eastern africa. in altitude, this kind of germplasm and the unique way to make it ready for human consumption may also contribute to reduce deforestation for fuel wood, while montane forests usually occupy a small acreage (national research council, 1989). finally, it is worth noting that this group has a high number of phaseolin types (tohme et al, 1995b), indicating a high diversity in contrast to other andean landraces (beebe et al, 2001). evaluation of popping beans is continuing in peru (cruz-balarezo et al, 2009) and colombia (otálora et al, 2006), while 30 debouck et al genetic resources (2021), 2 (4), 21–43 table 3. examples of impact of ciat bean in-trust collection for the advance of knowledge. field output, problem solved references botany new species described salcedo-castaño et al (2011) plant taxonomy taxonomic status of bean species re-assessed schmit et al (1996) review of the genus and species freytag and debouck (2002) agricultural botany founder effect of bean domestication schinkel and gepts (1988) definition of a 3rd gene pool in lima bean motta-aldana et al (2010) fifth case of domestication in the genus schmit and debouck (1991) crop evolution recombination between gene pools gioia et al (2013) phylogeography past trans-isthmic migrations of wild bean chacón-sánchez et al (2007) plant breeding gene pools/ races of common bean defined singh et al (1991a) plant pathology inheritance of ant/als resistance genes gonçalves-vidigal et al (2011) coevolution of als in bean gene pools guzmán et al (1995) plant virology resistance to clover yellow vein virus hart and griffiths (2014) entomology resistance to bean bruchids cardona et al (1990) resistance to bean weevil kamfwa et al (2018) plant genetics common bean genomic map schmutz et al (2014) common bean genome history & evolution rendón-anaya et al (2017) inheritance of pod dehiscence parker et al (2020) inheritance of leaf mutation garrido et al (1991) plant biotechnology genetic transformation in tepary bean dillen et al (1997) plant physiology flowering response to daylength white and laing (1989) identification of phosphorus-efficient genotypes beebe et al (1997) low phosphorus tolerance in bean rao (2001) variation in photosynthetic activity lynch et al (1992) plant root physiology tolerance to nacl salinity in early growth bayuelo-jiménez et al (2002) plant microbiology coevolution of rhizobium etli aguilar et al (2004) human nutrition content in micronutrients such as iron beebe et al (2000a) phaseolin type and digestibility montoya et al (2008) archaeology crop domestication and ancient diet piperno and dillehay (2008) intellectual property protection rebuttal of an undue crop utility patent pallottini et al (2004) the inheritance of the trait is being investigated (campa et al, 2011; yuste-lisbona et al, 2012). serving the breeders community and beyond as shown in figure 3 and table 3, distribution has been significant to a high diversity of users, going beyond ciat breeding activities in colombia and in eastern africa. the trend that in some countries dry bean consumption is declining (khoury et al, 2014) (contradictory to health and global environment benefits: foyer et al (2016) may mean fewer requests for that kind of germplasm but an increased interest into snap bean, often of andean origin (myers and baggett, 1999). with the development of urban gardening, snap bean might be on the rise, either through the planting of old heirloom varieties (kaplan and kaplan, 1992; zeven, 1997) or new ones. in ciat, the priority was on dry bean, little on snap bean for the tropics, but with possibilities of using a wide range of resistance sources developed for the former commodity (silbernagel et al, 1991). the changing fate of the popping beans, even in countries of origin over the last forty years, shows the ever-changing nature of markets. thus, examples of unpredictability abound, indicating for the genebank to focus on diversity per se, independently from immediate and local interests. this example of success brings a strong message to focus scarce resources, at a time when there is risk of repeating previous work because many disciplinary continuums have been broken. thus, it seems of paramount importance to document at accession level what is already known: phaseolin type, alleles of allozyme, rapd markers, scars, ssrs, also evaluation data (trait, location, strain as applicable). individual accessions should be linked with references and supporting documents. keeping in mind that one third of the collection has not been evaluated, there is still a lot of work for pathologists, entomologists and virologists. evaluations were done on up to 23,000 accessions only for anthracnose, angular leaf spot and common bacterial blight; for the other limiting pests the figures are much lower (hidalgo and beebe, 1997). the reaction should be reported at each accession level, and not restricted to the best performers. evaluation genetic resources (2021), 2 (4), 21–43 impact of a bean collection 31 should obviously capitalize on knowledge generated by previous protocols. for example, on bruchids, it is likely that evaluation of the rest of cultivated common bean germplasm will lead to nowhere (van schoonhoven and cardona, 1982), because domestication occurred elsewhere (chacón-sánchez et al, 2005; bitocchi et al, 2013; kwak et al, 2009). but the right arcelin can be picked by screening for the protein or the gene(s) involved instead of testing thousands of accessions in contact with the insects. from previous experience, it seems likely that genebanks will face periodic shortages in skilled, highly specialized staff (a challenge also mentioned by fu (2017). this is a recurrent limitation for germplasm evaluation (thus resulting in limited use of the collection in the future). sending the core collection or more accessions abroad for specialized evaluations is an option, although perhaps not as fast as having it evaluated by a multidisciplinary team as done at ciat in the 1970s. incidentally, blocks of resistance genes (gonçalves-vidigal et al, 2020) that can be traced by molecular markers are giving an unexpected support to that approach. but in view of complex traits such as heat or drought tolerance, for which just a fraction of the entire collection has been evaluated, multi site evaluation of thousands of accessions seems extremely time-consuming and expensive. new evaluation schemes have to be designed and are a true challenge at the organ level (zhao et al, 2019), but not impossible when focused for example on pulvini-caused movements of leaflets in relation to solar radiation avoidance (thomas et al, 1983). for location-related abiotic stresses, geographical approaches (targeted towards the surviving germplasm where the stress has been present for thousands of years, and thus logically the wild forms) may help. but these gis approaches did not pick up outstanding wild forms under low phosphorus stress (eighteen accessions tested, beebe et al (1997), while there seems some promise for drought tolerance (eighty-six accessions tested, cortés and blair (2018). internally, these approaches requires the genebank to be strict on passport data accuracy (van hintum et al, 2011). however, this is not always possible; for example, accession g40001 with promise for heat tolerance is from a market in veracruz (suárez et al, 2020). given some intrinsic limitations of common bean, coming heat and drought stress in the tropics and subtropics (battisti and naylor, 2009; beebe et al, 2011) may be the opportunity to re-balance the collection towards the tepary and lima bean, more hardy crops in this regard (freeman, 1913; rachie, 1973), respectively). eventually, bean breeders may realize that they have five crops instead of one, each one with a different ecological head start (debouck, 1992). with the advances in marker assisted selection and genetic maps, it might be faster to correct a shortcoming in seed or growth habit in tepary than expecting the common bean to fully change its ecological background. discussion the afore-mentioned facts suggest the following points for discussion. first, one can ask whether this bean germplasm collection meets the expectations for which it was established. many sources of disease resistance were found (table 2), and one should note that in many cases the findings were unpredicted, and largely independent of geographic origin or gene pool. as well noted by harlan (1978), page 351) “resistance is where you find it”. for those diseases where no good sources of resistance have been found, in the light of a similar experience with the usda collection (e.g. the case of white mold: schwartz and singh (2013), it seems more a deficiency of the common bean crop species itself than a severe lack of representativeness. thus, the breeders turned logically to the wild forms and the secondary gene pools (debouck, 1999), where the collection provided some solutions but also means for the needed preliminary studies in taxonomy and wide crossing. this links with a second point: given the above evidence of return on investment and incompleteness of the task (hidalgo and beebe, 1997), it might be important to continue with evaluation, namely for abiotic stresses such as drought or heat caused by global warming, as these will impact on yield (lobell and gourdji, 2012; beebe et al, 2013). in view of the numbers of accessions and facing the need for developing novel evaluation schemes for abiotic stresses including a network of well characterized (climate, soil) experimental plots, it might be cost effective to cooperate with other bean germplasm repositories (e.g. instituto nacional de investigaciones forestales, agŕıcolas y pecuarias, tepatitlán, mexico; institut für pflanzengenetik und kulturpflanzenforschung, gatersleben, germany; usda, usa). incidentally, this cooperation might also include a reciprocative safety backup and the development of a novel database because it is a shared concern. third, to the question whether the genebank has made any impact, the answer came from bean breeding but also many actors in the global community. the figures of germplasm distribution for applied and basic research (table 3 and figure 4) have shown a vibrant research community worldwide adding value to the collection. they invite the genebank to a permanent capacity to respond to requests (because of the ‘on-line shopping syndrome’) but also to document these impacts. the question whether the collection will make any impact in the future should also consider technological innovations such as transgenesis and gene editing (e.g. using crispr-cas9) (doudna and charpentier, 2014). these approaches of genetic engineering bring new light on using diversity, as they have the potential to add a new function such as herbicide tolerance or improve an existing one, such as seed protein quality, beyond the trait offer of the primary gene pool (gepts, 2002). transformation in common bean has proven to be particularly difficult (jacobsen, 1999), and with limited success (aragão et al, 1998, 2002; estrada-navarrete et al, 2007). transformation seems 32 debouck et al genetic resources (2021), 2 (4), 21–43 quite possible in tepary bean (zambre et al, 2005), but apparently little exploited for tepary improvement. the crispr technology in soybean aims at editing genes involved in a biosynthetic pathway for seed oil quality, for herbicide tolerance, or changing photoperiod sensitivity (bandyopadhyay et al, 2020; xu et al, 2020). new technologies will continue to appear, but under currently available evidence and costs they seem likely to contribute to a wider and/or faster use of the collection rather than to replace it. finally, in this context, in order to continue to meet the broader expectations of human societies, the genebanks should fill gaps which were identified early on, in terms of geography, e.g. the northern andes: (hidalgo and beebe, 1997; beebe and debouck, 2019); or in terms of biological coverage (ramı́rez-villegas et al, 2010, 2020). wild forms and wild species should thus be better represented in the collection, with due consideration to the regeneration capacity and disclosure of the potential. there are two points here: first, given the possibilities opened by comparative mapping in the phaseoleae (schmutz et al, 2014; vlasova et al, 2016; garcia et al, 2021; moghaddam et al, 2021) and by gene editing (bhatta and malla, 2020; ku and ha, 2020), it may be time to think beyond direct interspecific hybridization for the use of alien germplasm. in that sense, species of clade a that may represent half of the genus (delgado-salinas et al, 2006; porch et al, 2013; debouck, 2021) may be opportunities of genes to imitate and/or to regulate differently instead of genes to transfer. but given the speed of the technological development in breeding (hickey et al, 2019), the action should be initiated now with the most threatened habitats (williams et al, 2007), species (goettsch et al, 2021), or unpredictable conditions, or timeconsuming work. this leads to a second point, as forsaking millenary crops (mamidi et al, 2011) does not improve humankind’s food security. the four other bean crops mean four more opportunities for plant breeding. following the diversity criteria prevailing during the establishment of the common bean collection, similar efforts should be carried out for these bean crops. the change experienced by soybean from an oriental soy sauce in north america in 1767 into an animal feeding and agro-industrial crop in just one hundred years (hymowitz and bernard, 1991) is a strong message to not lose options. along the concept of a societal insurance provided by crop genetic resources (gepts, 2006), keeping more crops alive goes in line with productive, sustainable and locally adapted agriculture and, as a consequence, with reducing rural poverty and increasing appreciation towards indigenous cultures. more than ever before, genebanks should continue to be the reserve of all options. concluding remarks figure 1 presented breeding challenges in latin america in a time sequence, which were largely met by use of genetic resources assembled and evaluated over the last fifty years. the same germplasm collections allowed inheritance studies and the improvement of bean breeding methods, when looking for combining ability, tolerance to abiotic stress or tagging a resistance. the coming storms in areas of bean production (e.g. increased demand due to demography in eastern africa, extinction of crop wild relatives in mesoamerica, drought in 60% of bean growing areas worldwide) are resetting the timing to meet all breeding challenges at once and soon, but they also involve the genebanks to have the genetic solutions ready on the shelf or on the screen (or both). the contributions of the bean collections to advance knowledge on the nature, structure and evolution of phaseolus genetic resources can now help the genebanks to check two extinctions: the extinction of populations in the wild, and the extinction of knowledge about cultivated diversity. buying time on these two fronts will be difficult for genebanks, but the continuing improvement of conservation methods and efficiencies will contribute to find and enable the human talents for these daunting tasks. the above history shows many ways forward to ensure global food security in uncertain times. acknowledgments the authors warmly thank the following individuals for providing or confirming specific data about introduction and/or distribution of accessions: sandra albarraćın, alejandro borrero, juan carlos guerrero, ángela hernández, rigoberto hidalgo, dimary libreros, celia lima, carmenza llano, josefina mart́ınez, daniel eduardo salazar, orlando toro (deceased), alba marina torres and eliana urquijo. the invitation by peter wenzl to the first author to write down a few memories is fully acknowledged. the first author expresses deep gratitude to the following institutions for support during four decades: administration générale de la coopération au développement, bundesministerium für wirtschaftliche zusammenarbeit und entwicklung, the department for international development, european union, global crop diversity trust, international board for plant genetic resources, international center for tropical agriculture, united states agency for international development, united states department of agriculture, and the world bank. the agricultural research services of the following countries are fully acknowledged for support in the field work and subsequent germplasm introduction: argentina, bolivia, chile, colombia, costa rica, ecuador, el salvador, guatemala, mexico, panama and peru. author contributions dgd conceptualized and wrote the paper. dgd collated the data about use and impact for the first period of the genebank, while ms covered the most recent period. lgs compiled the data about germplasm distribution. ms and lgs re-checked the data of accession numbers. all authors read, revised and approved the manuscript. genetic resources (2021), 2 (4), 21–43 impact of a bean collection 33 conflict of interest statement the first author has been responsible of ciat genebank, as head of the genetic resources unit (1996-2009) and as leader of the genetic resources program (20092016), with over seventy staff members operating in five experimental stations and three labs, for three germplasm collections (bean, cassava and tropical forages) of over 67,000 accessions. the second author has been responsible for all genebank operations in 2017 to date, which include the regeneration of bean and tropical forages collections in the experimental stations and the conservation in the labs. the third author has been responsible since 2009 for all operations related to seed conservation of the bean and tropical forages collections, which include germplasm distribution. references abawi, g. s., provvidenti, r., crosier, d. c., and hunter, j. e. 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(2013). ques, a new phaseolus vulgaris genotype resistant to common bean weevils, contains the arcelin-8 allele coding for new lectin-related variants. theor. appl. genet 126, 647–661. doi: https://doi.org/ 10.1007/s00122-012-2008-2 zaumeyer, w. j. and thomas, h. r. (1957). a monographic study of bean diseases and methods for their control volume 868 of technical bulletin. (washington, d.c.) 255p. doi: https://doi.org/10. 22004/ag.econ.169625 zeven, a. c. (1997). the introduction of the common bean (phaseolus vulgaris l.) into western europe and the phenotypic variation of dry beans collected in the netherlands in 1946. euphytica 94, 319–328. doi: https://doi.org/10.1023/a:1002940220241 zhao, c., zhang, y., du, j., duo, x., wen, w., gu, s., wang, j., and fan, j. (2019). crop phenomics: current status and perspectives. front. plant sci 10, 1–16. doi: https://doi.org/10.3389/fpls.2019.00714 zimmerer, k. s. (1992). biological diversity and local development. mountain res. dev 12, 47–61. doi: https://doi.org/10.2307/3673747 https://doi.org/10.1094/pd-80-0650 https://doi.org/10.1094/pd-80-0650 https://doi.org/10.1186/1471-2229-12-136 https://doi.org/10.1186/1471-2229-12-136 https://doi.org/10.1007/s00122-004-1910-7 https://doi.org/10.1007/s00122-004-1910-7 https://doi.org/10.1094/phyto-75-1032 https://doi.org/10.1094/phyto-75-1032 https://doi.org/10.1007/s00122-012-2008-2 https://doi.org/10.1007/s00122-012-2008-2 https://doi.org/10.22004/ag.econ.169625 https://doi.org/10.22004/ag.econ.169625 https://doi.org/10.1023/a:1002940220241 https://doi.org/10.3389/fpls.2019.00714 https://doi.org/10.2307/3673747 the purpose for establishing a collection of genetic resources at ciat how the phaseolus collection was assembled the diversity in the bean collection and its use distribution of the bean collection impact of the bean genetic resources collection the other impact: the contribution to knowledge knowledge often overlooked, although associated with crop germplasm for millennia serving the breeders community and beyond discussion concluding remarks author contributions conflict of interest statement original article genetic resources (2025), 6 (12), 73–82 doi: 10.46265/genresj.bpgx5961 https://www.genresj.org issn: 2708-3764 received: 24.01.2025 | accepted: 07.07.2025 | published online: 26.09.2025 exploring the situation of transboundary breeds in europe for their effective management and conservation abstract: geographical distribution plays a crucial role in the effectiveness of breeding and conservation programmes, especially for livestock breeds with a small population size. among these, transboundary breeds present unique challenges and opportunities for conservation efforts. this study specifically examines the case of transboundary breeds in europe and the associated challenges. population and descriptive data were sourced from the domestic animal diversity information system (dad-is) to assess their current state of monitoring and management. the analysis revealed that 42% of the 6,460 national breed populations reported in europe are transboundary, with 25% occurring exclusively within the region (europe). alarmingly, 85% of european transboundary breeds are classified as ‘at risk’ or have an ‘unknown’ conservation status, a fact that further accentuates the urgent need for improved sustainable management. this paper identifies key data gaps, for instance related to common understanding of concepts used by managers, and proposes improvements to enhance the monitoring, conservation and management of transboundary breeds in europe. keywords: transboundary breeds, dad-is, conservation, animal genetic resources, livestock diversity citation: charvolin, e., tsiokos, d., leroy, g. and ligda, c. (2025) “exploring the situation of transboundary breeds in europe for their effective management and conservation”, genetic resources, 6(12), pp. 73–82. doi: 10.46265/genresj.bpgx5961. © copyright 2025 the authors. this is an open access article distributed under the terms of the creative commons attribution license (cc by 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. eléonore charvolina,†,*, dimitrios tsiokosb,†,*, grégoire leroyc and christina ligdad a université paris-saclay, inrae, agroparistech, gabi, 78350 jouy-en-josas, france b hellenic agricultural organization–dimitra, rias, 58100 paralimni giannitsa, greece c food and agriculture organization of the united nations, viale delle terme di caracalla, 00153 roma, italy d hellenic agricultural organization–dimitra, vri, 57001 thermi-thessaloniki, greece * corresponding authors: eléonore charvolin (eleonore.charvolinlemaire@inrae.fr), dimitrios tsiokos (tsiokosd@gmail.com) † equal contribution, listed in alphabetical order introduction livestock diversity in europe has been shaped by the collective management and selection practices of farmers, evolving within the region’s political, economic and social environment. in europe, systematic performance recording, animal identification and pedigree recording have contributed to the development of numerous livestock breeds (fao, 2015). however, changes in market demand, environmental factors and political instability continue to impact farmers’ selection decisions, directly influencing farmers’ preferences and have a direct impact on a breed’s population size (cao et al, 2021; verrier et al, 2015). a variety of migration, introgression and isolation events over time have influenced the domestic diversity within and among countries (leroy et al, 2015). while some breeds remain confined to specific geographical areas, others are distributed across multiple countries or even globally. it is estimated that, on a global scale, half of all breeds are transboundary, i.e. shared between at least two different countries (fao, 2024a; see box 1 for definitions). transboundary breeds (tbs) may be native to their current locations or introduced as exotic breeds, recently or a long time ago, sufficient to be considered adapted to the local environment (box 1). the conservation of animal genetic resources (angr) for food and agriculture became a priority in the late 20th century, driven by concerns over the loss of genetic diversity. this was motivated by observant breeders in some countries, who realized the risk early on. the next steps were taken by scientists, providing solid scientific evidence and contributing to enhanced awareness concerning the threats to global biodiversity. the understanding that the disruption of https://doi.org/10.46265/genresj.bpgx5961 mailto:https://doi.org/10.46265/genresj.apnr6909%0d?subject= https://www.genresj.org https://doi.org/10.46265/genresj.bpgx5961 mailto:eleonore.charvolin-lemaire%40inrae.fr?subject= mailto:eleonore.charvolin-lemaire%40inrae.fr?subject= mailto:tsiokosd%40gmail.com?subject= genetic resources (2025), 6(12), 73–8274 charvolin et al historical links between breeders and the breeds developed by their ancestors had led to a decline in those breeds’ population was the driving force for taking actions to interrupt this trend. threats such as indiscriminate crossbreeding, the introduction of exotic breeds, weak institutional policies and economic challenges continue to endanger genetic diversity (fao, 2015). conservation efforts encompass a wide range of activities, including awareness raising, strengthening governance, adding value to natural resources and implementing conservation schemes (ligda et al, 2013; sponenberg et al, 2019). genetic diversity losses and conservation issues are not limited to local breeds, as international breeds can also be subject to genetic erosion phenomena (ablondi et al, 2022). relevant strategies for angr management also concern international breeds due to their importance in ensuring food security and public health globally. sustainable management of both local and international breeds is essential for global food security and agricultural resilience (lefevre et al, 2024). europe initiated conservation efforts in the late 1970s; however, global consensus on preservation of livestock diversity only emerged after the publication of the first report on the state of the world’s animal genetic resources for food and agriculture (fao, 2007a). this led to the development of the global plan of action for animal genetic resources (gpa) (fao, 2007b). this framework outlines four strategic priorities: (1) characterization, inventory and monitoring of the breeds, (2) sustainable use and development, (3) conservation, and (4) policies, institutions and capacity building. the domestic animal diversity information system (dad-is), developed by the food and agriculture organization of the un (fao), serves as a comprehensive database supporting these conservation and management efforts worldwide. dad-is contains information on breed characteristics, uses, geographic distribution and demographics, and images and tools for generating reports. the recorded information, based on agreed definitions on terms and classification criteria and provided by nominated national coordinators, enables the monitoring of breeds at global, regional and national levels (polack et al, 2022). moreover, dad-is is the data source for two un sustainable development goals (sdg) indicators (2.5.1b and 2.5.2), as approved by the fao commission on genetic resources for food and agriculture (cgrfa) (fao, 2011; fao, 2013). in particular, sdg indicator 2.5.2 takes into account the proportion of breeds considered at risk of extinction, on the basis of their population size estimation. initially focusing on local breeds, the focus of the indicator is currently extended to transboundary ones (fao, 2024b). efficient breeding and conservation programmes for breeds with small population sizes are particularly challenging due to several reasons (biscarini et al, 2015). these breeds often face geographic constraints, policy limitations and economic disadvantages, creating a cycle that restricts their development. however, the presence of such breeds across multiple countries presents opportunities for collaborative management. understanding breed distribution across europe, along with different environments and breeding conditions, can improve resilience assessments and utilization of genetic resources. advances in molecular genetics enable systematic genomic studies that can further support the classification and management of tbs. breed (fao, 2000): is a homogenous, subspecific group of individuals with definable and identifiable external characters that enable it to be separated by visual appraisal from other similarly defined groups within the same species. alternatively, it is a homogeneous group where geographical separation from phenotypically similar groups has led to general acceptance of its separate identity. national breed population (nbp) (fao, 2021): the nbp refers to the animals belonging to one specific breed in one country. in case it is a local breed, occurring only in one country, it represents the breed. in case it is part of a transboundary breed, occurring in more than one country, it represents only a part of a breed. adaptedness classification (fao, 2015): the dad-is adaptedness classification differentiates exotic breeds from native/indigenous breeds and from locally adapted breeds. the latter correspond, in the classification, to non-native breeds that have become adapted to the local environment and/or production system. locally adapted breeds have been initially defined as breeds of exotic origin imported into the national territory from another country that show limited genetic relationship with the original population. later the definition has been broadened as breeds which have been in the country for a sufficient time to be genetically adapted to one or more of the traditional production systems or environments in the country. native breed (fao, 2001): (also termed indigenous breeds, autochthonous) originating from, adapted to and utilized in a particular geographical region,  form a subset of the locally adapted breed (broader definition). it refers to a breed in its country of origin (i.e. the country where the breed was created originally from genetic material that was available when the initial breed development commenced). it is important to note that a breed may be a native breed in more than one country depending on the history of the breed. box 1. definitions of breed and breed classification concepts genetic resources (2025), 6(12), 73–82 status of transboundary breeds in europe 75 definitions of specific terms used in this article are given to help the reader understand the situation of tbs (box 1). this study aims to explore the current status of tbs in europe using dad-is data. it provides information on the distribution of tbs around europe, their risk status at local and regional level and adaptedness classifications. the outcomes reveal the complexity of factors that impact the decisions concerning the management of tbs and focusses on certain areas for further analysis, using a case study approach. material and methods data for this analysis were extracted from fao’s dadis using the ‘data export’ tool. the data used include the descriptive and population data files, as well as the transboundary list file. the study included all 31 registered species in the 45 countries classified as ‘europe and caucasus’ (fao, 2007b). all data was provided by the national coordinators (ncs) appointed by the countries. the analysis was based on the population and historical data available as of 8 august 2023. the analysis was built on the national breed populations (nbps), as defined in box 1. the descriptive data included information on the risk status, geographic and adaptedness classification provided by dad-is. the population data file contained the information on the population size of the breeds for all years with recorded data. based on this information, an nbp was classified into the risk status categories. an nbp was reported as tb when this nbp was linked (by the nc) to a breed in the tb list provided in dad-is. this is done automatically by dad-is, without taking into consideration the number of nbps linked to the transboundary name. dad-is classified nbps as local (reported in only one country), regional transboundary (reported as tb, only by countries from a given region, e.g. europe), or international transboundary (reported as tb by countries from multiple regions). breeds were further classified by dad-is as native, locally adapted, or exotic, based on reported data (box 1). statistical comparisons were made to assess patterns in breed distribution, risk status, and adaptedness. associations between categorical variables were analyzed using the chisquare test via the crosstabs procedure in spss version 27.0 (ibm corp., 2020). statistical significance was set at p < 0.001. data gaps and inconsistencies were analyzed to identify possible reporting issues and areas for improvement. results assessment of the current situation among the 6,460 nbps analyzed, 3,761 (58.2%) are local breeds, while 2,699 (41.8%) are transboundary. of the transboundary nbps (figure 1), 674 (25%) are european tbs (found only within europe), and 2,025 (31.3%) are international tbs (present in at least one non-european region). these findings highlight the importance of coordinated conservation strategies within europe. the presence of tbs across multiple countries suggests opportunities for shared conservation programmes. tbs are reported by all countries in europe, as shown in figure 2. the tb share among nbps exhibits considerable variation, ranging from 11,6% (spain) to 77,1% (ireland). no significant correlation is found between the share of tbs among nbps and the number of nbps reported by the countries. the number of countries that declare a specific tb linked to their nbp, may reflect the dynamics of cooperation among figure 1. repartition of the european national breed populations (nbps) following their geographical classification, their corresponding number of nbps (in brackets, the corresponding proportions). data source dad-is. genetic resources (2025), 6(12), 73–8276 charvolin et al the countries. however, further information on the historical evolution of the nbps, the ongoing breeding or conservation programmes, organizational and social aspects is required. figures 3a and 3b provide information on the distribution of regional tbs between countries, in mammalian and avian species. in figure 3a, the results are presented separately for the five mammalian species (cattle, goat, horse, pig and sheep), for which breed-related information is reported by almost all countries in the european region (cattle and sheep in 100% of countries, goats in 93%, and horses and pigs in 91%) (fao, 2007a). similarly, the results in figure 3b concern avian species, for which breed-related information is reported by 50% or more european countries. these figures present a general overview of the distribution of tbs at species level and do not infer a species impact on the number of countries linked to a tb. figure 2. number of national breed populations (nbps) and percentage of transboundary breeds (tbs) declared per european country, ordered by increasing share of tbs. figure 3a (mammalian species) shows that a significant number of tbs is linked to only one nbp. this percentage varies between 25.9% (21 nbp) for sheep and 50% (7 nbp) for swine. in cattle, 13 (41.9%) of the 31 european tbps are reported by only one country. similar trends are observed in avian species (figure 3b); however, this is referred to an overall smaller number of tbs and lower number of countries reporting breed data on these species. those results underline a substantial gap between breeds classified as tbs according to dad-is rules and the fact that those breeds are actually reported by more than one country. the fact that an nbp has not been linked to an existing tb name could be attributed to a different perception of the concept of tb; however, several other reasons may also exist. therefore, a case study approach would be required to better understand the situation and propose a step forward. figure 3. regional transboundary breeds reported by one, two or three, and four or more countries across mammalian (a) and avian species (b). genetic resources (2025), 6(12), 73–82 status of transboundary breeds in europe 77 adaptedness classification considering the classification by adaptedness, from the total of 2,699 registered (regional and international) nbps, 15% were classified as native, 10% as locally adapted and 36% as exotic (table 1). the remaining 38% nbps were not classified in any category, as no relevant data has been reported by any country. the substantial number of nbps with no information on adaptedness classification in europe (260 nbps only reported in europe) indicate that further progress is still needed in this domain. the high percentage of unclassified breeds suggests the need for clearer reporting standards across countries. nevertheless, compared to the corresponding figures at global level (45% of non-available classification, fao 2023), the results of the european region are more complete. the high percentage of non-classified nbps is attributed to several capacity or policy-related factors, reflecting the organization of angr management in each reporting country and the specific considerations on the definition of terms, at table 1. number and percentage of international and regional national breed populations linked to transboundary breeds in europe in dad-is (classified as native/locally adapted/exotic) country level. nevertheless, this gap may hinder the efficient management of angr, as the missing information could result in misleading reports and biased decisions. from the remaining, 26% (177 nbps) are classified as exotic, thus they are considered as introduced from another country and have not been developed for sufficient time in the country. compared to international ones, european tbs display a larger proportion of locally adapted and native breeds (p < 0,0001), which could be partially explained by the fact that locally adapted and native breeds are more regionally distributed than the exotic ones, which have a wider global expansion. the distribution of nbps in adaptedness classes in european countries can be considered indicative of the current picture of breeds; however, an in-depth analysis of relevant case studies is needed. the data show that in 14 countries no information is available concerning the adaptedness classification of their recorded nbps (linked to tb), while in 21 countries, more than 80% of their nbps have recorded information on the adaptedness classification (figure 4). figure 4. adaptedness classification of national breed populations linked to transboundary breeds (tb) in europe breed classification (adaptedness) breed classification (geographic) international regional (europe) total no (%) no (%) no (%) native 225 (11) 184 (27) 409 (15) locally adapted 218 (10) 53 (7) 271 (10) exotic 809 (39) 177 (26) 986 (36) non-available 773 (38) 260 (38) 1,033 (38) total 2,025 (100) 674 (100) 2,699 (100) genetic resources (2025), 6(12), 73–8278 charvolin et al risk status specific thresholds differentiated by species are defined in various countries through national and international regulations that provide the framework for angr management. the current analysis is based on the local risk status, following fao's classification system, which is recognized as the global reference according to cgrfa (fao, 2013). in the case of tbs, in dad-is the risk status is estimated at local, regional and global levels, provided that the country has been reporting population data for the past ten years. the estimation of risk status at different levels, in the case of tbs, could support their efficient conservation management, as different approaches are needed when a tb is at risk in one country, but not at risk in other(s), or when the tb is at risk everywhere. among the 674 nbps found in more than one european country, 44.2% were considered at risk in at least one country (table 2). moreover, considering that the existence of sufficient recorded data and knowledge of population trends is a key point for the efficient management of breeds, 45% of table 2. local risk status of all national breed populations related to tbs reported by at least two countries in europe. breed classification (geographic)   at risk cryoconserved only extinct not at risk unknown total international count 844 2 108 230 841 2,025 row n % 41.7 0.1 5.3 11.4 41.5 100.0 regional count 298 1 33 39 303 674 row n % 44.2 0.1 4.9 5.8 45.0 100.0 total count 1,142 3 141 269 1,144 2,699 row n % 42.3 0.1 5.2 10.0 42.4 100.0 nbps in the ‘unknown’ category, i.e. without population data provided over the last ten years, could be interpreted as a first sign of being at risk. therefore, this could be considered an early indicator of the current or potential risk at national level of the nearly 90% of nbps that are transboundary (reported only in europe). however, a more in-depth analysis of these data is needed, as nbps (from tbs) could be at risk in one country and not in others, as previously mentioned. therefore, further examining the data in table 3, the nbp’s risk status was calculated under different combinations for the five species considered in this study. in table 3, it is shown that 42% of goat and 30% of sheep nbps linked to a tb, were reported at risk in all countries. when the ‘unknown’ category is added, this percentage increased to 75% and 70%, respectively. the higher percentage of sheep and goat nbps that were at risk in all countries could be explained, at least to some extent, by the different evolution of sheep and goat populations compared to cattle and pigs, for which intensification has been more widespread. table 3. local risk status of transboundary breeds present in more than one country, (%) by species not at risk in at least one country (%) at risk in all countries (%) at risk or unknown or extinct in all countries (%) cattle 11.76 5.88 88.24 goat 8.33 41.67 91.67 horse 22.73 9.09 77.27 pig 14.29 14.29 85.71 sheep 26.67 30.00 73.33 total 21.19 22.88 78.81 genetic resources (2025), 6(12), 73–82 status of transboundary breeds in europe 79 the diversity of cases is presented with selected examples in table 4. the hutsul horse reported as tb by eight countries, is at risk in six countries and unknown in the remaining two, due to lack of data. the hutsul horse is categorized as native in hungary, austria and poland, while it is defined as locally adapted in slovakia and czechia, and exotic in germany. the field was not filled in the remaining two countries. the tb is at risk at regional level. the case of precoce sheep, reported by france (native), spain (locally adapted) and portugal (exotic) is a similar case, as the breed is at risk at national and regional levels. however, the case of the ouessant sheep – present in six countries, native in france and exotic in the netherlands, germany and czechia (no classification in belgium and denmark) – differs as it is at risk in all countries, but the breed is not at risk at regional level. the podolian cattle represents a case where the breed is developed in the country of origin (italy), bred in high numbers, while a small population (at risk) is bred in another country (serbia). in the dad-is data analyzed, there was no information on the links and exchanges between breeders’ associations or on the genetic differences between nbps. discussion this study, based on the data reported in dad-is, provides a general overview of the status of tbs in europe and reflects the quality of data and level of recorded information. in europe, nbps linked to tbs account for a large part (42%) of nbps reported. this number could reach up to 80% of nbps in some countries, with varied percentages across countries. the interpretation of this variation is not straightforward, as various factors contribute to this picture. angr have evolved under specific conditions (physical, technical, social, political and organizational) in each country, forming the current breeds and links among countries. furthermore, angr management is under a country’s sovereignty, and national decisions and measures are in accordance with global (fao) and european guidelines. approaches to identify the link between nbps and tbs may differ among countries according to national needs and perceptions of terms. our results highlight that, depending on the species, a percentage between 20% and 50% of european tbs are reported by only a single country. this raises concerns about data accuracy and consistency. although this issue appears less significant at the global level – where only 17% of tbs are reported by a single country – it remains noteworthy in the european context. in europe, several such breeds are not linked to the tb list by their country of origin. examples include the turopolje pig from croatia, the asino sardo donkey from italy and the olkusz sheep from poland. in some cases, this cannot be considered a reporting gap, as it is a justified decision by the nc. such decisions could be based on specific national policies and priorities. certain countries could have chosen to classify their native breeds as local, given that global indicators mainly focus on local breeds. table 4. transboundary breed cases examples species transboundary breed name local breed name country sdg local risk status adaptedness regional risk status horse hutsul hucuł poland at risk native at risk hutsul romania unknown no info hucuł slovakia at risk locally adapted hucuł hungary at risk native huzule germany at risk exotic gutsul ukraine at risk no info huzule austria at risk native huculsky kun czechia at risk locally adapted cattle podolian podolica italy not at risk native not at risk podolian serbia at risk locally adapted sheep precoce merino precoz spain at risk locally adapted at risk merina precoce portugal at risk exotic mérinos précoce france unknown native sheep ouessant ouessant france at risk native not at risk           ouessant belgium at risk no info ouessant netherlands at risk exotic ouessant schaf germany at risk exotic kesantská ovce czechia at risk exotic ouessant denmark at risk no info genetic resources (2025), 6(12), 73–8280 charvolin et al the global indicators employed to assess goal attainment may introduce biases in data entry into global databases. these biases can also arise from the implementation of national indicators or adherence to national legislation. an illustrative example of a national rules-based decision is the case of the icelandic horse, which is reported by several countries, even though iceland itself does not link it to the tb list. this is due to national legislation prohibiting the re-importation of icelandic horses, which results in the national population being considered genetically isolated (campana et al, 2011). in addition to these cases, certain widely used industrial pig and poultry lines – such as the topigs lignée e (tn tempo) pig line, currently reported only by the netherlands – may appear underreported due to limited data exchange between private breeders and ncs. it cannot be excluded that in a number of cases, a breed in the tb list is actually present in only one country; however, it is likely that the greater part of this gap is caused by underreporting in countries. to address this issue, ncs could be encouraged to review the breeds listed in supplemental table 1 – particularly those listed in the first column (tb reported by only one country) – and assess whether nbps from their own country should also be linked to this list. complementarily, fao, in collaboration with ncs and relevant stakeholders, should undertake efforts to clarify the definitions and utilization of breed classifications. this includes revisiting the criteria used to associate nbps with tb names, to ensure greater consistency and completeness in reporting. several actions initiated within the framework of the european regional focal points for animal genetic resources (erfp) aim to facilitate exchange among countries and promote agreement on common perspectives. at the same time, however, it is important to recognize that the existing variation among countries could also be beneficial, as it reflects diverse needs and priorities. genomics could play a key role in the management of tbs and provide strong scientific evidence to justify further steps. the developments in genetics and the wide availability of genomic tools could reveal genetic diversity between nbps that have evolved in different environments and have been bred under separate breeding programmes for certain time periods. whole genome sequencing (wgs) could offer valuable information on the demographic history of populations to support decisions relevant to the management of tbs. thus, the management of tbs can be approached considering the existing definitions of breeds. according to the definition used by fao (fao, 1999), a breed is not a simple genetic concept, but it also has a social dimension. breeds are developed by the farmers’ breeding practices, which are not exclusively technically driven. several exogenous factors have an impact on them. in certain cases, such factors could radically change the environment in which the farmers work. generalized guidelines and rules cannot be easily applied in the case of tbs. a common understanding of the term ‘transboundary breed’, based on both genetic and social parameters, could help in developing appropriate approaches. however, one should be very sceptical in defining specific criteria that could be used in all cases, as the decision is not clearly technical, but also political, to a large extent. besides the aspect of demographic data quality, which results in an unknown risk status, the outcomes of our analysis highlight certain inconsistencies related to the approach followed in setting the tb groups, as shown by the number of tb groups, to which only one nbp is linked. taking this into account, the results can be considered as a first step in choosing breed cases that could be further analyzed, incorporating additional information. by examining tbs through case studies, either in neighbouring countries or across more widely spread breeds, useful conclusions could be drawn that may serve as guiding principles on a broader level. the knowledge of whether breeders cooperate already, i.e. through the exchange of breeding animals, is important for grouping tb cases and could be essential for making decisions in terms of ex situ conservation. mainstream breeds that are exported to other countries and are raised under separate breeding programmes, or with continuous import of breeding animals or semen, fall into this category; however, further elements are needed to decide on implementing common breeding programmes. which criteria could be used to consider these nbps as the same breed? furthermore, changes in european borders over the past 25 years, which have resulted in some tbs being native to multiple countries with different local risk status, present an interesting case study, linking genetics with economic, social and political changes. the level (local, regional or global) at which the risk status is estimated is a crucial point for tb management. the relevance of risk status at regional level is questionable when two (or more) nbps have been developed separately for years. besides that, the risk status classification is one of the critical points leading to reluctance in linking a nbp to an existing transboundary name. this is demonstrated in the current analysis by the number of nbps that are declared as transboundary by only one country (listed in supplemental table 1). the various combinations of risk status at local and regional levels could be the starting point of the case study approach, following the general approach presented in figure 5. supplemental table 1 provides the list of european tbs that could be examined following this general approach. conclusions this article provides new evidence on the status of tbs in europe, assessing the quality of data and the frequency of updates to population data and relevant fields, as reported in dad-is. these outcomes could be useful in improving data quality and population management by enhancing data exchange and communication among countries. data analysis revealed several inconsistencies, as is the case with tbs linked to only one nbp, which opens the discussion on the definitions of tbs and the criteria applied to link a nbp to a tb. under which conditions would it be feasible to establish unified criteria, including historical data and genetic information? the lack of standardized definitions and consistent risk classifications across countries poses significant challenges for tb management. collaborative breeding programmes, genetic studies and enhanced data sharing could improve conservation outcomes. advances in genomics can further clarify genetic relationships among nbps, supporting more effective conservation planning. as discussed above, tbs cannot be examined exclusively through demographic data and genetic information, as several technical, social and political aspects shape future management opportunities. thus, a case study approach is recommended for the analysis of breeds, either in situ or ex situ, when they genetic resources (2025), 6(12), 73–82 status of transboundary breeds in europe 81 participate in common breeding programmes, frequently exchange breeding animals, or share a common history and/ or environment. this discussion is in accordance with the recommendations of the animal genetic resources strategy for europe, that promotes in situ and ex situ strategies for tbs (erfp, 2021). specific actions are foreseen to improve the knowledge on tbs, support the exchange between actors involved in the conservation and breeding programmes of these breeds and promote cooperation in this field (erfp, 2021). in this complex context, the advances in genomics and the progress of relevant research could further enrich existing knowledge on tbs and support their sustainable management. supplemental data supplemental table 1. distribution of european transboundary breeds (tbs) and their risk status acknowledgements this work was supported by the european regional focal point for animal genetic resources (erfp) and realized in the frame of the task force on transboundary breeds (https://www.animalgeneticresources.net/wp-content/ uploads/2023/09/6d_erfp_ga_2024_tf-tb.pdf). the authors acknowledge the financial contributions of european countries to erfp to continue its activities and support the in situ and ex situ conservation and sustainable figure 5. questions and actions depending on transboundary breeds’ risk status at different levels, depending on the national breed population’s status (nbps) and the impact on the calculation of the indicators for the sustainable development goals (sdgs). use of animal genetic resources (angr) and facilitate the implementation of fao’s global plan of action for angr in europe. author contributions all the authors have conceptualized the analyses and drafted the manuscript. eleonore charvolin and dimitrios tsiokos performed the data analyses. all the authors have substantially contributed to the interpretation, drafting, revision and final version of the manuscript. data availability data used in this study were extracted from fao dadis and are publicly available (https://www.fao.org/dad-is/ data/en/). conflict of interest statement the authors declare that they have no conflicts of interest.  references ablondi, m., sabbioni, a., stocco, g., cipolat-gotet, c., dadousis, c., kaam, j. t. v., finocchiaro r., summer a. 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(2025). the french inrae biological resource center for pome fruits and roses: plant and dna collections of traditional and research genetic resources. genetic resources (s2), 135–146. doi: 10.46265/ genresj.iiet1610. © copyright 2025 the authors. this is an open access article distributed under the terms of the creative commons attribution license (cc by 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. introduction the french national research institute for agriculture, food and the environment (inrae) has historically hosted collections of traditional pome fruit genetic resources in angers, france. cultivars of apple (malus mill. spp.), pear (pyrus l. spp.) and quince (cydonia ∗corresponding author: alix pernet (alix.pernet@inrae.fr) † deceased tourn. ex mill. spp.) were mainly introduced in the collections in the 1950s and 1960s from french nurseries, horticultural societies and botanical gardens. botanical species were also introduced at this time from various european botanical gardens. most of the accessions of cider apple, perry pear, apple and pear rootstocks came from field surveys carried out in the same years. in addition, the collections have been continuously enriched for breeding and for distinctness, received: 31.10.2024 accepted: 05.03.2025 published online: 04.04.2025 https://www.genresj.org https://www.doi.org/10.46265/genresj.iiet1610 https://www.genresj.org https://www.doi.org/10.46265/genresj.iiet1610 mailto:alix.pernet@inrae.fr 136 feugey et al genetic resources (2025), (s2), 135–146 uniformity and stability (dus) testing activities, as well as for the conservation of heritage genetic resources. in particular, a duplication of old french varieties – identified as at risk of disappearing following a national genetic resources survey – was carried out in the 2000s. the pome fruit collection of research genetic resources began to be built up in the 1980s with the creation of haploids, dihaploids and aneuploids. this collection was then considerably enriched since the 2000s thanks to the genetic mapping progenies that have served as references in a series of european projects. the rose (rosa l. spp.) collection was launched in 2003 when the rosa genus was chosen as a model ornamental plant due to its economic importance and biological characteristics among ornamental woody plants. since then, research has focused on understanding the key processes involved in quality development in ornamental plants such as plant architecture, flowering and disease resistance. this required the introduction and creation of original research genetic material, mainly progenies for genetic mapping. due to their high importance in research, these mapping progenies have been preserved for further studies. these genetic resources are now being used to support national and international scientific collaborations. in addition, a dna bank of wild individuals of rosa gallica l. and traditional varieties conserved in rose gardens was established to study the evolution of the genetic variability in the rosa genus. the inrae ‘pome fruits and roses’ biological resources centre (rosepom brc) that gathers these collections is now hosted by the research institute on horticulture and seeds and the horticultural experimental unit. collection description for clarity and simplicity, accessions conserved in rosepom are classified into two categories: traditional genetic resources and breeding/research genetic resources. these terms are defined in accordance with the classification proposed by the multi-crop passport descriptors (mcpd v.2.1) (alercia et al, 2015) for the biological status of accessions. traditional genetic resources include: (1) wild relative species (code 100), i.e. presumed wild specimens collected in a wild environment; (2) traditional cultivars (code 300), comprising heritage cultivars (french or foreign old cultivars of heritage interest), other old cultivars and botanical accessions of species (i.e. presumed wild specimens grown in botanical gardens, sometimes originating from seeds collected in other botanical gardens where some natural hybridization may have occurred between different conserved accessions, including old varieties); and (3) advanced or improved cultivars (code 500), which are cultivars obtained after 1945. note that botanical accessions of species may or may not be declared of heritage interest, depending on the criteria used to define ‘heritage interest’. these criteria are currently under discussion in france. breeding/research genetic resources (code 400) include, among others, unnamed breeders’ material (codes 410 to 420), cytogenetic stocks (code 422), and mapping populations (code 423). note that we prefer to use the term ’mapping progenies’ rather than ’mapping populations’, since a progeny is the result of a cross between two parents, whereas a population may consist of several unrelated individuals. in summary, rosepom’s collections include traditional resources of agricultural, industrial, economic, scientific, social and cultural interest as well as research resources, much of which consist of material for genetic and genomic studies. more specifically, rosepom conserves approximately 8,800 reproductive clonal accessions (accessions conserved as plants in the field with the capacity to participate in the reproduction process), mainly from three genera, malus, pyrus and rosa (figure 1), each represented by multiple species. this includes around 3,500 traditional accessions (cultivars, botanical accessions and wild relatives) and 5,300 breeding/research accessions (breeding unnamed progenitors, research accessions composed of cytogenetic stocks and mapping progenies). rosepom conserves also several thousand additional non-reproductive accessions, in the form of leaves or dna only, particularly for the rosa genus. pome fruits the pome fruits collection contains 4,300 apple, 2,300 pear and 60 quince accessions. these reproductive accessions have been introduced from different geographical origins and according to their use in fruit production, as rootstocks, for ornamental use or for research purposes. they therefore represent a large genetic diversity. traditional conserved genetic resources include: (1) wild accessions of relative species of malus, pyrus, cydonia from all over the world including 47 taxa of western and eastern pyrus species, (2) botanical accessions of malus, pyrus, cydonia, (3) old or new cultivars of rootstocks of malus, pyrus, cydonia, (4) old or new cultivars of dessert or cider apple (malus domestica borkh.), dessert european pear (pyrus communis l.), perry pear (pyrus nivalis jacq.), nashi (pyrus pyrifolia (burm.f.) nakai) and quince (cydonia oblonga mill.), and (5) old or new cultivars of ornamental apple and pear (various species and many interspecific cultivars). among these, nearly 1,500 accessions are old cultivars from france (840 apple accessions, 640 pear accessions), old cultivars from other countries in europe and some well-known old cultivars from america, asia and africa. in addition, a upov international reference collection of nearly 1,000 accessions of recent apple and pear cultivars complements the rosepom resources. resources originating from breeding and prebreeding programmes include over 1,000 accessions of material collecting traits of agronomic interest, in particular, (1) for apple, resistance to scab (venturia inaequalis (cooke) winter), fire blight (erwinia amylovora (burgenetic resources (2025), (s2), 135–146 pome fruits and roses brc for the future 137 figure 1. reproductive collections: species groups and types. repartition of the number of accessions of rose, apple, pear and quince according to the type of resources (traditional or breeding/research). rill) winslow et al.) and powdery mildew (podosphaera leucotricha (ellis & everhart) salmon), fruit texture, (2) for pear, resistance to scab, fire blight and pear psylla (cacopsylla pyricola (förster), and (3) for rootstocks, dwarfing, resistance to fire blight and abiotic stresses. it also includes 16 intergeneric hybrids between pyrus, malus and cydonia. resources originating from research programmes include original material, in particular 46 accessions of haploids and double haploids and 1,400 accessions of mapping progenies segregating for disease and pest resistance. for apple, the two most important mapping progenies conserved by rosepom were created by crossing genotypes exhibiting different quantitative trait loci architecture (identified thanks to previous not conserved mapping progenies) for apple scab resistance: (1) ‘tn10-8’ hybrid crossed by the cultivar ’fiesta’ (260 accessions), and (2) ‘discovery’ crossed by ‘tn10-8’ hybrid (149 accessions). for pear, rosepom conserves in particular a mapping progeny segregating for resistance to pear psylla and obtained by crossing the interspecific pyrus bretschneideri rehd. × pyrus communis l. hybrid pear3, resistant to pear psylla, with the susceptible european pear cultivar ’moonglow’ (234 accessions). from the point of view of a classification based on the type of use, among the accessions of cultivars and unnamed breeding material, there are 320 rootstock accessions (of which nearly 200 of malus, 80 of pyrus and 45 of cydonia), 4,800 accessions of dessert or cider apple, dessert european pear, perry pear, nashi and dessert quince, and 120 accessions of ornamental apple and pear. roses the rose collection conserves traditional biological resources (mostly in a non-reproductive form) and research genetic resources (in reproductive and nonreproductive forms). traditional resources include wild roses, botanical roses and cultivated varieties. three thousand seven hundred (3,700) wild specimens are mainly of the species r. gallica, collected from more than 80 locations spread over 54 municipalities in france, with in general, 8 individuals per plot, depending on the size of the plot. three sites, near toulouse, france, were sampled more densely. around 1,500 rosa multiflora thunb. accessions were collected in semi-natural/sown (mcpd code 130) sites in france. botanical roses and cultivars 138 feugey et al genetic resources (2025), (s2), 135–146 were sampled from 2004 to 2018 in french rose gardens and the sangerhausen rosarium (germany). sampled cultivars were mostly bred during the 19th and 20th centuries. the high genetic diversity of these accessions enables research teams to assess the impact of evolutionary history, natural selection and human activity on the rosa genus. research resources are made up of mapping progenies or accessions selected for their particular characteristics. more specifically, these genetic resources include three diploid pseudo-f1 progenies linked by the use of the same male parent, an accession, abbreviated as rw, which is an interspecific hybrid of rosa wichurana crép., conserved in the jardin de bagatelle in paris, france. the three female parents are: rosa chinensis jacq. ‘old blush’, introduced into the united kingdom by james colvill around 1795 as ‘parsons’ pink china’, the ‘fairy’, polyantha variety bred in the united kingdom in 1932 by ann and john bentall, and ‘h190’, a dihaploid obtained by haploidization of tetraploid rosa ‘meirilocra’, commercially known as ‘zambra’ bred in 1980 by marie-louise meilland (meynet et al, 1994). the size of the three progenies is roughly 1,050, 100 and 590 offspring. these segregating progenies, whose parents have a contrasting phenotype for different morphological traits (recurrent blooming ability, flower duplication, shrub habit) or disease resistance, can be used to answer questions about the genetic determinism of traits of interest. conservation conservation types the main infrastructures (figure 2) used by rosepom for the conservation of accessions are fields (15.5ha, including 1.5ha protected by alt’carpo nets, nets designed to prevent the entry of codling moth), insect-proof tunnels (480m2), freezers at -20◦c and three cryopreservation tanks (total volume of 1,400l). rosepom also has access to glasshouses, a container yard, in vitro culture laboratories, cold chambers and -80◦c freezers. reproductive resources are conserved as plants in the field or in pots in insect-proof tunnels, as shoots in in vitro culture and as budwood in liquid nitrogen (cryopreservation). non-reproductive material such as leaves or petals are generally freeze-dried and stored at room temperature or in a cold chamber; dna extracts are stored in freezers at -20◦c. depending on various criteria, including in particular the value of interest, the rarity of the biological material and the type of biological material to be conserved, the conservation method or combination of conservation methods is defined according to four categories: (1) high level of conservation (at least two specimens) using a combination of conservation systems with special care for the specimen, (2) high level of conservation (two specimens) with one type of conservation system and special care for the specimen, (3) low level of conservation (one specimen) with special care for the specimen (i.e. propagation to be planned if the specimen is in danger of being lost), (4) low level of conservation (one specimen) without propagation even if the specimen is in danger of being lost. pome fruits for pome fruits, the highest level of conservation is currently used for old french varieties, which are part of the french national heritage, with: (1) a specimen in an orchard protected by alt’carpo nets, used to protect trees from the insects that transmit fire blight (one of the main causes of apple and pear trees loss), (2) another specimen in an insect-proof tunnel, which provides protection from damage by pests and diseases transmitted by insects, such as pear decline (candidatus phytoplasma pyri seemüller & schneider) transmitted by pear psylla, and (3) ideally a specimen in cryopreservation. cryopreservation is in progress for apples, though it remains a lengthy process requiring special care during the dehydration and freezing steps, which depend on the variety. for pears, the success rate is very genotype-dependant and is therefore not currently used. high-level conservation is also applied to specific apple and pear research resources, such as haploids and doubled haploids. these accessions, which tend to grow poorly in orchards, are maintained in in vitro culture chambers as well as in the field. conversely, the lowest level of conservation is currently used for some progeny collections where the loss of some accessions is less detrimental to the overall collection. in the orchard, trees are planted in the field with a distance of 4m between rows. space between trees within rows ranges from 1.2m for those used only for conservation, to 2.5m between plants in the row for trees used both for conservation and other purposes (observation of the fruit and architecture of the trees, use as female parent in cross-breeding for research or pre-breeding, production of budwood). depending on the intended use beyond conservation, trees are managed with minimal pruning and without fruit thinning or according to industrial fruit production methods or pruned specifically to provide budwood. orchards are irrigated, except when planted in naturally wet soils, and trellised, except for trees pruned for budwood production. trees are fertilized with organic fertilizers, except when the aim is precise fruit characterization – in this case, mineral fertilizers are applied to the soil. in all cases, mineral foliar fertilizers are applied. trees are subject to preventive or curative phytosanitary treatment for diseases such as scab, powdery mildew and pests, in particular rosy aphid (dysaphis plantaginea (passerini)) and pear psylla, in accordance with sustainable agriculture standards. potted plants are grown in 3l pots for apples and 7.5l pots for pears and maintained in screened tunnels. plants are spaced 25cm apart and heavily pruned to keep them small (about 1m high). automatic watering in the tunnels is scheduled twice a day at fixed times, with frequency, amount and method varying according genetic resources (2025), (s2), 135–146 pome fruits and roses brc for the future 139 figure 2. infrastructures used for the conservation of the accessions: a, field; b, orchards under alt’carpo nets; c, insect-proof tunnels; d, cryotanks; e, in vitro chamber for the conservation of living plant material; f, freezers at -20◦c for the conservation of non-reproductive material. to the season and weather conditions. phytosanitary treatments are applied when pests or diseases threaten the plant’s survival. in addition to conserving accessions in our facilities, the preservation of old french cultivars, is managed at the national level through a national network for pome fruits genetic resources. the aim is twofold: first, to ensure that each heritage cultivar is conserved in at least two locations by different institutions; and, second, to ensure that some heritage cultivars are not conserved in too many copies, in order to optimize human and material resources. roses for roses, shrubs are planted on agricultural tarpaulins in the field with 3.5m between rows and 1 or 1.5m between plants in the row. the large space between rows was chosen to accommodate farm equipment used for pome fruits. annual pruning is carried out when winter temperatures become stable, i.e. between late november and early january, followed by a copper treatment to protect the plant from fungal diseases. soil analysis is carried out to adjust the availability of nutrients to the needs of the plants. fertilizers are applied to support the flowering period. a powdery mildew treatment in early spring and one or two aphicides per season are applied to these resources. maintaining the edges of the agricultural tarpaulins and the base of the roses represents many hours of work. these plots are not irrigated. 140 feugey et al genetic resources (2025), (s2), 135–146 traditional resources are conserved at rosepom only in a non-reproductive form, such as leaves or dna. the wild accessions are conserved as reproductive forms in wild habitats, which in france are under the aegis of national botanical conservatories. traditional varieties are being preserved in rose gardens, which are owned by the public sector or by private enthusiasts. health monitoring for pome fruits, plant health monitoring is carried out throughout the year, but more intensively between april and october. it mainly involves visual inspection, but may also include analyses to confirm a diagnosis. the pathogens observed are mainly regulated organisms, including fire blight, european canker (neonectria ditissima (tulasne & c. tulasne) samuels & rossman) and pear decline. for roses, field plants are regularly inspected for the absence of visible symptoms. identity validation after propagation and authentication for pome fruits, after each cycle of propagation of the accessions, each plant planted in the field or in pots for conservation is tested with a set of five microsatellite (ssr) markers to check that the molecular identity of the plant obtained after propagation corresponds to the original plant. in the case of mutants, a visual comparison of the tree and fruit of the original plant and the new plant is necessary. this process generally takes one year (if the ssr control is sufficient) to three or four years (if it is necessary to check the fruit). the original trees are kept until all these checks are made. collaborative projects at european and international levels have enabled the comparison of molecular profile varieties from different collections in europe and the united states, leading to the assignment of a unique code to each variety. this work helped identify duplicates of a variety represented by different accessions under different names and to determine the most probable varietal name of an accession with the same profile. this does not replace the historical authentication of varieties (i.e. checking the accuracy of the given variety name) but improves the knowledge of the identity of the accessions (durel et al, 2023). for roses, new plants are tested after propagation, where necessary with ssr markers, to validate their conformity to the mother plant. authentication of old cultivars is based on historical data and the expertise of rose garden managers, but it is rarely fully certain. accession description molecular and phenotypic characterization covers both traditional and breeding/research resources and is essential to ensure their trueness-to-type. to date, molecular characterization has mainly been carried out using ssr markers revealed by capillary electrophoresis or sequencing. additionally, single nucleotide polymorphism (snp) markers are now being used by research projects and revealed by high-throughput technologies. so far, 68% of pome fruit accessions and 41% of rose accessions have been genotyped. rosepom carries out a part of the characterizations and also centralizes data generated by research teams using the rosepom’s genetic resources or supplying rosepom with new resources and associated data. for pome fruits, rosepom uses a minimum list of descriptors for the management of pome fruit collections, including phenotypic characteristics (such as flowering time and harvest time) and simple morphological characteristics (such as fruit colour, shape and size, and tree habit). characterization is based on the european cooperative programme for plant genetic resources (ecpgr) reference documents (lateur et al, 2022a,b) and the upov guidelines for the distinctness, uniformity and stability of apple (upov, 2023) and pear (upov, 2000). phenotypic characterization is carried out for certain characteristics (disease resistance, flowering, architecture, fruit quality, etc.) in the field, in the greenhouse or the laboratory. for roses, the first characteristics used to help verify the identity of an accession are the colour of the flower, the number of petals and the plant’s habit. many other traits of morphological, disease susceptibility or biochemical nature, for example, can be phenotyped through research projects. the data obtained is then centralized by the rosepom information system. distribution this material is regularly used in research and breeding programmes and is also distributed to thirdparty organizations and the international scientific community. around 1,000 to 1,500 accessions are distributed per year mainly through collaborative projects. depending on the availability of material, rosepom will distribute, in accordance with national and international regulations in force: (1) plant material in the form of plants or samples (grafts, cutting, pollen, leaves, petals, fruits, etc.), (2) extracted plant material such as dna, and (3) data associated with this plant material, such as phenotypic or molecular data. the distribution service is available to public or private research bodies, institutes or breeders mainly in the frame of cooperative projects. it is also available to public or private actors in biodiversity conservation (conservatories managed by amateur associations or local authorities, nurseries specialized in the sale of old cultivars). rosepom offers the opportunity to obtain resources on request by emailing crb-rosepom-contact@inrae.fr. requests must be made in advance, considering legal and administrative constraints and the nature of the material requested. distributions are made after the signature of a contract between inrae and the recipient, with different requirements for the use of the resource, depending on the nature of the resource and the use genetic resources (2025), (s2), 135–146 pome fruits and roses brc for the future 141 planned by the recipient. rosepom does not guarantee the availability or quantity of requested material. quality management systems rosepom’s quality management system has been iso 9001:2015 certified since february 2023. the certification covers the organization of activities related to the acquisition, conservation, characterization and distribution of rosepom’s genetic resources and associated data. the aim is to effectively meet the needs of its users. it ensures the traceability of living plant material, nonreproductive plant material and associated data. the orchard is managed in accordance with the iso 14001 environmental management system and the french ‘vergers ecoresponsables’ label. use of the resources in breeding rosepom collections have long been used as a source of germplasm for pre-breeding and breeding pome fruit programmes. these programmes have resulted in the release of 36 new plant breeders’ rights (pbr) varieties. pre-breeding programmes are currently being carried out in partnership with cep-innovation and novadi breeding companies. rosepom’s wild species and old apple and pear cultivars have long been tested for resistance to diseases and pests, particularly scab, fire blight, powdery mildew and pear psylla, and the more interesting accessions have been used to introduce resistance genes and qtls coming from different genetic backgrounds by crossing with modern cultivars. the improved genetic stocks obtained have been introduced into rosepom’s breeding material collections for use in the development of new varieties of dessert apple, cider apple and pear. collections from research programmes, particularly mapping progenies established to study pest and disease resistance and fruit quality, have also been used to select improved material for dessert apple and pear, with the knowledge gained from these mapping progenies allowing the use of marker-assisted selection to pyramiding resistances to diseases and pests western and eastern pyrus species from rosepom have been used in rootstock breeding to produce rootstocks more tolerant to global warming (six taxa including: p. cordata desv. , p. amygdaliformis vill., p. amygdaliformis var. persica (pers.) bornm., p. elaeagrifolia pall., p. syriaca boiss.), biotic stress (p. calleryana decne. and p. betulifolia bunge) or to induce dwarfing of scions (p. nivalis jacq.). perry pear (p. nivalis jacq.) cultivars conserved by rosepom have been used in the breeding of dwarfing rootstocks, as cytochemical analyses have shown that this compartment is divided into two sections: one of triploid genotypes, with a high vigour segregation in their progenies, and one of diploid genotypes, which are very homogeneous in terms of vigour. this triploid compartment has been used to create dwarfing rootstocks. the cultivar collections of apple and pear varieties (dessert apple, cider apple, pear, rootstocks and ornamental apple and pear) have been used as reference material for dus testing for official variety registration and pbr at the national and european levels. thanks to these large collections, inrae is an entrusted examination office to carry out the dus technical examination on behalf of the community plant variety office (cpvo). in research identity, diversity, relatedness and traits of interest are elucidated by characterizing the resources of rosepom. pome fruits characterizing genetic diversity. more than 20,000 apple cultivars are documented worldwide. an indepth analysis of the genetic diversity, structure and relatedness of old and recent apple cultivars conserved in rosepom and in institutional and associated germplasm collections at the french and european levels has been carried out by comparing more than 3,000 accessions from more than 20 european countries, usa and canada, using ssr genotypic profiles (leforestier et al, 2015; suprun et al, 2015; lassois et al, 2016; urrestarazu et al, 2016; durel et al, 2023). the highlighted main characteristics are a weak geographic structure and an important gene flow on a european scale, resulting from the numerous exchanges of graftwood over the centuries. a similar approach is also currently developed for european pear (denancé et al, 2019; bassil et al, 2023; durel et al, 2023). an apple core collection (287 accessions) has been developed from rosepom (lassois et al, 2016) and further phenotyped for various traits (lopez et al, 2015) including scab and fire blight resistance. further, large multi-generation pedigrees were reconstructed from very old founders to elite cultivars thanks to a large snp dataset, enlightening the history of apple empirical selection and the unexpected major role of a very limited number of founders such as the french cultivar ‘reinette franche’ and ‘margil’ from the renaissance period (muranty et al, 2020; howard et al, 2021). the amazing empirical selection of a high frequency of triploid cultivars (~20% of old apple cultivars are triploids) was also deciphered thanks to snp-based pedigree analyses (howard et al, 2023). thanks to this work, inrae’s elite breeding population is now connected back to old founders with an accurate description of the transmission of genomic fragments across generations, giving a complete picture of haplotype sharing among all individuals. genetic mapping of disease/pest resistance factors. identifying new resistance genes/qtls in pome fruit is an important goal to help breeding, especially for those pathosystems not yet sufficiently explored. thanks to the conserved mapping progenies, numerous qtls for pear resistance against scab, fire blight and psylla were identified (montanari et al, 2015, 2016b; perchepied et al, 2015, 2016). in apple, a major resistance gene 142 feugey et al genetic resources (2025), (s2), 135–146 against rosy apple aphids was fine-mapped thanks to plant accessions conserved in rosepom (dall’agata et al, 2018) with further development of tightly linked markers useful for breeding. significant qtl (epistatic) interactions have been observed in both apple and pear progenies, which may correspond to the combined effect of favourable alleles for two or three genes along a metabolic pathway to promote strong resistance (van de weg et al (2018) for apple/fire blight, perchepied et al (2016) for pear/psylla). in traditional accessions, attempted genome-wide association studies (gwas) for both apple scab and fire blight have so far been rather unsuccessful, although scab tests have been conducted with various strains in the greenhouse to account for v. inaequalis genetic diversity. for the v. inaequalisapple interaction, numerous low-frequency gene-forgene relationships in the core collection studied may have hampered qtl detection and may require a redesign of this collection. in contrast, gwas performed for flowering and harvest dates or for eco-physiological traits successfully identified genomic regions controlling these traits (urrestarazu et al, 2017; coupel-ledru et al, 2022). three scab resistance qtls identified in the ‘tn108’ x ‘fiesta’ progeny were subjected to in-depth analysis with the ultimate goal of cloning them and analyzing the metabolic pathway they control, in particular by looking for accurate co-localizations of qtls controlling either disease resistance or metabolic compounds (lapous et al, 2023). one (qt1) exhibits a specific interaction with v. inaequalis strains and has been shown to finely co-localize with the major scab resistance gene rvi6/vf. for the other two (qf11 and qf17), haplotype-sharing analysis allowed us to trace them in various genetic backgrounds along the reconstructed pedigree. in particular, qf17, initially mapped at almost the same position in two unrelated backgrounds, actually corresponded to the same snpbased haploblock inherited from an as-yet-unknown common (and certainly ancient) ancestor (durel, 2019). the conserved progenies also allowed a very original work to be carried out, analyzing the combination of genetic (intrinsic) and plant resistance inducer (pri)induced resistances to diversify/complement apple disease control methods. putative synergistic or antagonistic effects between the two types of resistance were investigated using a genetic mapping approach. the f1 population ‘tn10-8’ x ‘fiesta’ was genotyped and phenotyped for scab and fire blight resistance without and with pri pretreatment (with acibenzolar-s-methyl – asm -, a functional analogue of salicylic acid). qtls (including qt1, qf11, and qf17) were detected in both situations, but with a rather limited impact of asm on qtls effect, indicating a mainly additive effect of both resistance mechanisms (bénéjam et al, 2021, 2024). in addition, other traits of interest were phenotyped on apple and pear mapping progenies, namely fruit texture/colour and sublethal genes (ben-sadok et al, 2015; montanari et al, 2016a; moriya et al, 2017). apple genome sequencing. one of the double haploid accessions conserved by rosepom, the double haploid ‘golden delicious’ line (gddh13), also coded x9273, obtained at inra in 1963, has been used, thanks to the homozygosity of this line, to produce a high-quality de novo assembly of the apple genome (daccord et al, 2017). roses characterizing genetic diversity. in the cultivated compartment, with more than 30,000 varieties, rose breeding activity has been very intensive, especially since the 19th century when old varieties of various origins and wild accessions were hybridized. the biological resources of 1,228 garden roses (botanical roses and old varieties) stored in rosepom (leaves and dna) were genetically characterized using 32 microsatellite primer pairs (ssr). the genetic structure was revealed. the study of its evolution during the 19th century showed that the genetic background of european rose hybrids displayed a shift from an old european genetic background to an asian one (liorzou et al, 2016). this result is consistent with the introgression of old chinese garden roses into european hybrids, especially for their continuous flowering. indeed, the genotyping of the copia allele of the roksn gene, a homologue of tfl1 responsible for continuous flowering, on 270 accessions showed that this allele was selected in europe during the 19th century (soufflet-freslon et al, 2021). all this work was carried out using an interdisciplinary approach, collaborating with historians to answer the same questions through both genetic resources and archival research, while considering the historical construction of rose diversity. the 20th century was marked by the intensification of specific uses, such as cut and pot roses and the globalization of rose production and markets. these specificities were studied in a larger interdisciplinary approach with geographers, sociologists, economists and historians. accessions were collected from rose gardens and private companies to constitute a large sample representative of time periods, usages and kinds of breeders. the first results on 1,796 accessions genotyped with 23 microsatellite primer pairs showed a decrease in genetic diversity during the 20th century, in line with the dominance of tea hybrids. cut roses appear to be a subset of garden roses. in the wild compartment, 219 rosa gallica populations represented by 901 plants collected in france by rosepom or by associated research teams and partners throughout europe were genotyped by ssr sequencing of amplified fragments from 29 ssr primer pairs together with 717 cultivated accessions in order to investigate the diversity structure and origin of french populations. diversity in france is much more structured than elsewhere in europe, which can reveal multiple origins (pawula, 2023). genetic resources (2025), (s2), 135–146 pome fruits and roses brc for the future 143 furthermore, the identification of clones and sports (vegetative mutants), thanks to genotyping and phenotyping characterizations, is in progress. genetic mapping of traits of interest. with research genetic resources, genetic maps have been developed using ssr or snp markers, allowing to anchor the genome sequence of old blush (hibrand-saint-oyant et al, 2018) and to identify regions with important genes controlling traits of interest, such as floral characteristics, fragrance, disease resistance (lopezarias et al, 2020) and prickle development (zhou et al, 2020). for example, a study on the fragrance components (more than 30 components) was carried out on two pseudo-f1 populations conserved in rosepom’s fields. three genes were identified for the production of: geraniol, rhnudx1-1 located on linkage group 2 (magnard et al, 2015); 2-phenylethanol, rhpaas located on linkage group 6 (roccia et al, 2019); and farnesol, rhnudx1-2 located on linkage group 7 (sun et al, 2020). all of these research projects have greatly enriched rosepom’s collections, either in terms of biological material or associated data. these resources and associated data are valuable for expertise, e.g. in dus testing for pome fruits, or for supporting urban management decisions through knowledge of wild r. gallica populations. outreach from an outreach point of view, rosepom is regularly presented to various audiences, including the general public, schoolchildren, students, professionals and scientists from other disciplines, such as historians, who see rosepom as a living archive. this is achieved through visits, stands at trade fairs and conferences. participation in networks and working groups rosepom is one of the inrae biological resource centers that have joined forces with other french public institutions (cirad – french agricultural research centre for international development, ird – french national research institute for sustainable development, and institut agro – french institute for higher education & research in food, agriculture & the environment) in the brc4plants pillar (bergheaud et al, 2025) of the national agronomic resources research infrastructure (agrobrc-rare). rosepom, as a member of the brc4plants pillar, is linked to the national structure coordinating plant genetic resources (duval et al, 2023). since 2023, the pome fruit collection has been one of the collections for which inrae is recognized as an official manager of plant genetic resources for food and agriculture by the french ministry of agriculture. this collection is also labelled as a ‘national collection’ by the conservatoire des collections végétales spécialisées (conservatory of specialized plant collections). at the national level, rosepom leads a national cooperative network on pome fruit genetic resources involving public and private partners, in accordance with the strategy drawn up as part of the national charter for the conservation of genetic resources. it is also involved as an expert in the national cooperation network on roses, whose members are french rose gardens. at the international level, rosepom is involved in the european cooperative programme for plant genetic resources (ecpgr) malus/pyrus working group, which helps to define common standards and protocols for these species. it is also involved in the ecpgr cryopreservation working group. conclusion the rosepom brc plays an important role in the conservation of genetic resources of major fruit and ornamental plants, especially pome fruits. it has acquired considerable expertise in understanding the diversity of these genetic resources, both wild and cultivated. the conservation and knowledge of these genetic resources have proved very useful for three main activities: breeding, research and dus testing. interest in these resources is growing as crops are subject to many changes, in particular due to new regulations on the use of phytosanitary products, the emergence of pathogens and pests, and climate change. however, the conservation of these resources is costly in terms of human and material resources and faces multiple constraints. in order to maintain the collections in good condition despite these challenges, the management of all stages of the process must be carefully organized. the quality management system helps to achieve this objective. as highlighted by engels et al (2024), collaborative networks are of primary importance for an efficient genetic resource conservation system. the organization in a network with different parties at the national and european levels makes it possible to share experiences, reflections and work to reduce the technical and financial constraints and optimize the process of conservation and valorization of these resources. nevertheless, in order to enhance this activity of common interest for society, we emphasize the need to continuously raise awareness among the general public, researchers and policymakers of the importance of these genetic resources in view of their potential use in the future. author contributions writing – original: draft ap and lf, with contributions from all co-authors. rose collections were described especially by ap, ff, tt, ac. pome fruits collections were described especially by lf, ced, mhs, kg, alh. photos were provided by kg and tt. writing editing: all authors, especially ap, lf, hm, fl. review editing:ap and lf. 144 feugey et al genetic resources (2025), (s2), 135–146 conflict of interest statement the authors have no conflicts of interest to report. acknowledgements the authors would like to thank all those who contribute on a daily basis to the smooth running of rosepom. the authors especially thank the horticultural experimental unit for providing the fields needed to maintain the genetic resources and the phenotic platform for providing the greenhouses and insect-proof tunnels. the authors would also like to thank the anonymous reviewers and the editors for their helpful comments and editing. references alercia, a., diulgheroff, s., and mackay, m. 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(2025) “novel germplasm of tepary and other phaseolus bean wild relatives from dry areas of southwestern usa”, genetic resources, 6(12), pp. 131–152. doi: 10.46265/genresj.lsbj4572. © copyright 2025 the authors. this is an open access article distributed under the terms of the creative commons attribution license (cc by 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. daniel g. deboucka,*, richard c. prattb, sarah dohlec, timothy porchd, marcela santaellaa, luis guillermo santosa and milan o. urbane agenetic resources program, international center for tropical agriculture (ciat), palmira, colombia bnew mexico state university, department of plant and environmental sciences, las cruces, new mexico, usa cunited states department of agriculture, agriculture research service (usda-ars), pullman, washington, usa dunited states department of agriculture, agriculture research service (usda-ars), mayagüez, puerto rico ebean program, international center for tropical agriculture (ciat), palmira, colombia now at international center for biosaline agriculture (icba), dubai, united arab emirates * corresponding author: daniel debouck (d.debouck@cgiar.org) introduction the megadrought of western north america with effects extending south to the central american dry corridor is a current and historic climatic feature (williams et al, 2020; mckinnon et al, 2021; ipcc, 2023; chen et al, 2025). that water-limited area is a huge arc extending in the north from saskatchewan (canada) to north dakota and nebraska south to new mexico (usa), chihuahua and zacatecas (mexico) and ending in eastern guatemala. the present combination of record high temperatures, prolonged drought and limited water resources can have profound implications for agricultural systems. increasingly, growing urban areas and agriculture will compete for ever scarcer fresh-water resources. farmers in remote areas will likely seek grains with sufficient value (e.g. barley for breweries, beans for export to mexico and central america, quinoa for specialty markets) to cover production and postharvest costs such as transportation. high-value grains capable of providing more stable income with a lower water requirement will be essential. crops and cultivars highly resistant to drought and heat stresses are now high on the agenda of agronomists and breeders in that area (parker et al, 2023; silber-coats et al, 2025), but also in other arid regions of the world e.g. africa (assefa et al, 2019). while the vast american arc aforementioned currently has many introduced crops, tepary bean (phaseolus acutifolius asa gray) has long been known as a native drought and heat tolerant crop (freeman, 1912) as it was grown by the pre-columbian peoples of the southwest (carter, 1945; kaplan, 1956); seeds have been dated by accelerator mass spectrometry to at least 2,000 years before present (kaplan and lynch, 1999). in addition to drought https://doi.org/10.46265/genresj.lsbj4572 https://www.genresj.org http://10.46265/genresj.lsbj4572 mailto:d.debouck%40cgiar.org?subject= genetic resources (2025), 6(12), 131–152132 debouck et al (parsons and howe, 1984; barrera et al, 2024) and high temperature tolerance (lin and markhart iii, 1996; cruz et al, 2023), tepary is resistant to several diseases (ashy stem blight: miklas et al, 1998; common bacterial blight: coyne et al, 1963; bean golden mosaic virus (later shown to be bean golden yellow mosaic virus): miklas and santiago, 1996; bean golden yellow mosaic virus: porch et al, 2021; bean common mosaic necrosis virus: bornowski et al, 2023; rust: miklas and stavely, 1998) and pests (bruchids: shade et al, 1987; jiménez et al, 2017; thrip and leafhopper: porch and estévez de jensen, 2024). further, some accessions of p. acutifolius are tolerant to low temperatures (souter et al, 2017) and salinity (bayuelo-jiménez et al, 2002). not surprisingly, there have been many attempts to transfer the useful traits of tepary into the common bean through interspecific hybridization (pratt and nabhan, 1988), but with limited success because of the genetic distance between them (debouck, 1999; barrera et al, 2022). however, the technological context of bean breeding is changing, given the development of the genome sequences for both species, marker assisted selection, and genomic technologies (schmutz et al, 2014; moghaddham et al, 2021; parker et al, 2023; wang et al, 2024). genome editing through the crispr-cas 9 of 2012 and subsequent improvements are also quickly changing the field of potential pathways for cultivar development (bandyopadhyay et al, 2020; jha et al, 2022; singh et al, 2024). there are a lot of possibilities given the high level of synteny between tepary and common bean (gujaria-verma et al, 2016; moghaddham et al, 2021). transfer of candidate genes from common bean for highly heritable traits such as seed size, seed colour, growth habit, and disease and pest resistance, may prove more expedient than attempting to transfer complex polygenic traits such as heat and/or drought tolerance from tepary into common bean. incidentally, the approach to breed tepary itself is increasingly being considered in its traditional areas of cultivation (pratt et al, 2023) but also in sub-saharan africa (mwale et al, 2020) and in the tropics (porch et al, 2024). so, it might be faster to breed tepary itself with molecular markers developed using the reference genome sequences of both species, based on the high level of synteny between them. further, the food processing industry may offer opportunities for bean seed types outside the current market classes (voysest and dessert, 1991) and the traditional ways of cooking. these factors are likely contributing to the increasing trend in p. acutifolius germplasm requests from the united states department of agriculture (usda) genebank (dohle, 2024). the success of these new approaches depends heavily on the availability of genetic variability in tepary bean. unfortunately, a lot of landraces went extinct, first when the spaniards introduced new irrigation techniques into mesoamerica in the 16th century onwards, and second in the 1930s onwards when water pumps with fuel-based engines changed the watering systems in the southwest and other parts of aridoamerica (nabhan and felger, 1978; pratt et al, 2023). some cultivated germplasm has been collected from the historic range of tepary cultivation from northern arizona (carter, 1945; nabhan and felger, 1978) to guanacaste in costa rica (debouck, 1992), and it is conserved in several genebanks. once internal duplicates are identified, there are about 100 different landraces of tepary bean in the major germplasm collections for that crop: usda, the international center for tropical agriculture (ciat) and inifap (instituto nacional de investigaciones forestales, agrícolas y pecuarias of mexico). given this inadequate representation of the intraspecific genetic variation in the genebanks and their obligation to anticipate breeders’ needs instead of reacting to them, it is imperative to expand the reservoir of available genetic diversity. the greatest benefit will come from the two wild forms of tepary (often named var. acutifolius asa gray and var. tenuifolius asa gray: delgado-salinas, 1985). the distribution of these wild forms extends from the southwestern usa (central arizona, southern new mexico, and the trans-pecos region of texas) to michoacán, mexico (nabhan and felger, 1978; debouck, 2021). while there might be ecological reasons for the recognition of these two varieties (pratt and nabhan, 1988), genetic evidence indicates that it is still an open question (muñoz-florez et al, 2006; blair et al, 2012). a sister species and wild relative of tepary, p. montanus brandegee (synonym p. parvifolius freytag), strikingly similar in appearance to, but separated from var. tenuifolius based on biochemical (florez-ramos et al, 2004) and molecular evidence (zink and nagl, 1998a; muñoz-florez et al, 2006; blair et al, 2012), extends from southeastern arizona down to jalapa, guatemala through several regions of the pacific side of mexico (debouck, 2021). because of its presence in the chiricahua mountains (debouck, 2019), an additional question is whether p. montanus is present in western new mexico (the arizona-new mexico state border line likely not being an ecological barrier, at least up to the continental divide). another group of wild beans and with some genetic relationship with common bean is section rugosi (zink and nagl, 1998b; delgado-salinas et al, 2006). it includes p. angustissimus asa gray, p. carterae freytag & debouck and p. filiformis bentham (freytag and debouck, 2002; dohle et al, 2019). gene transfer to common bean from these relatives seems very difficult (maréchal and baudoin, 1978). which traits could be of interest for introgression into tepary or into common bean? wild teparies and these rugosi species might be tolerant to drought, salinity and freezing temperatures (bayuelo-jiménez et al, 2002; balasubramanian et al, 2004). in conclusion, the main purpose of this project is to increase the representation of wild teparies and species of the section rugosi from new mexico, where collection to date has been inadequate, in the usda collection and later that of ciat. further, given the rapid progress in comparative pangenome analysis (e.g. khan et al, 2024), it might be good foresight to collect other phaseolus species from new mexico as the opportunity arises in the field. at the start of this project in mid-2023 there were two accessions of wild p. acutifolius (pi 640990, a collection by oliver wendel norvell of november 1969 and pi 702622, a collection by richard pratt of october 2017; see also figure 1) and two accessions of p. angustissimus (pi 535272, a collection by russ buhrow and pi 535273, a collection by george frederick freytag) from new mexico in the genebanks of usda-pullman and ciat-palmira. accordingly, a joint exploration was carried out in fall 2023. unfortunately, as it often happens in desert areas, 2023 was a special year with below normal, erratic and late rainfalls in the counties of interest in southern new mexico. however, several populations of wild teparies yielded some seeds from dried genetic resources (2025), 6(12), 131–152 new wild tepary bean germplasm from sw usa 133 plants of the previous year, as did return visits to three sites (namely big burro mountains and two sites in the organ mountains) with delayed flowering (debouck et al, 2023). those collections have been successfully increased in the glasshouse of the usda pullman during the spring of 2024 (s. dohle, personal communication, 2024). finally, the finding of a new species of rhizobium in nodules of p. filiformis tolerant to salinity and high temperatures in baja california (rocha et al, 2020) justifies the continued sampling (initiated in 2023) of rhizosphere microorganisms in wild teparies and rugosi species to capture effective nitrogen fixation under these abiotic stresses. materials and methods populations of target species the team used two primary kinds of information to decide where to sample: the study of herbaria (identified by their acronyms: thiers, 2023) keeping samples of phaseolus sensu stricto and the sightings posted on inaturalist (https://www. inaturalist.org/). the former source by personal visits to 86 herbaria collections since 1978 (debouck, 2021, p. 102103) and the study of 16 herbaria through the southwestern environmental information network (seinet) portal (https://swbiodiversity.org/seinet/collections/list.php/) in april 2023 (supplemental material 1) gave a total of 189 populations for the state of new mexico for six species (p. acutifolius (29 populations), p. angustissimus (67), p. filiformis (9), p. grayanus (37), p. maculatus (40) and p. parvulus (7)) covering a period of field collecting between 1849–2014 (supplemental table 1). for the 29 populations of wild tepary, the three coordinates were provided by the collector(s) only for three locations (one by global positioning system (gps)). the second source of information provided gps coordinates and a colour picture collected by citizens in the year 2024, and some in 2023. in addition, photos and descriptions of habitat preferences of wild teparies were provided to several area hikers. they subsequently reported to us possible sightings during their hikes. these multiple strategies allowed us to find concordance between prospective collection sites and areas where (more) favourable seasonal rainfall patterns had occurred. on the other hand, germplasm of the type localities from original descriptions was also taken into account, given its importance for future taxonomic and genomic studies. for new mexico, this representation of type materials in genebanks refers to p. acutifolius var. tenuifolius (‘near the copper mines, new mexico’ in october 1851), p. angustissimus (‘hill-sides above doña ana’ in july 1851), p. grayanus (‘san luís mountains’ in september 1893) and p. parvulus (‘pinos altos mountains’ in august 1880) (wooton and standley, 1915; freytag and debouck, 2002). timing of collection in collecting in the northern chihuahuan desert and surrounding dry areas (dick-peddie, 1993; cornett, 2013), it was critically important to know how the populations revealed by the two approaches aforementioned had their flowering and pod setting affected by the north american monsoon, in essence variable from year to year (nolin and hall-mckim 2006; reichenbacher and peachey, 2025). based on the experience of 2023, monitoring of rain accumulation and distribution was started in july 2024 through late september 2024 for an area covering southwestern new mexico (roughly south of 33° 15’ latitude north and from 106° 27’ longitude west) extending beyond the state border with arizona to include cochise and greenlee counties. that area was the one containing most populations of target species and the object of the grants. rainfall information provided by the us drought monitor (https://www.droughtmonitor. unl.edu) (supplemental figure 1) was complemented with invaluable field visits and consultations with staff of the us forest service (fs) and the bureau of land management (blm). such field visits indicated that the peloncillo, the florida and the southern black range mountains (almost no rain) would not be fruitful in terms of seed production, and to concentrate exploration instead in 2024 in the gila region (see figure 1). for example, information provided by a ranger from the southwestern research station (srs) near portal, arizona, on 23 september 2024 that water was still running in turkey creek while not in cave creek in the chiricahua mountains was key for the team to decide to cross the border into arizona in search of p. montanus (see discussion). implements used during field work were indicated elsewhere (debouck, 1988; moss and guarino, 1995). gps coordinates were obtained from a garmin gpsmap 62s receiver and checked against a second gps receiver (garmin etrex 32x) for any significant deviation (that did not happen: see table 3 in debouck et al, 2025), while primary elevation readings were provided by a barometric altimeter thommen 3d-16 (0-5000). the application onx backcountry (onx maps inc., missoula, montana) installed on the smartphone of one participant gave further validation to these direct readings, as well as valuable data such as offline maps, names of places and landmarks, and property ownership. trying to get the right geographic coordinates was doubly important: first, to have the possibility of getting back to the same population for any additional sampling in the same or subsequent season, and second, to monitor the fate of these populations over time. many collections by edward lee greene, henry hurd rusby, elmer ottis wooton and charles wright in the 1850s–1900s lack accurate data about location, making our ability to monitor changes since these early observations difficult, if not impossible. further, ensuring the exact coordinates is critically important in relation to future germplasm evaluation for stresses related to location such as drought, extreme temperatures or soil limitations (salinity, nutrient deficiency or excess in minor elements). for managing the time at the collection sites and to improve sampling, communication was critical, and two pairs of radios (‘walkie-talkies’) were found useful to link members of the team looking for plants in different parts of a single location. sampling and data collection it was a deliberate strategy to sample as many plants as possible at a single site and therefore the team often worked in pairs or individually for half an hour (often longer) in search of additional plants in a population. the reason for this strategy aimed at collecting genetic diversity lies in the cleistogamous reproduction of tepary (lord and kohorn, 1986). the sampling of the populations should also be targeted at the conservation of rare alleles. the example of resistance to bruchids in wild common bean (osborn et al, 1986, 1988) – where plants having the right arcelin causing antibiosis were less than 20% in the original https://www.inaturalist.org/ https://www.inaturalist.org/ https://swbiodiversity.org/seinet/collections/list.php/ https://www.droughtmonitor.unl.edu https://www.droughtmonitor.unl.edu genetic resources (2025), 6(12), 131–152134 debouck et al populations – clearly advocates for thorough sampling. the data taken at collection sites follow the guidelines proposed elsewhere (debouck, 1988; moss and guarino, 1995), and were reflected in the labels of the herbarium vouchers (supplemental material 2). vegetation types were described based on the classification of castetter (1956), dick-peddie (1993) and o’kane (2025). vernacular names of plants followed hitchcock (1971a,b) and dodson’s guide (2012). gps coordinates were checked against the atlas and gazetteer of arizona (delorme, 2008) and new mexico (delorme, 2012) and web-based topographic maps provided by caltopo – backcountry mapping (https://caltopo.com/) and the us geological survey (https://www.usgs.gov/programs/ national-geospatial-program/topographic-maps). these sources and the comprehensive reference guide of place names in new mexico produced by julyan (1998) enable the checking of place names. results general in 2024, a total of 28 populations were found, including two disclosed in 2023 (table 1 and detailed information about each population in supplemental material 2) for six species (phaseolus acutifolius and its two variants, p. angustissimus, p. filiformis, p. grayanus, p. maculatus and p. montanus, the latter in southeastern arizona) during a 9-day exploration. two populations of wild tepary disclosed in 2023 (#3387, #3390) were revisited in 2024 (successfully) to collect more seed for germplasm conservation; for all other populations found in 2023, prior scouting visits indicated no plant development due to lack of rains (and verified for populations #3392 and #3396). seeds were collected for conservation for all populations except #3407 (too early), and herbarium specimens were collected for all except #3420 and #3421 (too late) (see supplemental table 2). table 1. populations found in chronological order (those highlighted in grey refer to populations found in 2023 for which seeds were sought and found in 2024). collection no. species latitude n longitude w elevation (masl) date 3406 acutifolius 32° 17’ 48.4” 106° 36’ 38.4” 1,565 3-oct-2024 3407 acutifolius 32° 18’ 19.0” 106° 35’ 32.0” 1,740 3-oct-2024 3408 acutifolius 32° 18’ 44.4” 106° 34’ 43.2” 1,859 3-oct-2024 3387 acutifolius 32° 22’ 08.6” 106° 33’ 34.7” 1,750 4-oct-2024 3409 acutifolius 32° 21’ 50.8” 106° 33’ 55.5” 1,893 4-oct-2024 3390 acutifolius 32° 20’ 16.6” 106° 35’ 10.7” 1,757 5-oct-2024 3410 acutifolius 32° 17’ 33.5” 106° 35’ 40.2” 1,639 5-oct-2024 3411 filiformis 32° 17’ 34.0” 106° 35’ 39.1” 1,647 5-oct-2024 3412 acutifolius 32° 02’ 18.8” 106° 57’ 23.0” 1,288 6-oct-2024 3413 acutifolius 31° 53’ 13.6” 109° 12’ 30.6” 1,663 7-oct-2024 3414 montanus 31° 53’ 13.6” 109° 12’ 30.6” 1,663 7-oct-2024 3415 grayanus 31° 54’ 33.2” 109° 15’ 09.3” 1,967 7-oct-2024 3416 montanus 31° 54’ 33.1” 109° 15’ 09.7” 1,964 7-oct-2024 3417 montanus 31° 55’ 44.6” 109° 13’ 10.4” 1,693 7-oct-2024 3418 acutifolius 31° 55’ 44.6” 109° 13’ 10.4” 1,693 7-oct-2024 3419 maculatus 32° 39’ 08.3” 108° 31’ 56.2” 1,781 8-oct-2024 3420 acutifolius 32° 39’ 01.3” 108° 31’ 56.8” 1,790 8-oct-2024 3421 acutifolius 32° 47’ 17.8” 108° 29’ 38.2” 1,518 8-oct-2024 3422 acutifolius 32° 51’ 04.8” 108° 35’ 26.0” 1,327 8-oct-2024 3423 acutifolius 33° 02’ 59.0” 108° 30’ 03.4” 1,535 9-oct-2024 3424 angustissimus 32° 57’ 57.6” 108° 33’ 48.2” 1,412 9-oct-2024 3425 maculatus 32° 53’ 32.7” 108° 14’ 06.4” 1,991 10-oct-2024 3426 grayanus 33° 07’ 1.3” 108° 11’ 57.5” 2,248 10-oct-2024 3427 maculatus 33° 13’ 46.2” 108° 15’ 52.3” 1,744 10-oct-2024 3428 acutifolius 33° 13’ 35.9” 108° 16’ 11.5” 1,795 10-oct-2024 3429 acutifolius 33° 10’ 45.0” 108° 12’ 19.2” 1,698 10-oct-2024 3430 acutifolius 33° 05’ 16.6” 109° 05’ 21.8” 1,827 11-oct-2024 3431 acutifolius 32° 57’ 04.6” 108° 57’ 35.8” 1,918 11-oct-2024 https://caltopo.com/ https://www.usgs.gov/programs/national-geospatial-program/topographic-maps https://www.usgs.gov/programs/national-geospatial-program/topographic-maps genetic resources (2025), 6(12), 131–152 new wild tepary bean germplasm from sw usa 135 seeds were taken to the usda ars national plant germplasm system greenhouses located in pullman, washington, for increase, while 96 herbarium specimens were deposited at the new mexico state university (nmsu) biology herbarium (nmc) for conservation and further distribution. plants from seeds for all the annual species collected during the fall 2024 plant exploration are currently growing in the greenhouses at usda pullman (supplemental figure 2). populations may be accessioned and available for distribution likely from 2026 onwards. as can be seen in figure 1, the collections of 2023 and 2024 resulted in a significant increase in the usda collection of wild tepary. samples of nodules or soil around the rhizosphere of the collected plants were obtained for all populations (except two wild tepary populations #3407 and #3409; supplemental table 2). soil and microbe samples are conserved at the new mexico state university, las cruces, new mexico, usa. figure 1. map showing the number and location of accessions of wild tepary (phaseolus acutifolius) in the usda collection before (red dots) and after (blue dots) the explorations of 2023 and 2024. more information about the collections made east of las cruces, new mexico, usa, can be found in supplemental figure 3. per species phaseolus acutifolius in 2024, 18 populations were found, most of them at seed dispersal stage. some populations were noted with relatively broad leaflets (#3387, #3390, #3406, #3407, #3408, #3409, #3410, #3412), while others displayed very narrow ones (#3413, #3418, #3422, #3423, #3428, #3429, #3430, #3431). two populations (#3420, #3421) were found with all leaves completely dry, making it impossible to prepare good herbarium specimens. in addition to variation in leaf shape, seed colour and size varied (figure 2). the smallest 100-seed weight was for #3413 at 1.7g, and the largest was for #3408 and #3412, both at 3.5g. one population (#3428) was found on the slope close to the caves in the gila cliff dwellings national monument. this seems to be the northernmost latitude for p. acutifolius in new mexico, genetic resources (2025), 6(12), 131–152136 debouck et al and apparently, upon evidence available to us, the first record for catron county. an inaturalist report indicated the presence of wild tepary in the aden lava flow wilderness study area, doña ana county, new mexico; before reaching the given coordinates, population #3412 (figure 3 top) was identified and sampled for seed and herbarium specimens. this population is of particular interest because of its location being between the organ and the florida mountains and its low elevation (1,210m barometric). for reasons related to proximity with las cruces and distance between transects, the distribution on the western slope of the organ mountains (supplemental figure 3) is better sampled as compared to other areas (figure 1). figure 1 also shows the positive return of scouting in august–september, in line with moisture level predicted in the gila region by the us drought monitor (supplemental figure 1). wild teparies were found in open, quite diverse habitats, from desert treeless grasslands (figure 4 top) to pine juniper woodland (figure 4 bottom). they were found in chaparral-like habitat (figure 5 top) or in dry stream beds (figure 5 bottom). these habitats match with those reported by allred and jercinovic (2020) and alexander (2025), perhaps with the exception of the ponderosa pineoak community, because our sampling has not yet been targeted towards higher elevations (not enough rain in the black range in 2023 and 2024!). figure 2. closeups of seeds of phaseolus acutifolius asa gray after field harvest. top row: population #3406 (all scale bars in mm; note the difference in lower row), right #3410. middle row: left #3422, right # 3428. lower row: left # 3430, right #3431 (all photographs by sd). genetic resources (2025), 6(12), 131–152 new wild tepary bean germplasm from sw usa 137 figure 3. wild tepary phaseolus acutifolius asa gray. top: habitat of #3412, aden lava flow wilderness area (photo dgd); in absence of cattle grazing, tepary plants (foreground and middle right, arrow) can reach significant development. lower left: #3430 with narrow leaflets, the lateral ones lobed at base (arrow) (photo sd); lower right: #3406, dry pods as found in most populations at this time; the one to the left with eight seeds (photo sd). genetic resources (2025), 6(12), 131–152138 debouck et al figure 4. wild phaseolus acutifolius habitats. top: habitat of population #3406, a desert grassland at sierra vista tank in organ mountains, doña ana county, new mexico; a few stems can be seen on the opuntia at foreground (arrow). the rough topography makes the entrance of cattle difficult, while big rocks reduce the effects of drying winds and help collect a bit of air moisture. bottom: habitat of population #3423, a pine juniper woodland with scattered oaks in turkey creek in brushy canyon, grant county, new mexico (photos dgd). genetic resources (2025), 6(12), 131–152 new wild tepary bean germplasm from sw usa 139 figure 5. wild phaseolus acutifolius habitats. top: habitat of population #3387, open low oak chaparral, aguirre spring, organ mountains, doña ana county, new mexico. bottom: habitat of population #3396, a dry wash along red rock road in big burro mountains, grant county, new mexico. arrows mark where plants were found in 2023 (photos dgd). genetic resources (2025), 6(12), 131–152140 debouck et al phaseolus angustissimus one population (#3424) was found east of gila, grant county, new mexico (there was a previous collection in the area: 4 miles east of gila, bear creek canyon by bassett maguire 11664, 23 may 1935, kept at the herbarium of the new york botanical garden (ny) with sprawling stems and very narrow leaflets (figure 6 lower right); several stems were cut to the base of the plant most likely due to grazing by cattle and/or nibbling by deer. the roadside location (figure 6 top) perhaps saved this population from being completely wiped out by grazing. while this population was found thanks to an inaturalist report of september 2024, another population of p. angustissimus similarly reported from a spot inside silver city was not found. the habitat of population #3424 matches with the one reported by jason alexander (2025). figure 6. phaseolus angustissimus asa gray, population #3424. top: habitat, a desert scrubland with few scattered junipers and soil almost half bare; the yellow arrows mark where plants thrive (photo sd). lower left: close-up of a late purple flower (photo sd). lower right: a plant with sprawling stems in roadside gully (photo dgd). genetic resources (2025), 6(12), 131–152 new wild tepary bean germplasm from sw usa 141 phaseolus filiformis one small population (#3411; figure 7) was found (very close to a wild tepary #3410) in the central-southern part of the organ mountains, doña ana county, new mexico; because of the location (cuates canyon, after an unsuccessful search in achenbach canyon, same mountainous range), it might be a new record for the organ mountains. it was found at seed dispersal stage (figure 7 lower right). the population #3393 found in 2023 in rockhound state park, luna county, new mexico, was visited again for seed in 2024, but because of lack of rains not a single plant was seen (nor for its sympatric wild tepary #3392). the two populations found so far (#3393 and #3411) fall within the diversity of dry and open habitats reported by allred and jercinovic (2020) and alexander (2025). figure 7. phaseolus filiformis bentham, population #3411. top: habitat in cuates canyon of organ mountains, the arrow marking where the plants thrive. lower left: a late green branch, where all leaflets are quite parallel to sun rays because of very active pulvini, thus difficult to detect. lower right: an almost dry stem on opuntia with wrinkled leaflets, twisted pods (arrow) and seeds already dispersed (all photos sd). genetic resources (2025), 6(12), 131–152142 debouck et al phaseolus grayanus two populations (#3415, #3426) were found at green and mature pod stage, often in altitude pine woodland (figure 8 top). that habitat is one of those reported by allred and jercinovic (2020) and alexander (2025). many vines were seen without any raceme (figure 8 lower left), and the low pod productivity may reflect the low amount of rain at these sites in 2024. if flowering is not triggered, more products of photosynthesis will go into the tuberous root (figure 8 lower right) as the survival strategy of this pluriannual species. figure 8. phaseolus grayanus wooton & standley. top: habitat of population #3415, a pine forest in cochise county, arizona (photo dgd). lower left: if left ungrazed, dense mats of sprawling vines can be seen as in population #3426 in pine woodland, grant county, new mexico (photo dgd). lower right: a 4–5-year-old root (20cm long, diameter 15mm) of plant in population #3426 (photo sd). genetic resources (2025), 6(12), 131–152 new wild tepary bean germplasm from sw usa 143 phaseolus maculatus all three populations (#3419, #3425 and #3427) showed stems with completely dried, tan whitish leaflets, perhaps due to lack of rains in september or scarcity of water in the immediate rocky environment. p. maculatus is normally a pluriannual prostrate legume of the grasslands of the chihuahuan desert (gentry, 1957). its abundant biomass of palatable shoots explains its extinction in these flatlands due to cattle grazing, while it survives on steep rocky slopes (figure 9 top). roadsides (#3419) and pine-oak (#3425) communities were two of the habitats mentioned by alexander (2025) and one by allred and jercinovic (2020). in contrast to wild teparies or p. filiformis (figure 7 lower right), pods in p. maculatus dehisce less abruptly (figure 9 lower right). damage due to seed weevils (coleoptera brentidae subfamily apioninae, j. king, personal communication, 2024) was seen in population #3427. figure 9. phaseolus maculatus scheele. top: habitat of population #3427 at gila cliff dwellings, catron county, new mexico, a rocky outcrop above a small riverine plain now converted into a parking lot; arrows mark plants with dried sprawling stems (photo dgd). lower left: population #3419 on a cliff west of red rock, grant county, new mexico: note the active pulvini putting the leaflets in an upright position (photo sd). lower right: population #3427 with pods at maturity with 1–3 globose seeds (arrow) (photo sd). genetic resources (2025), 6(12), 131–152144 debouck et al phaseolus montanus three small populations (#3414, #3416, figure 10, and #3417) were found in 2024, all in cochise county in southeastern arizona, while none were identified in new mexico. as explained below, the team had to enter into cochise co., in the chiricahua mountains, to verify the presence of the species, and to investigate the species habitat in order to address the question about its presence in new mexico. the plants were found at flowering and green pod stages, often intermixed with p. acutifolius var. tenuifolius with very narrow leaflets. it was thus important to verify some of the discriminant traits, namely in leaflets (freytag and debouck, 2002, page 174) and pods (brandegee, 1893, page 130), as flowers were not present on all plants (figure 11). confirming field observations made in durango in 1978, and in guerrero and jalapa both in 1987 (reported by freytag and debouck, 2002), the plants do not exceed 60cm in height and have narrow leaflets (figure 11) with active pulvini that make their identification challenging in the field. figure 10. phaseolus montanus brandegee. top: habitat of population #3414 (shared with p. acutifolius # 3413) (photo dgd). bottom: leaflets of population #3416 in a shady spot; comparing with figure 3 lower left, no lobed leaflets are present (photo sd). genetic resources (2025), 6(12), 131–152 new wild tepary bean germplasm from sw usa 145 figure 11. traits used to help identify phaseolus acutifolius var. tenuifolius (left) and p. montanus (right). top row: trifoliolate leaves, #3423 (photo sd) and #3414 (photo lgs). middle two rows: abaxial faces of lateral leaflets; upper: #3413 (photo sd), with arrow marking the formation of an external lobe, and lower: #3414 (photo lgs). lower row: flowers, #3390 and #3416, the greenish tip of the keel serving as indication of scale (photos dgd). bottom row: mature pods before shattering: #3428 left (photo sd) and #3417 right (photo sd) (note four developing seeds against eight in p. acutifolius, seven in photograph in upper corner left). genetic resources (2025), 6(12), 131–152146 debouck et al phaseolus parvulus one small population (#3395) was found in 2023, in the pinos altos range ne of silver city, grant, nm. given the lack of accuracy in the original species description (‘in the pinos altos mountains, new mexico’, greene, 1881, page 217), it could be considered as falling within the range of the type specimen. together with a few forbs of compositae and scattered grasses, it thrives on organic soils in the undergrowth of old ponderosa pine forest (figure 12); this matches with the habitat mentioned by allred and jercinovic (2020) and one of the three habitats indicated by alexander (2025). figure 12. phaseolus parvulus greene. top: habitat of population #3395; bottom: three fully developed plants, the one in the centre with an open pod, scale 5cm long (photos dgd). genetic resources (2025), 6(12), 131–152 new wild tepary bean germplasm from sw usa 147 discussion these results suggest the following points for discussion, namely on purpose, newness, diversity and prospects. first, with the results of 2023, all six species (p. acutifolius, p. angustissimus, p. filiformis, p. grayanus, p. maculatus and p. parvulus) reported for the state of new mexico (wooton and standley, 1915; freytag and debouck, 2002; allred and jercinovic, 2020; alexander, 2025) have been found, and some germplasm has been secured for the usda genebank. importantly, the team has learned about vegetation type and microhabitats of each taxon to more readily find additional populations in the future. while the collecting priority was on wild teparies, some germplasm was found for the other species, and this is important for the genebanks serving broader interests in different disciplines (e.g. plant taxonomy, ecophysiology, genomics). in this regard, arizona has the same six species and in addition, p. montanus and p. ritensis jones. we concur with allred and jercinovic (2020) (page 452) that p. ritensis has not been found yet in new mexico, and the same for p. montanus. this observation links with a second point that goes beyond settling a floristic question, given the unique value of p. montanus for reciprocal breeding of common and tepary beans (barrera et al, 2022). finding this taxon was debated between the members of the team during preparation, and explained the brief entry into arizona. we had one record, a collection by jacob corwin blumer #1676 made in 1907 on paradise slope in the chiricahua mountains in the northeastern extreme of the madrean archipelago (figure 1 in van devender et al, 2013). it was actually a mixed collection of p. acutifolius var. tenuifolius (specimens studied at the herbaria of ariz, f, isc, mo, nmc and ny1) and of p. montanus (specimens studied at the herbaria of cas, k, l and min2) (debouck, 2019). before arriving to the paradise slope not far from the southwestern research station, we found the two species (#3413 and #3414) almost growing side by side. this close proximity repeated itself eastwards from the locality of paradise (with #3417 as p. montanus and #3418 as p. acutifolius), while population #3416 of p. montanus was found close to p. grayanus #3415. a collection made by howard scott gentry #6472 in sinaloa, mexico in 1941 (annotated by one of us in ny; debouck, 2019) also showed that the two species can be found at the same spot much further south. but the fact that p. montanus is found alone in many places from guerrero, southern mexico, south to jalapa, eastern guatemala (freytag and debouck, 2002; debouck, 2021) would argue against it being a mere morphological segregant of p. acutifolius var. tenuifolius. clearly, this close proximity requires further investigation. a third point relates to sampling diversity of wild teparies in southern new mexico, where a clearer picture starts to appear thanks to our field work. extremes in elevation are so far: 1,288m for #3412 and 1,918m for #3431, and extremes in latitude: 31° 30’ 56.8” for #3400 (from 2023 1 ariz, university of arizona, usa; f, field museum of natural history, usa; isc, iowa state university, usa; mo, missouri botanical garden, usa; nmc, new mexico state university, usa; ny, new york botanical garden, usa 2 cas, california academy of sciences, usa; k, royal botanic garden, kew, uk; l, naturalis biodiversity center, the netherlands; min, university of minnesota, usa in the peloncillo mountains) and 33° 13’ 35.9” for #3428 (from 2024 in the gila cliff dwellings national monument). the records from herbaria indicated the following range in elevation: the specimen collected by elmer ottis wooton 528 (kept at ny) was found at 1,370m and the specimen collected by j travis columbus 1588 (kept at nmcr) was found at 1,980m. so, we have put the limit a bit further towards lower elevation (the collection #3412 of aden lava flow wilderness). likewise, the study of herbarium specimens indicated a collection (nmc-15801) by eo wooton sn in august 1902 ‘mangas springs; near silver city’ as the northernmost location (approxim. 32° 51’), so it seems that we have pushed the limit a bit further north. but the western slope of the organ mountains apart (supplemental figure 3), our sampling is still unequal and scanty (figure 1), not because of lack of records – though variously documented (29 wild tepary populations out of 189 of phaseolus records: supplemental table 1), but because of the heavy dependence on intensity and timing of the monsoon rainfall patterns. this uncertainty is common in north american deserts (beck and haase, 1969; larson and larson, 1997; nolin and hall-mckim, 2006; reichenbacher and peachey, 2025) and raises the point of how sampling could be improved. one can mention the critical importance of local scouting during august and early september, while the us drought monitor gave only a broad picture (supplemental figure 1). in this regard, given the lack of meteorological stations in the wilderness of new mexico, the information provided by rangers of the bureau of land management and of the us forest service was extremely valuable, while inaturalists and informal hikers’ observations gave a 50% chance of accurate data about wild p. acutifolius, the rest being other legumes such as galactia (for example, for gallinas canyon ne of san lorenzo in the mimbres watershed). the help by inaturalists can perhaps be made more effective if genebanks put on their websites macrophotos of flowers and pods, cumulating many traits for an accurate identification of target species (figure 11). finally, as noted in the first collection year (2023), grazing in protected areas may be a threat to wild teparies because the soil seed bank might not recover sufficiently to ensure the long-term survival of the populations in the context of the drying southwest. as a suggestion, from our field work, populations of wild teparies might be selected for the bureau of land management or the us forest service to launch a pilot project of in situ conservation, more precisely to address many questions related to the soil seed bank. concluding, the field work of 2023 and 2024 resulted in a significant increase of representation of wild tepary germplasm in genebanks, adding 22 new accessions from diverse habitats of the southwest (new mexico, arizona). that ecological diversity may announce an important diversity to be disclosed at the genetic level, thus opening up new prospects for breeding of that crop. supplemental data supplemental material 1. list of museums of natural history and herbaria where specimens were annotated. supplemental material 2. detailed information about each population found. supplemental table 1. numbers of populations by species (verified) and by county of new mexico. https://www.genresj.org/index.php/grj/article/view/genresj.lsbj4572/suppdata286 https://www.genresj.org/index.php/grj/article/view/genresj.lsbj4572/suppdata286 https://www.genresj.org/index.php/grj/article/view/genresj.lsbj4572/suppdata286 genetic resources (2025), 6(12), 131–152148 debouck et al supplemental table 2. results about the collection of seed, herbarium specimens and nodules/ soil samples of the immediate rhizosphere. supplemental figure 1. map of new mexico indicating drought prediction as compared to moisture expected across that state at that date. supplemental figure 2. photographs of seedlings during the seed increase process at usda pullman. supplemental figure 3. satellite map of the organ mountains showing the progress of sampling of wild tepary populations. author contributions this germplasm exploration was originally conceived after a distance workshop during covid19 lockdown in which participated scientists of the three institutions. for the first (2023) and second (2024) explorations, rp brought the experience and information about suitability of areas from the scouting prior to the field work. he provided many edits and several references about tepary research. sd did the soil sampling in 2024 and collected material of the rhizosphere as well. she added names of several locations of individual collections and provided many high-quality photographs. rp and sd rechecked names of places for all collections reported in supplemental material 2. ms and tp participated very actively in the seed collection in 2023 and 2024, respectively. tp provided several insights about tepary breeding and genomics and several references about tepary research. lgs prepared the herbarium specimens in 2023; in 2024 he provided the data about the second gps recording, photographs and the handling of herbarium specimens. mou did the soil sampling in 2023 and contributed the rhizosphere samples as well. dgd participated into the identification of populations and collection of data. he did the literature review, wrote the initial draft of the paper and integrated all edits by co-authors. all authors, who were in the field in 2023 and 2024, contributed to the population sampling, read, revised and approved the manuscript. acknowledgements the field work in 2023 was possible thanks to an usda/ ars national plant germplasm system plant exploration office funding for plant exploration in new mexico for phaseolus spp. and the field work in arizona and new mexico in 2024 was possible thanks to the project ‘plant exploration in new mexico to collect wild species of phaseolus acutifolius and phaseolus filiformis germplasm for crop improvement’ sponsored by the usda/ars award no. 58-2090-4-042. the authors extend special thanks to dr anne frances and the reviewers for considering the proposal, and the phaseolus crop germplasm committee for their letter of support. additional funding was provided by the plant and environmental sciences department of new mexico state university, the genetic resources program of ciat, and the agriculture research service of usda. permits to collect herbarium specimens, seed for germplasm conservation and samples of microorganisms and soil were kindly and swiftly granted by the forest service of usda, the bureau of land management of the department of interior and the land trust of the state of new mexico. the authors express deep gratitude to britton bourland (usda) for help on the maps. the help and interest of the following persons: josh bachman (nmsu), stephen beebe (ciat), geoff bender (srs), esteban bolaños (ciat), nury escobar (ciat), sara fuentes soriano (nmsu), lois grant (nmsu-retired), gabriela guerrero florez (wsu), anowar islam (nmsu), joanie king (nmsu), juan david libreros (ciat), claudia maldonado (ciat), carla olson (usda), erin riordan (arizona-sonora desert museum), zachary rogers (nmsu), kirsten romig (blm), fermin salas (nps), john jairo sánchez (ciat), joe tohme (ciat), eliana urquijo (ciat), carlos urrea (unl), marilyn warburton (usda) and peter wenzl (ciat) at different steps of these explorations are deeply acknowledged. the authors express gratitude to a regional director of usda for interest and to the editor for helping to improve the manuscript. conflict of interest statement the authors are all interested in increasing knowledge about the native bean species of new mexico, and adding genetic diversity into usda and ciat genebanks, specifically of wild teparies. sd is currently the curator of the usda phaseolus collection and responsible for the bean genebank at the western plant introduction station of usda, pullman, washington. rp is professor at new mexico state university and has investigated the agronomy and ecology of tepary and relatives since the 1980s. tp is a bean breeder and researcher of the agricultural research service based at the tropical agricultural research station in mayagüez, puerto rico, and has launched tepary breeding since the 2000s. ms is the manager of the genebank of future seeds of the alliance of bioversity international and ciat, based in palmira, colombia, and oversees all operations for the seed collections of phaseolus beans and tropical forages. lgs is curator of the bean collection kept in the genebank of future seeds in palmira, colombia. mou, now at the international center for biosaline agriculture, worked as the bean physiology leader in ciat, with interest to develop new technologies to measure tolerance to heat, drought, low phosphorus and high aluminium in crop plants. dgd, ciat emeritus, has been responsible for ciat genebank in 1996–2017; now retired he continues writing and sharing information about crops of neotropical origin. ethics statement the primary objective of this collaborative project involving usda, nmsu and ciat (the concerned branch of the alliance of bioversity international and ciat) was to increase the genetic diversity in the usda germplasm collection. therefore, it was of utmost importance that the different materials were collected in full knowledge of the authorities and with the appropriate permits, and the role of sarah dohle, being staff member of the agricultural research service of usda, was key in this regard. where permits were required on public land, they were obtained as follows: • new mexico bureau of land management, reference no. 6850 (9300) laura hronec, acting deputy state director division of lands and resources (2023 and 2024) • new mexico national forest, inter agency courtesy provided by jessie willett, new mexico zone contracting officer (2023 and 2024) https://www.genresj.org/index.php/grj/article/view/genresj.lsbj4572/suppdata286 https://www.genresj.org/index.php/grj/article/view/genresj.lsbj4572/suppdata286 https://www.genresj.org/index.php/grj/article/view/genresj.lsbj4572/suppdata286 https://www.genresj.org/index.php/grj/article/view/genresj.lsbj4572/suppdata286 genetic resources (2025), 6(12), 131–152 new wild tepary bean germplasm from sw usa 149 • new mexico state parks, research permit #017 (2023) and #027 (2024) provided by robert stokes, program support bureau chief • arizona apache national forest, inter agency courtesy permission provided by trace douglas timber management officer for apache-sitgreaves nfs, alpine and springerville ranger districts (2024) • arizona coronado national forest, inter agency courtesy permission provided by douglas ruppel, district ranger douglas ranger district (2024). at the gila cliff dwellings national monument, an in-person permission was provided by fermin salas, superintendent of gila cliff dwellings national monument, national park service, and the staff of the national monument very kindly accompanied the collecting team along the official path within the monument. references alexander, j. a. 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