Vorgabe neu Journal of Applied Botany and Food Quality 80, 135 - 137 (2006) 1Institute for Plant Sciences, Division for Plant Physiology, Karl-Franzens-University of Graz, Austria 2Institute for Plant Sciences, Division for Botany, Karl-Franzens-University of Graz, Austria Assessment of the genetic diversity of native apple cultivars in the south eastern ranges of the Alps with three selected microsatellite loci Stephan Monschein1, Martin Grube2, Dieter Grill1 (Received June 6, 2006) Summary The regional diversity of native apple cultivars in parts of the south eastern ranges of the Alps (Styria, Austria and northern parts of Slovenia) was examined. As the application of conventional pomo- logical methods to characterise cultivars may sometimes be am- biguous, we regard the application of molecular methods to be essential for thorough cultivar diversity assessments. Five hundred samples were collected from different climatic and edaphic regions and analysed using three selected microsatellite loci. With this approach we were able to distinguish 190 named varieties at which we chose 50 as reference varieties. The high diversity of native races suggests that the Southern alpine ranges represent a „hot spot“ of cultivar diversity. This can be attributed to historical effects and the local persistence of a traditional management practice with orchards of widely spaced and old-grown trees of various races. Because these „old“ native races could harbour interesting genetic traits (pathogen resistance, taste, etc.) that will be important in future food production, measures for their conservation are overdue. Our approach will not only show which local cultivars/genotypes require rapid action for their protection, but due to the international nature of our project we can also show which old and untraceable local names in different languages correspond with the same cultivars. Introduction Representing one the oldest and most widely cultivated temperate fruit crops, apple (Malus x domestica Borkh.) developed into a great diversity of cultivars. Of these, only a few are grown in mass pro- duction to fit the requirements of the international market of today. In former times each country or region maintained an own local stock of cultivars (JANICK et al., 1996). From these, ecological types could be selected which were particularly adapted to local environments and requirements. These old varieties and land races have been used by humans since hundreds of years and therefore hold a special position in the cultural landscape heritage of any region where apple has a history of cultivation. However, for complex reasons the genetic diversity of the domesticated apples has decreased dramatically in the last few years (HOKANSON et al., 2001). The colline to montane elevations of the south eastern Alpine borders are a perfect example for a historically rich diversity of apple culti- vars. Originating from Styria (Austria), which might be regarded as “hot-spot” of apple diversity, apple cultivation was extended towards Slovenia, northern Italy and parts of Croatia during the times of the Habsburgian empire. Some of the races were better adapted than others to grow at different altitudes, to withstand high light, to resist massive damage by pathogens, etc. A tremendous number of names of these cultivars are found in the literature (ROLFF, 2001; HARTMANN, 2003; GRILL and KEPPEL, 2005), or can be collected in interviews with local farmers. Since there has never been a comprehensive and consistent collection of cultivars for comparison, the naming practice resulted in many confusion, especially with rarely grown cultivars. E.g. old horticultural names were sometimes replaced by informal local names, which persisted through times, or wrong determinations could result in the dissemination of shoots with wrong names. The taxonomic chaos of informal vernacular names lead to a considerable uncertainty about estimates of cultivar diversity in the south-eastern alpine ranges. Because native apple cultivars represent a rich stock of exploitable genetic resources, the conservation of old and local cultivars should be seen as an important task to ensure a sustainable environment. It is clear that a general screening of the diversity of old local apple varieties is an indispensable first step before an effective conservation management can be outlined (HODGKIN et al., 2001). However, and especially in the case of apple, the traditional determination methods, including measurements and objective descriptions of fruit and tree characteristics, are time-consuming and not always consistent (KENIS et al., 2001). This is due to the variability in their fruit para- meters (e. g. colour, shape, tightness), which is strongly influenced by ecological and physiological parameters, such as soil composition, exposition, climatic fluctuations. To complement the morphological characterisation, isoenzymes and alloenzymes have previously been included (ROYO and ITOIZ, 2004), but their patterns may again de- pend on environmental factors. The emergence of new PCR-based molecular markers, such as randomly amplified polymorphic DNA (RAPDs), simple sequence repeats (SSRs), and amplified fragment length polymorphisms (AFLPs), has created the opportunity for fine- scale genetic characterizations of germplasm collections that were previously impossible (HOKANSON et al., 1998). An alternative method for characterisation and identification of old native apple cultivars which is independent from changing environ- mental influences is provided by the anlaysis of length variation at microsatellite loci. By their polymorphic nature, microsatellites became popular for various genetic approaches (genomic mapping, study of genomic instability in cancer cells, population genetics, forensics and conservation biology; SHINDE et al., 2003). Micro- satellites, or simple sequence repeats (SSRs), are short stretches of DNA, consisting of tandemly repeated nucleotide units. The high levels of variation in the number of repeats are thought to arise from replication slippage, i.e. the transient dissociation of the replicating DNA strands followed by misaligned reassociation (ELLEGREN, 2004). A general method for the detection of polymorphic micro- satellites is based on PCR amplification using a unique pair of primers flanking the simple sequence repeats (WEBER and MAY, 1989). As they are uniformly distributed, hypervariable, codominant and abundant in most genomes, they are interesting for studies of apple (GIANFRANCESCHI et al., 1998), and can be used to gain deeper insights in the distribution of phenotypic traits (by QTL analysis) or for an assessment of cultivar diversity. The aim of this study is to examine the genetic diversity of native apple cultivars by means of microsatellite length variations and to clarify some naming problems of apple varieties in the area of investigation. Materials and methods For the estimation of the genetic diversity of native apple cultivars in the area of investigation (Styria and northern parts of Slovenia) a basic inquiry was carried out. We sent out a standardised question- naire to all agricultural schools to obtain information about the cultivars composing these special cultural landscape. From these collected data we selected varieties for genetic analysis by micro- satellites according to specified criteria, as follows: 1. the age of the trees (80 - 100 years old), 2. unclear local names, 3. special usage of the fruits (such as dried fruit), 4. occurrence at relatively high alti- tudes (> 1300 m) or 5. such varieties, which are not yet cultivated in a living bank of genetic resources (arboretum). Varieties determined by pomological experts (S. Bernkopf, Linz and H. Keppel, Graz) were analysed as references for comparison. From the selected trees for microsatellite analyses we collected young leaves for DNA extraction. The leaves were frozen in liquid nitrogen immediately after harvesting. The lyophilised leaves (Hetosicc Freeze Dryer Type CD 4, Heto Lab Equipment, Birkerod, Denmark) were ground to powder by the Micro-Dismembrator II (B. Braun Biotech International GmbH, Meisungen, Germany) and stored in humidity proof plastic vials at -25°C until DNA extraction. The DNA was isolated from 17 mg of leaf powder using the DNeasy® Plant Mini Kit (Qiagen, Vienna). For the genetic characterisation of the native apple cultivars by simple sequence repeats we used three different polymorphic primer com- binations with high expected heterozygosity (forward and reverse primer), which represent different linkage groups (GIANFRANCESCHI et al., 1998): CH01F02, CH02C06 and CH02D12. The forward primer of each primer pair was labelled at the 5´-end with a fluorescent dye (FAM, HEX or NED). The amplification of the three selected micro- satellite loci by PCR followed the conditions published by GIAN- FRANCESCHI et al. (1998). The specific amplified DNA fragments (microsatellites) were run on a ABI PRISM® 310 Genetic Analyser. The detected fragment lengths were calibrated by a fluorescent labelled size standard (Genescan 500 ROX) which was added to each sample. Sizing of fragments was done with GeneScan®Analysis Software Version 3.1. Because lengths from each microsatellite locus was uniform within an cultivar, differences in the allelic compositions were used for characterisation and identification of old native apple cultivars. Results and discussion Based on the primary inquiry of the native apple cultivars by standardised questionnaires and after selection of relevant varieties (see above) we examined 500 trees by microsatellite analysis. The examined apple cultivars are characterised by their allelic compo- sition at the three selected SSR loci. From the 500 DNA-extractions, which were sampled evenly across the area of investigation, we could differentiate 190 varieties. The application of only three selected microsatellite loci can be regarded as a cost efficient method for a screening of regional apple cultivar diversity. To characterise unknown cultivars and to clarify the complex sy- nonymy, it is necessary to compare the allelic length of reference samples of varieties with that of cultivars with dubious names. The determination of such references is a difficult task. The selection of samples, which are to become reference samples in the future was carried out in accordance of microsatellite data with morphological fruit specifications (e. g. colour, shape, tightness), and expert knowledge for local varieties (S. Bernkopf, Linz and H. Keppel, Graz). Of the 190 differentiated samples we selected 50 as reference varieties (Tab. 1). This reference data base will allow a secure iden- tification of apples by their allelic composition. The allelic composition of the cultivars interpreted according to LIEBHARD et al. (2002) as follows. In cases where only one fragment is visible, the allel can be homozygous or an null allel is involved (eg. Ananasrenette at locus CH01F02). In the latter case the individual Tab. 1: Allelic composition of 50 reference apple cultivars (nomenclature according to GRILL and KEPPEL, 2005) Cultivar SSR name CH01F02 CH02C06 CH02D12 Ananasrenette 183 241:249 190:198 Baumanns Renette 169:179 215:227 182:198 Berner Rosenapfel 181 233:249 198:206 Boikenapfel 179 215:233 198:200 Champagner Renette 183 249 190:200 Charlamowsky 171 205:251 176:198 Coulons Renette 181:183 227:237 180:210 Damasonrenette 179:183 233:243:253 182:198 Danziger Kantapfel 169 229:251 180:198 Elise Rathke 169:183 249 180:190 Geflammter Kardinal 169:179 215:227:247 182:198:210 Geheimrat Dr. Oldenburg 189 249 176:190 Gehrers Rambur 169 215:227:249 198 Gelber Bellefleur 173:179 233:249 194:198 Goldrenette von Blenheim 183 241:249:257 198 Grahams Jubiläumsapfel 173 249 176:198 Graue Herbstrenette 179:183 233:243 182:198:200 Gravensteiner 181:183 215:249 198:212 Großer Rheinischer Bohnapfel 179:183 215:249 194:198 Grüner Stettiner 169 215:233:255 180:194:198 Hagedorn 179:183 215:251 176:198 Harberts Renette 183:185 241:249 180:198 Haslinger 169:179:183 249 176:182:190 Ilzer Rosenapfel 169 259 182:198 Jakob Lebel 179:187:195 227:237 176:182:198 Jonathan 169:179 233:249 190:198 Kalterer Böhmer 169 229:231 182:206 Kanada Renette 179 227:247 182:190:198 Kardinal Graf Galen 169 249 176:206 Karmeliter Renette 179:183 257 198 Klöcher Maschanzker 169:181 243:249 190:198:206 Kronprinz Rudolf 169:183 241:249 176:198 Landsberger Renette 183:189 237:251 176:190 Lavanttaler Bananenapfel 183 249 206:210 London Pepping 179 215 176:206 Odenwälder 169:193 249 176:198 Rheinischer Krummstiel 169:181 237:249 176:198 Rote Schafnase 169 229:231 198:204 Roter Herbstkalvill 173 249 182:198 Roter Trierer Weinapfel 169 247 180:182 Sauergrauech 181:183 233:247 198 Schmidberger Renette 169:183 249 198:210 Schöner von Boskoop 183 227:237 180:194 Signe Tillisch 189 215:251 176 Steirischer Maschanzker 169:181 249 198:206 Steirischer Passamaner 169:171 215:231 184:198 Wagener Apfel 169:181 249 180:206 Weißer Klarapfel 181:187 215:249 178:198 Weißer Winterkalvill 181 249 182:198 Wintergoldparmäne 183 237:249 198:210 136 Stephan Monschein, Martin Grube, Dieter Grill could be heterozygous and the other allel is not detectable because of mutations in the priming site and prevention of annealing. Two allels represent an diploid heterozygous individual (or a triploid with null allel). Three allels at the same locus of one individual could indicate a triploid heterozygous cultivar or for multilocus SSR (e. g. Damasonrenette at locus CH02C06). Our data confirm a high diversity of extant apple cultivars in Styria and parts of Slovenia. We could also resolve several nomenclatural problems with our data (Tab. 2). For example the cultivar „Baumanns Renette“ displays the same allelic composition as the collections „Kranzler“, „Türken rot“, „Nikolausapfel“ and „Baumanova reneta“, which represent vernacular names for the regular cultivar term “Bau- manns Renette”. A posteriori inspection of pomological characters confirmed the molecular approaches. Any slight differences between the phenotypical descriptions seem to be within the range of modi- fications of each race. Tab. 2: Examples for discovered synonyms (regular cultivar names are given in bold letters, GRILL and KEPPEL, 2005) Cultivar SSR name CH01F02 CH02C06 CH02D12 Baumanns Renette 169:179 215:227 182:198 Kranzler 169:179 215:227 182:198 Türken rot 169:179 215:227 182:198 Nikolausapfel 169:179 215:227 182:198 Baumanova reneta 169:179 215:227 182:198 Haslinger 169:179:183 249 176:182:190 Steirischer Pogatschenapfel 169:179:183 249 176:182:190 Roter Pogatschenapfel 169:179:183 249 176:182:190 Haselapfel 169:179:183 249 176:182:190 Breitschädel 169:179:183 249 176:182:190 Pogacarica 169:179:183 249 176:182:190 Roter Herbstkalvill 173 249 182:198 Himbeerapfel 173 249 182:198 Erdbeerapfel 173 249 182:198 Klachlapfel 173 249 182:198 Roter Paradiesapfel 173 249 182:198 Herzapfel 173 249 182:198 Klingler 173 249 182:198 Tschepperer 173 249 182:198 Rdeci jesenski kalvil 173 249 182:198 Weißer Klarapfel 181:187 215:249 178:198 Butterapfel 181:187 215:249 178:198 In parallel to SSR-typing of characterised apple varieties their cultivation in an arboretum as a living bank of genetic resources is planned. This will facilitate the conservation and dissemination of genetically valuable native apple cultivars. Another anticipated en- deavour is a transregional alignment of the obtained microsatellite data with other research institutes. Acknowledgements This research was funded by the INTERREG III A programm between Austia and Slovenia and a “Bund-Bundesländer-Kooperation”. We thank Karin Herbinger and Melanie Hofer for the collection of apple leaf material, Herbert Keppel and Siegfried Bernkopf for their scien- tific support. References ELLEGREN, H., 2004: Microsatellites: Simple sequences with complex evolution. Nature Rev. Genet. 5, 435-445. GIANFRANCESCHI, L., SEGLIAS, N., TARCHINI, R., KOMJANC, M., GESSLER, C., 1998: Simple sequence repeats for the genetic analysis of apple. Theor. Appl. Genet. 96, 1069-1076. GRILL, D., KEPPEL, H., 2005: Alte Apfel- und Birnensorten für den Streu- obstbau. Leopold Stocker Verlag, Graz-Stuttgart. HARTMANN, W., 2003: Farbatlas alte Obstsorten. Verlag Eugen Ulmer, Stuttgart. HODGKIN, T., ROVIGLIONI, R., DE VICENTE, M.C., DUDNIK, N., 2001: Molecular methods in the conservation and use of plant genetic resources. Acta Horticulturae 546, 107-118. HOKANSON, S.C., SZEWC-MCFADDEN, A.K., LAMBOY, W.F., MCFERSON, J.R., 1998: Microsatellite (SSR) markers reveal genetic identities, genetic diversity and relationships in a Malus x domestica borkh. core subset collection. Theor. Appl. Genet. 97, 671-683. HOKANSON, S.C., LAMBOY, W.F., SZEWC-MCFADDEN, A.K., MCFERSON, J.R., 2001: Microsatellite (SSR) variation in a collection of Malus (apple) species and hybrids. Euphytica 118, 281-294. JANICK, J., CUMMINS, J.N., BROWN, S.K., HEMMAT, M., 1996: Apples. In: Janick, J., Moore, J.N. (eds.), Fruit Breeding, Volume I: Tree and Tropical Fruits, 1-77. John Wiley & Sons Inc., New York. KENIS, K., PAUWELS, E., VAN HOUTVINCK, N., KEULEMANS, J., 2001: The use of microsatellites to establish unique fingerprints for apple cultivars and some of their descendants. Acta Horticulturae 546, 427-431. LIEBHARD, R., GIANFRANCESCHI, L., KOLLER, B., RYDER, C.D., TARCHINI, R., VAN DE WEG, E., GESSLER, C., 2002: Development and charac- terisation of 140 new microsatellites in apple (Malus x domestica Borkh.). Molecular Breeding 10, 217-241. ROLFF, J.H., 2001: Der Apfel, Sortennamen und Synonyme. Band 1. Selbst- verlag Johann-Heinrich Rolff, Kiefersfelden. ROYO, J.B., ITOIZ, R., 2004: Evaluation of the discriminance capacity of RAPD, isoenzymes and morphologic markers in apple (Malus x domestica Borkh.) and the congruence among classifications. Genet. Res. Crop Evolut. 51, 153-160. SHINDE, D., LAI, Y., SUN, F., ARNHEIM, N., 2003: Taq DNA polymerase slippage mutation rates measured by PCR and quasi-likelihood analysis: (CA/GT)n and (A/T)n microsatellites. Nucleic Acids Res. 31, 974- 980. WEBER, J. L., MAY, P. E., 1989: Abundant class of human DNA polymorphism which can be typed using the polymerase chain reaction. Amer. J. Human Genet. 44, 388-396. Address of the authors: Mag. Stephan Monschein (corresponding author), Institute for Plant Sciences, Division for Plant Physiology, Karl-Franzens-University of Graz, Schubert- straße 51, A-8010 Graz, Austria. Ao. Univ.-Prof. Mag. Dr. Martin Grube, Institute for Plant Sciences, Division for Botany, Karl-Franzens-University of Graz, Holteigasse 6, A-8010 Graz, Austria. Univ.-Prof. Dr. Dieter Grill, Institute for Plant Sciences, Division for Plant Physiology, Karl-Franzens-University of Graz, Schubertstraße 51, A-8010 Graz, Austria. 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