Impaginato 215 Adv. Hort. Sci., 2022 36(3): 215­226 DOI: 10.36253/ahsc­13045 Genetic and ampelographic characteriza­ tion of grapevine accessions maintained in the Lebanese national collection A. Chehade 1, L. Chalak 2, J. Merheb 2, A. Elbitar 3, E. Rmeily 4, N. Madi 2, M. Massaad 2 (*) 1 Lebanese Agricultural Research Institute, Tal Aamara, Department of Plant Biotechnology, Plant Genetic Resources Unit, P.O. Box 287, Zahleh, Lebanon. 2 The Lebanese University, Faculty of Agronomy, Dekwane, Beirut, Lebanon. 3 Lebanese Agricultural Research Institute, Tal Aamara, Department of Plant Biotechnology, Plant Tissue Culture Unit, P.O. Box 287, Zahleh, Lebanon. 4 Lebanese Agricultural Research Institute, Tal Aamara, Department of Pomology and Viticulture, P.O. Box 287, Zahleh, Lebanon. Key words: Genetic diversity, ISSR markers, Lebanon, local germplasm, Vitis vine‐ fera L. Abstract: Safeguarding grapevine biodiversity is one of the main concerns in viticulture today. Management of ex situ collections requires a comprehensive characterization of the conserved germplasm to provide basic material for selection and breeding programs. In this study, the local grapevine germplasm conserved at the national collection of the Lebanese Agricultural Research Institute and composed of 43 accessions,was submitted to a genetic and ampel­ ographic study. Nine ISSR primers, previously developed in grapevine, were used in this study. These primers generated a total of 51 bands, of which 77.7% were polymorphic allowing the differentiation of 41 genetic profiles vs. one case of synonymy that was recorded among three accessions. Ampelographic characterization was conducted using a set of 33 descriptors established by the International Office of Vine and Wine and related to leaf, bunch and berry. Principal component analysis identified 12 descriptors i.e. veraison date, matu­ rity date, berry length, upper and lower vein pigmentation, bunch density, bunch weight, sugar content at harvesting, flesh of juiciness, berry weight, flesh firmness and color skin, as being the most discriminating descriptors. The corre­ lation between the ISSR clustering and the ampelographic one was not signifi­ cant (r=0.26) because of the divergence of accessions groups, except for the three accessions synonymy case which was confirmed in both dendrograms. Finally, this comprehensive evaluation of the existing local gene pool of grapevine revealed a substantial diversity. It would further allow the promo­ tion of the valuable accessions directly through multiplication scheme, and their sustainable utilization in genetic improvement programs. 1. Introduction One of the main concerns for public research in viticulture today is the (*) Corresponding author: mmassaad1@gmail.com Citation: CHEHADE A., CHALAK L., RMEILY E., ELBITAR A., MERHEB J., MADI N., MASSAAD M., 2022 ­ Genetic and ampelographic characterization of grapevine accessions maintained in the Lebanese national collection. ­ Adv. Hort. Sci., 36(3): 215­ 226. Copyright: © 2022 Chehade A., Chalak L., Rmeily E., Elbitar A., Merheb J., Madi N., Massaad M. This is an open access, peer reviewed article published by Firenze University Press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Competing Interests: The authors declare no competing interests. Received for publication 21 April 2022 Accepted for publication 24 August 2022 AHS Advances in Horticultural Science https://doi.org/10.36253/ahsc-13045 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ Adv. Hort. Sci., 2022 36(3): 215­226 216 need to safeguard grapevine biodiversity by estab­ lishing well managed national repositories to ensure germplasm availability to breeders, researchers and farmers (Nass et al., 2012). There are presently around 130 grapevine germplasm collections across the word (Dettweiler et al., 2000). One of the first grapevine germplasm collections is the one estab­ lished in Spain in 1950, which contains more than 1066 accessions (Ortiz et al., 2004). In France, more than 2,200 varieties originally collected from 35 countries are grouped together at the National Institute of Agronomic Research (Tessier et al., 1999). Similar collections of grapevine were also established USA and in Latin America containing grapevine acces­ sions collected all over the world (Martinez et al., 2003). Grapevine is one of the oldest fruit crops growing in the Eastern Mediterranean region includ­ ing Lebanon (Zohary, 2003). The country was among the first worldwide to have implemented vineyards which gradually became a traditional culture in Lebanon for the production of both table grapes and wine (Chalak et al., 2016). Today viticulture occupies the eighth rank in the agricultural sector in the coun­ try, with a total production area of 9,240 hectares and an annual production of about 89,000 tons of table grapes (FA0, 2010; FAOSTAT, 2019) vs. 3,000 hectares dedicated to wine grape with an annual pro­ duction of approximately 10,000 tones (Rahal, 2015). In addition, about 800 hectares of vineyards are intended for the production of Lebanese Arak (Roby, 2003). Commercial plantations of table grapes have long been constituted of four local varieties, com­ monly named ‘Tfayfihi’, ‘Baitamouni’, ‘Maghdouche’ and ‘Obeidy’. More recently, the new plantations in the country are mostly constituted of improved vari­ eties imported from Europe and the United States. Around 77 varieties are imported to Lebanon. Nevertheless, the major share went mostly to ‘Cabernet Sauvignon Blanc’, ‘Syrah’, ‘Viognier’, ‘Chardonnay’ and ‘Merlot’ which represent 65% of the total saplings quantities imported during 2012­ 2014 (Tabaja, 2015). The long history of viticulture in Lebanon suggests the existence of a large indigenous germplasm associated to a wide range of traditional varieties that are well adapted to the various agro­cli­ matic conditions of the country (Chalak et al., 2016). In Lebanon and despite the increasing interest on the conservation and characterization of plant genetic resources in general, only few studies have addressed the local germplasm of grapevine, although it is threatened by various anthropogenic pressures including in particular the progressive replacement of local varieties by more advantageous improved for­ eign varieties that are regularly imported into the country (Riachi, 1998; Madi, 2007; Chalak et al., 2016; Rahme, 2016). To face the threat of genetic erosion and avoid the loss of traditional germplasm, the Lebanese Agricultural Research Institute (LARI, Tal Amara station) established in 1998 a national grapevine collection containing numerous local acces­ sions collected from different production areas across the country (Madi, 2007). Within the environmental change and the challenging predictions for the Eastern Mediterranean region including Lebanon (Santillán et al., 2019), there is a growing need to address the local genetic resources such as traditional varieties which are recognized to be more adapted to harsh conditions and having better tolerance to vari­ ous biotic and abiotic stresses (FAO, 2015). Added to this, the wine industry is witnessing a new trend towards the utilization of the local traditional varieties in the production of typical and well prized wines. Therefore clonal selection, with the respect to specific traits, has become the most important way to improve the quality of grape cultivars. As a consequence, there is a need for reliable and precise methods of clonal characterization for further use by breeders, nurseries and industries (Galet, 1998; Moreno et al., 1998). Ampelography is a scien­ tific methodology that has long been the single method used for the characterization of grapevine phenotype, based on the description of different morphological, phenological and pomological charac­ ters (Galet, 1998; Sabir et al., 2009; Laucou et al., 2011). In addition to the ampelograhic description, in order to discriminate the varieties, synonyms, homonyms and the variation among the accessions, molecular methods has become more frequently used especially markers based on Polymerase chain reaction techniques (Riaz et al., 2012; Madhumati, 2014). Among the molecular approaches, the ISSR (Inter Sequence Simple Repeat) has being evaluated for its usefulness in grapevine cultivar identification and in assessing genetic diversity of grapevine germplasms (Moreno et al., 1998; Dhanorkar et al., 2005; Santiago et al., 2005; This et al., 2006; Sabir et al., 2009; Seyedimoradi et al., 2012; Choudhary et al., 2014; Castro et al., 2016). This study aimed to characterize the 43 local grape accessions preserved at the Lebanese National Collection of Grapevine at LARI using both ISSR mark­ ers and ampelographic descriptors. This work was conducted in an attempt to evaluate the diversity of the local germplasm for further actions of conserva­ Chehade et al. ‐ Genetic diversity in Lebanese grapevine germplasm 217 tion and sustainable utilization of grapevine genetic resources in diversification strategies. 2. Materials and Methods Plant material A total of 43 accessions of traditional grapevine varieties growing in the Lebanese national collection located in Tal Amara, Bekaa (N 33° 49’ 59”, E 36° 0’ 0”, 908 m a.s.l.) established in 1998, were considered in this study (Table 1). Accessions initially consisted of farmer’s local varieties surveyed and collected across the Bekaa region (Lebanon). The vine cultivars were grafted with four duplications on B41 root­ stocks which were planted directly in the collection land a year before the grafting process under the same growing conditions using horizontal trellis train­ ing system with the spaces 2.75 x 2.75 m. The site of plantation is characterized by a clay fertile soil and an average of precipitation ranging between 600 to 700 mm/year. For each accession, young leaves were sampled from a single selected plant and stored at ­ 20°C for the DNA extraction and molecular analysis. On the other hand, samples of ten leaves, five clus­ ters and 20 fully mature berries were taken from the same selected plant of each accession between August and October 2020 to undergo the ampelo­ graphic characterization. Molecular characterization Total genomic DNA was extracted from 300 mg of young leaf tissue after been crushed to a fine powder with liquid nitrogen and stored at ­20°C until use. DNA extraction of grapevine landraces was per­ formed according to (Doyle and Doyle, 1990) proto­ col with minor modifications (Kafkas et al., 2006). DNA concentration was measured by Biospec­nano (Shimadzu corp) and stored at ­20 C until further analysis. Nine ISSR primers: UBC807, UBC812, UBC815, UBC816, UBC818, UBC828, UBC864, UBC868 and UBC880 (Table 2) were selected and Table 1 ­ Sites of 43 collected samples of traditional cultivars grapevine growing in the national germplasm collection at the Lebanese Agricultural Research Institute (LARI­Tal Amara) Cultivars growing in the national germplasm collection at LARI Sites of collected samples Region Ainouni Tamnin Bekaa Central Al Mir, Ari, Ashlamish Ahmar, Asouad, Bakhouri, Bourji, Bzaz Al­Anzi, Maksasi, Mariami, Moukh Al­Baghel, Oubaidi, Souri Nabi Ayla Bekaa Central Amarcani Abiad, Amarcani Asouad, Baitamouni, Baitamouni Asouad, Gbaii, Maghdoushi, Nih, Tfaifihi, Zaghzaghani Niha Bekaa Central Amarcani Souri Ablah Bekaa Central Arasani, Souri Zahlaoui Saghbin Bekaa Ouest Ashlamish Abiad, Rins Rayak Bekaa Central Houzairani, Khoudri, Zaitouni El­Rafid Bekaa Ouest Karn Al Ghazal, Maghdoushi Mgaddad, Misky, NABI, Raha, Samaani Abiad, Samani Asouad, Souri Asouad, Souri Mhayyar, Yafaoui, Asali, Boulbouli Asouad, Gnoubi Unknown Site Bekaa Table 2 ­ The resulting discriminating power, number of polymorphic and monomorphic, the size of bands of ISSR primers, percentage of polymorphism and the polymorphic information content Primer Size range (bp) Number amplified bands Number of polymorphic bands Number of monomorphic bands Polymorphism (%) PD PIC UBC812 350­1000 7 6 1 85.7 0.64 0.33 UBC818 300­700 7 6 1 85.7 0.87 0.59 UBC815 400­650 4 3 1 75 0.62 0.6 UBC828 550­800 3 2 1 66.6 0.44 0.45 UBC864 280­700 7 6 1 85.7 0.9 0.48 UBC868 225­750 4 2 2 50 0.3 0.27 UBC816 300­900 5 4 1 80 0.64 0.64 UBC807 250­800 7 6 1 85.7 0.84 0.47 UBC880 125­600 7 6 1 85.7 0.85 0.58 Total 125­1000 51 41 10 ­­­­­ ­­­­­ ­­­­­ Mean 5.6 4.5 1.1 77.7 0.67 0.49 Adv. Hort. Sci., 2022 36(3): 215­226 218 used in this study based on their good results for amplification and high power of discrimination on grapevine (Seyedimoradi et al., 2012). The ISSR (Inter Simple Sequence Repeat) amplifi­ cation was carried out as per (Sabir et al., 2009), using 20 µl reaction mixture containing 2 µl of 10 X PCR buffer (750 mM Tris­HCl pH 8.8; 0.1% Tween­20), 0.2 mM dNTP, 2 mM MgCl2; 200 mM Primer; 50 ng genomic DNA and 1 U Taq DNA polymerase (MBI Fermentas Inc, Hanover, MD­21076, USA). The ampli­ fication program consisted of 94°C for 4 minutes fol­ lowed by 35 cycles of 94°C for 1minute, 52°C for 1.05 minutes, 72°C for 2 minutes and a final extension of 8 minutes at 72°C. The amplified DNA was visualized on the 3% agarose gel. Ampelographic characterization Thirty­three major morphological descriptors selected from the descriptors list previously devel­ oped by the OIV (Organisation International de la Vigne et du Vin) (OIV, 2017) were used in this study (Tables 3, 4, 5). They include 17 qualitative descrip­ Table 3 ­ Descriptive statistics analysis of quantitative morphological descriptors of 43 grapevine accessions Table 4 ­ Leaf descriptors notation and their frequency distribution among the 43 studied grapevine accessions Descriptors Mean Minimum Maximum Least significant difference (LSD) Leaf length (cm) 9.77 7.84 11.26 1.06 Leaf width (cm) 13.29 11.4 15.74 1.35 Petiole (cm) 7.92 4.24 11.12 0.99 Number of lobes 4.89 3.00 5.00 0.09 Bunch weight (g) 576 203 1186 121 Bunch length (cm) 21.76 14.93 30.6 3.60 Bunch width (cm) 13.62 8.15 21.23 2.33 Berry weight (g) 5.7 0.94 9.95 0.47 Berry length (cm) 2.24 1.50 3.40 0.12 Berry width (cm) 1.75 1.16 2.54 0.08 Pedicel length (cm) 1.01 0.49 1.53 0.11 Seed number 2.51 1.00 4.00 0.16 Acidity at veraison (g/L) 2.82 1.35 6.10 0.89 Brix at veraison (◦Brix) 15.67 11.2 18.00 1.77 Acidity at harvesting (g/L) 1.98 1.2 3.10 0.43 Brix at harvesting (◦Brix) 18.38 13.6 23.00 2.13 Descriptors ­Leaf Notation and frequency (Number of accessions) Shape of blade Wedge­shaped (31) ­ pentagonal (3) ­ circular (9) Shape of teeth Both side straight (3) ­ both side convex (1) ­ mixture between both side straight and both side convex (39) Anthocyanin coloration of main veins on upper side Absent (25) ­ only on the petiolar point (10) ­ up to the 1st bifurcation (5) ­ up to the 2nd bifurcation (2) ­ beyond the 2nd bifurcation (1) Anthocyanin coloration of main veins on lower side Absent (16) ­ only on the petiolar point (17) ­ up to the 1st bifurcation (5) ­ up to the 2nd bifurcation (1) ­ beyond the 2nd bifurcation (4) Prostrate hairs on main veins on lower side None or very low (21) ­ low (11) ­ medium (7) ­ high (4) Prostrate hairs on main veins on upper side Absent (40) ­ present (3) Density of prostrate hairs on petiole None or very low (38) ­ low (1) ­ medium (2) ­ high (2) Opening of petiole sinus Wide open (13) ­ open (26) ­ closed (3) ­ overlapping (1) Leaf Length Short (13) ­ medium (30) Leaf width Narrow (10) ­ medium (27) ­ wide (6) Petiole length Short (8) ­ medium (25) ­ long (10) Number of lobes Three lobes (1) ­ five lobes (42) Chehade et al. ‐ Genetic diversity in Lebanese grapevine germplasm 219 tors and 16 quantitative descriptors related to the leaf (12 descriptors), bunch (5 descriptors) and berry (16 descriptors). Data analysis To assess the information given by ISSR markers, the following parameters were calculated as follow: number of alleles per locus, power of discrimination (PD= 1 ­Σ gi2, where gi is the frequency of the ith genotype), polymorphism information content (PIC= 1­∑ (Pi)2, where Pi is the proportion of samples carry­ ing the ith allele of a particular locus) (Botstein et al., 1980). Genetic distances were calculated according to Jaccard. Trees clustering the data with the unweighted pair­group method (UPGMA) with SAHN­ clustering and tree programs of PAST software (Kriege et al., 2014). Qualitative characteristics have been described and scored. For quantitative descriptors, the mean was calculated. To assess the degree of similarity between the units tested and understand the rela­ tionships between them, the data were subjected to a principle component analysis (PCA) in order to con­ dense the quantitative and qualitative descriptors in a small number of synthetic components (Saporta, 1990). Thus, the degree of contribution of each of the characters to the total variation was calculated in order to indicate the most relevant characters. Hierarchical Cluster Analyses was executed using Euclidean distance to classify cultivars into different groups based on morphological evaluation. LSD test (SAS Institute Inc, 1995) was done in purpose to com­ pare means of quantitative characters between dif­ ferent accessions. The correlation between molecular and ampelographic clustering was studied by per­ forming a Mantel test using past program. 3. Results ISSR markers analysis The nine ISSR markers (UBC807, UBC812, UBC815, Table 5 ­ Bunch and berry descriptors notation and their frequency distribution among the 43 studied grapevine accessions Descriptors Notation and frequency (Number of accessions) Bunch descriptors Bunch shape Cylindrical (21) ­ conical (15) ­ funnel shaped (7) Bunch density Loose (5) ­medium (25) ­ dense (13) Bunch weight Very low (4) ­ low (13) ­ medium (13) ­ high (8) ­ very high (5) Bunch length Short (3) ­ medium (6) ­ long (26) ­ very long (8) Bunch width Narrow (13) ­ medium (21) ­ wide (8) ­ very wide (1) Berry descriptors Berry shape Obloid (2) ­ globose (9) ­ broad ellipsoid (11) ­ narrow ellipsoid (6) ­ cylindric (1) ­ obtuse ovoid (7) ­ ovoid (3) ­ obovoid (3) ­ horn shaped (1) Color of skin Green yellow (27) ­ rose (2) ­ red (1) ­ dark red violet (8) ­ bleu black (5) Thickness of skin Very thin (2) ­ thin (17) ­ medium (17) ­ thick (5) ­ very thick (2) Firmness of flesh Soft (6) ­ slightly firm (27) ­ firm (10) Juiciness of flesh Slightly juicy (7) ­ medium juicy (27) ­ very juicy (9) Veraison time Very early (8) ­ early (3) ­ medium (2) ­ late (24) ­ very late (6) Veraison acidity (TA) Low (5) ­ medium (32) ­ high (6) Veraison brix Low (6) ­ medium (27) ­ high (10) Harvesting time Very early (3) ­ early (4) ­ medium (7) ­ late (20) ­ very late (9) Harvesting acidity (TA) Low (6) ­ medium (30) ­ high (7) Harvesting brix Low (6) ­ medium (31) ­ high (6) Berry weight Very low (4) ­ low (6) ­ medium (26) ­ high (5) ­ very high (2) Berry length Short (7) ­ medium (16) ­ long (15) ­ very long (5) Berry width Very narrow (1) ­ narrow (21) ­ medium (20) ­ wide (1) Length of pedicel Very short (1) ­ short (3) ­ medium (5) ­ long (7) ­ very long (9) Seed number One (2) ­ two (24) ­ three (11) ­ four (6) 220 Adv. Hort. Sci., 2022 36(3): 215­226 UBC816, UBC818, UBC828, UBC864, UBC868 and UBC880) showed distinct polymorphism among the 43 different grapevine accessions of this study (Table 2). A total of 41 polymorphic bands were detected. The size of amplified products ranged from 125 bp to 1000 bp. The calculated discriminating power (PD) was between 0.3 (UBC868) and 0.9 (UBC864), indi­ cating a high diversity of the loci and confirming the efficiency of these primers in studying the polymor­ phism of the Lebanese grapevine germplasm. The polymorphic information content (PIC) value varied between 0.27 (i.e. UBC868) and 0.64 (i.e. UBC816) with an average of 0.49. Number of polymorphic bands varied between two (e.g. UBC868) to six (e.g. UBC812). Eight primers had one monomorphic band, while UBC868 generated two monomorphic bands. Genetic clustering of the accessions The allelic diversity data was used to produce a dendrogram by using distance matrix­UPGMA, thus revealing the genetic relationship among grapevine accessions (Fig. 1). The dendogram constructed on the base of the amplification product of ISSR primers of the different accessions showed 41 different mole­ cular patterns. Tfaifihi variety indicated a unique marker with the primer UBC812 (305 bp). One case of synonymy was observed between Gbaii, Arasani and Gnoubi cultivars and they are very close for the most discriminating ampelographic traits. The ISSR analysis showed four distinct groups: G1, G2, G3 and G4 at the distance of 0.67 of similarity including 6, 26, 2, and 9 cultivars respectively. The first group G1 consists of six accessions ‘Baitmouni Asouad’, ‘Zaitouni, ‘Asouad’, ‘Ainouni’, ‘Moukh Al­Baghel’ and ‘Karn Al Ghazal’ and have a nine alleles in common with six primers (UBC807, UBC812, UBC816, UBC864, UBC868 and UBC880). The Largest group (G2) gathers 26 grapevine acces­ sions, shared only five alleles in common with UBC807, UBC812 and UBC818. This group can be divided into 4 sub­groups: G2.1 contains six acces­ sions, ‘Nabi’, ‘Misky’, ‘Asali’, ‘Bourji’, ‘Oubaidi’ and ‘Baitamouni’. G2.2 comprises 11 accessions, with two cases of close similarity detected between ‘Souri Zahlaoui’ and ‘Khoudri’, and between ‘Yafaoui’ and ‘Souri Mhayyar’, at 0.94 similarity levels. Only one case of synonymy was observed between three dif­ ferent accessions initially collected from the south of Lebanon under different vernacular names and pre­ senting the same genetic profile: ‘Gnoubi’, ‘Gbaii’, ‘Arasani’. G2.3 consists of four accessions, with a close similarity detected between ‘Bzaz Al­Anzi’ and ‘Amarcani Souri’ at a similiraty level of 0.96. G2.4 includes five accessions; ‘Bakhouri’, ‘Amarcani Abiad’, ‘Maghdoushi Mgaddad’, ‘Maksasi’ and ‘Ashalamish Abiad’. The third group (G3) consists only of two accessions: ‘Raha’ and ‘Tfaifihi’ had 20 alleles in common with all the nine ISSR markers. The fourth group (G4) consists of nine accessions: ‘Souri Asouad’, ‘Boulbouli Asouad’, ‘Houzairani’, ‘Amarcani Asouad’, ‘Mariami’, ‘Ari’, ‘Zaghzaghani’, ‘Samani Asouad’ and ‘Samaani Abiad’, shared only two alleles in common with UBC815 and UBC864 primers. Fig. 1 ­ Hierarchical clustering analysis of the 43 Lebanese grape­ vine cultivars, based on 9 ISSR primers using Jaccard distance and UPGMA. Chehade et al. ‐ Genetic diversity in Lebanese grapevine germplasm 221 Ampelographic description A total of 43 cultivars were studied. Locale names were commonly given by farmers or nurseries based on berry colour or on the country of origine (Asouad, Souri Aswad, Amarcani Abiad) and maturity date (Houzairani). For each cultivar, a descriptive list was established with 33 morphological traits related to the leaf, bunch and berry (Tables 3, 4, 5). Leaf description. The majority of accessions have five lobes except ‘Souri Zahlaoui’ accession which only have three lobes. Thirty­one accessions present­ ed a wedge­shaped leaf form (e.g. ‘Ari’, ‘Bourji’, ‘Gbaii’), while only nine accessions had circular leaf (e.g. ‘Raha’, ‘Bakhouri’), and the rest had a pentago­ nal leaf form (e.g. ‘Arasani’, ‘Asali’, ‘Souri Mhayyar’) (Table 4). Most of the accessions shared same teeth form with both sides straight and both sides convex except for the ‘Oubaidi’ that possesses a both side convex teeth form, and three accessions (‘Boulbouli Asouad’, ‘Nabi’ and ‘Samani Asouad’) which had a both side straight teeth form (Table 4, Fig. 2). Anthocyanin coloration of main veins on both the upper and the lower side of blade varied from absent (e.g. ‘Al Mir’), limited to the petiolar point (e.g. ‘Arasani’), extended to the first bifurcation (e.g. ‘Khodre’), and beyond the second burifaction (e.g. ‘Zaghzaghani’). The opening of the petiole sinus varied from over­ lapping sinus (e.g. ‘Zaitouni’), closed (e.g. ‘Souri Asouad’), open (e.g. ‘Misky’, ‘Rins’), to wide open sinus (e.g. ’Gbaii’, ‘Souri Zahlaoui’). The density of prostrate hairs between main veins on both sides of the blade and on the petiole varied widely from absent (e.g. Ainouni), low (e.g. ‘Bakhouri’, ‘Zaghzaghani’), medium (e.g. ‘Zaitouni’, ‘Ari’), and high (e.g. ‘Tfaifihi’, ‘Al Mir’). Only three accessions present prostrate hairs on the upper side of the main blade veins (‘Rins’, ‘Souri Mhayyar’, and ‘Tfaifihi’). Leaves generally presented an average length between 7.84 (i.e. ‘Souri Zahlaoui’) and 11.26 cm (i.e. Ari) with LSD 1.06 and width between 11.4 (i.e. ‘Rins’) and 15.74 cm (i.e. ‘Ashlamish Ahmar’). Petiole length average was between 4.24 (i.e. ‘Bzaz Al­ Aanzi’) and 11.12 cm (i.e. ‘Baitamouni Asouad’) (Table 3). Bunch description. Bunch characteristics investi­ gated showed a great diversity among the accessions studied. Almost 48.8% of the accessions had cylindri­ cal bunch (e.g. ‘Ainouni’, ‘Houzairani’), 34.9% were conical (e.g. ‘Boulbouli Asouad’, ‘Maksasi’) and 16.3% of the accessions had a funnel shaped cluster (e.g. ‘Arasani, Raha’) (Table 5). Both ‘Samani Asouad’ and ‘Samaani Abiad’ had the heaviest bunch weight, with an average of 1150 g approximately. For the remain­ ing accessions, bunch weight ranged from 1090 g (i.e. ‘Rins’) to 203 g (i.e. ‘Amarcani Abiad’). Most of the accessions had a bunch length ranging between 14.93 (i.e. ‘Maghdoushi Mgaddad’) and 30.6 cm (i.e. ‘Rins’) with 3.6 of LSD. Bunch width ranged between a minimum of 8.15 cm (i.e. ‘Bourji’) and a maximum of 21.23 cm (i.e. ‘Samaani Abiad’) (Table 3, Fig. 3). Berry description. Berry’s external appearance presented an important diversity among accessions (Table 5). For the shape, 11 accessions had a broad ellipsoid berry shape (e.g. ‘Ainouni’, ‘Arasani’, ‘Khoudri’), nine accessions had a globose berry form (e.g. ‘Ari’, ‘Tfaifihi’), and the remaining accessions had obvoid (e.g. ‘Al Mir’, ‘Souri Mhayyar’), narrow ellipsoid (e.g. ‘Gbaii’, ‘Souri Asouad’), obloid (e.g. ‘Asali’, ‘Samani Asouad’), ovoid (e.g. ‘Misky’, ‘Ashlamish Ahmar’, ‘Yafaoui’), obtuse ovoid (e.g. ‘Maghdoushi’, ‘Asouad’, ‘Souri Zahlaoui’, ‘Zaitouni’), cylindical (e.g. ‘Bzaz Al­Anzi’) and horn berry shaped (‘Karn Al­Ghazal’). As to skin color, most of the acces­ sions had green yellow berries (e.g. ‘Mekssese’, ‘Yafawi’, ‘Karn Al Ghazal’). While ‘Houzairani’ diverged from others with its red berries, eight acces­ sions had a dark red violet berry color and five other accessions shared the bleu black color. Only the rose color was found in ‘Ashlamish Ahmar’ and ‘Tfaifihi’ accessions. Fig. 2 ­ Example of variability of leaf shape. Fig. 3 ­ Example of variability of bunch shape. Adv. Hort. Sci., 2022 36(3): 215­226 222 A wide range of variability was also found for the berry quantitative descriptors (Table 3). ‘Asouad’ and ‘Asali’ accessions were outstanding with their signifi­ cantly heavier berries (9.95 g and 9.69 g respectively) while ‘Ashlamish Abiad’ had only 0.94 g, as berry average weight. For the rest of the accessions, berry weight ranged from 8.3 g in ‘Baitamouni Asouad’ to 1.71 g in ‘Amarcani Abiad’. On the other hand, ‘Karn Al Ghazal’ accession was distinguished with its long berries (3.4 cm) and its long pedicel (1.53 cm) while ‘Asali’ accession had the largest berries (2.54 cm). Principal component analysis The first three components presented 31.9% of the total variation of the different descriptors (Table 6). The first component consisted of 12.2% of the total variation and included berry skin color, firmness of flesh, bunch weight, harvesting and veraison times, in addition to the brix level at harvesting. Thus, the first component was dominated by the fruit characteristics more than the leaf ones. The second component represented 10.9% of the total variation and included four variables namely berry weight and length, leaf lower vein pigmentation and lower face pilosity. The third component was characterized by a percentage of variation of 8.8% and was dominated by the flesh juiciness and the bunch density. Based on this validation through PCA, this shortlist of dis­ criminating descriptors may be further considered to study the relationships between the grapevine acces­ sions. Ampelographic accession clustering The dendogram illustrating the relationship among the 43 studied grapevine accessions was con­ structed on the 12 most discriminating descriptors as validated by PCA. Four groups were differentiated at the distance ­17 of similarity using Euclidean distance (Fig. 4). The first group (G1) consisted of eight acces­ sions of different skin color but sharing same verai­ son date and the same sugar content at harvesting time. The second and the largest group (G2) consist­ ed of 13 accessions, sharing all the same bunch char­ acteristics in terms of density, juiciness in addition to the common sugar content at harvesting time. Moreover, these accessions started the stage of veraison almost at the same date around the 5th of September. The third group (G3) contained 12 acces­ sions, characterized by medium green berries. A case of close similarity was found between three acces­ sions of different vernacular names ‘Gnoubi’, ‘Gbaii’ and ‘Arasani’. The fourth group (G4) clustered 10 accessions of which eight accessions with dark red violet (e.g. ‘Asali’) to bleu black (e.g. ‘Mariami’) berries, and two accessions with green yellow berries (‘Rins’ and ‘Samaani Abiad’). All these accessions have common leaf characteristics and intermediate sugar content at maturity time. Table 6 ­ The first three components of the principal component analysis involving the 33 ampelographic traits and per­ formed for the 43 accessions of the national grapevine germplasm collection Variable Factor 1 Factor 2 Factor 3 Leaf form ­0.14042 0.423101 ­0.00785 Number of lobes ­0.07649 ­0.08669 0.136368 Teeth form ­0.15343 ­0.29052 0.301802 Upper vein pigmentation ­0.14288 ­0.25638 ­0.45432 Lower vein pigmentation 0.069659 ­0.63447 ­0.19775 Lower face pilosity ­0.03692 0.627619 ­0.00083 Upper face pilosity 0.17382 0.179676 0.009038 Petiole pilosity 0.27927 ­0.3332 0.328737 Overlapping of petiole ­0.00076 0.475518 0.226222 Berry shape ­0.0347 ­0.00661 0.439309 Color skin 0.511841 ­0.13872 ­0.09665 Skin thickness 0.013082 0.133863 0.46865 Firmness of flesh 0.519899 0.152408 0.095108 Juiciness of flesh ­0.27819 0.150394 ­0.55995 Seeds number ­0.01707 ­0.0642 ­0.44797 Bunch form ­0.14446 0.507152 ­0.03091 Bunch density 0.21315 0.362343 ­0.6217 Bunch weight 0.598172 0.272448 ­0.44281 Bunch length 0.344545 0.046891 ­0.35337 Bunch width 0.459819 0.390514 ­0.32888 Leaf length 0.306676 0.272602 0.398343 Leaf width 0.41474 0.04784 0.18026 Petiole length 0.436157 0.049669 0.154936 Pedicel length 0.269837 ­0.46508 0.060371 Berry weight 0.437611 ­0.53381 ­0.29825 Berry length 0.253778 ­0.65709 0.106556 Berry width ­0.13315 ­0.30376 ­0.35394 Veraison time 0.789894 0.04527 0.01873 Harvesting time 0.667212 0.098136 ­0.22776 Acidity at veraison ­0.42158 ­0.18862 ­0.28567 Brix at veraison ­0.49511 0.304928 ­0.05688 Brix at harvesting ­0.57237 0.156018 ­0.1208 Acidity harvesting ­0.44026 ­0.21603 ­0.20417 Percentage total variation 12.2% 10.9% 8.8% The characters in bold are discriminant. Chehade et al. ‐ Genetic diversity in Lebanese grapevine germplasm 223 4. Discussion and Conclusions In our study encompassing 43 local accessions, 51 bands were generated from nine primers, out of which 41 were polymorphic. Similarly, 55 polymor­ phic bands were obtained in Palestine by Basheer­ Salimia, (2015) in studying 36 grape accessions using 17 ISSR markers; while 69 polymorphic bands were obtained by Seyedimoradi et al. (2012) in studying 21 local Iranian grapevine cultivars using 10 ISSR primers. Our results related to polymorphism rate (50­85%) and size of amplified bands (125­1000bp) are close to the ones previously reported in Portugal (Castro et al., 2016), Turkey (Sabir et al., 2009), Egypt (Hassan et al., 2011), Iran (Seyedimoradi et al., 2012), and Palestine (Basheer­Salimia, 2015). Larger frag­ ments (300­1500 bp) were generated by the same ISSR markers with values up to 1500 bp in India (Dhanorkar et al., 2005) and 2500 bp in Turkey (Sabir et al., 2009). Moreover, our results indicated a dis­ crimination power value ranging from 0.3 (UBC868) and 0.9 (UBC864) and PIC value varying between 0.27 (i.e.UBC868) and 0.64 (i.e.UBC816) with an average of 0.49, which was similar to the results obtained in Iran by Seyedimoradi et al. (2012). Our ISSR results revealed an important genetic diversity within the national collection of grapevine germplasm. About 41 different molecular profiles were clearly differentiated within the 43 accessions, with only one case of synonymy found between three accessions (‘Arasani’, ‘Jbai’ and ‘Jnoubi’). These findings certainly confirm one more time the efficien­ cy of these ISSR markers in investigating the genetic variability of grapevine. It also indicates the efficiency of the initial survey and collection of local accessions in establishing this national collection of grapevine germplasm with only 5% synonymy. In Spain, the molecular characterization of grapevine accessions of the national gene bank at Alcalá de Henares allowed the differentiation of 177 accessions (30%) over 621 initially collected (Ortiz et al., 2004). Furthermore, the SSR analysis of the Eastern European cultivars led to the differentiation of only 659 unique profiles over 997 accessions studied (Maul et al., 2015). In Italy, Cipriani et al. (2010) reported 200 groups of syn­ onyms vs. 774 unique genotypes out of 1005 grapevine accessions studied. Also, Lopes et al. (1999), studying 49 supposed different cultivars from the Portuguese grapevine national collection of Terciera (Azores Island) through microsatellite mark­ ers, detected only 36 different profiles after deter­ mining the synonym cases. Along with the genetic differentiation of the grapevine accessions, an ampelographic description was carried out for the 43 Lebanese accessions using 33 descriptors recommended by the OIV, and mostly related to various descriptors including yield compo­ nents like bunch and berries dimensions and weight (Kara, 1990; Ortiz et al., 2004; Santiago et al., 2005; OIV, 2007; Akram et al., 2019). Mature leaf descrip­ tors did not vary significantly among the Lebanese accessions. Most of these accessions had leaves with Fig. 4 ­ Hierarchical clustering analysis of the 43 Lebanese grape­ vine cultivars, based on the most 12 discriminant morphological traits, using Euclidean distance and Ward method. Adv. Hort. Sci., 2022 36(3): 215­226 224 five lobes similarly to the results reported in other studies (Ecevit and Kelen, 1999; Chalak et al., 2016). On the other hand, our results revealed a large vari­ ability of bunch shape, weight, density, as well as for the berry shape, color and size, similarly to the results reported in previous works in the Eastern Mediterranean (Sabir et al., 2009; Biniari and Stavrakaki, 2016). On the other hand, the multivari­ ate analysis was efficient to analyze large data gener­ ated in our study by qualitative and quantitative descriptors to further identify patterns and relation­ ship among powerful statistical techniques. PCA allowed to extract the most discriminating descriptors, e.g. veraison date, maturity date, berry length, upper vein pigmentation, lower vein pigmen­ tation, bunch density, bunch weight, brix at harvest­ ing, juiciness of flesh, berry weight and firmness of flesh and skin color. Our results were in accordance with those previously obtained by Riachi (1998), Madi (2007) and Chalak et al. (2016) confirming one more the stability of the discriminating descriptors over years. This also in accordance with Leao et al. (2010, 2011) who reported the stability of the dis­ criminating characters for two consecutive years. Phenotypic clustering allowed differentiate the 43 accessions studied in four main groups. The syn­ onymy case first revealed between the three acces­ sions ‘Gbaii’, ‘Gnoubi’, and ‘Arasani’ by the ISSR clus­ tering was also confirmed by using ampelographic. This is the minimum rate for verifying synonyms and for clone selection processes (Cervera et al., 2002). Furthermore, many cases of close phenotypic sim­ ilarity, first revealed by Madi (2007), were also recorded in this study. It is worthy to note that some of our accessions were also described in other coun­ tries. This is the case of ‘Bzaz Al Anzi’ reported in Egypt by Hassan et al. (2011) and presenting almost similar ampelographic characteristics of the bunch and the berries. When comparing the two dendograms generated apart by the molecular and ameplographic descrip­ tors, the Mantel test indicated a weak correlation (r = 0.26, data not shown) between them. This reflects different structure for the accessions clustering whether it is generated by molecular or ampelo­ graphic descriptors. Exception is made to the case of synonymy between the three accessions ‘Gnoubi’, ‘Gbaii’, and ‘Arasani’, which was confirmed by both, ampelographic and molecular clustering. This dis­ crepancy between the two clustering types was also reported in other studies in grapevine collections (Knezović et al., 2017) and other fruit crops (Talebi et al., 2008; Zdunic et al., 2008). On the other hand, considering some key ameplo­ graphic descriptors, it was possible to categorize the existing diversity of the collected Lebanese germplasm according to the phenological stage, and according to berry skin color. In five groups of matu­ rity time; with 27 accessions had green yellow berries, only one accession with red berries, two accessions with rose berries, five accessions with bleu black berries and eight accessions with dark red violet berries. Surprisingly, similar ratios between green and red accessions were also reported in Canary Islands and Madeira by Marsal et al. (2019). Additionally, most of the accessions studied had crunchy berries with thick skin and very low juice content. This indicates the potentiality of using these accessions for table grapes rather than wine grapes. Only three accessions had thin skin, juicy flesh and high sugar content and, therefore, may be tested for fermentation. Such descriptive ampelographic char­ acterization of the berries is hopefully necessary for evaluating grapevine accessions conserved in field genebanks with respect to their usage as table grape or wine grape (Sabir et al., 2009; Ates et al., 2011; Basheer­Salimia, 2015). This diversity upon the 43 grapevine accessions conserved at the Lebanese national collection of grapevine as revealed by ISSR markers and ampelo­ graphic descriptors indicate the efficiency of sam­ pling/collecting strategy conducted in 1998 in the Bekaa and Chouf areas. Nevertheless, additional grapevine varieties grow­ ing locally under different vernacular names, which do not appear in the national grapevine germplasm collection, were recently assessed in their growing site by (Chalak et al., 2016). Therefore, it is strongly recommended to further extend the survey and mis­ sion collection to cover multiple grapevine produc­ tion areas, particularly the North, the South and of Mount of Lebanon in order to enrich the national col­ lection. Combining both molecular makers and ampelographic descriptors as it was shown in this study, would be very useful in optimizing the collec­ tion of accessions by clarifying the mislabeling cases, understanding the genetic distances among acces­ sions, and setting up a descriptive assessment for these accessions. The comprehensive evaluation of the existing local gene pool of grapevine would fur­ ther allow the sustainable utilization of the valuable accessions directly for multiplication in certified nurs­ Chehade et al. ‐ Genetic diversity in Lebanese grapevine germplasm 225 eries and also in further breeding programs. Acknowledgements This study is part of the research activities of the Plant Genetic Resources Unit funded by the Lebanese Agricultural Research Institute (LARI). Authors would like to thank Dr. Michel Afram, President Director General of LARI for his valuable support. They also thank Mr. Joe­Assaad Touma from Chateau St Thomas for their valuable guidance and support. 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