Molecular Identification and Genetic Relationships of Palestinian Grapevine Cultivars

Rezq Basheer-Salimia • Silvia Lorenzi • Fadi Batarseh • Paula Moreno-Sanz • Francesco Emanuelli • M. Stella Grando

R. Basheer-SalimiaDepartment of Plant Production and Protection, Faculty of Agriculture, Hebron University, West-Bank, Palestine

S. Lorenzi • F. Batarseh • P. Moreno-Sanz • F. Emanuelli • M. S. GrandoResearch and Innovation Centre, Fondazione Edmund Mach (FEM), Via E. Mach 1, 38010 San Michele all’Adige, Italye-mail: f.batarseh@hotmail.com

F. BatarsehCremisan Salesian Convents, Bethlehem, Palestine

Abstract Palestine has a wide range of agro-ecological concerns and hosts a large variety of plants. Grapes are part of the cultural heritage and provide an indispensable food ingredient. Local cultivars have been traditionally identified on the basis of morphological traits, geographical origin, or names of the vineyard owner; therefore, the occurrence of homonymy, synonymy, and misnaming significantly prevents their valorization. DNA profiling by 22 common SSR markers was used to characterize 43 putative cultivars grown mainly for local table grape consumption at the southern highland regions of West-Bank, to further evaluate genetic diversity and relationships of the population. Consistent matching of SSR markers with grapevines cultivated in neighboring countries or maintained in European germplasm collections was found for 8 of the 21 different non-redundant genotypes discovered, suggesting possible synonyms as well as the occurrence of breeding selections formerly developed in the USA. Genetic relationships inerred from SSR markers clearly assigned Palestinian cultivars to the Proles orientalis sub- pr. Antasiatica ancestral population, and they even remarked the connection between local resources and cultivars generated from international table grape breeding.

This study supports the value of collection and conservation of vines endemic to a region of immense historical importance for viticulture.

Keywords Vitis vinifera • Table grapevine • SSRs • Palestine • Genetic relationship

Introduction

Grapevine (Vitis vinifera L.) is one of the most ancient cultivated fruit trees in the world. In Palestine, its plantation goes back at least 4000 years [1], and currently it is considered as the second important fruit crop after olive in terms of both areas covered as well as economic returns [2]. Due to the unique geographical and ecological environment for growing high-quality fruits, grape growing and production are still limited to the southern part of West-Bank, especially Hebron and Bethlehem areas, where it represents an indispensable food ingredient [molasses, raisins, fruit roll (malban), vinegar, and juice are produced], with a total cultivated area of about 7,600 ha [3]. With the establishment of the Islamic civilization and the Islamic conquests (700 AD till now), Palestinian viticulture focused more on cultivation of table grape varieties [4, 5], mainly through introduction of new varieties from other countries such as Yemen, Saudi Arabia, Egypt, Syria, and Lebanon. In fact, many existing varieties have been named based on their geographical origins. To date, Palestinian grapes rely heavily on the use of anciently introduced regional cultivars, which could have also locally given origin to cultivars (landraces), for which a large number of homonymous and synonymous designations, as well as the occurrence of misnaming, may exist. In addition, grape cultivars have been traditionally

identified on the basis of characteristics such as fruit skin color, local geographical origin, names of the vineyard owner, and cluster shape. Moreover, a significant reduction in the Palestinian grapevine genetic diversity occurred because of the phylloxera pest [6] and urbanization, thereby resulting in the disappearance of some old local varieties. Hence, there is a crucial necessity of a clear discrimination between these landraces with the aim of characterizing existing cultivars, conserving the plant genetic resources, guaranteeing the trueness-to-type of the propagation materials and, in addition, using this characterized plant material for future crop improvement [7]. Molecular characterization is now the favored way to quantify variation within germplasm samples. Studies on local grapevine cultivars and their wild relatives have been undertaken with a range of molecular marker technologies [8–10]. Microsatellites (simple sequence repeats, SSR), because of their polymorphism, reproducibility, and codominant nature, have become the markers of choice for compilation, standardization, and exchange of information concerning grapevine genetic resources [11]. Application of SSR markers to several surveys of large germplasm collections [12–14] provided insight into phylogenetic relationships among grapevine varieties; however, almost none of the investigations included Palestinian varieties, neither at morphological nor at molecular level. The main goals of the present research were to determine the number of genetically different cultivars that were actually collected in Palestine, infer possible cases of synonymy and homonymy, identify possible matches with SSR profiles of grape cultivars reported in international databases and literature, and evaluate the genetic relationships of the characterized cultivars.

Materials and Methods

Plant Material

Young leaves were collected from 43 assumed diverse grape cultivars throughout the southern highland (Hebron and Bethlehem) regions of West-Bank, Palestine (Table 1; Fig. 1). The climate of the region is atypical Mediterranean, with mild temperatures (18–25 °C), rainy weather (300–600 mm/year) in autumn and winter, and hot, dry summers. The color of the berry skin of each accession was determined according to OIV code 225 (http://www.oiv.int/). The degree of sugar content of the berry and the firmness of flesh were subjectively scaled based on refractometer and penetrometer measurements, respectively. Reported information about agronomic and culture conditions of some cultivars was collected from the Faculty of Agriculture, Hebron University records.

SSR Genotyping

Genomic DNA was extracted from young leaves using the DNeasy Plant Mini Kit (QIAGEN Inc.). The 43 accessions were first genotyped at nine microsatellite loci combined in four multiplex polymerase chain reactions (PCRs) as follows: VVS2, VVMD32 and VVMD28; VVMD27 and VVMD7; VrZAG62 and VrZAG79; VVMD5 and VVMD25. Primer sequences and nomenclature are reported in Refs. [15–18]. Next, non-redundant genotypes were analyzed at further 13 SSR loci [19], combined in five multiplex PCRs: VVIQ52, VVMD24, and VVIN16; VVIV37 and VVIH54; VMC4F8, VVMD21, and VVIN73; VVIP31 and VVIB01; VMC4F3.1, VVIV67, and VMC1B11. The forward primer of each marker was labeled with a fluorescent dye (6-FAM, NED, or VIC). Markers used within the same multiplex PCR were tagged with different dyes to allow their identification. Simultaneous PCR amplifications were carried out in a final volume of 12.5 ll containing 10 ng of genomic DNA, 0.25 mM of each dNTPs, 2 mM MgCl 2 , and 1.5 U Taq DNA Polymerase (Gold Taq Ò ). Depending on the locus, primer concentrations ranged from 0.2 to 0.6 lM. Reactions were performed on a GeneAmp PCR System 9700 (Applied Biosystems, Foster City, CA) using a hot start of 95 °C for 7 min, 30 amplification cycles of 45 s at 95 °C, 1 min at 54 °C, 30 s at 72 °C, and a final extension step of 1 h at 72 °C. PCR products (0.5 ll) were mixed with 9.3 ll of formamide and 0.2 ll of the GeneScan TM

500 ROX Ò Size Standard (Applied Biosystems). DNA fragments were denatured (2 min at 95 °C) and separated by size using capillary electrophoresis on an ABI 3130 Genetic Analyzer (Applied Biosystems). Subsequently, GeneMapper v3.5 (Applied Biosystems) was used for the estimation of allele sizes. Genomic DNA from the common cultivar Pinot noir was included in the analysis procedure to harmonize SSR allele sizes and compare molecular genotypes among different studies and databases.

Genetic Diversity and Relationships

The genetic diversity of the Palestinian population of grapevine varieties was measured as mean number of alleles per locus (a), number of effective alleles (A e ), levels of observed (H o ), and expected (H e ) heterozygosity. These calculations together with values of PI (the probability that two unrelated individuals in the population share the same genotype) were performed using GenAlex 6.5 [20]. The genetic profiles at 22 SSR loci were further subjected to cluster analysis together with homologous profiles of 39

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Fig. 1 Map showing the collecting sites

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accessions belonging to the Fondazione Edmund Mach germplasm collection (ITA 362). These last were shown to represent the classical eco-geographic grouping of grape cultivars [21] within a population of 330 unique genotypes of V. vinifera [14]. An unweighted neighbor-joining tree based on the simple matching dissimilarity matrix with 10,000 bootstrap replicates was constructed using the Darwin software package v5.0148 [22]. The SSR genotype of three grape rootstock varieties was used as an outgroup.

Results and Discussion

Historical Palestine is located between the continents Asia, Europe, and Africa, and between the eastern Mediterranean Sea, Red Sea, Dead Sea, and Jordan River. Despite its small size, this location has a wide range of agro-ecological concerns and hosts a large variety of plants [23], including grapevines. Plant samples analyzed in this study were collected from putative diverse cultivars throughout the main grape producing localities at the southern highland regions of West-Bank, Palestine. These cultivars are grown mainly for local table grape consumption.

SSR Marker-Based Characterization and Genetic Diversity

Of the 43 samples analyzed, 21 unique genetic profiles were detected using the nine SSR markers chosen from the European Vitis Database [24] (Table 2). The standard set of markers proved a high discriminating capacity for grapevine varieties and this was supported in the present study by a cumulative probability of identity (PI) of 8.9 9 10 -11 (Table 4). After pair-wise comparison, 12 genetic profiles resulted represented by only one accession in the whole population, while 9 SSR genotypes were common between 2 and 7 different accessions. A low genetic variation was found especially for 18 accessions which revealed only three different SSR genotypes. The list of accessions and their respective SSR genotypes is shown in Table 1. Accessions having the SSR genotype no. 1 were all characterized by green-yellow colored fruits, early ripening, well-developed seeds, drought resistance, and successful cultivation under a wide variety of soil types, and were also most suitable for the production of raisins and processing in general. They mainly presented a high degree of sugar content and soft-medium flesh firmness. These accessions are most widely grown in Palestine. The three Zaini accessions likely received their names based on the appealing shape of the clusters, as ‘‘Zain’’ in the Arabic language reflects beauty and sweetness. These three accessions vary slightly in the names ascribed to them, likely reflecting differences in local dialects or farmer descriptions. The names also reflect some minor phenotypic differences that may have arisen due to particular local growing conditions. Furthermore, the three Dabuki accessions were likely named based on their perceived sweetness because ‘‘Dabuki’’ in Arabic also means sweetness. Concerning the accession Salti Khdari, it was introduced from the city of Salt in Jordan, less than 70 km from Hebron, and possesses very similar characteristics to Zaini and Dabuki accessions. Few slight differences observed in this putative cultivar include a stronger green color, a firmer cluster texture, and lower sugar content. Regarding the final accession, Baluti Abiad, the name is derived from the shape and color of the fruit. That is, ‘‘Baluti’’ means the shape of the oak fruit and ‘‘Abiad’’ means white (i.e., green in the lexicon of grape descriptors). SSR genotype no. 2 was observed for accessions Malikat Libnan, Jandali, Jandali HabaTawila, Jandali Haba Kurawi, and Jandali-Beitlahim which are almost very similar phenotypically except for the size and shape of the cluster and berries. The observed phenotypic differences among these accessions are probably related to varying environmental growing conditions. Malikat Libnan (The Queen of Lebanon) possibly received its name based on the beautiful arrangement of the berries within the cluster and also the sugar content of the grapes. Alternatively, this cultivar may have been introduced from the neighboring country of Lebanon (Libnan in Arabic). One distinguishing characteristic observed in all accessions in this genotype was the development of a stylar scar (small, corky area remaining on the apex of a berry after the style abscises following fertilization). SSR genotype no. 3 was common to six accessions, three named based on ‘‘Marawi’’ and three derived from ‘‘Hamadani’’, in which all accessions possessed similar shape and color of the berries, soft to medium berries’ firmness, cluster shape, ripening time, and juiciness. The Hamadani accessions revealed a slightly crispy texture of berries and a higher number of seeds, whereas the Marawi accessions were characterized by almost an absence of crispness in the texture of the berries and a lower number of seeds. SSR genotype no. 4 was found for Halawani and Halawani Haba Kabera which are almost very similar phenotypically. Both are characterized by colored skin, large berry size, well-developed seeds, very firm, high crispness, and late ripening. They likely received their names based on the sweetness of the berry juiciness, as ‘‘Halawani’’ in the Arabic language reflects sweetness. The second accession followed by Haba Kabera, which means in the Arabic language ‘‘big berries’’, is likely reflecting differences in local dialects or farmer descriptions. SSR genotype no. 5 was common to Aswad Baladi and Dabuki Aswad Baladi accessions. ‘‘Aswad’’ and ‘‘Baladi’’ means in the Arabic language ‘‘black skin color’’ and

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6

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‘‘local cultivar’’, respectively. Dabuki means ‘‘sweeter’’ over the first accession and this is likely the only minor phenotypic difference between the two accessions that can be reported. SSR genotype no. 6 was found for three accessions: Shami Mtartash ‘‘Mlawwan’’, Shami Aswad, and Shami. Their names likely reflect differences in local dialects or farmer descriptions mainly for the skin color hue (greenish black, blue black, and black, respectively). Furthermore, the ‘‘Shami’’ name may be related to a possible introduction of the cultivars from Syria, neighboring country called Sham in Arabic. SSR genotype no. 12 was found for two accessions, Mtartash-Fhessi and Fhaisi, which are similar phenotypically except for the spotted (speckled) berries typically brown-red color of the first accession (‘‘Mtartash’’ means in Arabic having many scattered colored spots). In addition, Fhaisi is the name of the city of Fhess in the neighboring country Jordan, suggesting a possible origin of the cultivars. SSR genotype no. 14 was common to Betooni and Baluti accessions. Betooni accession might have been firstly introduced to the city of Betoonia nearby Rammalla and then transferred to the southern region of Palestine. The Baluti name, instead, may relate to the elongated berries shape that looks like oak fruits and is a distinguishable trait between these accessions besides a lighter skin color. SSR genotype no. 16 was observed for Darawishi and Sheukhi accessions. Darawishi comes from the name of the Palestinian man, ‘‘Darwish’’, who first introduced this accession from Lebanon and cultivated it in Sheukh ‘‘a small village at Hebron Governorate’’. Therefore, these accessions are still named based on the original introduction history and are likely synonyms. Polymorphism of the nine reference loci was moderate in the population with a number of alleles per locus ranging from 5 for VrZAG79 to 10 for VVMD5 (mean 7.6) for a total of 68 alleles. Gene diversity, quantified as the expected heterozygosity, was comparable to that of grapevine populations of similar size from different European regions analyzed by Sefc et al, [9]. Likewise excess of heterozygotes was observed at some loci (Table 4). The European Vitis Database (http://www.eu-vitis.de) and data from the literature were consulted for searching identical genetic profiles; eight complete matchings were found (Table 2). This was an important step since it revealed that some genetic resources collected in the Palestinian territories are represented at least in one European germplasm repository, whereas other cultivars may be synonyms of grapevine varieties cultivated in neighboring

countries. In this study, seven putative varieties, three of which called Dabuki (accessions no. 5, 26, and 34) and one Salti Khdari (no. 15), showed the same SSR genotype of a ‘‘Dabouki’’ accession reported in the database. Dabouki is a variety registered in the national catalogs of France, Spain, and Portugal for growing table grape and has the ID #3309 in the Vitis International Variety Catalog (VIVC, www.vivc.de) where Armenia is indicated as its country of origin. Salti Khdari was actually among the many synonyms reported in VIVC for this cultivar. The cultivar Mtartash (accession no. 13), instead, displayed a unique SSR profile among the Palestinian accessions studied (genotype no. 13). The profile matched that of an ‘‘Emperor’’ accession in the database. Consistent with morphological characteristics, it may be a synonym. The table grape Emperor was originated in the USA, and is registered and cultivated in Italy, Croatia and Portugal under the VIVC #3904 code. Based on the molecular markers, the accession Halawani Mutar (accession no. 41) would correspond to the variety Red Globe, a breeding selection developed by H.P.

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Olmo and A. Koyama at California Agricultural Experiment Station (USA) in the late 1950s from the cross of (Hunisia 9 Emperor) 9 (Hunisia 9 Emperor 9 Nocera). Halawani means beauty and sweetness, while Mutar is referred to a very good smell which looks like perfume. The use of local words describing the main characteristics of the cultivars, instead of the international names, may represent an ethno-linguistic phenomenon which contributes to the cultivation and use of a variety after its introduction. Besides other regions in the world, Red Globe variety (VIVC #9972) is cultivated in Southern Europe countries (France, Spain, Portugal, and Italy). The fourth cultivar identified by searching the SSR database was the accession called Fahace (accession no. 40). The genetic profile of this sample at nine markers was the same described for grapevine accessions of the widely grown table grape variety Ahmeur bou Ahmeur also found in several germplasm collections, including the Sodea germplasm collection of Morocco [25]. For this variety (VIVC #140), thought to be originated from Algeria, dozens of different synonyms exist. Four genetic profiles (genotypes no. 4, 6, 8, and 14) were consistent with the molecular markers reported in the literature for local grapevine cultivars of the Holy Land by Klein et al. [26], which are maintained in the Sataf collection near Jerusalem. The study characterized 24 vine accessions at 6 of the 9 loci applied in the present investigation and was therefore enough informative for the comparison of genotypes. Full matching was found between the molecular profile of the Halawani and the Hilwani accessions from the two collections. Similarly, identical genetic profiles were revealed for the accessions called Shami in both surveys. Zeini Sheik from the Sataf collection shared the same SSR markers than a Bairuti accession in our study and finally both investigations reported identical profiles for Betooni/Bituni and Baluti accessions. In total, Benjamin et al. [26] detected

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22 different genetic profiles of which only few may have been represented in our survey. This would confirm the value of further collection and conservation of vines endemic to the area between the Jordan River and the Mediterranean Sea, a region of immense historical importance for viticulture and viniculture.

Genetic Relationships

The set of 21 distinct genotypes identified based on the ‘‘core set’’ of 9 SSR markers was further characterized using 13 additional SSRs (Tables 3, 4) to support a genetic relationship analysis including genotypic data of grapevines from different origins. The last were represented by 36 SSR profiles of cultivars sorted from the four subpopulations identified within V. vinifera germplasm through a structural analysis of 733 diverse accessions by Emanuelli et al. [14]. Since this clustering substantially agrees with the classification of eco-geographic variation proposed by Negrul [21], the ancestral population of origin of Palestinian cultivars was thus inferred. The neighbor-joining unweighted tree, built based on a dissimilarities matrix calculated from SSR alleles, is shown in Fig. 2. According to their origin, most Palestinian genotypes formed a well-defined cluster close to the groups of cultivars within the Proles orientalis subpr. Antasiatica. The accession Roomi Aswad Haba Tawila (genotype no. 11) clustered very closely with Flame seedless and Calmeria, two seedless grape varieties related to Sultanina and generated by cross breeding in the USA. A more distinct branching, but still assigned to the Proles orientalis group, was formed by the putative Emperor cultivar (genotype 13), the putative Red Globe cultivar (genotype 19) and genotype 20 (SPS seedless accession). This finding well supports the identification results, since Emperor and Red Globe, being close relatives, are expected to cluster together.

Conclusion

Domestication of grapevine is suggested to have taken place in the south Caucasus between the Caspian and Black Seas about 8000 years ago [27]. From this area, cultivated forms would have been spread gradually by humans into south to the western side of the Fertile Crescent, Egypt, and the Jordan Valley by 5000 years ago [28]. The collection, molecular characterization, and conservation of local table grape vines reported here provide the first picture of the genetic diversity of the area between the Jordan River and the Mediterranean Sea, and enable us to include additional cultivars into the studies of V. vinifera and grape growing culture evolution.

Acknowledgments We thank VIS—Volunteers for International Development, Italian NGO who coordinated and funded research activities with the contribution of the CEI—Italian Bishops Conference and the Province of Trento. We are also grateful to Salvatore Maule and Enzo Mescalchin (FEM) for providing some plant materials. This work was carried out in the framework of COST Action FA1003 ‘‘East–West Collaboration for Grapevine Diversity Exploration and Mobilization of Adaptive Traits for Breeding’’.

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