Art15_Ercisli.indd Molecular characterization of autochthonous grapevine cultivars by SSR Journal of Applied Botany and Food Quality 85, 224 - 228 (2012) 1Department of Horticulture, Agriculture Faculty, Ataturk University, Erzurum, Turkey 2Ankara University, Biotechnology Institute, Ankara, Turkey Genetic characterization and relatedness among autochthonous grapevine cultivars from Northeast Turkey by Simple Sequence Repeats (SSR) Y. Hizarci1, S. Ercisli1*, C. Yuksel2, A. Ergul2 (Received October 10, 2012) * Corresponding author Summary 25 autochthonous grapevine cultivars from Northeast Anatolia in Turkey together with two well-known standard cultivars, Cabernet Sauvignon and Merlot were fi ngerprinted using six pairs of SSR primers to assess their genetic diversity and relatedness. All six SSR primers produced successful amplifi cations and revealed DNA polymorphisms that were subsequently used to assess genetic relatedness of the cultivars. A total of 52 alleles were detected with a mean value of 8.67 alleles per locus indicating allele richness. The average expected heterozygosity (He) and observed heterozygosity (Ho) were 0.759 and 0.809, respectively. Considering the number of alleles generated, the highest number was observed in VVS2 loci (14 alleles/locus), while the lowest in VrZAG83 loci (5 alleles/ locus). The Unweighted Pair-Group Method with Arithmetic mean (UPGMA) dendrogram constructed based on the SSR data yielded two main clusters. First cluster included only cv. Kibris and the second cluster included rest of the cultivars including Cabernet Sauvignon and Merlot. The results showed that SSR markers have proved to be an effi cient tool for fi ngerprinting grapevine cultivars and conducting genetic diversity studies in grapevine. Introduction Turkey, located at the junction of two main plant gene centers, has a very old and rich grapevine germplasm including over 1500 cultivars. Archaeological excavations also confi rm that grapevine cultivation is a very old tradition in Anatolia dating back to 4000 BC (SELLI et al., 2007). Each grape-growing region in Turkey has particular local grapevine cultivars which differ from each other in color, taste, shape, bunch density etc. There is also a wide variation in terms of synonymous cultivars in each region and correct identifi cation of these cultivars is of great importance in cultivar standardization and determination of total cultivar number (ERGUL et al., 2006). Northeast Anatolia region in Turkey has older and special grapevine cultivation. There were a lot of churches in this region and grape berylliums of different shapes can be seen on the walls of churches. MCGOVERN (2008) stated that the region, together with other regions of the South Caucasus and Anatolia, is the cradle of domestication and primary viticulture in the Old World. In this region, Yusufeli district is the main traditional viticulture area including over 30 very old and local grapevine cultivars. This area is also very rich in terms of wild grapes (Vitis vinifera ssp. sylvestris). In Christian period, these cultivars were used for wine-making. However, at present, wine production is not practiced because of the conservative life style of the people in this region. Hence, the cultivars previously used for wine-making are currently used in juice production. Northeast Anatolia region differs greatly from the other regions in Turkey in terms of climatic characteristics and thus it is expected that the grape germplasm of this region would have economically important adaptive traits that can potentially be incorporated into grape breeding programs in future. During the last decade, the genomic resources that are available to the grapevine research community have increased enormously in parallel to a renewed interest in grapevine (Vitis vinifera L.) germplasm resources and analysis of genetic diversity in grapes. It is well recognized that genetic variation is invaluable for crop improvement and understanding gene function, and this fact applies to grapevine as well (THIS et al., 2006). Knowledge of the genetic diversity and relationships among the grapevine cultivars is important for recognizing gene pools, identifying pitfalls in germplasm collections, and developing effective conservation and management strategies (ERGUL et al., 2006). The grapevine is highly heterozygous and propagation by cuttings or layers maintains their heterozygosity. Therefore, morphological classifi cations provide rough guidelines, while molecular evaluations provide further insight into the genetic structure and differentiation within and among taxa which is useful for geneticists, plant breeders, and gene bank managers (PAPANNA et al., 2009) Lots of studies have investigated the genetic variation within grapevine by using different molecular marker systems such as Random Amplifi cation of Polymorphic DNA (RAPD) (BENJAK et al., 2005), Amplifi ed Fragment Length Polymorphism (AFLP) (DOULATI BANEH et al., 2007), Inter Simple Sequence Repeats (ISSR) (ARGADE et al., 2009), Single Nucleotide Polymorphism (SNP) (PINDO et al., 2008) and SSR (DE MATTIA et al., 2009; LEAO et al., 2009). Among these marker systems, SSRs have been widely used by researchers to determine the genetic diversity within grapevine cultivars. Six so-called “core set” SSR markers (i.e., VVS2, VVMD5, VVMD7, VVMD27, VrZAG62, and VrZAG79) are recommended for the direct comparison of results from different laboratories (THIS et al., 2004). In Northeast part of Turkey, there are several specifi c grape germplasms and Coruh Valley is one of the most important grape germplasm centers of this region. There are a number of autochthonous grapevine cultivars which were used many centuries ago. Therefore, it can be valuable to characterize this germplasm both morphologically and genetically. In this study, an attempt to assess the level of genetic diversity and genetic relationship among 25 autochthonous grapevine cultivars from Northeast Anatolia in Turkey has been made. It is expected that the information presented here would be useful for selection and more effi cient utilization of this germplasm in grape breeding programs in future. Material and methods Plant material Leaf samples of 25 autochthonous grapevine cultivars used in this study were collected from Yusufeli district in Northeast Anatolia region in Turkey. A total of 25 grapevine cultivars to gether with two reference cultivars, Cabernet Sauvignon and Merlot were included in SSR analysis. Some important ampelographic traits of these cultivars provided by the International Union for the Protection of New Varieties of Plants (UPOV) are presented in Tab. 1. Molecular characterization of autochthonous grapevine cultivars by SSR 225 DNA extraction Genomic DNA was extracted from young leaf tissue using the Wizard® Genomic DNA Purifi cation Kit (Promega, Madison, WI) according to the instructions provided by the manufac turer. Subsequently, a RNAse treatment was performed on the eluted DNA samples. Purity and concentration of the DNA were checked both on 1% (w/v) agarose gels and by NanoDrop® ND-1000 Spectrophotometer. SSR analysis Six SSR markers (i.e., VVS2, VVMD5, VVMD7, VVMD27, VrZAG62, and VrZAG79) were used in Polymerase Chain Reaction (PCR) studies. PCR was conducted in a volume of 10 µL and contained 15 ng genomic DNA, 5 pmol of each primer, 0.5 mM dNTP, 0.5 unit GoTaq DNA polymerase (Promega), 1.5 mM MgCl2 and 2 µL 5X buffer. The forward primers were labeled with WellRED fl uorescent dyes D2 (black), D3 (green) and D4 (blue) (Pro ligo, Paris, France). Reactions without DNA were included as negative controls. PCR amplifi cation was performed by using the Biometra® PCR System. The amplifi cation condi tions consisted of an initial denaturation step of 3 mins at 94°C, followed by 35 cycles of 1 min at 94°C, 1 min at 52-56°C and 2 mins at 72°C with a fi nal extension at 72°C for 10 mins. The PCR products were fi rst separated on a 3% (w/v) agarose gel run at 80 V for 2 hrs. The gel was then stained with ethidium bromide at a concentration of 10 mg/mL. A DNA ladder (100 bp) (Promega) was used for the approximate quantifi cation of the bands. The amplifi cation products were visualized under UV light, and their sizes were estimated relative to the DNA ladder. For further determination of polymorphisms, the PCR prod ucts were run on CEQTM 8800 XL Capillary Genetic Analysis System (Beckman Coulter, Fullerton, CA). The analyses were repeated at least twice to ensure reproducibility of the results. Allele sizes were determined for each SSR locus using the Beckman CEQTM Frag ment Analysis software. In each run, Cabernet Sauvignon and Merlot cultivars were included as reference cultivars. Genetic analysis The genetic analysis program “IDENTITY” 1.0 (WAGNER and SEFC, 1999) was used according to PAETKAU et al. (1995) for the calculation of number of alleles, allele frequency, expected and observed heterozygosity, estimated frequency of null alleles, and probability of identity per locus. Genetic dissimilarity was determined by the program “MICROSAT ” (version 1.5) (MINCH et al., 1995) using proportion of shared alleles, which was calculated by using “ps (option 1 - (ps))”, as described by BOWCOCK et al. (1994). The results were then converted to a similarity matrix, and a dendrogram was constructed with the UPGMA method (SNEATH and SOKAL, 1973) using the software NTSYS-pc (Numerical Taxonomy and Multiware Analy sis System,version 2.0) (ROHLF, 1988). Results and discussion The study presents SSR analysis results of a total of 27 grapevine cultivars comprising 25 autochthonous grapevine cultivars sampled from Northeast Anatolia and two reference cultivars Cabernet Sauvignon and Merlot, to determine genetic diversity and relatedness among them. As shown in Tab. 2, a total of 52 alleles ranging from 5 to 14 alleles per locus with a mean value of 8.67 alleles per locus were detected. Polymorphic bands were obtained with all primers. VVS2 loci were the most polymorphic among the six loci, with the highest effective number of alleles (14 alleles), followed by the loci of VrZAG79 (11 alleles) and VVMD27 (9 alleles). The VrZAG83 loci generated the lowest number of alleles (5 alleles) (Tab. 2). The mean He and Ho were determined as 0.759 and 0.809 over six loci, respectively. Among the six loci, the Ho values were the highest (0.889) in VVS2 loci indicating high genetic diversity while the lowest (0.593) in VrZAG83 loci (Tab. 2). Among the six loci, only VVMD7 generated more than two alleles (tri-allelic loci) in two varieties (Karul and Nanebur) (Tab. 3). In present study, the frequency of null alleles (r) in VVS2 and Tab. 1: Basic descriptive characteristics of 25 grapevine cultivars used in this study. Cultivar Utility Berry Berry Color shape Alvan Juice Dark red-violet Round Artvin Juice Green-yellow Ovate Beyaz Istanbul Table Green-yellow Elliptic Beyaz turfanda Table Green-Yellow Ovate Ciklap Juice Green-Yellow Round Erik Table Yellow Ovate Gelin parmagi Juice Green-Yellow Narrow elliptic Gines Table Dark red-violet Round Goh Juice Blue-black Round Hatkul Juice Blue-black Round Kara turfanda Juice Blue-black Round Karul Table Green-Yellow Narrow elliptic Keci memesi Juice Dark red-violet Narrow elliptic Kibris Table Dark red-violet Ovate Kirmizi Istanbul Table Rose Ovate Kiskinbur Juice Rose Round Kokulu Juice Blue-black Round Kutuk Juice Green-Yellow Ovate Mandagozu Juice Blue-black Elliptic Mezarlik Juice Yellow Round Nanebur Juice Dark red-violet Elliptic Razaki Table Dark red-violet Ovate Sulu Kurta Juice Dark red-violet Round Tokat Table Green-yellow Round Yag Juice Dark red-violet Round Tab. 2: Number of alleles, allele range (bp), expected heterozygosity, observed heterozygosity, and probability of identity values of grapevine cultivars. Loci N He Ho PI r VVMD7 6 0.756 0.852 0.186 -0.0547 VVMD27 9 0.748 0.852 0.134 -0.0592 VrZAG79 11 0.831 0.852 0.088 -0.0112 VVMD24 7 0.763 0.815 0.150 -0.0296 VVS2 14 0.903 0.889 0.032 0.0072 VrZAG83 5 0.612 0.593 0.330 0.0119 Total 52 4.613 4.853 Average 8.67 0.759 0.809 N: number of alleles; Ho: observed heterozygosity; He: expected hetero- zygosity; PI: probability; r: null allele frequencies 226 Y. Hizarci, S. Ercisli, C. Yuksel, A. Ergul Molecular characterization of autochthonous grapevine cultivars by SSR VrZAG83 loci was positive, but these low values suggest the absence of null alleles (Tab. 2). The most informative loci, with regards to the probability of identity (PI), were VVS2 and VrZAG79 which generated 14 alleles (PI: 0.032) and 11 alleles (PI: 0.088), respectively, whereas the least in- formative locus was VrZAG83 which generated 5 alleles (PI: 0.330) (Tab. 2). Allele sizes (bp) of 27 cultivars from six SSR loci are shown in Tab. 3. The most frequent alleles were 246 (30.38%) in VVMD7, 185 (44.44%) in VVMD27, 246 (27.78%) in VrZAG79, 207 (38.89%) in VVMD24, 143 (18.52%) in VVS2 and (53.70%) in VrZAG83, respectively. The number of microsatellite different genotypes ranged from 8 (VrZAG83) to 21 (VVS2) with an average of 13.1 and a total of 79. The genetic similarity ranged from 0.17 to 0.92 among cultivars and the cultivars Kutuk and Yag; Kokulu and Yag as well as Kibris and Cabernet Sauvignon were found to be more distant at 0.17 similari- ty ratio (Fig. 1). According to genetic similarity ratio, cultivars Kirmizi Istanbul and Razaki were the closest (0.92) (Fig. 1). The average similarity ratio was 0.53 indicating high genetic diversity among cultivars. To elucidate the genetic relationship among grapevine cultivars, a dendrogram generated from the UPGMA cluster analysis over six SSR loci classifi ed 27 cultivars into two main groups depicted in Fig. 1. A single cultivar (cv. Kibris) was clustered separately from the remaining cultivars. The second main cluster included 24 autochthonous cultivars and the two reference cultivars. This cluster was further divided into two subgroups. The fi rst subgroup comprised the two reference cultivars (Cabernet Sauvignon and Merlot) and two autochthonous cultivars (cvs. Mandagozu and Beyaz Istanbul). In this subgroup reference cultivars clustered separately from the other two cultivars. The second subgroup comprised the remaining 22 autochthonous cultivars. In this study we report for the fi rst time the use of SSR markers for assessing genetic relatedness among 25 autochthonous grapevine cultivars from Northeastern Turkey. The results obtained in the present study show that microsatellites can be effectively used for fi ngerprinting purposes in grapevine. In fact, all tested microsatellite primer pairs produced various levels of amplifi cations. The mean value of 8.67 alleles per locus obtained with the microsatellites that produced polymorphic amplifi cation patterns is consistent with the reported data of 4-16 alleles per locus in V. vinifera germplasms assessed with microsatellites in different countries (BOWERS et al., 1996; FATAHI et al., 2003; MARTIN et al., 2003; MARTINEZ et al., 2006; TANGOLAR et al., 2009). The high levels of within-group variation and the simple genetic structure observed in the dendrogram probably suggest a complex history of development of grapevine cultivars in Northeast Anatolia. Introduction and spread of wild and semi-domesticated grapes, especially from its native Near Eastern range, domestication of indigenous wild grape, natural hybridization between indigenous and introduced vines, and human selection may have contributed to this high variation. The cv. Kibris was clustered separately on dendrogram. If we look at Tab. 3, we can see that this cultivar has unique alleles in at least fi ve loci. This cultivar was brought to Coruh Tab. 3: Allele sizes, in base pairs, of six microsatellites loci from 27 grapevine cultivars. Cultivar VVMD7 VVMD27 VrZAG79 VVMD24 VVS2 VrZAG83 Artvin 238:246 181:185 248:256 207:211 139:143 185:191 Alvan 238:246 185:189 244:246 207:207 145:149 191:191 Beyaz Istanbul 236:246 185:185 244:252 207:211 135:151 185:197 Beyaz turfanda 238:246 185:191 244:246 207:215 135:141 185:191 Ciklap 238:246 179:185 240:246 207:215 139:151 191:191 Erik 236:244 179:181 248:256 207:207 139:149 185:185 Gelin parmagi 238:246 181:185 246:250 217:217 125:149 191:191 Gines 236:242 183:185 246:246 207:215 133:145 191:191 Goh 238:246 181:185 248:256 207:215 143:143 191:191 Hatkul 238:246 179:185 244:244 205:217 123:143 191:191 Kara turfanda 236:244 179:185 246:250 205:217 135:149 185:191 Karul 230:238:246 179:185 238:246 201:207 135:143 187:191 Keci memesi 240:246 185:189 242:246 205:217 141:145 185:191 Kibris 238:238 195:195 246:248 205:211 159:159 185:191 Kirmizi Istanbul 236:244 181:185 248:256 207:211 137:143 185:191 Kiskinbur 238:246 185:185 248:250 205:215 125:143 185:191 Kokulu 238:246 179:183 234:244 205:207 123:151 185:187 Kutuk 238:246 175:179 244:248 205:207 133:137 185:185 Mandagozu 236:246 185:185 238:248 207:211 131:133 191:197 Mezarlik 236:244 183:185 244:248 207:215 133:133 181:185 Nanebur 238:242:246 179:185 240:248 207:209 139:143 185:191 Razaki 236:244 181:185 248:256 207:211 139:143 185:191 Sulu Kurta 246:246 181:185 246:250 217:217 123:149 191:191 Tokat 244:244 177:185 244:246 205:207 137:143 185:191 Yag 236:244 181:185 246:250 217:217 125:149 191:191 Cabernet Sauvig. 236:236 175:189 246:246 207:217 139:151 197:197 Merlot 236:244 189:191 258:258 207:211 139:151 191:197 Molecular characterization of autochthonous grapevine cultivars by SSR 227 Valley from Cyprus Island and due to the protective position of the island, the genetic structure of the cultivar may have been protec- ted. Based on the number of alleles generated and probability of identity values, the most informative loci were VVS2 (14 alleles per locus, PI:0.032) and VrZAG79 (11 alleles per locus, PI: 0.088), whereas the least informative locus was VrZAG83 generated (5 alleles per locus, PI: 0.330). Previously, among the six SSR primers, the highest number of alleles was obtained from VVS2 primer (FATAHİ et al., 2003; NUÑEZ et al., 2004; SELLİ et al., 2007; TANGOLAR et al., 2009) while the lowest number of alleles was detected in VrZAG83 primer (SEFC et al., 2000; SNOUSSI et al., 2004; TANGOLAR et al., 2009). Microsatellites have been becoming the marker of choice for fi ngerprinting and genetic diversity studies in a wide range of living organisms. The approach described in this paper shows that microsatellite analysis is a powerful tool for characterization of grapevine cultivars as well. Taken together with their co-dominant nature and reproducibility, SSR markers are very useful for the analysis of genetic diversity, genomic mapping and marker-assisted selection in many plant species compared to many other marker systems. In this study, the mean Ho level was slightly higher than that of He (80.9% for Ho and 75.9% for He). Comparable observed heterozygosity levels were reported between 74.3-85.5% in different grape-cultivated areas in the world (BOWERS et al., 1996; SEFC et al., 2000; FATAHI et al., 2003; MARTINEZ et al., 2006; TANGOLAR et al., 2009). Such high levels of heterozygosity are commonly observed among clonally propagated, outbreeding, perennial species since they are favoured during selection and are known to confer greater adaptability, vigour and productivity on clonal varieties (ARADHYA et al., 1998; SEFC et al., 2000). Grapes, being an outbreeding species, have highly heterozygous cultivars, carry a heavy genetic load and suffer from severe inbreeding depression (OLMO, 1976). Our fi ndings also indicated that there was a high genetic distance value among cultivars ranging from 0.17 to 0.92. Considering the environmental conditions of the region, it is expected that the grapevine germplasm in the region would have economically important adaptive traits that can potentially be incorporated into grapevine breeding programs. Hence, it is expected that the results of this study will assist current grapevine breeding efforts in Turkey as well as maintain the genetic integrity of the genetic resources. In summary, the gene pool of cultivated grapes surveyed in Northeast Anatolia has signifi cant amounts of genetic variation. In regard to germplasm management, our results show that the germplasm collection is highly variable and most variation is common to all genetic groups identifi ed. 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