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Microsatellite fingerprinting of homonymous grapevine (Vitis vinifera L.) varieties in neighboring regions of South-East Turkey
Scientia Horticulturae 114 (2007) 164–169 www.elsevier.com/locate/scihorti
Microsatellite fingerprinting of homonymous grapevine (Vitis vinifera L.) varieties in neighboring regions of South-East Turkey Hu¨seyin Karatas¸ a, Dilek Deg˘irmenci b, Riccardo Velasco c, Silvia Vezzulli c, C¸ag˘rı Bodur d, Y. Sabit Ag˘aog˘lu b,* a
Dicle University, Faculty of Agriculture, Department of Horticulture, Diyarbaky´r, Turkey Ankara University, Faculty of Agriculture, Department of Horticulture, Ankara, Turkey c Istituto Agrario di San Michele all’Adige, San Michele a/Adige (TN), Italy d Middle East Technical University, Department of Biology, Ankara, Turkey
b
Received 24 January 2007; received in revised form 26 June 2007; accepted 6 July 2007
Abstract Genotyping of Turkish grapevine (Vitis vinifera L.) germplasm was characterized by use of six highly polymorphic microsatellite loci (VVS2, VVMD5, VVMD7, VVMD27, VrZAG62, VrZAG79). In this study we aimed to clarify the relationships between homonymous varieties coming from different regions. Our results showed a large degree of genetic variability among most of the homonymous cultivars. The number of alleles per locus ranged from 10 to 21, and gene diversity (expected heterozygosity) values ranged from 0.85 to 0.93. Cultivars presenting the same names of Sergi karası (sampled from S¸anlıurfa and Gaziantep), Yediveren (sampled from S¸anlıurfa, Gaziantep, and National Germplasm Repository Vineyard in Tekirdag˘) and Serpenekıran (sampled from S¸anlıurfa and Gaziantep) were clustered together, or very close to each other, in a phenogram. Moreover, the alleles at the six microsatellite loci analyzed were found to be similar in terms of base pairs within each of these three closely positioned varieties. However, all the other cultivars failed to show a suitable clustering pattern when comparing their DNA profiles and names. Similarly named cultivars were not generally grouped together in the phenogram. On the other hand, we detected a tendency for differently named homonymous grape cultivars to cluster together. # 2007 Elsevier B.V. All rights reserved. Keywords: Vitis vinifera L.; Microsatellite; Turkish grapevine germplasm
1. Introduction Grapevine (Vitis vinifera L.) is one of the oldest and most important perennial crops in the world. Alleweld et al. (1990) estimated the existence of about 14,000 cultivars, with numerous synonyms and occasional use of the same or similar names for genetically different cultivars. Anatolia has long been linked with the origin of viticulture and wine making, especially in its eastern and southeastern regions to which the earlier authors commonly ascribe its origin. In Turkey, a large grape germplasm, consisting of about 1200 accessions, is conserved and has so far been transferred from the different ecological zones of the country to the National Germplasm
* Corresponding author. Tel.: +90 317 05 50; fax: +90 312 91 19. E-mail address: [email protected] (Y.S. Ag˘aog˘lu). 0304-4238/$ – see front matter # 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2007.07.001
Repository Vineyard in Tekirdag˘ (C ¸ elik et al., 2000; Ergu¨l et al., 2002). Truness-to-type is necessary when planting vineyards, making wine, managing germplasm collections, choosing parents for controlled crosses and legally protecting new cultivars. The large number of grapevine cultivars and clones makes the corrected identification and characterization a challege. Traditional ampelography (from the Greek ampelos-grapevine and graphos-description), analysing and comparing morphological characters to identify cultivars, is not sufficiently reliable and consistent due to environmental factors, individual plant biology, and plant growth stage (Lamboy and Alpha, 1998; Sefc et al., 1998, 1999; Fatahi et al., 2003). Up-to-now in Turkey, varietal identifications have been carried out with ampelographic studies through a few isoenzymatic approaches. On the other hand, a DNA-based identification has been performed for a limited number of
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grapevine cultivars (Ag˘aog˘lu and Ergu¨l, 1999a,b; Ag˘aog˘lu et al., 2000; Ergu¨l, 2000; Ergu¨l and Ag˘aog˘lu, 2001; Ergu¨l et al., 2002; Atak, 2003; Karatas¸ and Ag˘aog˘lu, 2006). For a profitable exploitation of the germplasm in future, breeding and MAS (marker assisted selection) programs, the genetic identification and characterization of grapevine cultivars represent a basic requirement. Compared to different molecular markers, SSRs (simple sequence repeats) provide a unique genetic profile for every cultivar, permitting unambiguous identification that is not affected by enviroment, disease or farming methods (Meredith, 2001). Owing to their high degree of polymorphism, reproducibility and codominant nature, microsatellite markers have been favoured and widely employed as powerful and versatile molecular tools. Nowadays, more than 500 grape SSRs are publicly available (Thomas and Scott, 1993; Bowers et al., 1996, 1999; Sefc et al., 1999; Scott et al., 2000; Di Gaspero et al., 2000; Di Gaspero et al., 2005; Merdinog˘lu et al., 2005). Traditionally assigned to non-coding genomic regions, additional 405 ‘‘functional’’ SSRs have recently been identified in a grape EST collection (Moser et al., 2005). Microsatellite markers have extensively been used for varietal characterization (Botta et al., 1995; Zulini et al., 2005; Fatahi et al., 2003; Hvarleva et al., 2004; Costantini et al., 2005) and for rootstock identification (Lin and Walker, 1998). Pedigree (Meredith et al., 1996; Sefc et al., 1998) and parantage analysis (Sefc et al., 1997; Vouillamoz et al., 2004) has also been reported. Since SSRs have been revealed fully informative and solid markers, they have definitely been involved in mapping studies (Grando et al., 2003; Riaz et al., 2004; AdamBlondon et al., 2004; Doligez et al., 2006). Moreover, these markers have been used for identification of chimaeras of grapes (Franks et al., 2002; Riaz et al., 2002; Hocquigny et al., 2004; Zulini et al., 2005). A novel SSR application has finally concerned the authentication of varietal wines (Siret et al., 2000). The aim of the present research was to achieve the genetic discrimination of ancient homonymous grapevine varieties in neighboring regions of South-East Turkey. Genotypes, grown under the same name in three different cities, were collected to evaluate their genetic diversity by using a minimal standard SSR marker set.
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Table 1 Grapevine genotypes studied with SSR markers N
Sample name
Collection
Berry colour
Use (table/ raisin/wine)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39
C ¸ ilores¸ (U) C ¸ ilores¸ (G) C ¸ ilores¸ (T–U) C ¸ ilorut (U) C ¸ ilorut (G) C ¸ ilorut (T–U) Ho¨nu¨su¨ (U) Ho¨nu¨su¨ (G) Ho¨nu¨su¨ (T–G) Dımıs¸kı (U) Dımıs¸kı (G) Dımıs¸kı (T–G) Kabarcık (U) Kabarcık (G) Kabarcık (T–G) Ku¨lahi (U) Ku¨lahi (G) Hatunparmag˘ı (U) Hatunparmag˘ı (G) Hatunparmag˘ı (T–G) Sergi karası (U) Sergi karası (G) Kızlartahtası (G) Kızlartahtası (U) Azezi (G) Azezi (U) Yediveren (U) Yediveren (G) Yediveren (T–G) Serpenekıran (U) Sepenekıran (G) Gu¨lgu¨lu¨ (U) Gu¨lgu¨lu¨ (G) Kızılbanki (G) Kızılbanki (U) Horoz karası (U) Horoz karası (G) Muhammediye (U) Muhammediye (G)
U G T–U U G T–U U G T–G U G T–G U G T–G U G U G T–G U G G U G U U G T–G U G U G G U U G U G
White White White White White White Red Red Red White White White White White White White White White White White Red Red White White White White Red Red Red White White Pink Pink Pink Pink Red Red White White
Table Table Table Table Table Table Table Table Table Table Table Table Table Table Table Table Table Table, raisin Table, raisin Table, raisin Raisin Raisin Table Table Table Table Table Table Table Table Table Table Table Table Table Table, raisin, wine Table, raisin, wine Table Table
U: S¸anlıurfa; G: Gaziantep; T: Tekirdag˘ (National Germplasm Repository Vineyard); T–G: National Germplasm Repository Vineyard samples that were previously brought from Gaziantep; T–U: National Germplasm Repository Vineyard samples that were previously brought from S¸anlıurfa city.
2.2. Microsatellite analysis 2. Materials and methods 2.1. Grapevine genotypes and DNA extraction Microsatellite analysis was carried out on 39 grapevine genotypes; 16 samples, showing the same name, were collected from both Gaziantep (G) and S¸anlıurfa (U) provinces, and one small group (7 samples), containing individuals coming from the ‘‘National Germplasm Repository Vineyard’’, was collected in Tekirdag˘ (T) province. The cultivars used in this study are listed in Table 1. The Pinot noir genotype was added as a well known and reference cultivar (This et al., 2004). DNA was isolated from young leaves as described by Lodhi et al. (1994).
In order to allow a comparison among internationally grown homonymous varieties, a minimal standard SSR marker set was considered (This et al., 2004). This set consists of six highly polimorphic loci as follows: VVS2 (Thomas and Scott, 1993), VVMD5 (Bowers et al., 1996), VVMD7 and VVMD27 (Bowers et al., 1999), VrZAG62 and VrZAG79 (Sefc et al., 1999). For each SSR locus, annealing temperatures and allele size ranges are shown in Table 2. Genomic DNA was amplified by the polymerase chain reaction (PCR) according to the following conditions: 20 ng of DNA template, 1 PCR reaction buffer (Qiagen), 1.5 mM MgCl2, 0.2 mM for each dNTP, 0.5 mM forward and reverse primer, 0.25 Unit HotStartTaq DNA polymerase (Qiagen) and
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Table 2 The minimal standard SSR marker set used for grapevine genotyping Locus
Annealing temperature (8C)
Allele size range (bp)
N
He
Ho
VVS2 VVMD5 VVMD7 VVMD27 VrZAG62 VrZAG79
51 52 51 52 52 52
119–137 222–250 230–256 161–191 138–204 220–256
15 10 10 14 21 18
0.85 0.89 0.92 0.86 0.91 0.93
0.48 0.42 0.47 0.85 0.73 0.62
14.66
0.89
0.60
Mean
N: number of alleles; He: expected heterozygosity; Ho: observed heterozygosity.
milliQ water to 12.5 ml PCR final volume. PCR thermocycling reactions were performed with a 15-min initial denaturation/ activation step, followed by 35 cycles at 94 8C for 45 s, annealing temperature (Ta) for 45 s, and 72 8C for 1 min 30 s, with a final extension step of 7 min at 72 8C. PCR products were assessed by gel electrophoresis in 1.5% agarose, visualized by means of Syber Gold probe. The Mass ruler DNA ladder mix (Fermentas, Life Sciences) was used for their quantification. Capillary electrophoresis of PCR products was performed on ABI PRISM1 3100 Genetic Analyzer (Applied Biosystems Inc.). First, 0.5 ml of suitably diluted PCR products were added to a mixture containing 9.4 ml of Hi-Di Formamide and 0.1 ml of Genescan1-500 ROX Size Standard (Applied Biosystems Inc.) and then injected prior to denaturation at 95 8C for 2 min. Allele identification was performed by using GeneScan v3.7 software (Applied Biosystems Inc.); automatic size calling of peak positions was double-checked by Genotyper v3.7 software (Applied Biosystems Inc.). 2.3. Statistical analysis Heterozygosities, allele numbers and frequencies were estimated for each microsatellite locus using Hardy–Weinberg equilibrium option of Arlequin v2.000 software (Schneider et al., 2000). Phenogram was obtained by the drawgram program in PHYLIP v3.6 software (Felsenstein, 1988) using the DLR distance matrix. Genotype likelihood ratio distance (DLR) is based on the assignment test that was described by Paetkau et al. (1997). DLR distances were calculated by using Doh assignment test calculator which is freely available at http:// www.biology.ualberta.ca/jbrzusto/Doh.php. 3. Results and discussion In this study, homonymous grapevine cultivars in neighbouring regions were genotyped with a minimal standard SSR marker set for their discrimination. Microsatellite profiles of homonymous cultivars are presented in Table 3. Each homonymous variety had got the same name in three different cities. However, cultivars presenting the same designation were found to have shown different morphological characters. We investigated the genetic similarity between them by microsatellite analysis.
Within the 39 grapevine cultivars obtained from S¸anlıurfa, Gaziantep and ‘‘National Germplasm Repository Vineyard’’ of Tekirdag˘, we detected a total of 88 alleles at the 6 studied SSR loci. In case of 1-bp shift between the reported (This et al., 2004) reference cultivar SSR profile and our actual results at a given locus, the corresponding Turkish genotype profiles were also adjusted. In this way, possible misscoring due to technical differences led to a correct allele size assessment. Allele numbers, expected and observed heterozygosities are shown in Table 2. We found the mean allele number per locus as 14.66. The most polymorphic microsatellite was VrZAG62 (21 alleles) and the least polymorphic ones were VVMD5 and VVMD7 (10 alleles). Expected heterozygosity (gene diversity) levels of the studied loci ranged from 0.85 (locus VVS2) to 0.93 (locus VrZAG79). The lowest observed heterozygosity was detected at VVMD5 locus with 0.42 and the highest one at VVMD27 with 0.85. We found that the observed proportions of heterozygous individuals (observed heterozygosities) were significantly ( p < 0.05) lower than the expected ones at 5 out of 6 microsatellite loci, when considering all the 39 samples as one population. Only VVMD27 locus did not give a significant deviation from Hardy–Weinberg equilibrium. However, when we tested the deviation from Hardy–Weinberg equilibrium for 16 single populations, containing cultivars with the same variety names, we did not detect any significant deviation at p = 0.05 level. The excess genic diversity that we observed in heterozygosity values for the whole population of 39 grapevine cultivars clearly results from the presence of population structure. These 39 cultivars correspond to 16 different homonymous grapevine variety names, collected from 3 different cities in Turkey. Expected heterozygosities are significantly higher than the observed ones because these grapevine cultivars are not expected to behave as one population. In order to further characterize the structure of Turkish grapevine gene pool, a phenogram based on the genetic similarity of investigated homonymous varieties was constructed using the homonymous cultivars as operational taxonomic units (Fig. 1). However, it is important to underline that the results of this phenetic analysis cannot be used to draw conclusions with regard to the degree of kinship between the cultivars since clusters illustrate similarity rather than kinship (Sefc et al., 1999; Pellorone et al., 2001). Only 4 out of 16 grapevine varieties showed phenogram positioning consistent with their names (i.e. the samples having the same variety names clustered together or very close to each other in the phenogram) when considering the samples collected from S¸anlıurfa and Gaziantep. When we considered seven cases that contain sampling from all the three locations (S¸anlıurfa, Gaziantep, and National Germplasm Repository Vineyard in Tekirdag˘), we noticed that four out of seven cases showed congruent positioning with their names. The homonymous grapevine cultivars C¸ilorut, Ho¨nu¨su¨, Gu¨lgu¨lu¨, Horoz karası, Kızılbanki, Kızlartahtası, and Ku¨lahi, which that were collected from different locations did not show a clustering pattern consistent with their variety names. The first two varieties in this group, C ¸ ilorut and Ho¨nu¨su¨, came from S¸anlıurfa, Gaziantep and National Germplasm Repository
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Table 3 Genetic profile of 16 Turkish homonymous Vitis vinifera L. cultivars analyzed at 6 highly polymorphic microsatellite loci (allele sizes are given as base pairs) Population
Homonymous cultivar name
VVS2
VVMD5
VVMD7
1
C¸ilores¸ (U) C¸ilores¸ (G) C¸ilores¸ (T–U)
129 129 123
129 129 123
222 224 248
222 224 248
230 230 248
250 250 248
181 181 181
191 191 181
198 198 204
202 202 204
224 224 242
248 248 256
2
Cilorut (U) C¸ilorut (G) C¸ilorut (T–U)
131 139 131
139 149 141
232 228 228
232 228 228
244 248 248
250 256 248
191 177 175
191 191 191
146 156 160
146 156 160
248 220 238
248 220 254
3
Ho¨nu¨su¨ (U) Ho¨nu¨su¨ (G) Ho¨nu¨su¨ (T–G)
139 137 139
145 137 139
228 222 234
228 236 234
240 244 248
240 248 248
169 175 177
189 191 181
140 150 188
150 150 194
234 238 256
240 248 256
4
Dımıs¸kı (U) Dımıs¸kı (G) Dımıs¸kı (T–G)
129 141 129
129 141 153
224 228 232
224 234 236
230 230 248
250 250 248
181 181 175
185 185 191
200 160 192
204 160 204
248 226 254
248 244 256
5
Kabarcık (U) Kabarcık (G) Kabarcık (T–G)
129 129 129
145 139 139
222 232 232
222 232 232
240 234 234
240 250 250
191 163 163
191 167 167
188 150 150
204 160 160
238 234 234
248 246 246
6
Ku¨lahi (U) Ku¨lahi (G)
129 141
129 141
222 228
222 234
250 250
250 250
193 175
193 193
154 160
158 160
248 224
248 242
7
Hatunparmag˘ı (U) Hatunparmag˘ı (G) Hatunparmag˘ı (T–G)
131 131 131
131 139 131
222 222 234
230 224 234
248 240 248
248 248 248
183 181 181
191 189 191
150 146 188
160 150 200
248 254 238
248 256 244
8
Sergi karası (U) Sergi karası (G)
131 131
137 151
222 222
234 234
248 250
248 250
175 165
177 175
196 192
200 200
236 236
246 246
9
Kızlartahtası (G) Kızlartahtası (U)
125 121
125 121
232 224
238 224
246 250
252 250
175 181
175 185
188 184
192 200
254 248
254 248
10
Azezi (G) Azezi (U)
129 129
129 129
224 222
232 222
248 248
248 248
181 183
191 191
190 142
202 154
248 224
254 248
11
Yediveren (U) Yediveren (G) Yediveren (T–G)
147 147 125
151 151 125
250 228 250
250 228 250
238 238 238
238 250 238
175 175 175
175 191 191
188 202 146
202 202 160
222 228 224
230 234 250
12
Serpenekıran (U) Sepenekıran (G)
119 129
129 129
232 232
232 250
244 248
250 248
165 165
173 175
150 150
160 160
226 234
232 242
13
Gu¨lgu¨lu¨ (U) Gu¨lgu¨lu¨ (G)
129 129
149 131
228 232
250 232
248 238
248 248
177 169
191 179
144 150
158 154
244 238
254 242
14
Kızılbanki (G) Kızılbanki (U)
125 129
135 129
250 234
250 234
244 244
250 250
179 169
181 179
190 138
190 150
246 240
246 240
15
Horoz karası (U) Horoz karası (G)
129 131
129 139
222 234
228 248
248 248
248 248
183 181
193 191
200 160
204 160
248 236
248 236
16
Muhammediye (U) Muhammediye (G)
129 147
129 147
232 232
250 232
248 234
250 240
179 177
191 193
154 152
154 160
256 222
256 232
Vineyard in Tekirdag˘. The remaining five varieties were collected from S¸anlıurfa and Gaziantep. In this group, the grapevine cultivars with the same variety name were clustered far from each other in the phenogram. The three varieties Azezi, Dımıs¸kı, and Muhammediye also showed a non-consistent pattern with their names. Dımıs¸kı samples were collected from S¸anlıurfa, Gaziantep, and National Germplasm Repository Vineyard in Tekirdag˘, Azezi and Muhammediye from S¸anlıurfa and Gaziantep. The cultivars with the same name were not grouped together in the phenogram even if they were not as far away to each other as were the previously described seven varieties.
VVMD27
VrZAG62
VrZAG79
Genotypes with the same name of Sergi karası (from S¸anlıurfa and Gaziantep) and Yediveren (from S¸anlıurfa, Gaziantep, and National Germplasm Repository Vineyard in Tekirdag˘) clustered together. Serpenekıran varieties collected from S¸anlıurfa and Gaziantep were positioned very close to each other in the phenogram. Moreover, the alleles at the six SSR loci analyzed were found to be similar in terms of base pairs within each of these three closely positioned varieties. When analyzing the positions of three Kabarcık varieties, we observed that Kabarcık samples, collected from Gaziantep and National Germplasm Repository Vineyard in Tekirdag˘
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H. Karatas¸ et al. / Scientia Horticulturae 114 (2007) 164–169
Fig. 1. Phenogram of the 16 homonymous grapevine varieties.
(cultivars were brought from Gaziantep to the National Germplasm Repository Vineyard), were grouped together. However, the other Kabarcık genotype coming from S¸anlıurfa clustered far from the other two. Moreover, the SSR allele sizes of these three genotypes were identical between the two cultivars that clustered together, but different for the third cultivar that was collected from S¸anlıurfa. Hatunparmag˘ı homonymous genotypes coming from Gaziantep and National Germplasm Repository Vineyard in Tekirdag˘ clustered close to each other, but the genotype from S¸anlıurfa was positioned far from the other two in the phenogram. Similar microsatellite alleles were detected for C¸ilores¸ homonymous grapevine cultivars from S¸anlıurfa and Gaziantep and they were grouped together in the phenogram. However, the National Germplasm Repository Vineyard cultivar that was brought from S¸anlıurfa was located far from these two cultivars in the phenogram. The overall positioning in the phenogram reveals that the grapevine varieties showing the same names are not genetically identical based on microsatellites. It is obvious that the varieties cultivated in different ecological conditions of Turkey have attained different genetic profiles during the time. This differentiation among cultivars with the same names was
also increased by the high mutation rates of microsatellites. What we observed when analyzing the phenogram was actually the general tendency of cultivars of the same regions to group together rather than genotypes belonging to the same variety. Naming of the homonymous grapevine genotypes is a major problem in Turkish grapevine cultivation. The current study indicates how serious the situation is. Similarly named cultivars are generally not grouped together. On the other hand, we could say that differently named homonymous grape cultivars are clustered together. Thus, we have to choose the genetic characteristics of certain cultivars as representatives of that variety and name the other ones as relatives. Nowadays, no genetic profile of our studied Turkish variety names is reported in a publicly available SSR-based grapevine database. Before any decisions, additional molecular markers and morphometric studies conducted on Turkish grapevines should be taken into account along with these microsatellite results. Another limit in interpretation of our results was the uncertainty about representing correctly the homonymous cultivars by only one genotype. We suspect that there are several somatic mutants of the same homonymous grapevine cultivar. Hence, it would be useful to analyze more than one genotype to represent a homonymous cultivar in future studies. In conclusion, we obtained a very high allelic polymorphism among genotypes expected to be different (having different variety names) or between genotypes that were supposed to have the same variety name. The sources of these differences observed among samples with the same varietal name collected from three different ecological regions could be summarized as follows. First, these homonymous grapevine varieties cultivated in different environments for many years, and those transferred to the National Germplasm Repository Vineyard could be inappropriately named. Second, changes in genetic backgrounds of these varieties may be caused by somatic mutations resulted from the effects of continuous vegetative reproduction and environmental factors. Turkey is a very rich country in terms of homonymous grape varieties which results from the ancien tradition of grape cultivation in Anatolia, which began approximately 7000–8000 years ago. We are of the opinion that it is crucial to preserve this genetic potential by describing a reasonable nomenclature and determining the relationships among these varieties through DNA-based markers. References Adam-Blondon, A.F., Roux, C., Claux, D., Butterlin, G., Merdinog˘lu, D., This, P., 2004. Mapping 245 SSR markers on the Vitis vinifera genome: a tool for grape genetics. Theor. Appl. Genet. 109, 1017–1027. Ag˘aog˘lu, Y.S., Ergu¨l, A., 1999a. Genetic identification in ecotypes of Amasya grape cultivar by RAPD markers. In: 3rd Natl. Hortic. Congr., Kızılcahamam/Ankara, Tu¨rkiye, September 14–17, 1999, pp. 372–386. Ag˘aog˘lu, Y. S., Marasalı, B., Ergu¨l, A., 2000. Molecular characterization on the grapevine (Vitis vinifera L. cvs.) by RAPD (random amplified polymorphic) technique. Ankara University Research Fond. 96-11-01-02 no.lu Report of Project Result, Ankara.
H. Karatas¸ et al. / Scientia Horticulturae 114 (2007) 164–169 Ag˘aog˘lu, Y.S., Ergu¨l, A., 1999b.In: Identification of some Turkish table grape cultivars (Vitis vinifera L.) by random amplified polymorphic DNA (RAPD), Deutsch.-Tu¨rkische Agrarfoschung Symposium. Giessen, Germany, pp. 201– 206. Alleweld, G., Spiegel-Roy, P., Reisch, B., 1990. Grapes (Vitis). In: Moore, I.N., Ballington, J.L. (Eds.), Genetic Resources of Temprate Fruit and Nut Crops. Acta Hortic. 290, 291–337. Atak, A., 2003. Identification of some grape cultivars hybrids and their parents by RAPD technique. MSc Thesis (unpublished) [Tr, en]. Ankara University, Ankara, Tu¨rkiye. Botta, R., Scott, N.S., Eynard, I., Thomas, M.R., 1995. Evaluation of microsatellite sequence-tagged site markers for characterizing Vitis vinifera cultivars. Vitis 34 (2), 99–102. Bowers, J.E., Dangl, G.S., Meredith, C.P., 1999. Development and characterization of additional microsatellite DNA markers for grape. Am. J. Enol. Vitic. 50, 243–246. Bowers, J.E., Dangl, G.S., Vignani, R., Meredith, C.P., 1996. Isolation and characterization of new polymorphic simple sequence repeat loci in grape (Vitis vinifera L.). Genome 39, 628–633. Costantini, L., Monaco, A., Vouillamoz, J.F., Forlani, M., Grando, M.S., 2005. Genetic relationships among homonymous Vitis vinifera cultivars from Campania (Italy). Vitis 44, 25–34. C ¸ elik, H., Marasalı, B., So¨ylemezog˘lu, G., Tangolar, S., Gu¨ndu¨z, M., 2000. ¨ retim Hedefleri (Future Prospect in Turkish Viticulture). Bag˘cılıkta U TMMOB Ziraat. Mu¨h. Odası, Tu¨rkiye Ziraat Mu¨hendislig˘i V. Teknik Kongresi, 17–21 Ocak 2000, Ankara [Tr]. Bildiriler 2, 645–678. Di Gaspero, G., Peterlunger, E., Testolin, R., Edwards, K.J., Cipriani, G., 2000. Conservation of microsatellite loci within the genus Vitis. Theor. Appl. Genet. 101, 301–308. Di Gaspero, G., Cipriani, G., Marazzo, M.T., Andreetta, D., Castro, M.J.P., Peterlunger, E., Testolin, R., 2005. Isolation of (AC)n-microsatellites in Vitis vinifera L. and analysis of geneticbackground in grapevines under marker assisted selection. Mol. Breed. 15, 11–20. Doligez, A., Adam-Blondon, A.F., Cipriani, G., Di Gaspero, G., Laucou, V., Merdinog˘lu, D., Meredith, C.P., Riaz, S., Roux, C., This, T., 2006. An integrated SSR map of grapevine based on five mapping populations. Theor. Appl. Genet. 113, 369–382. Ergu¨l, A., 2000. Molecular characterization of grapevine (Vitis vinifera L. cvs.) by genomic DNA fingerprinting. PhD Thesis (unpublished) [Tr, en]. Ankara University, Ankara, Tu¨rkiye. Ergu¨l, A., Agaog˘lu, Y.S., 2001. Molecular similarity analysis of some grapevine rootstocks from different nursery in Turkey. J. Agric. Sci. 7(2), 141–143 [Tr, en]. Ankara, Tu¨rkiye. Ergu¨l, A., Marasalı, B., Ag˘aog˘lu, Y.S., 2002. Molecular discrimination and identification of some Turkish grape cultivars (Vitis vinifera L.) by RAPD markers. Vitis 41 (3), 159–160. Fatahi, R., Ebadi, A., Bassil, N., Mehlenbacher, S.A., Zamani, Z., 2003. Characterization of Iranian grapevine cultivars using microsatellite markers. Vitis 42 (4), 185–192. Felsenstein, J., 1988. Phylogenies from molecular sequences—inference and reliability. Annu. Rev. Genet. 22, 521–565. Franks, T., Botta, R., Thomas, M.R., 2002. Chimerisim in grapevines: implications for cultivar identitiy, ancestry, and genetic improvement. Theor. Appl. Genet. 104, 192–199. Grando, M.S., Bellin, D., Edwards, K.J., Pozzi, C., Stefanini, M., Velasco, R., 2003. Molecular linkage maps of Vitis vinifera L. and Vitis riparia Mchx. Theor. Appl. Genet. 106, 1213–1224. Hocquigny, S., Pelsy, F., Dumas, V., Kindt, S., Heloir, M.-C., Merdinog˘lu, D., 2004. Diversification within grapevine cultivars goes through chimeric states. Genome 47, 579–589. Hvarleva, T., Rusanov, K., Lefort, F., Tsvetkov, I., Atanassov, A., Atanassov, I., 2004. Genotyping of Bulgarian Vitis vinifera L. cultivars by microsattellite analysis. Vitis 43 (1), 27–34. Karatas¸, H., Ag˘aog˘lu, Y.S., 2006. Molecular analysis of Diyarbakır region’s grapevine germplasm by RAPD (Random Amplified Polimorphic DNA) technique. 8. Deutsch-Turkische Agrarforsching Symposium, 4–8 October, 2005. Braunschweig, Germany, 411–421.
169
Lamboy, W.F., Alpha, C.G., 1998. Using simple sequence repeats (SSRs) for DNA fingerprinting germ plasmaccessions of grape (Vitis. L. species). J. Am. Soc. Hortic. Sci. 123, 182–188. Lin, H., Walker, M.A., 1998. Identifying grape rootstocks with simple sequence repeat (SSR) DNA markers. Am. J. Enol. Vitic. 49, 403–407. Lodhi, M.A., Daly, M.J., Ye, G.-N., Weeden, N.F., Reisch, B.I., 1994. A Simple and efficient method for DNA extraction from grapevine cultivars and Vitis species. Plant Mol. Biol. Reptr. 12 (1), 6–13. Merdinog˘lu, D., Butterlin, G., Bevilacqua, L., Chiquet, V., Adam-Blondon, A.F., Decroocq, S., 2005. Development and characterization of a large set of microsatellite markers in grapevine (Vitis vinifera L.) suitable for multiplex PCR. Mol. Breed. 15, 349–366. Meredith, C., 2001. Grapevine genetics: probing the past and facing the future. Agriculturae Conspectus Sientificus 66, 21–25. Meredith, C., Dangl, G.S., Bowers, J.E., 1996. Clarifying the identitiy of some California winegrapes by DNA profilling. Riv. Vitic. Enol. 49 (1), 65–68. Moser, C., Segala, C., Fontana, P., Salakhudtinov, I., Gatto, P., Pindo, M., Zyprian, E., Toepfer, R., Grando, M.S., Velasco, R., 2005. Comparative analysis of expressed sequence tags from different organs of Vitis vinifera L. Funct. Integr. Genomics 5, 208–217. Paetkau, D., Waits, L.P., Clarkson, P.L., Craighead, L., Strobeck, C., 1997. An empirical evaluation of genetic distance statistics using microsatellite data from bear (Ursidae) populations. Genetics 147, 1943–1957. Pellorone, F., Edwards, K., Thomas, M., 2001. Grapevine micosatellite repeats: isolation, characterization and use for genotyping of grape germplasm from Southern Italy. Vitis 41, 83–87. Riaz, S., Dangl, G.S., Edwards, K.J., Meredith, C.P., 2004. A microsatellite marker based framework linkage map of Vitis vinifera L. Theor. Appl. Genet. 108, 864–872. Riaz, S., Garrisson, K.E., Dangl, G.S., Boursiquot, J.M., Meredith, J.P., 2002. Genetic divergence and chimerism within ancient asexually propagated winegrape cultivars. J. Am. Soc. Hortic. Sci. 127, 508–514. Schneider, S., Roessli, D., Excoffier, L., 2000. Arlequin Ver. 2.000: A Software for Population Genetics Data Analysis. Genetics and Biometry Laboratory, University of Geneva, Switzerland. Scott, K.D., Eggler, P., Seaton, G., Rosetto, M., Ablett, E.M., Lee, L.S., Henry, R.J., 2000. Analysis of SSRs derived from grape ESTs. Theor. Appl. Genet. 100, 723–726. Sefc, K.M., Regner, F., Turetschek, E., Glo¨ssl, J., Steinkellner, H., 1999. Identification of microsatellite sequences in Vitis riparia and their applicability for genotyping of different Vitis species. Genome 42, 367–373. Sefc, K.M., Steinkellner, H., Glo¨ssl, J., Kampfer, S., Regner, F., 1998. Reconstruction of a grapevine pedigree by microsatellite analysis. Theor. Appl. Genet. 97, 227–231. Sefc, K.M., Steinkellner, H., Wagner, H.W., Glo¨ssl, J., Regner, F., 1997. Application of microsatellite markers to parentage studies in grapevine. Vitis 36 (4), 179–183. Siret, R., Boursiquot, J.M., Merle, M.H., Cabanis, J.C., This, P., 2000. Toward the authentication of varietal wines by the analysis of grape (Vitis vinifera L.) residual DNA in must and wine using microsatellite markers. J. Agric. Food Chem. 48, 5035–5040. This, P., Jung, A., Boccacci, P., Borrego, J., Botta, R., Costantini, L., Crespan, M., Dangl, G.S., Eisenheld, C., Ferreira-Monteiro, F., Grando, M.S., Iba´n˜ez, J., Lacombe, T., Laucou, V., Magalha˜es, R., Meredith, C.P., Milani, N., Peterlunger, E., Regner, F., Zulini, L., Maul, E., 2004. Development of a standard set of microsatellite references alleles for identification of grape cultivars. Theor. Appl. Genet. 109, 1048–1058. Thomas, M.R., Scott, N.S., 1993. Microsatellite repeats in grapevine reveal DNA polimorphisms when analysed as sequence-tagged sites (STSs). Theor. Appl. Genet. 86, 985–990. Vouillamoz, J.F., Maigre, D., Meredith, C.P., 2004. Identity and parentage of two Alpine grape cultivars from Switzerland (Vitis vinifera L. Lafnetscha and Himbertscha). Vitis 43 (2), 81–87. Zulini, L., Fabro, E., Peterlunger, E., 2005. Characterisation of the grapevine cultivar Picolit by means of morphological descriptors and molecular markers. Vitis 44 (1), 35–38.
Microsatellite fingerprinting of homonymous grapevine (Vitis vinifera L.) varieties in neighboring regions of South-East Turkey Hu¨seyin Karatas¸ a, Dilek Deg˘irmenci b, Riccardo Velasco c, Silvia Vezzulli c, C¸ag˘rı Bodur d, Y. Sabit Ag˘aog˘lu b,* a
Dicle University, Faculty of Agriculture, Department of Horticulture, Diyarbaky´r, Turkey Ankara University, Faculty of Agriculture, Department of Horticulture, Ankara, Turkey c Istituto Agrario di San Michele all’Adige, San Michele a/Adige (TN), Italy d Middle East Technical University, Department of Biology, Ankara, Turkey
b
Received 24 January 2007; received in revised form 26 June 2007; accepted 6 July 2007
Abstract Genotyping of Turkish grapevine (Vitis vinifera L.) germplasm was characterized by use of six highly polymorphic microsatellite loci (VVS2, VVMD5, VVMD7, VVMD27, VrZAG62, VrZAG79). In this study we aimed to clarify the relationships between homonymous varieties coming from different regions. Our results showed a large degree of genetic variability among most of the homonymous cultivars. The number of alleles per locus ranged from 10 to 21, and gene diversity (expected heterozygosity) values ranged from 0.85 to 0.93. Cultivars presenting the same names of Sergi karası (sampled from S¸anlıurfa and Gaziantep), Yediveren (sampled from S¸anlıurfa, Gaziantep, and National Germplasm Repository Vineyard in Tekirdag˘) and Serpenekıran (sampled from S¸anlıurfa and Gaziantep) were clustered together, or very close to each other, in a phenogram. Moreover, the alleles at the six microsatellite loci analyzed were found to be similar in terms of base pairs within each of these three closely positioned varieties. However, all the other cultivars failed to show a suitable clustering pattern when comparing their DNA profiles and names. Similarly named cultivars were not generally grouped together in the phenogram. On the other hand, we detected a tendency for differently named homonymous grape cultivars to cluster together. # 2007 Elsevier B.V. All rights reserved. Keywords: Vitis vinifera L.; Microsatellite; Turkish grapevine germplasm
1. Introduction Grapevine (Vitis vinifera L.) is one of the oldest and most important perennial crops in the world. Alleweld et al. (1990) estimated the existence of about 14,000 cultivars, with numerous synonyms and occasional use of the same or similar names for genetically different cultivars. Anatolia has long been linked with the origin of viticulture and wine making, especially in its eastern and southeastern regions to which the earlier authors commonly ascribe its origin. In Turkey, a large grape germplasm, consisting of about 1200 accessions, is conserved and has so far been transferred from the different ecological zones of the country to the National Germplasm
* Corresponding author. Tel.: +90 317 05 50; fax: +90 312 91 19. E-mail address: [email protected] (Y.S. Ag˘aog˘lu). 0304-4238/$ – see front matter # 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2007.07.001
Repository Vineyard in Tekirdag˘ (C ¸ elik et al., 2000; Ergu¨l et al., 2002). Truness-to-type is necessary when planting vineyards, making wine, managing germplasm collections, choosing parents for controlled crosses and legally protecting new cultivars. The large number of grapevine cultivars and clones makes the corrected identification and characterization a challege. Traditional ampelography (from the Greek ampelos-grapevine and graphos-description), analysing and comparing morphological characters to identify cultivars, is not sufficiently reliable and consistent due to environmental factors, individual plant biology, and plant growth stage (Lamboy and Alpha, 1998; Sefc et al., 1998, 1999; Fatahi et al., 2003). Up-to-now in Turkey, varietal identifications have been carried out with ampelographic studies through a few isoenzymatic approaches. On the other hand, a DNA-based identification has been performed for a limited number of
H. Karatas¸ et al. / Scientia Horticulturae 114 (2007) 164–169
grapevine cultivars (Ag˘aog˘lu and Ergu¨l, 1999a,b; Ag˘aog˘lu et al., 2000; Ergu¨l, 2000; Ergu¨l and Ag˘aog˘lu, 2001; Ergu¨l et al., 2002; Atak, 2003; Karatas¸ and Ag˘aog˘lu, 2006). For a profitable exploitation of the germplasm in future, breeding and MAS (marker assisted selection) programs, the genetic identification and characterization of grapevine cultivars represent a basic requirement. Compared to different molecular markers, SSRs (simple sequence repeats) provide a unique genetic profile for every cultivar, permitting unambiguous identification that is not affected by enviroment, disease or farming methods (Meredith, 2001). Owing to their high degree of polymorphism, reproducibility and codominant nature, microsatellite markers have been favoured and widely employed as powerful and versatile molecular tools. Nowadays, more than 500 grape SSRs are publicly available (Thomas and Scott, 1993; Bowers et al., 1996, 1999; Sefc et al., 1999; Scott et al., 2000; Di Gaspero et al., 2000; Di Gaspero et al., 2005; Merdinog˘lu et al., 2005). Traditionally assigned to non-coding genomic regions, additional 405 ‘‘functional’’ SSRs have recently been identified in a grape EST collection (Moser et al., 2005). Microsatellite markers have extensively been used for varietal characterization (Botta et al., 1995; Zulini et al., 2005; Fatahi et al., 2003; Hvarleva et al., 2004; Costantini et al., 2005) and for rootstock identification (Lin and Walker, 1998). Pedigree (Meredith et al., 1996; Sefc et al., 1998) and parantage analysis (Sefc et al., 1997; Vouillamoz et al., 2004) has also been reported. Since SSRs have been revealed fully informative and solid markers, they have definitely been involved in mapping studies (Grando et al., 2003; Riaz et al., 2004; AdamBlondon et al., 2004; Doligez et al., 2006). Moreover, these markers have been used for identification of chimaeras of grapes (Franks et al., 2002; Riaz et al., 2002; Hocquigny et al., 2004; Zulini et al., 2005). A novel SSR application has finally concerned the authentication of varietal wines (Siret et al., 2000). The aim of the present research was to achieve the genetic discrimination of ancient homonymous grapevine varieties in neighboring regions of South-East Turkey. Genotypes, grown under the same name in three different cities, were collected to evaluate their genetic diversity by using a minimal standard SSR marker set.
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Table 1 Grapevine genotypes studied with SSR markers N
Sample name
Collection
Berry colour
Use (table/ raisin/wine)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39
C ¸ ilores¸ (U) C ¸ ilores¸ (G) C ¸ ilores¸ (T–U) C ¸ ilorut (U) C ¸ ilorut (G) C ¸ ilorut (T–U) Ho¨nu¨su¨ (U) Ho¨nu¨su¨ (G) Ho¨nu¨su¨ (T–G) Dımıs¸kı (U) Dımıs¸kı (G) Dımıs¸kı (T–G) Kabarcık (U) Kabarcık (G) Kabarcık (T–G) Ku¨lahi (U) Ku¨lahi (G) Hatunparmag˘ı (U) Hatunparmag˘ı (G) Hatunparmag˘ı (T–G) Sergi karası (U) Sergi karası (G) Kızlartahtası (G) Kızlartahtası (U) Azezi (G) Azezi (U) Yediveren (U) Yediveren (G) Yediveren (T–G) Serpenekıran (U) Sepenekıran (G) Gu¨lgu¨lu¨ (U) Gu¨lgu¨lu¨ (G) Kızılbanki (G) Kızılbanki (U) Horoz karası (U) Horoz karası (G) Muhammediye (U) Muhammediye (G)
U G T–U U G T–U U G T–G U G T–G U G T–G U G U G T–G U G G U G U U G T–G U G U G G U U G U G
White White White White White White Red Red Red White White White White White White White White White White White Red Red White White White White Red Red Red White White Pink Pink Pink Pink Red Red White White
Table Table Table Table Table Table Table Table Table Table Table Table Table Table Table Table Table Table, raisin Table, raisin Table, raisin Raisin Raisin Table Table Table Table Table Table Table Table Table Table Table Table Table Table, raisin, wine Table, raisin, wine Table Table
U: S¸anlıurfa; G: Gaziantep; T: Tekirdag˘ (National Germplasm Repository Vineyard); T–G: National Germplasm Repository Vineyard samples that were previously brought from Gaziantep; T–U: National Germplasm Repository Vineyard samples that were previously brought from S¸anlıurfa city.
2.2. Microsatellite analysis 2. Materials and methods 2.1. Grapevine genotypes and DNA extraction Microsatellite analysis was carried out on 39 grapevine genotypes; 16 samples, showing the same name, were collected from both Gaziantep (G) and S¸anlıurfa (U) provinces, and one small group (7 samples), containing individuals coming from the ‘‘National Germplasm Repository Vineyard’’, was collected in Tekirdag˘ (T) province. The cultivars used in this study are listed in Table 1. The Pinot noir genotype was added as a well known and reference cultivar (This et al., 2004). DNA was isolated from young leaves as described by Lodhi et al. (1994).
In order to allow a comparison among internationally grown homonymous varieties, a minimal standard SSR marker set was considered (This et al., 2004). This set consists of six highly polimorphic loci as follows: VVS2 (Thomas and Scott, 1993), VVMD5 (Bowers et al., 1996), VVMD7 and VVMD27 (Bowers et al., 1999), VrZAG62 and VrZAG79 (Sefc et al., 1999). For each SSR locus, annealing temperatures and allele size ranges are shown in Table 2. Genomic DNA was amplified by the polymerase chain reaction (PCR) according to the following conditions: 20 ng of DNA template, 1 PCR reaction buffer (Qiagen), 1.5 mM MgCl2, 0.2 mM for each dNTP, 0.5 mM forward and reverse primer, 0.25 Unit HotStartTaq DNA polymerase (Qiagen) and
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Table 2 The minimal standard SSR marker set used for grapevine genotyping Locus
Annealing temperature (8C)
Allele size range (bp)
N
He
Ho
VVS2 VVMD5 VVMD7 VVMD27 VrZAG62 VrZAG79
51 52 51 52 52 52
119–137 222–250 230–256 161–191 138–204 220–256
15 10 10 14 21 18
0.85 0.89 0.92 0.86 0.91 0.93
0.48 0.42 0.47 0.85 0.73 0.62
14.66
0.89
0.60
Mean
N: number of alleles; He: expected heterozygosity; Ho: observed heterozygosity.
milliQ water to 12.5 ml PCR final volume. PCR thermocycling reactions were performed with a 15-min initial denaturation/ activation step, followed by 35 cycles at 94 8C for 45 s, annealing temperature (Ta) for 45 s, and 72 8C for 1 min 30 s, with a final extension step of 7 min at 72 8C. PCR products were assessed by gel electrophoresis in 1.5% agarose, visualized by means of Syber Gold probe. The Mass ruler DNA ladder mix (Fermentas, Life Sciences) was used for their quantification. Capillary electrophoresis of PCR products was performed on ABI PRISM1 3100 Genetic Analyzer (Applied Biosystems Inc.). First, 0.5 ml of suitably diluted PCR products were added to a mixture containing 9.4 ml of Hi-Di Formamide and 0.1 ml of Genescan1-500 ROX Size Standard (Applied Biosystems Inc.) and then injected prior to denaturation at 95 8C for 2 min. Allele identification was performed by using GeneScan v3.7 software (Applied Biosystems Inc.); automatic size calling of peak positions was double-checked by Genotyper v3.7 software (Applied Biosystems Inc.). 2.3. Statistical analysis Heterozygosities, allele numbers and frequencies were estimated for each microsatellite locus using Hardy–Weinberg equilibrium option of Arlequin v2.000 software (Schneider et al., 2000). Phenogram was obtained by the drawgram program in PHYLIP v3.6 software (Felsenstein, 1988) using the DLR distance matrix. Genotype likelihood ratio distance (DLR) is based on the assignment test that was described by Paetkau et al. (1997). DLR distances were calculated by using Doh assignment test calculator which is freely available at http:// www.biology.ualberta.ca/jbrzusto/Doh.php. 3. Results and discussion In this study, homonymous grapevine cultivars in neighbouring regions were genotyped with a minimal standard SSR marker set for their discrimination. Microsatellite profiles of homonymous cultivars are presented in Table 3. Each homonymous variety had got the same name in three different cities. However, cultivars presenting the same designation were found to have shown different morphological characters. We investigated the genetic similarity between them by microsatellite analysis.
Within the 39 grapevine cultivars obtained from S¸anlıurfa, Gaziantep and ‘‘National Germplasm Repository Vineyard’’ of Tekirdag˘, we detected a total of 88 alleles at the 6 studied SSR loci. In case of 1-bp shift between the reported (This et al., 2004) reference cultivar SSR profile and our actual results at a given locus, the corresponding Turkish genotype profiles were also adjusted. In this way, possible misscoring due to technical differences led to a correct allele size assessment. Allele numbers, expected and observed heterozygosities are shown in Table 2. We found the mean allele number per locus as 14.66. The most polymorphic microsatellite was VrZAG62 (21 alleles) and the least polymorphic ones were VVMD5 and VVMD7 (10 alleles). Expected heterozygosity (gene diversity) levels of the studied loci ranged from 0.85 (locus VVS2) to 0.93 (locus VrZAG79). The lowest observed heterozygosity was detected at VVMD5 locus with 0.42 and the highest one at VVMD27 with 0.85. We found that the observed proportions of heterozygous individuals (observed heterozygosities) were significantly ( p < 0.05) lower than the expected ones at 5 out of 6 microsatellite loci, when considering all the 39 samples as one population. Only VVMD27 locus did not give a significant deviation from Hardy–Weinberg equilibrium. However, when we tested the deviation from Hardy–Weinberg equilibrium for 16 single populations, containing cultivars with the same variety names, we did not detect any significant deviation at p = 0.05 level. The excess genic diversity that we observed in heterozygosity values for the whole population of 39 grapevine cultivars clearly results from the presence of population structure. These 39 cultivars correspond to 16 different homonymous grapevine variety names, collected from 3 different cities in Turkey. Expected heterozygosities are significantly higher than the observed ones because these grapevine cultivars are not expected to behave as one population. In order to further characterize the structure of Turkish grapevine gene pool, a phenogram based on the genetic similarity of investigated homonymous varieties was constructed using the homonymous cultivars as operational taxonomic units (Fig. 1). However, it is important to underline that the results of this phenetic analysis cannot be used to draw conclusions with regard to the degree of kinship between the cultivars since clusters illustrate similarity rather than kinship (Sefc et al., 1999; Pellorone et al., 2001). Only 4 out of 16 grapevine varieties showed phenogram positioning consistent with their names (i.e. the samples having the same variety names clustered together or very close to each other in the phenogram) when considering the samples collected from S¸anlıurfa and Gaziantep. When we considered seven cases that contain sampling from all the three locations (S¸anlıurfa, Gaziantep, and National Germplasm Repository Vineyard in Tekirdag˘), we noticed that four out of seven cases showed congruent positioning with their names. The homonymous grapevine cultivars C¸ilorut, Ho¨nu¨su¨, Gu¨lgu¨lu¨, Horoz karası, Kızılbanki, Kızlartahtası, and Ku¨lahi, which that were collected from different locations did not show a clustering pattern consistent with their variety names. The first two varieties in this group, C ¸ ilorut and Ho¨nu¨su¨, came from S¸anlıurfa, Gaziantep and National Germplasm Repository
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Table 3 Genetic profile of 16 Turkish homonymous Vitis vinifera L. cultivars analyzed at 6 highly polymorphic microsatellite loci (allele sizes are given as base pairs) Population
Homonymous cultivar name
VVS2
VVMD5
VVMD7
1
C¸ilores¸ (U) C¸ilores¸ (G) C¸ilores¸ (T–U)
129 129 123
129 129 123
222 224 248
222 224 248
230 230 248
250 250 248
181 181 181
191 191 181
198 198 204
202 202 204
224 224 242
248 248 256
2
Cilorut (U) C¸ilorut (G) C¸ilorut (T–U)
131 139 131
139 149 141
232 228 228
232 228 228
244 248 248
250 256 248
191 177 175
191 191 191
146 156 160
146 156 160
248 220 238
248 220 254
3
Ho¨nu¨su¨ (U) Ho¨nu¨su¨ (G) Ho¨nu¨su¨ (T–G)
139 137 139
145 137 139
228 222 234
228 236 234
240 244 248
240 248 248
169 175 177
189 191 181
140 150 188
150 150 194
234 238 256
240 248 256
4
Dımıs¸kı (U) Dımıs¸kı (G) Dımıs¸kı (T–G)
129 141 129
129 141 153
224 228 232
224 234 236
230 230 248
250 250 248
181 181 175
185 185 191
200 160 192
204 160 204
248 226 254
248 244 256
5
Kabarcık (U) Kabarcık (G) Kabarcık (T–G)
129 129 129
145 139 139
222 232 232
222 232 232
240 234 234
240 250 250
191 163 163
191 167 167
188 150 150
204 160 160
238 234 234
248 246 246
6
Ku¨lahi (U) Ku¨lahi (G)
129 141
129 141
222 228
222 234
250 250
250 250
193 175
193 193
154 160
158 160
248 224
248 242
7
Hatunparmag˘ı (U) Hatunparmag˘ı (G) Hatunparmag˘ı (T–G)
131 131 131
131 139 131
222 222 234
230 224 234
248 240 248
248 248 248
183 181 181
191 189 191
150 146 188
160 150 200
248 254 238
248 256 244
8
Sergi karası (U) Sergi karası (G)
131 131
137 151
222 222
234 234
248 250
248 250
175 165
177 175
196 192
200 200
236 236
246 246
9
Kızlartahtası (G) Kızlartahtası (U)
125 121
125 121
232 224
238 224
246 250
252 250
175 181
175 185
188 184
192 200
254 248
254 248
10
Azezi (G) Azezi (U)
129 129
129 129
224 222
232 222
248 248
248 248
181 183
191 191
190 142
202 154
248 224
254 248
11
Yediveren (U) Yediveren (G) Yediveren (T–G)
147 147 125
151 151 125
250 228 250
250 228 250
238 238 238
238 250 238
175 175 175
175 191 191
188 202 146
202 202 160
222 228 224
230 234 250
12
Serpenekıran (U) Sepenekıran (G)
119 129
129 129
232 232
232 250
244 248
250 248
165 165
173 175
150 150
160 160
226 234
232 242
13
Gu¨lgu¨lu¨ (U) Gu¨lgu¨lu¨ (G)
129 129
149 131
228 232
250 232
248 238
248 248
177 169
191 179
144 150
158 154
244 238
254 242
14
Kızılbanki (G) Kızılbanki (U)
125 129
135 129
250 234
250 234
244 244
250 250
179 169
181 179
190 138
190 150
246 240
246 240
15
Horoz karası (U) Horoz karası (G)
129 131
129 139
222 234
228 248
248 248
248 248
183 181
193 191
200 160
204 160
248 236
248 236
16
Muhammediye (U) Muhammediye (G)
129 147
129 147
232 232
250 232
248 234
250 240
179 177
191 193
154 152
154 160
256 222
256 232
Vineyard in Tekirdag˘. The remaining five varieties were collected from S¸anlıurfa and Gaziantep. In this group, the grapevine cultivars with the same variety name were clustered far from each other in the phenogram. The three varieties Azezi, Dımıs¸kı, and Muhammediye also showed a non-consistent pattern with their names. Dımıs¸kı samples were collected from S¸anlıurfa, Gaziantep, and National Germplasm Repository Vineyard in Tekirdag˘, Azezi and Muhammediye from S¸anlıurfa and Gaziantep. The cultivars with the same name were not grouped together in the phenogram even if they were not as far away to each other as were the previously described seven varieties.
VVMD27
VrZAG62
VrZAG79
Genotypes with the same name of Sergi karası (from S¸anlıurfa and Gaziantep) and Yediveren (from S¸anlıurfa, Gaziantep, and National Germplasm Repository Vineyard in Tekirdag˘) clustered together. Serpenekıran varieties collected from S¸anlıurfa and Gaziantep were positioned very close to each other in the phenogram. Moreover, the alleles at the six SSR loci analyzed were found to be similar in terms of base pairs within each of these three closely positioned varieties. When analyzing the positions of three Kabarcık varieties, we observed that Kabarcık samples, collected from Gaziantep and National Germplasm Repository Vineyard in Tekirdag˘
168
H. Karatas¸ et al. / Scientia Horticulturae 114 (2007) 164–169
Fig. 1. Phenogram of the 16 homonymous grapevine varieties.
(cultivars were brought from Gaziantep to the National Germplasm Repository Vineyard), were grouped together. However, the other Kabarcık genotype coming from S¸anlıurfa clustered far from the other two. Moreover, the SSR allele sizes of these three genotypes were identical between the two cultivars that clustered together, but different for the third cultivar that was collected from S¸anlıurfa. Hatunparmag˘ı homonymous genotypes coming from Gaziantep and National Germplasm Repository Vineyard in Tekirdag˘ clustered close to each other, but the genotype from S¸anlıurfa was positioned far from the other two in the phenogram. Similar microsatellite alleles were detected for C¸ilores¸ homonymous grapevine cultivars from S¸anlıurfa and Gaziantep and they were grouped together in the phenogram. However, the National Germplasm Repository Vineyard cultivar that was brought from S¸anlıurfa was located far from these two cultivars in the phenogram. The overall positioning in the phenogram reveals that the grapevine varieties showing the same names are not genetically identical based on microsatellites. It is obvious that the varieties cultivated in different ecological conditions of Turkey have attained different genetic profiles during the time. This differentiation among cultivars with the same names was
also increased by the high mutation rates of microsatellites. What we observed when analyzing the phenogram was actually the general tendency of cultivars of the same regions to group together rather than genotypes belonging to the same variety. Naming of the homonymous grapevine genotypes is a major problem in Turkish grapevine cultivation. The current study indicates how serious the situation is. Similarly named cultivars are generally not grouped together. On the other hand, we could say that differently named homonymous grape cultivars are clustered together. Thus, we have to choose the genetic characteristics of certain cultivars as representatives of that variety and name the other ones as relatives. Nowadays, no genetic profile of our studied Turkish variety names is reported in a publicly available SSR-based grapevine database. Before any decisions, additional molecular markers and morphometric studies conducted on Turkish grapevines should be taken into account along with these microsatellite results. Another limit in interpretation of our results was the uncertainty about representing correctly the homonymous cultivars by only one genotype. We suspect that there are several somatic mutants of the same homonymous grapevine cultivar. Hence, it would be useful to analyze more than one genotype to represent a homonymous cultivar in future studies. In conclusion, we obtained a very high allelic polymorphism among genotypes expected to be different (having different variety names) or between genotypes that were supposed to have the same variety name. The sources of these differences observed among samples with the same varietal name collected from three different ecological regions could be summarized as follows. First, these homonymous grapevine varieties cultivated in different environments for many years, and those transferred to the National Germplasm Repository Vineyard could be inappropriately named. Second, changes in genetic backgrounds of these varieties may be caused by somatic mutations resulted from the effects of continuous vegetative reproduction and environmental factors. Turkey is a very rich country in terms of homonymous grape varieties which results from the ancien tradition of grape cultivation in Anatolia, which began approximately 7000–8000 years ago. We are of the opinion that it is crucial to preserve this genetic potential by describing a reasonable nomenclature and determining the relationships among these varieties through DNA-based markers. References Adam-Blondon, A.F., Roux, C., Claux, D., Butterlin, G., Merdinog˘lu, D., This, P., 2004. Mapping 245 SSR markers on the Vitis vinifera genome: a tool for grape genetics. Theor. Appl. Genet. 109, 1017–1027. Ag˘aog˘lu, Y.S., Ergu¨l, A., 1999a. Genetic identification in ecotypes of Amasya grape cultivar by RAPD markers. In: 3rd Natl. Hortic. Congr., Kızılcahamam/Ankara, Tu¨rkiye, September 14–17, 1999, pp. 372–386. Ag˘aog˘lu, Y. S., Marasalı, B., Ergu¨l, A., 2000. Molecular characterization on the grapevine (Vitis vinifera L. cvs.) by RAPD (random amplified polymorphic) technique. Ankara University Research Fond. 96-11-01-02 no.lu Report of Project Result, Ankara.
H. Karatas¸ et al. / Scientia Horticulturae 114 (2007) 164–169 Ag˘aog˘lu, Y.S., Ergu¨l, A., 1999b.In: Identification of some Turkish table grape cultivars (Vitis vinifera L.) by random amplified polymorphic DNA (RAPD), Deutsch.-Tu¨rkische Agrarfoschung Symposium. Giessen, Germany, pp. 201– 206. Alleweld, G., Spiegel-Roy, P., Reisch, B., 1990. Grapes (Vitis). In: Moore, I.N., Ballington, J.L. (Eds.), Genetic Resources of Temprate Fruit and Nut Crops. Acta Hortic. 290, 291–337. Atak, A., 2003. Identification of some grape cultivars hybrids and their parents by RAPD technique. MSc Thesis (unpublished) [Tr, en]. Ankara University, Ankara, Tu¨rkiye. Botta, R., Scott, N.S., Eynard, I., Thomas, M.R., 1995. Evaluation of microsatellite sequence-tagged site markers for characterizing Vitis vinifera cultivars. Vitis 34 (2), 99–102. Bowers, J.E., Dangl, G.S., Meredith, C.P., 1999. Development and characterization of additional microsatellite DNA markers for grape. Am. J. Enol. Vitic. 50, 243–246. Bowers, J.E., Dangl, G.S., Vignani, R., Meredith, C.P., 1996. Isolation and characterization of new polymorphic simple sequence repeat loci in grape (Vitis vinifera L.). Genome 39, 628–633. Costantini, L., Monaco, A., Vouillamoz, J.F., Forlani, M., Grando, M.S., 2005. Genetic relationships among homonymous Vitis vinifera cultivars from Campania (Italy). Vitis 44, 25–34. C ¸ elik, H., Marasalı, B., So¨ylemezog˘lu, G., Tangolar, S., Gu¨ndu¨z, M., 2000. ¨ retim Hedefleri (Future Prospect in Turkish Viticulture). Bag˘cılıkta U TMMOB Ziraat. Mu¨h. Odası, Tu¨rkiye Ziraat Mu¨hendislig˘i V. Teknik Kongresi, 17–21 Ocak 2000, Ankara [Tr]. Bildiriler 2, 645–678. Di Gaspero, G., Peterlunger, E., Testolin, R., Edwards, K.J., Cipriani, G., 2000. Conservation of microsatellite loci within the genus Vitis. Theor. Appl. Genet. 101, 301–308. Di Gaspero, G., Cipriani, G., Marazzo, M.T., Andreetta, D., Castro, M.J.P., Peterlunger, E., Testolin, R., 2005. Isolation of (AC)n-microsatellites in Vitis vinifera L. and analysis of geneticbackground in grapevines under marker assisted selection. Mol. Breed. 15, 11–20. Doligez, A., Adam-Blondon, A.F., Cipriani, G., Di Gaspero, G., Laucou, V., Merdinog˘lu, D., Meredith, C.P., Riaz, S., Roux, C., This, T., 2006. An integrated SSR map of grapevine based on five mapping populations. Theor. Appl. Genet. 113, 369–382. Ergu¨l, A., 2000. Molecular characterization of grapevine (Vitis vinifera L. cvs.) by genomic DNA fingerprinting. PhD Thesis (unpublished) [Tr, en]. Ankara University, Ankara, Tu¨rkiye. Ergu¨l, A., Agaog˘lu, Y.S., 2001. Molecular similarity analysis of some grapevine rootstocks from different nursery in Turkey. J. Agric. Sci. 7(2), 141–143 [Tr, en]. Ankara, Tu¨rkiye. Ergu¨l, A., Marasalı, B., Ag˘aog˘lu, Y.S., 2002. Molecular discrimination and identification of some Turkish grape cultivars (Vitis vinifera L.) by RAPD markers. Vitis 41 (3), 159–160. Fatahi, R., Ebadi, A., Bassil, N., Mehlenbacher, S.A., Zamani, Z., 2003. Characterization of Iranian grapevine cultivars using microsatellite markers. Vitis 42 (4), 185–192. Felsenstein, J., 1988. Phylogenies from molecular sequences—inference and reliability. Annu. Rev. Genet. 22, 521–565. Franks, T., Botta, R., Thomas, M.R., 2002. Chimerisim in grapevines: implications for cultivar identitiy, ancestry, and genetic improvement. Theor. Appl. Genet. 104, 192–199. Grando, M.S., Bellin, D., Edwards, K.J., Pozzi, C., Stefanini, M., Velasco, R., 2003. Molecular linkage maps of Vitis vinifera L. and Vitis riparia Mchx. Theor. Appl. Genet. 106, 1213–1224. Hocquigny, S., Pelsy, F., Dumas, V., Kindt, S., Heloir, M.-C., Merdinog˘lu, D., 2004. Diversification within grapevine cultivars goes through chimeric states. Genome 47, 579–589. Hvarleva, T., Rusanov, K., Lefort, F., Tsvetkov, I., Atanassov, A., Atanassov, I., 2004. Genotyping of Bulgarian Vitis vinifera L. cultivars by microsattellite analysis. Vitis 43 (1), 27–34. Karatas¸, H., Ag˘aog˘lu, Y.S., 2006. Molecular analysis of Diyarbakır region’s grapevine germplasm by RAPD (Random Amplified Polimorphic DNA) technique. 8. Deutsch-Turkische Agrarforsching Symposium, 4–8 October, 2005. Braunschweig, Germany, 411–421.
169
Lamboy, W.F., Alpha, C.G., 1998. Using simple sequence repeats (SSRs) for DNA fingerprinting germ plasmaccessions of grape (Vitis. L. species). J. Am. Soc. Hortic. Sci. 123, 182–188. Lin, H., Walker, M.A., 1998. Identifying grape rootstocks with simple sequence repeat (SSR) DNA markers. Am. J. Enol. Vitic. 49, 403–407. Lodhi, M.A., Daly, M.J., Ye, G.-N., Weeden, N.F., Reisch, B.I., 1994. A Simple and efficient method for DNA extraction from grapevine cultivars and Vitis species. Plant Mol. Biol. Reptr. 12 (1), 6–13. Merdinog˘lu, D., Butterlin, G., Bevilacqua, L., Chiquet, V., Adam-Blondon, A.F., Decroocq, S., 2005. Development and characterization of a large set of microsatellite markers in grapevine (Vitis vinifera L.) suitable for multiplex PCR. Mol. Breed. 15, 349–366. Meredith, C., 2001. Grapevine genetics: probing the past and facing the future. Agriculturae Conspectus Sientificus 66, 21–25. Meredith, C., Dangl, G.S., Bowers, J.E., 1996. Clarifying the identitiy of some California winegrapes by DNA profilling. Riv. Vitic. Enol. 49 (1), 65–68. Moser, C., Segala, C., Fontana, P., Salakhudtinov, I., Gatto, P., Pindo, M., Zyprian, E., Toepfer, R., Grando, M.S., Velasco, R., 2005. Comparative analysis of expressed sequence tags from different organs of Vitis vinifera L. Funct. Integr. Genomics 5, 208–217. Paetkau, D., Waits, L.P., Clarkson, P.L., Craighead, L., Strobeck, C., 1997. An empirical evaluation of genetic distance statistics using microsatellite data from bear (Ursidae) populations. Genetics 147, 1943–1957. Pellorone, F., Edwards, K., Thomas, M., 2001. Grapevine micosatellite repeats: isolation, characterization and use for genotyping of grape germplasm from Southern Italy. Vitis 41, 83–87. Riaz, S., Dangl, G.S., Edwards, K.J., Meredith, C.P., 2004. A microsatellite marker based framework linkage map of Vitis vinifera L. Theor. Appl. Genet. 108, 864–872. Riaz, S., Garrisson, K.E., Dangl, G.S., Boursiquot, J.M., Meredith, J.P., 2002. Genetic divergence and chimerism within ancient asexually propagated winegrape cultivars. J. Am. Soc. Hortic. Sci. 127, 508–514. Schneider, S., Roessli, D., Excoffier, L., 2000. Arlequin Ver. 2.000: A Software for Population Genetics Data Analysis. Genetics and Biometry Laboratory, University of Geneva, Switzerland. Scott, K.D., Eggler, P., Seaton, G., Rosetto, M., Ablett, E.M., Lee, L.S., Henry, R.J., 2000. Analysis of SSRs derived from grape ESTs. Theor. Appl. Genet. 100, 723–726. Sefc, K.M., Regner, F., Turetschek, E., Glo¨ssl, J., Steinkellner, H., 1999. Identification of microsatellite sequences in Vitis riparia and their applicability for genotyping of different Vitis species. Genome 42, 367–373. Sefc, K.M., Steinkellner, H., Glo¨ssl, J., Kampfer, S., Regner, F., 1998. Reconstruction of a grapevine pedigree by microsatellite analysis. Theor. Appl. Genet. 97, 227–231. Sefc, K.M., Steinkellner, H., Wagner, H.W., Glo¨ssl, J., Regner, F., 1997. Application of microsatellite markers to parentage studies in grapevine. Vitis 36 (4), 179–183. Siret, R., Boursiquot, J.M., Merle, M.H., Cabanis, J.C., This, P., 2000. Toward the authentication of varietal wines by the analysis of grape (Vitis vinifera L.) residual DNA in must and wine using microsatellite markers. J. Agric. Food Chem. 48, 5035–5040. This, P., Jung, A., Boccacci, P., Borrego, J., Botta, R., Costantini, L., Crespan, M., Dangl, G.S., Eisenheld, C., Ferreira-Monteiro, F., Grando, M.S., Iba´n˜ez, J., Lacombe, T., Laucou, V., Magalha˜es, R., Meredith, C.P., Milani, N., Peterlunger, E., Regner, F., Zulini, L., Maul, E., 2004. Development of a standard set of microsatellite references alleles for identification of grape cultivars. Theor. Appl. Genet. 109, 1048–1058. Thomas, M.R., Scott, N.S., 1993. Microsatellite repeats in grapevine reveal DNA polimorphisms when analysed as sequence-tagged sites (STSs). Theor. Appl. Genet. 86, 985–990. Vouillamoz, J.F., Maigre, D., Meredith, C.P., 2004. Identity and parentage of two Alpine grape cultivars from Switzerland (Vitis vinifera L. Lafnetscha and Himbertscha). Vitis 43 (2), 81–87. Zulini, L., Fabro, E., Peterlunger, E., 2005. Characterisation of the grapevine cultivar Picolit by means of morphological descriptors and molecular markers. Vitis 44 (1), 35–38.