Microsatellite fingerprinting of homonymous grapevine (Vitis vinifera L.)

varieties in neighboring regions of South-East Turkey

Huseyin Karatas a, Dilek Degirmenci b, Riccardo Velasco c, Silvia Vezzulli c,Cagrı Bodur d, Y. Sabit Agaoglu b,*

a Dicle University, Faculty of Agriculture, Department of Horticulture, Diyarbakyr, Turkeyb Ankara University, Faculty of Agriculture, Department of Horticulture, Ankara, Turkey

c Istituto Agrario di San Michele all’Adige, San Michele a/Adige (TN), Italyd Middle East Technical University, Department of Biology, Ankara, Turkey

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 Sanlıurfa and Gaziantep), Yediveren (sampled from Sanlıurfa, Gaziantep, and National Germplasm Repository

Vineyard in Tekirdag) and Serpenekıran (sampled from Sanlıurfa 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.

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Scientia Horticulturae 114 (2007) 164–169

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: agaoglu@agri.ankara.edu.tr (Y.S. Agaoglu).

0304-4238/$ – see front matter # 2007 Elsevier B.V. All rights reserved.

doi:10.1016/j.scienta.2007.07.001

Repository Vineyard in Tekirdag (Celik et al., 2000; Ergul

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 ampe-

los-grapevine and graphos-description), analysing and compar-

ing 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

Table 1

Grapevine genotypes studied with SSR markers

N Sample name Collection Berry

colour

Use (table/

raisin/wine)

1 Cilores (U) U White Table

2 Cilores (G) G White Table

3 Cilores (T–U) T–U White Table

4 Cilorut (U) U White Table

5 Cilorut (G) G White Table

6 Cilorut (T–U) T–U White Table

7 Honusu (U) U Red Table

8 Honusu (G) G Red Table

9 Honusu (T–G) T–G Red Table

10 Dımıskı (U) U White Table

11 Dımıskı (G) G White Table

12 Dımıskı (T–G) T–G White Table

13 Kabarcık (U) U White Table

14 Kabarcık (G) G White Table

15 Kabarcık (T–G) T–G White Table

16 Kulahi (U) U White Table

17 Kulahi (G) G White Table

18 Hatunparmagı (U) U White Table, raisin

19 Hatunparmagı (G) G White Table, raisin

20 Hatunparmagı (T–G) T–G White Table, raisin

21 Sergi karası (U) U Red Raisin

22 Sergi karası (G) G Red Raisin

23 Kızlartahtası (G) G White Table

24 Kızlartahtası (U) U White Table

25 Azezi (G) G White Table

26 Azezi (U) U White Table

27 Yediveren (U) U Red Table

28 Yediveren (G) G Red Table

29 Yediveren (T–G) T–G Red Table

30 Serpenekıran (U) U White Table

31 Sepenekıran (G) G White Table

32 Gulgulu (U) U Pink Table

33 Gulgulu (G) G Pink Table

34 Kızılbanki (G) G Pink Table

35 Kızılbanki (U) U Pink Table

36 Horoz karası (U) U Red Table, raisin, wine

37 Horoz karası (G) G Red Table, raisin, wine

38 Muhammediye (U) U White Table

39 Muhammediye (G) G White Table

U: Sanlıurfa; 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 Sanlıurfa city.

H. Karatas et al. / Scientia Horticulturae 114 (2007) 164–169 165

grapevine cultivars (Agaoglu and Ergul, 1999a,b; Agaoglu

et al., 2000; Ergul, 2000; Ergul and Agaoglu, 2001; Ergul et al.,

2002; Atak, 2003; Karatas and Agaoglu, 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; Merdinoglu 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; Adam-

Blondon 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.

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 Sanlıurfa (U)

provinces, and one small group (7 samples), containing

individuals coming from the ‘‘National Germplasm Reposi-

tory 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).

2.2. Microsatellite analysis

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

Table 2

The minimal standard SSR marker set used for grapevine genotyping

Locus Annealing

temperature (8C)

Allele size

range (bp)

N He Ho

VVS2 51 119–137 15 0.85 0.48

VVMD5 52 222–250 10 0.89 0.42

VVMD7 51 230–256 10 0.92 0.47

VVMD27 52 161–191 14 0.86 0.85

VrZAG62 52 138–204 21 0.91 0.73

VrZAG79 52 220–256 18 0.93 0.62

Mean 14.66 0.89 0.60

N: number of alleles; He: expected heterozygosity; Ho: observed heterozygosity.

H. Karatas et al. / Scientia Horticulturae 114 (2007) 164–169166

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 neigh-

bouring 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 micro-

satellite analysis.

Within the 39 grapevine cultivars obtained from Sanlıurfa,

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 con-

structed 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 Sanlıurfa and Gaziantep. When we considered

seven cases that contain sampling from all the three locations

(Sanlıurfa, 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 Cilorut, Honusu,

Gulgulu, Horoz karası, Kızılbanki, Kızlartahtası, and Kulahi,

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, Cilorut and Honusu, came from

Sanlıurfa, Gaziantep and National Germplasm Repository

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 VVMD27 VrZAG62 VrZAG79

1 Cilores (U) 129 129 222 222 230 250 181 191 198 202 224 248

Cilores (G) 129 129 224 224 230 250 181 191 198 202 224 248

Cilores (T–U) 123 123 248 248 248 248 181 181 204 204 242 256

2 Cilorut (U) 131 139 232 232 244 250 191 191 146 146 248 248

Cilorut (G) 139 149 228 228 248 256 177 191 156 156 220 220

Cilorut (T–U) 131 141 228 228 248 248 175 191 160 160 238 254

3 Honusu (U) 139 145 228 228 240 240 169 189 140 150 234 240

Honusu (G) 137 137 222 236 244 248 175 191 150 150 238 248

Honusu (T–G) 139 139 234 234 248 248 177 181 188 194 256 256

4 Dımıskı (U) 129 129 224 224 230 250 181 185 200 204 248 248

Dımıskı (G) 141 141 228 234 230 250 181 185 160 160 226 244

Dımıskı (T–G) 129 153 232 236 248 248 175 191 192 204 254 256

5 Kabarcık (U) 129 145 222 222 240 240 191 191 188 204 238 248

Kabarcık (G) 129 139 232 232 234 250 163 167 150 160 234 246

Kabarcık (T–G) 129 139 232 232 234 250 163 167 150 160 234 246

6 Kulahi (U) 129 129 222 222 250 250 193 193 154 158 248 248

Kulahi (G) 141 141 228 234 250 250 175 193 160 160 224 242

7 Hatunparmagı (U) 131 131 222 230 248 248 183 191 150 160 248 248

Hatunparmagı (G) 131 139 222 224 240 248 181 189 146 150 254 256

Hatunparmagı (T–G) 131 131 234 234 248 248 181 191 188 200 238 244

8 Sergi karası (U) 131 137 222 234 248 248 175 177 196 200 236 246

Sergi karası (G) 131 151 222 234 250 250 165 175 192 200 236 246

9 Kızlartahtası (G) 125 125 232 238 246 252 175 175 188 192 254 254

Kızlartahtası (U) 121 121 224 224 250 250 181 185 184 200 248 248

10 Azezi (G) 129 129 224 232 248 248 181 191 190 202 248 254

Azezi (U) 129 129 222 222 248 248 183 191 142 154 224 248

11 Yediveren (U) 147 151 250 250 238 238 175 175 188 202 222 230

Yediveren (G) 147 151 228 228 238 250 175 191 202 202 228 234

Yediveren (T–G) 125 125 250 250 238 238 175 191 146 160 224 250

12 Serpenekıran (U) 119 129 232 232 244 250 165 173 150 160 226 232

Sepenekıran (G) 129 129 232 250 248 248 165 175 150 160 234 242

13 Gulgulu (U) 129 149 228 250 248 248 177 191 144 158 244 254

Gulgulu (G) 129 131 232 232 238 248 169 179 150 154 238 242

14 Kızılbanki (G) 125 135 250 250 244 250 179 181 190 190 246 246

Kızılbanki (U) 129 129 234 234 244 250 169 179 138 150 240 240

15 Horoz karası (U) 129 129 222 228 248 248 183 193 200 204 248 248

Horoz karası (G) 131 139 234 248 248 248 181 191 160 160 236 236

16 Muhammediye (U) 129 129 232 250 248 250 179 191 154 154 256 256

Muhammediye (G) 147 147 232 232 234 240 177 193 152 160 222 232

H. Karatas et al. / Scientia Horticulturae 114 (2007) 164–169 167

Vineyard in Tekirdag. The remaining five varieties were

collected from Sanlıurfa 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, Dımıskı, and Muhammediye also

showed a non-consistent pattern with their names. Dımıskı

samples were collected from Sanlıurfa, Gaziantep, and

National Germplasm Repository Vineyard in Tekirdag, Azezi

and Muhammediye from Sanlıurfa 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.

Genotypes with the same name of Sergi karası (from

Sanlıurfa and Gaziantep) and Yediveren (from Sanlıurfa,

Gaziantep, and National Germplasm Repository Vineyard in

Tekirdag) clustered together. Serpenekıran varieties collected

from Sanlıurfa 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

Fig. 1. Phenogram of the 16 homonymous grapevine varieties.

H. Karatas et al. / Scientia Horticulturae 114 (2007) 164–169168

(cultivars were brought from Gaziantep to the National

Germplasm Repository Vineyard), were grouped together.

However, the other Kabarcık genotype coming from Sanlıurfa

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 Sanlıurfa. Hatunparmagı

homonymous genotypes coming from Gaziantep and National

Germplasm Repository Vineyard in Tekirdag clustered close to

each other, but the genotype from Sanlıurfa was positioned far

from the other two in the phenogram. Similar microsatellite

alleles were detected for Cilores homonymous grapevine

cultivars from Sanlıurfa and Gaziantep and they were grouped

together in the phenogram. However, the National Germplasm

Repository Vineyard cultivar that was brought from Sanlıurfa

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 representa-

tives 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 polymorph-

ism among genotypes expected to be different (having diffe-

rent 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 culti-

vated 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.

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