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1Dry Land Farming and Oasis Cropping Laboratory, Arid Land Institute, Km 22.5, Rte. El Djorf, 4119 Medenine, Tunisia; 2Unité de Chimie Biologique In-dustrielle, Gembloux Agro-Bio Tech, Passage des Déportés, 2, 5030 Gembloux, Belgium; and 3Instituto de Ciencias de la Vid y del Vino (CSIC, Universidad de La Rioja, Gobierno de La Rioja), Complejo Científico Tecnológico, C/Madre de Dios 51, 26006 Logroño, Spain.*Corresponding author (email: javier.ibanez@icvv.es; tel: 34-94 1299694; fax: 34-94 1299608)Acknowledgments: This research was supported by Agencia Española de Cooperación Internacional para el Desarrollo, Spain, within the framework of the projects A/023457/09 and A/031064/10. The authors thank all the staff of forest services in northwestern Tunisia for their support during the collec-tion of wild grapevine material and Silvia Hernáiz for technical assistance.Supplemental data is freely available with the online version of this article.Manuscript submitted Nov 2012, revised Apr 2013, Jul 2013, accepted Aug 2013Copyright © 2013 by the American Society for Enology and Viticulture. All rights reserved.doi: 10.5344/ajev.2013.12135

Research NoteGenetic Identification and Origin of Grapevine Cultivars

(Vitis vinifera L.) in Tunisia

Sana Ghaffari,1 Nejib Hasnaoui,2 Lalla Hasna Zinelabidine,3 Ali Ferchichi,1 José Miguel Martínez-Zapater,3 and Javier Ibáñez3*

Abstract: Nine nuclear microsatellite (SSR) markers were used to characterize 35 wild grapevines (Vitis vinifera subsp. sylvestris) prospected from northwestern Tunisia and 64 cultivated accessions (Vitis vinifera subsp. vinifera) maintained in the repository of the Arid Land Institute of Medenine (Tunisia). All analyzed SSR loci were polymor-phic, revealing 62 distinct genotypes, including 31 cultivated and 31 wild accessions. Some cases of synonymies, color sports, and homonymies were detected as well as matches with previously analyzed Tunisian samples and international cultivars. Chloroplast microsatellite analyses showed that chlorotype A was most abundant in wild samples (65%), whereas chlorotypes C and D were more frequent in cultivated genotypes (45% and 23% respec-tively). Genotypic analysis showed that both Tunisian wild and cultivated samples maintain high levels of genetic variation and high average posterior probabilities of assignment to their group of origin. This is in agreement with the estimated low gene flow between cultivated and wild forms, revealing that most cultivated accessions do not derive directly from the local wild populations but could correspond to materials introduced from different locations or derived from spontaneous hybridizations among them. However, we could not discard the hypothesis that a few analyzed samples could arise from hybridization events between wild and cultivated grapevines.

Key words: chlorotypes, grapevine cultivars, nuclear microsatellites, wild grapevines, Vitis vinifera

Grapevine (Vitis vinifera L.) is one of the oldest, most ex-tensively cultivated and economically important fruit crops in the world. The wild subspecies (Vitis vinifera subsp. sylves-tris) is believed to be the ancestor of present cultivars (Negrul 1938). In Tunisia, the first historical record of viticulture dates back to 6000 bc (Zohary and Hopf 2000), and northwestern Tunisia still contains plants of the wild subspecies V. vinifera L. ssp. sylvestris (Harbi Ben Slimane 1999, Riahi et al. 2010). They represent ecotypes with large morphological variabil-ity, but their presence remains very limited and is especially threatened by various environmental and anthropogenic fac-tors (Harbi Ben Slimane 1999).

For cultivated grapevines, a rich germplasm of autochtho-nous cultivars is grown from north to south and seems to be well adapted to different local climatic conditions. Neverthe-less, there are many problems related to cultivar identification (Harbi Ben Slimane 1999). There are now three national re-positories holding cultivated grapevines in Tunisia. Collection of cultivated V. vinifera started in the 1990s, which led to a first germplasm collection in 1998 at the Institut National de Recherche Agronomique de Tunis (INRAT) that was am-pelographically (Harbi Ben Slimane 1999) and genetically characterized (Snoussi et al. 2004). A second collection was established at the Centre de Biotechnologie de Borj Cedria (CBBC). Genetic diversity of this collection was examined using molecular markers, particularly SSRs (Riahi et al. 2010, Zoghlami et al. 2009). The CBBC collection was duplicated in 2008 at the Arid Land Institute of Medenine (IRA) to con-duct physiological studies and to collect some new accessions directly from vineyards.

Here we report the genetic identification and character-ization of the cultivated grapevines from the IRA collection as well as wild samples from northern Tunisia, with nine selected nuclear and four chloroplast microsatellite (SSR) markers. This set of nuclear markers has proved suitable for grapevine characterization, including the demanding tests for the evaluation of distinctness, uniformity, and stability (DUS) (Ibáñez et al. 2009b, Vélez and Ibáñez 2012). Data obtained have been used to clarify homonyms and synonyms to overcome the existing confusion in Tunisian grapevine nomenclature, particularly in the IRA grapevine collection, and to evaluate the genetic diversity and relationships within and between the cultivated and wild groups.

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Materials and MethodsPlant material. Wild grapevine (V. vinifera subsp. sylves-

tris) leaf samples were collected from 35 plants located in the northwest region of Tunisia. The prospected region included the Atlas Mountains, which stretch from the Algerian bor-ders to the north Mediterranean Sea, where wild grapevine is secluded (Table 1). These plants were located in wetlands and forest ecosystems, close to rivers or brooks, and with the presence of characteristic tree species such as elm, poplar, fig, rough bindweed, dog rose and alder, on which wild grape-vines grow as lianas. Each sample location was identified by GPS and recorded photographically. Leaf samples from 64 cultivated grapevine accessions (V. vinifera subsp. vinif-era), maintained in the repository of the Arid Land Institute of Medenine (IRA), were included in this study (Table 1). When possible, each accession was sampled twice, from two different plants.

DNA extraction. DNA extractions were carried out from lyophilized young leaves (lyophilization at -40°C and 0.05 to 0.12 mbar for 72 hr) using the DNeasy Plant Mini Kit (Qia-gen, Valencia, CA) according to the manufacturer’s protocol. The resulting DNAs were quantified by visual comparison with lambda DNA on ethidium bromide stained agarose gels (0.8%) and by spectrophotometry (NanoDrop 2000; Thermo Scientific, Waltham, MA).

SSR genotyping. A set of nine previously described nuclear microsatellite loci, located in nine different linkage groups, was used: VVMD5, VVMD27, VVMD28, VVS2, ssrVrZAG29, ssrVrZAG62, ssrVrZAG67, ssrVrZAG83, and ssrVrZAG112. A multiplex PCR with the nine markers was used in addition to a second multiplex PCR with four pre-viously described chloroplast microsatellite loci: cpSSR3, cpSSR5, cpSSR10, and ccSSR9. One primer of each pair was f luorescently labeled with Dye Phosphoramidites (6-FAM, VIC, or NED). Amplified PCR fragments were re-solved in an ABI PRISM 310 Genetic Analyzer (Applied Biosystems, Foster City, CA), using LIZ 500 as an internal size standard. Automatic size calling of peak positions was double-checked using GeneMapper 4.1 software (Applied Biosystems). PCR and electrophoresis were according to Ibáñez et al. (2009a).

Genetic identification. Matching genotypes were identi-fied by pair-wise comparisons among all 99 individuals based on their multilocus SSR profile. GenAlEx 6.501 software (Peakall and Smouse 2006) was used for genotype matching analyses. Accessions with the same name and genotype were considered duplicates; accessions with different names that fully matched at the nine loci were considered synonyms, sports, or mislabeled accessions. Accessions with the same name and different genotype were considered homonyms or mislabeled accessions. Only one representative of each geno-type was included in subsequent analyses of genetic diversity and population structure. These genotypes were also used to detect putative identities and synonymies with published Mediterranean and international well-known varieties (El Oualkadi et al. 2009, Laiadi et al. 2009, Riahi et al. 2010, Zinelabidine et al. 2010, Zoghlami et al. 2009).

Analysis of genetic diversity. Analyses of population genetics were separately done for each group of wild and cultivated grapevines. Allele frequencies (pi), observed het-erozygosity (Ho), expected heterozygosity (He), probability of identity (PI = Σ pi

4 + Σ [2pi pj]2), estimation of null allele fre-quency from the heterozygote deficiency (r), and paternity ex-clusion probability (Q) were estimated using IDENTITY 4.0 software (Wagner and Sefc 1999). The mean values of each parameter were compared using t test (ver. 5.04; GraphPad Prism, San Diego, CA) to verify the statistical significance of the mean differences between the two groups. A p value <0.05 was considered statistically significant.

Excess and deficiency of heterozygotes and genic and genotypic differentiation, distribution of alleles, and test-ing for Hardy-Weinberg equilibrium were calculated using GENEPOP 4.1 (Rousset 2008). Multilocus FST values were obtained with ARLEQUIN 3.11 (Excoffier et al. 2005). Sta-tistical significance was estimated by permutation analysis using 10,000 replications. Genetic distances were calculated as 1-proportion of shared alleles, using MICROSAT 1.5d (Minch 1997). The genetic distance matrix was used for a principal coordinate analysis, using GenAlEx 6.501 (Peakall and Smouse 2006).

A Bayesian assignment method, as implemented in the software NEWHYBRIDS (ver. 1.1 beta) (Anderson and Thompson 2002), was used to identify putative pure indi-viduals and distinguish among different hybrid types. The software computes the posterior probability that each indi-vidual in the sample belongs to each of the different catego-ries, referred by the software as: Pure Cultivated, Pure Wild, F1 (Cultivated x Wild), F2 (F1 x F1), Cultivated Backcross (Cultivated x F1), and Wild Backcross (Wild x F1). Tests were carried out for all nine loci with uniform priors and with a run of 100,000 iterations after a burn-in period of 100,000 itera-tions; multiple runs were conducted. This approach reduces the influence of low-frequency alleles, preventing sampling and genotyping errors in closely related populations.

Results and DiscussionGenetic identification. Tunisian cultivated and wild ac-

cessions were genotyped at nine nuclear microsatellite loci, generating 62 different genetic profiles for the 99 analyzed accessions (Supplemental Table 1). Among them, 31 cor-responded to cultivated accessions and 31 to wild samples. The probability of finding two different grapevines with the same genotype at all loci, calculated as the global probabil-ity of identity (PI), was 4.9 10-11. This low PI value reflects the usefulness of the chosen set of microsatellite markers for varietal identification. Four of the SSRs used (VVS2, VVMD5, VVMD27, and VrZAG62) belong to the core set adopted as descriptors by the OIV and one other (VVMD28) is included in several European projects. Their use facilitated the comparison of our genotyping data with other published genotypes or public databases, such as the European Vitis Database (www.eu-vitis.de).

Among the 31 cultivated genotypes, 18 were represented by a single accession name while 13 were found in more than

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Name Origin Color/usea Genotypeb

CultivatedAhmar Bou Ahmar Kerkennah R/T 1Amokrane - W/T 30Arbi Mornag W/T 21Arbi F - W/T 2Arich Abiadh Zarzis W/T 17Arich Ahmar Zarzis R/T 19Arich Djerba Djerba P/T 19Arich Dressé Baddar P/T 19Asli Kerkennah W/T-R 3Balta 1 Balta W/T 4Balta 2 Balta W/T 20Balta 3 Balta W/T 20Bezzoul Kalba Bidha Sfax W/T 5Bezzoul Khadem Rafraf R/T 17Behbahi Djebba W/T 6Beldi Baddar W/T 18Beldi Local Rafraf Rafraf W/T 7Beldi Sayeb Mornag W/T 20Bidh Hmem Baddar W/T 20Bidh Hmem Sfax W/T 21Blanc Djebba W/T 16Chaaraoui Djebba W/T 20Chaouch Djerba W/T 8Dabouki - W/T 9Dalia Kerkennah W/T 17Dalia Bou Ficha Bou Ficha W/T 10El Biodh Baddar W/T 11Farrani Rafraf W/T 20Garai - R/T 12Guelb Sardouk Gafsa DB/T 13Hammouri Gafsa R/T 28Hamri Kerkennah DR/T 29Hencha 1 Hencha W/T 6Hencha 2 Hencha W/T 20Jerbi Dgueche W/T 14Jerbi Kerkennah R/T 15Kahli Kerkennah B/T 17Khalt Abiadh Tozeur W/T 21Khalt Bou Chemma Gabès W/T 16Khalt Mdaouar Tozeur W/T 17Khamri Tozeur R/T-W 18Khedhiri 1 Bargou W/T 20Khedhiri 2 Bargou W/T 3Mahdoui Kerkennah W/T 20Mansouri Rafraf R/T 22Marsaoui Kerkennah W/T 21Medina Gabès P/T 19Meski Local Djerba W/T 20Meski Rafraf Rafraf W/T 21Mguergueb Djerba W/T 22

Name Origin Color/usea Genotypeb

CultivatedMuscat d’Alexandrie Zarzis W/T 23Neb Jmel Ben

GuerdaneDR/T 24

Oasis 46 Gafsa W/T 25Razegui Djerba W/T 20Reine de Vignes - W/T 26Rich Baba Sam Rafraf W/T 27Sakasli Djerba DB/T 28Saoudi - B/T 20Sfaxi Sfax W/T 29Siper Abiadh - W/T 19Sultanine - W/T 23Taferielt - B/T 30Tounsi Kerkennah W/T 20Turki Baddar W/T 31

WildAin Bakouch 1 Ain Bakouch na/- 32Ain Bakouch 2 Ain Bakouch na/- 32Ain Bakouch 3 Ain Bakouch na/- 33Ain Bakouch 4 Ain Bakouch na/- 33Ain Snoussi-Chaaba Chayba Ain Snoussi B/- 34Bellif 1 Bellif na/- 35Bellif 2 Bellif na/- 36Bellif 3 Bellif na/- 37Bellif 4 Bellif na/- 38Bellif 5 Bellif na/- 38Bellif 6 Bellif na/- 39Douard Dhwayfiya E Dmayen W/- 40E Rwayssiya 1 E Rwayssiya na/- 41E Rwayssiya 2 E Rwayssiya na/- 42E Rwayssiya 3 E Rwayssiya B/- 43E Rwayssiya 4 E Rwayssiya na/- 44Khorguelia 1 Khorguelia na/- 45Khorguelia 2 Khorguelia B/- 46Khorguelia 3 Khorguelia B/- 47Khorguelia 4 Khorguelia na/- 48Les Dunes de Tabarka 1 Tabarka na/- 49Les Dunes de Tabarka 2 Tabarka na/- 49Les Dunes de Tabarka 3 Tabarka na/- 50Les Dunes de Tabarka 4 Tabarka na/- 51Les Dunes de Tabarka 5 Tabarka na/- 52Ouchtata 1 Ouchtata na/- 53Ouchtata 2 Ouchtata W/- 54Oued E Nour 1 Oued E Nour B/- 55Oued E Nour 2 Oued E Nour B/- 56Oued E Nour 3 Oued E Nour na/- 57Oued E Nour 4 Oued E Nour na/- 58Oued E Nour 5 Oued E Nour na/- 59Oued E Nour 6 Oued E Nour na/- 60Oued E Nour 7 Oued E Nour na/- 61Oued E Nour 8 Oued E Nour na/- 62

Table 1 List of accessions analyzed, including the name of cultivated varieties or the location of wild individuals, the region of origin, berry color and use (specific to Tunisia), and assigned genotype number based on the nine nuclear SSR analyzed.

aBerry color: B: black, DB: dark blue, DR: dark red, R: red, P: pink, W: white (green/yellow). Berry use: T: table grapes, R: raisin, W: wine. na: no information availablebGenotype number; also see Supplemental Table 1.

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one accession. Genotype 20 was the most redundant and was found in 13 accessions (Table 2), while for nine genotypes only two matching accessions were found. Accessions with matching genotypes can be considered as synonyms, dupli-cates, sports, or just mislabeled accessions. Some of the acces-sions with identical genotypes have different berry color and are likely “color sports.” Color somatic variation is common in table grape, and it is relevant to consider variants as dif-ferent cultivars.

Some local names led to homonym accessions within Tu-nisian cultivated grapes. Eight cases of possible homonymies or mislabeled accessions were detected (Table 3), showing that similar names for Tunisian cultivars are not always good indicators for identical genotypes.

As frequently observed in other grapevine collections, the IRA collection contained numerous redundancies, hin-dering efficient conservation and use. Some of the observed redundancies had also been detected in the original CBBC collection; for example, Balta 2 and Balta 3 were confirmed to be the same accession, as reported previously (Snoussi et al. 2004, Zoghlami et al. 2009). In other cases, cultivars that were considered different in one study had identical geno-types in another study; for example, the three Arich cultivars (Djerba, Dressé, and Ahmar) were reported as genetically different in the CBBC collection (Zoghlami et al. 2009) but shared the same genotype for the nine SSR loci used in the current study. The comparison of the genetic profiles of ac-cessions with the same names in both collections, expected to be identical, revealed frequent discrepancies, likely resulting from mistakes during replication of plant material, transfer to establish the IRA collection, or laboratory genotyping (such as sampling, allele binning, lack of specific alleles amplifica-tion).

In order to provide some clarity, our data were compared to the SSR genotypes of Tunisian accessions previously pub-lished (Riahi et al. 2010, Zoghlami et al. 2009) and additional Maghrebian collections (El Oualkadi et al. 2009, Laiadi et al. 2009, Zinelabidine et al. 2010) (Supplemental Table 2). By comparison with the ICVV (Instituto de Ciencias de la Vid y del Vino) database and other public databases (Vitis Interna-tional Variety Catalogue, VIVC; http://www.vivc.de/), some genotypes could be identified as corresponding to known Mediterranean or international varieties, suggesting that the local names could represent new synonym denominations. Some denominations are conserved in two or more countries, with slight spelling differences, or conserving the meaning, as Muscat d’Alexandrie and Ahmar Bou Ahmar, which were found to be identical to Muscat of Alexandria and the Al-gerian classical cultivar Ahmeur bou Ahmeur, respectively. This last genotype also matched with Ahmar de Mascara and Muscat d’Alexandrie corresponded to the Algerian accession Muscat de Fandouk 1 (Laiadi et al. 2009) and the Moroccan accession Muscat Doukkala (Zinelabidine et al. 2010).

Table 2 Synonym cases found among the analyzed grapevine accessions.

GaSynonyms, duplicates (colorb)

Color sportsc

(colorb)Selected name

3 Asli (W)Khedhiri 2 (W)

Asli

6 Behbahi (W)Hencha 1 (W)

Behbahi

16 Blanc (W)Khalt Bou Chemma (W)

Khalt Bou Chemma

17 Arich Abiadh (W)Dalia (W)Khalt Mdaouar (W)

Bezzoul Khadem (R)Kahli (B)

Khalt Mdaouar

18 Khamri (R) Beldi (W) Khamri19 Arich Djerba (P)

Arich Dressé (P)Medina (P)

Arich Ahmar (R)Siper Abiadh (W)

Medina

20 Balta 2 (W)Balta 3 (W)Beldi Sayeb (W)Bidh Hmem (Baddar) (W)Chaaraoui (W)Farrani (W)Hencha 2 (W)Khedhiri 1 (W)Mahdoui (W)Meski Local (W)Razegui (W)Tounsi (W)

Saoudi (B) Mahdoui

21 Arbi (W)Bidh Hmem (Sfax) (W)Khalt Abiadh (W)Marsaoui (W)Meski Rafraf (W)

Meski Rafraf

22 Mguergueb (W) Mansouri (R) Mguergueb23 Muscat

d’Alexandrie (W)dMuscat d’Alexandrie

28 Sakasli (DB) Hammouri (R) Sakasli29 Sfaxi (W) Hamri (DR) Sfaxi30 Taferielt (B) Amokrane (W) Taferielt32 Ain Bakouch 1

Ain Bakouch 233 Ain Bakouch 3

Ain Bakouch 438 Bellif 4

Bellif 549 Les Dunes de Tabarka 1

Les Dunes de Tabarka 2aGenoype number; also see Supplemental Table 1.bBerry color: B: black, DB: dark blue, DR: dark red, R: red, P: pink, W: white (green/yellow).

cWithin any genotype, the most common color phenotype, in terms of number of names existing, is in the column “Synonyms, duplicates” and the remaining in the column “Color sports” without any implica-tion about which was the original color phenotype and which the somatic variant/s.

dAnd another mislabeled accession as Sultanine.

Table 3 Homonym cases found among the analyzed grapevine accessions.

Ga Name Ga Name17 Arich Abiadh 19 Arich Ahmar4 Balta 1 20 Balta 2/Balta 37 Beldi Local Rafraf 18 Beldi

20 Bidh Hmem (Baddar) 21 Bidh Hmem (Sfax)6 Hencha 1 20 Hencha 2

14 Jerbi (Dgueche) 15 Jerbi (Kerkennah)20 Khedhiri 1 3 Khedhiri 220 Meski Local 21 Meski Rafraf

aGenoype number; also see Supplemental Table 1.

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The genotype of accession Balta 1 matched with that of the Italian Axina de Tres Bias (Giannetto et al. 2010). This variety and Muscat à Petits Grains Blancs have been de-scribed as the parents of Muscat of Alexandria (Cipriani et al. 2010). Microsatellite data obtained in this work for Mus-cat d’Alexandrie and Balta 1 and published data for Muscat à Petits Grains Blancs (Vargas et al. 2009) are also compatible with such a cross. Both Balta 1 and Muscat d’Alexandrie carry chlorotype B, while Muscat à Petits Grains Blancs carries chlorotype D (Vargas et al. 2009), and so Balta 1 (Axina de Tres Bias) would be the female parent of Muscat of Alexandria. Axina de Tres Bias has been described as a synonym of the cultivar Heptakilo in Greece, Bouresla in Tunisia, and Centorotolli in Sicily (Lacombe et al. 2013), consistent with the material exchanges among these regions.

Bezzoul Kalba Bidha—identified as a synonym of Dat-tier de Beyrouth (El Oualkadi et al. 2009, Zinelabidine et al. 2010), Razaki (Riahi et al. 2010), and Afus Ali (VIVC)—is a well-known cultivar, proposed to originate from Lebanon (Galet 2000). The relevance of this cultivar in the Maghreb suggest this region as an alternative hypothesis for its geo-graphical origin. In this context, the varieties Bezzoul Kalba Bidha and Balta 1 have played an important role in the de-velopment of several other varieties. Afus Ali has been a parent in breeding programs for 149 crosses (VIVC), while Balta 1 was proposed as the putative parent of many Tunisian accessions (19 offspring) through spontaneous hybridizations (Zoghlami et al. 2009).

Accession Turki had an identical genotype to Italia, also known as Doña Sofia, Ideal, Pirovano 65, and Muscat d’Italie (Galet 2000). This Turki is a misname, as it is different from the known cultivar Turki, a described synonym of Medina (VIVC), which also matched with other studied accessions (genotype 19). The Reine de Vignes studied here is likely a misname, as it did not match with Koenigin der Weingaerten, which has 57 synonyms (VIVC). El Biodh was genotypically identical to the predominant Spanish cultivar Airén (Galet 2000). Airén was described to be synonymous of El Biod and Zerhouni in Morocco (Zinelabidine et al. 2010), and El Biodh (Tunisia) and El Biod (Morocco) were described as two clones of the same cultivar, as they differed just in one allele at locus VVMD7 after comparing 19 microsatellite loci (Riahi et al. 2010). Accession Chaouch fully matched with Chaouch Blanc proposed to originate in Turkey. Khalt Md-aour was genotypically identical to the Moroccan accessions Bezoul el Aouda and Sidi Taybi (Zinelabidine et al. 2010) and to Bezoul el Khadem (El Oualkadi et al. 2009) and Bezzoul el Khadem Rafraf (Riahi et al. 2010). Finally, the Dalia Bou Ficha was genotypically identical to Planta Fina from Spain, and Sfaxi matched with the table grape Ruby Seedless from the United States (Supplemental Table 2).

Results of genetic identification confirm that most of the 31 nonredundant cultivated genotypes presented here corre-sponded to varieties described in ampelographic literature as autochthonous Tunisian cultivars, including Arbi, Asli, Balta, Behbahi, Beldi, Dalia, Guelb Sardouk, Jerbi, Khalt, Mahdoui, Neb Jmal, and Oasis 46 (Galet 2000, VIVC). Other cultivars

such as Ahmar Bou Ahmar, Bazzoul Kalba Bidha, El Biodh, and Taferielt have been proposed to originate from Tunisia, Algeria, and/or Morocco, indicating that their cultivation is widespread in the Maghreb area (Galet 2000). Cultivars such as Chaouch, Dabouki, Khamri, Medina, Muscat d’Alexandrie, and Sakasli have been described as having different putative origins. Khamri is identified as originating from Tunisia or Azerbaijan and Muscat d’Alexandrie is cultivated worldwide and originated from Northern Africa (Galet 2000, VIVC). No references were found for Mguergueb and Rich Baba Sam.

Origin of Tunisian cultivars. Several genetic parameters were assessed separately for wild and cultivated grapevines. Nonsignificant differences were found for the average values of observed and expected heterozygosity between the culti-vated (0.774 and 0.714, respectively) and the wild (0.685 and 0.710, respectively) groups (Supplemental Table 3). These val-ues are high enough to support that both sample sets maintain high levels of genetic variation. Similar findings have been re-ported (Riahi et al. 2010, Snoussi et al. 2004, Zinelabidine et al. 2010, and Zoghlami et al. 2009). Tests for Hardy-Weinberg equilibrium revealed a deficit of heterozygotes only for wild grapevines at loci ZAG62 (p = 0.0234), VVS2 (p = 0.0063), and VVMD28 (p = 0.0190) at 0.05 level. No significant excess of heterozygotes was detected for wild and cultivated popu-lations at any locus. In agreement with the Hardy-Weinberg results, estimated frequency of null alleles (r) was negative for all the nine loci in the cultivated grapevines and was positive for wild accessions in six loci, although always <0.08. This could be due to the real existence of null alleles or to the reduced number of individuals within each wild population, causing genetic drift.

Furthermore, 18 alleles were detected only in the wild grapevines and another 18 only in the cultivated ones (private alleles, Supplemental Table 1), showing that each group practi-cally behaves as an independent genetic pool, and reflecting the need to maintain the genetic variability of these limited resources. In agreement with the high presence of private al-leles, the probability for differences in allele frequencies over all loci using Fisher’s method was highly significant (p < 0.01) for the comparison between wild and cultivated accessions, whereas results for the distribution of genotypes within the two populations closely correlated to the allele frequency values. The average value for FST = 0.11737 over all loci (p = 0.01) revealed a significant genetic differentiation between wild and cultivated accessions. This value is higher than that given else-where (Riahi et al. 2012): FST = 0.0747 between 95 samples of Maghrebian cultivars and 21 samples of Tunisian wild plants.

Four chloroplast microsatellite loci were analyzed to de-termine the chlorotypes of the 62 nonredundant genotypes (Supplemental Table 1). Chlorotypes were named according to Arroyo-García et al. (2006). Chlorotype A was infrequent in the analyzed cultivated accessions (19%) but predominant in wild accessions (65%). These results are similar to those reported in wild populations from the Iberian Peninsula (Arroyo-García et al. 2006), Morocco (Zinelabidine et al. 2010), and Tunisia (Riahi et al. 2010, Snoussi et al. 2004). Chlorotype C, detected in 19% of wild samples, was the most

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abundant in cultivated accessions (45%). Chlorotype B was only observed in the cultivated accessions, while chlorotype D was minor in both cultivated and wild accessions. Chlorotype C was previously reported to be present at higher frequencies among Near and Middle Eastern table grape cultivars (Arroyo-García et al. 2006) and in Maghrebian cultivars (Laiadi et al. 2009, Riahi et al. 2010, Zinelabidine et al. 2010). Thus, re-sults suggest at least two major origins for cultivars currently grown in Tunisia: one Eastern, related to the Near and Middle East, and characterized by chlorotypes C and D, and another Western, related to the Iberian Peninsula, Central European, and Northern Africa wild populations and characterized by chlorotype A (Arroyo-García et al. 2006). This is consistent with historical data that show a more intensive exchange of cultivars with the East rather than the West.

A principal coordinate analysis was conducted on the 62 nonredundant genotypes of wild and cultivated grapevines to visualize the distribution of genetic variation revealed by the nine nuclear microsatellite loci. The first two principal coor-dinates explained ~23% of the total variation (Figure 1). The PCoA plot reflected the genetic differentiation between wild and cultivated groups. Wild grapevines clustered in the left, while all but one cultivated grapevine (Bezzoul Kalba Bidha) clustered in the right. Bezzoul Kalba Bidha (genotype 5) also carried chlorotype A, supporting a closer genetic relationship with the wild group. PCo2 seems to differentiate two groups of cultivars. The upper group contains all cultivars bearing chlorotype A including Iberian cultivars such as Planta Fina and Airén as well as bred table grape cultivars like Italia. The lower group includes Muscat-related cultivars and those more strongly identified as Tunisian cultivars in different studies.

SSR profiles were also analyzed with the Bayesian ap-proach implemented in NEWHYBRIDS to evaluate the ge-netic constitution of cultivated and wild groups and to detect possible hybridization events. Although the number of mark-ers was low, all independent runs converged and produced identical results. A sharp distinction between the wild and

cultivated groups was confirmed and most samples were assigned to their group of origin (cultivated or wild) with a posterior probability ≥0.75. These numbers suggest that hybridization has been low between wild and domesticated grapevines, which have essentially remained reproductively isolated, in agreement with the results of genetic distances and principal coordinate analyses. Similar situations have been reported with Sardinian (Zecca et al. 2010) and Ibe-rian grapevines (De Andrés et al. 2012, Lopes et al. 2009) and with Northern African cultivars and some wild Tunisian samples (Riahi et al. 2012).

According to PCoA and NEWHYBRIDS, Bezzoul Kalba Bidha (genotype 5) contained a high wild genetic compo-nent. A similar situation was observed for Alvarinho and Merlot cultivars (Lopes et al. 2009). Bezzoul Kalba Bidha is locally considered one of the oldest Tunisian cultivars and is a synonym of the well-known Afus Ali. Given the relation-ship between this cultivar and Tunisian wild grapevines and considering its A chlorotype, the hypothesis that this grape could have arisen from Western wild grapevine populations such as Tunisian should be considered. Because domesticated grapevines are propagated vegetatively by cuttings, Bezzoul Kalba Bidha would have persisted unchanged over centuries, as with similar reported situations (Grassi et al. 2003, Riahi et al. 2010). This accession originated from Sfax, a Tunisian coastal city, where ancient wild grapevines have been found.

An interesting situation was also observed for Ahmar Bou Ahmar, Asli, and E Rwayssiya 3 (Supplemental Figure 1), which were only partially assigned to their populations of origin and were distributed among different hybrid frequency classes. These grapevines were located centrally in the PCoA (Figure 1, genotypes 1, 3, and 43, respectively). The wild ge-netic group could have been occasionally introgressed with cultivated forms, giving rise to complex introgression-derived hybrids (Zohary and Hopf 2000). Similar situations have been observed in other studies in the Iberian Peninsula (De An-drés et al. 2012) and in the Maghreb (Riahi et al. 2010). The presence of white berries in two wild plants also could be due to introgression from cultivated forms. Given the vegeta-tive propagation of cultivars, they would only be separated from wild forms by a limited number of generations (Arroyo-García et al. 2006, De Andrés et al. 2012, Grassi et al. 2003).

The genetic differentiation observed between wild and cultivated samples (FST = 0.11737) and the high number of private alleles within the cultivated grapevines also reflect that most cultivated accessions do not derive directly from the local wild populations but could correspond to materials introduced from different locations during historical times or derived from spontaneous hybridizations among them.

ConclusionThe results provide greater clarity on synonyms, duplicates,

misnaming, and homonyms. Tunisian accessions are often found in common with other countries in the area such as Morocco and Algeria. A characteristic feature of the analyzed accessions is the higher representation of table grape cultivars concomitant with a higher frequency of chlorotype C followed

Figure 1 Two-dimensional projection of the principal coordinate analysis of 62 grapevine genotypes based on nine SSR markers along the first two principal axes. Wild and cultivated populations represented respectively by triangles and circles. Chlorotypes are indicated by color (A: dark grey, B: white, C: light grey, and D: black). Numbers correspond to genotype numbers as defined in Table 1.

544 – Ghaffari et al.

Am. J. Enol. Vitic. 64:4 (2013)

by chlorotype D. Wild samples were mostly represented by chlorotype A. Although the number of nuclear microsatellites used is limited, results suggested both Eastern and Western origins for grapevine cultivars grown in Tunisia. The contribu-tion of wild grapevine to the regional cultivated pool is low, although it cannot be excluded in some particular cases. Fur-thermore, data analyses demonstrated a large genetic diversity in both wild and cultivated groups.

Literature CitedAnderson, E.C., and E.A. Thompson. 2002. A model-based method for

identifying species hybrids using multilocus genetic data. Genetics 160:1217-1229.

Arroyo-Garcia, R., et al. 2006. Multiple origins of cultivated grapevine (Vitis vinifera L. ssp. sativa) based on chloroplast DNA polymor-phisms. Mol. Ecol. 15:3707-3714.

Cipriani, G., et al. 2010. The SSR-based molecular profile of 1005 grapevine (Vitis vinifera L.) accessions uncovers new synonymy and parentages, and reveals a large admixture amongst varieties of different geographic origin. Theor. Appl. Genet. 121:1569-1585.

De Andrés, M.T., et al. 2012. Genetic diversity of wild grapevine populations in Spain and their genetic relationships with cultivated grapevines. Mol. Ecol. 21:800-816.

El Oualkadi, A., M. Ater, Z. Messaoudi, V. Laucou, J.M. Boursiquot, T. Lacombe, and P. This. 2009. Molecular characterization of Moroccan grapevine germplasm using SSR markers for the establishment of a reference collection. J. Int. Sci. Vigne Vin 43:135-148.

Excoffier, L., G. Laval, and S. Schneider. 2005. Arlequin 3.11: An integrated software package for population genetics data analysis. Evol. Bioinformatics Online 1:47-50.

Galet, P. 2000. Dictionnaire encyclopédique des cépages. Hachette, Paris.

Giannetto, S., R. Caruana, P. La Notte, A. Costacurta, and M. Crespan. 2010. A survey of Maltese grapevine germplasm using SSR markers. Am. J. Enol. Vitic. 61:419-424.

Grassi, F., M. Labra, S. Imazio, A. Spada, S. Sgorbati, A. Scienza, and F. Sala. 2003. Evidence of a secondary grapevine domestication centre detected by SSR analysis. Theor. Appl. Genet. 107:1315-1320.

Harbi Ben Slimane, M. 1999. Etude de la variabilité génétique des vignes autochtones cultivées et spontanées de Tunisie. Thesis, Faculty of Sciences, University of Tunis, El Manar.

Ibáñez, J., A.M. Vargas, M. Palancar, J. Borrego, and M.T. de Andrés. 2009a. Genetic relationships among table-grape varieties. Am. J. Enol. Vitic. 60:35-42.

Ibáñez, J., M. Vélez, M.T. de Andrés, and J. Borrego. 2009b. Molecu-lar markers for establishing distinctness in vegetatively propagated crops: A case study in grapevine. Theor. Appl. Genet. 119:1213-1222.

Lacombe, T., J.M. Boursiquot, V. Laucou, M. Di Vecchi-Staraz, J.P. Péros, and P. This. 2013. Large-scale parentage analysis in an ex-tended set of grapevine cultivars (Vitis vinifera L.). Theor. Appl. Genet. 126:401-414.

Laiadi, Z., M.M. Bentchikou, G. Bravo, F. Cabello, and J.M. Martínez Zapater. 2009. Molecular identification and genetic relationships of Algerian grapevine cultivars maintained at the germplasm collection of Skikda (Algeria). Vitis 48:25-32.

Lopes, M.S., D. Mendonça, M. Rodrigues dos Santos, J.E. Eiras-Dias, and A. da Cămara Machado. 2009. New insights on the genetic basis of Portuguese grapevine and on grapevine domestication. Genome 52:790-800.

Minch, E. 1997. Microsat Version 1.5b. Stanford University Medical Center, Stanford, CA.

Negrul, A.M. 1938. Evolution of cultivated forms of grapes. Academy of Sciences of the USSR 18:585-588.

Peakall, R., and P.E. Smouse. 2006. GenAlEx 6: Genetic analysis in Excel. Population genetic software for teaching and research. Mol. Ecol. Notes 6:288-295.

Riahi, L., V. Laucou, L. Le Cunff, N. Zoghlami, J.M. Boursiquot, T. Lacombe, K. El-Heit, A. Mliki, and P. This. 2010. Genetic structure and differentiation among grapevines (Vitis vinifera) accessions from Maghreb region. Genet. Resour. Crop Evol. 57:255-272.

Riahi, L., V. Laucou, L. Le Cunff, N. Zoghlami, J.M. Boursiquot, T. Lacombe, K. El-Heit, A. Mliki, and P. This. 2012. Highly polymor-phic nSSR markers: A useful tool to assess origin of North African cultivars and to provide additional proofs of secondary grapevine domestication events. Sci. Hortic. 141:53-60.

Rousset, F. 2008. genepop’007: A complete re-implementation of the genepop software for Windows and Linux. Mol. Ecol. Resour. 8:103-106.

Snoussi, H., M. Harbi Ben Slimane, L. Ruiz-García, J.M. Martínez-Zapater, and R. Arroyo-García. 2004. Genetic relationship among cultivated and wild grapevine accessions from Tunisia. Genome 47:1211-1219.

Vargas, A.M., M.T. de Andrés, J. Borrego, and J. Ibáñez. 2009. Pedi-grees of fifty table-grape cultivars. Am. J. Enol. Vitic. 60: 525-532.

Vélez, M.D., and J. Ibáñez. 2012. Assessment of the uniformity and stability of grapevine cultivars using a set of microsatellite markers. Euphytica 186:419-432.

Wagner, H.W., and K.M. Sefc. 1999. IDENTITY 4.0. Centre for Ap-plied Genetics, University of Agricultural Sciences, Vienna, Austria.

Zecca, G., F. De Mattia, G. Lovicu, M. Labra, F. Sala, and F. Grassi. 2010. Wild grapevine: silvestris, hybrids or cultivars that escaped from vineyards? Molecular evidence in Sardinia. Plant Biol. 12:558-562.

Zinelabidine, L.H., A. Haddioui, G. Bravo, R. Arroyo-García, and J.M. Martínez Zapater. 2010. Genetic origins of cultivated and wild grapevines from Morocco. Am. J. Enol. Vitic. 61:83-90.

Zoghlami, N., L. Riahi, V. Laucou, T. Lacombe, A. Mliki, A. Ghorbel, and P. This. 2009. Origin and genetic diversity of Tunisian grapes as revealed by microsatellite markers. Sci. Hort. 120:479-486.

Zohary, D., and M. Hopf. 2000. Domestication of Plants in the Old World: The Origin and Spread of Cultivated Plants in West Asia, Europe and Nile Valley. 3d ed. Oxford University Press, New York.