Genetic clustering and parentage analysis of Western Balkan grapevines (Vitis vinifera L.)

Vitis 54 (Special Issue), 67–72 (2015) Genetic clustering and parentage analysis of Western Balkan grapevines (Vitis vinifera L.) N. ŠTAJNER1), L. TOMIĆ), V. PROGAR1), T. POKORN1), T. LACOMBE2), V. LAUCOU2),J. M. BOURSIQUOT3), B. JAVORNIK1) and R. BACILIERI2) 1) University of Ljubljana, Biotechnical Faculty, Ljubljana, Slovenia 2) UMR AGAP, Equipe Diversité Adaptation et Amélioration de la Vigne, INRA, Montpellier, France 3) UMR AGAP, Equipe Diversité Adaptation et Amélioration de la Vigne, Montpellier SupAgro, Montpellier, France Correspondence to: Dr. R. BACILIERI, UMR AGAP, Equipe Diversité Adaptation et Amélioration de la Vigne, INRA, 2, place Pierre Viala, 34060 Montpellier Cedex 02, France. E-mail: [email protected] Summary A total of 90 grapevine samples collected in five countries of the Western Balkan region were evaluated for trueness-to-type and kinship relations based on comparative analysis with 1,130 grapevine genotypes held at the INRA "Domaine de Vassal" French Grape Germplasm Repository, using 14 microsatellite markers. In the context of the comparative analysis, twentyfour synonyms/counterparts and the putative parents for twelve Balkan accessions were identified. We discovered five pairs of homonyms, subsequently confirming the identity or parentage of three of them. Some of the examined accessions were identified either on the basis of the genotypes found in the literature, or through parentage relationships revealed in this study. For the remaining fifty accessions we were unable to establish either their pedigree or to identify them on the basis of SSR profiles available elsewhere. Finally, the Balkan genotypes that were not well classified by synonymy or parentage analysis were further studied with a Principal Coordinate Analysis to reveal genetic clustering within larger datasets of genotypes. The graphical display of the individual and group distances showed that about forty accessions (85 %) are structured within a group of Balkan and Eastern Europe genotypes and only a minor proportion resulted in admixed population assignment. K e y w o r d s : microsatellites; identity; synonyms; principal coordinate analysis. Introduction In recent decades, progress in genome sequencing has enabled the reporting of hundreds of polymorphic neutral markers, such as microsatellites, which have proven to be powerful tools for parental and kinship analysis, including with grapevines. The largest grapevine parentage analysis to date was performed on a dataset of 2,344 unique genotypes (LAUCOU et al. 2011) (held in the INRA "Domaine de Vassal" French Grape Germplasm Repository). The full parentage of 828 accessions was identified using 20 microsatellite loci (LACOMBE et al. 2013). The second largest study explored the parentage of 1,005 grapevines and the analysis of kinship uncovered 74 complete pedigrees, with both parents identified (CIPRIANI et al. 2010). However, other smaller parentage studies have been carried out in several countries (e.g. MENA et al. 2014). In our study, microsatellites were used to investigate the parentage and origin of Balkan grapevines collected in five Western Balkan countries - Slovenia (SVN), Serbia (SRB), Bosnia and Hercegovina (BIH), Macedonia (MKD) and Montenegro (MNE). To integrate and evaluate the SSR data of Balkan genotypes in a larger context, the dataset from a large scale parentage study (LACOMBE et al. 2013) was chosen for comparison. The dataset used in this work thus encompasses 1,130 genotypes from the INRA Domaine de Vassal grapevine repository (http://www1.montpellier.inra.fr/vassal; LACOMBE et al. 2013) and 90 genotypes from the Balkan region (http://vitis.atcglabs.com). Panels of synonyms/ counterparts and putative parentage-offspring trios were obtained, which are presented and related to some existing reports. The standardization and integration procedure of the two sets of data is described, together with issues related to the linking procedure. The discovered genetic clustering in comparison to the larger reference dataset, are discussed. Material and Methods Plant material and DNA analysis: The first step of this study was the identity and parentage analysis of grapevine genotypes from two recently published studies (LACOMBE et al. 2013, ŠTAJNER et al. 2013), using 7 microsatellite loci (VVMD5, VVMD7, VVMD25, VVMD27, VVMD28, VVMD32, VVS2) that were common to the two studies. The obtained results suggested that 90 samples from the Balkan region (Tab. 1) are interesting for further analysis with an additional set of loci: VVMD21, VVMD24 (BOWERS et al. 1999a), VVIp31, VVIv67, VVIp60, VVIn16, VVIb01 and VVIq52 (MERDINOGLU et al. 2005). Standardization of allele length and panel bins between the two datasets was carried out using common reference samples 'Cabernet-Sauvignon', 'Chardonnay', 'Merlot', 'Pinot Noir', 'Sultanine', and 'Touriga National'. The procedure of plant material sampling and DNA isolation was as described in 68 ŠTAJNER et al. ŠTAJNER et al. (2013) as well as PCR amplifications and genotyping of SSR loci. Data analysis: Amplified fragments were analysed and sized with GeneMapper software version 4.0 (Applied Biosystems, Carlsbad, California). All the pherograms were manually checked and the relative sizes of the amplified products were determined. Relative allele sizes were first standardized using six reference cultivars that were common to the two data sets; this procedure enabled us to jointly examine data from both sets of samples. To carry out this task, we wrote several custom R scripts for improved management of input/output data, dealing specifically with the normalization of SSR data from two different sets and with identity analysis. The scripts, with instructions for use are freely available from https://github. com/vp-uni-lj/ssr-link.git. The first step consisted in the identification of matching genotypes, i.e. counterparts between the two datasets. The synonymous genotypes obtained by the R scripts were also manually inspected. Data were subsequently analysed with Cervus 3.0 software (MARSHALL et al. 1998; KALINOWSKI et al. 2007). Parentage analysis was performed in two steps; 1) we ran a simulaTable 1 List of the 90 grapevine accessions from Western Balkans and their passport data Accession Name Country of sampling Prime Name Variety No. VIVC Accession Name Country of sampling Prime Name Variety No. VIVC Babić BIH Babic 844 Muskat Ruža SRB Muscat Rouge de Madere 8249 Bačka SRB - 21272 Muštoš Feher SRB Mustoasa de Maderat 8311 Bela Dinka SRB - 16848 Neznana Bela SVN - - Bela Zgodnja SVN Csaba Gyoengye 9166 Ohridsko Belo MKD Ohridsko Blanc 21403 Beli Medenac SRB - - Plavac Mali BIH Plavac Mali 9549 Belo zimsko SRB Opsimos Edessis 8789 Plavka BIH Plavina Crna 9557 Belovina MKD - 42060 Plovdina Crna SRB - - Bena BIH - 1129 Poljšakica SVN 17712 Blatina BIH Blatina 1454 Prokupac BIH Prokupac 9734 Crn Valand. Drenok MKD Parmak Cerven 8945 Prokupac SRB - - Čauš MKD Chaush Chernyi 7307 Prošip BIH 24630 Čauš Bel MKD Chaouch Blanc 10196 Prošip Bijela BIH - - Dobrogostina BIH Trbjan 12619 Radovača BIH 24598 Dolga Pentlja SVN Dolga Petla 17675 Radovača MNE Afus Ali 122 Dolgi Grozdi SVN Pelena, Vitovska Grganja 24459 Razaklija BIH - - Drenak BIH - - Rebula SVN Ribolla Gialla 10054 Drenak Beli SRB - 16564 Rebula Portalis SVN - - Drenak Crni SRB Hora 5423 Refosco SVN Refosco dal Peduncolo Rosso 9987 Drenjak BIH Ruderusa 10327 Refošk SVN Refosk Terrano 12374 Elezovka BIH - - Rezaklija BIH 24131 Furmint SRB Knipperle 6312 Ružica SRB Koevidinka 13727 Gavran SRB Gek Crni 5067 Ružica Mirisava SRB - - Gnet Kras SVN - 24599 Ružica V MNE Pamid 8899 Gnjet SVN Piccola Nera 9235 Ružica VI MNE - - Godominka SRB - 16003 Sipa SVN Osipka 17705 Grk BIH - - Slankamenka Crvena SRB Pamid 8899 Gročanka SRB 5069 Smederevka BIH Dimyat 5716 Harsleveli SRB Harslevelue 5314 Sremska Zelenika SRB 15934 Kadarka SRB - - Stanušina MKD Stanusina Crna 11994 Kadarka Bela SRB Kadarka Feher 5899 Stara Žilavka BIH Trbjan 12619 Kadarun BIH Kadarun 5900 Stari Rizling Montengro - - Kavčina SRB Koelner Blau 6344 Surac Plavi BIH - - Kolana SVN - - Šipon SVN Furmint 4292 Končanka MKD - 22962 Šljiva BIH Sljiva Hercegovacka 23206 Kratošija MNE Primitivo 9703 Tamjanika Crna SRB Tamjanika Crna 8057 Kreaca SRB Kreaca 6501 Trbljan Beli SRB Trbjan 12619 Krivača Bijela BIH Krivalja Bijela 10050 Trnjak BIH - - Krivaja SRB - - Trnjak Krupni BIH Ruderusa 10327 Krkošija BIH 16850 Urban Crveni SRB Urban 12781 Krkošija Šupljica SRB Kujundzusa Bela 6545 Vranac BIH Vranac 13179 Kujundžuša BIH Kujundzusa Bela 6545 Žametovka SVN Kavcina Crna 6047 Malvazija BIH - - Žilavka BIH Zilavka 13446 Manastirsko Belo MKD 24597 Žižak MNE - - Marburger SVN Neuburger 8501 Žlozder BIH Medna 7584 Menigovka BIH Italia 5582 Žunić SRB 15923 Genetic clustering and parentage analysis of Western Balkan grapevines 69 tion of parentage analysis to estimate the resolving power of a series of microsatellite loci and 2) parentage analysis was performed to test candidate parents against offspring and, for each offspring tested, to assign the most-likely candidate parent with a pre-determined level of confidence, or left it unassigned. LOD (logarithm (base 10) of odds) scores were assigned to each possible parent and parent pair. The genetic clustering of Balkan samples was obtained with a Principal Component Analysis (PCA) using the package adegenet implemented in R (JOMBART 2008). The internal structure of the data and quantitative variables were calculated for predetermined groups of Vassal samples based on STRUCTURE clustering as reported by BACILIERI et al. (2013). Results I d e n t i t y a n a l y s i s : Based on the identity analysis, twenty-four Balkan genotypes (Tab. 2) found a counterpart in the Vassal dataset. Two pairs of near synonyms differing in one allele only were additionally identified: 'Beli Medenac' and 'Lisztesfeher' #2808 differed by 2 bp at locus VVIp60 and 'Gročanka' and 'Grocanka' #3637 by 4 bp at locus VVIb01. In addition, the counterparts were found among published SSR data for seven accessions: 'Kadarka Bela' = 'Kadarka Byala' (DZHAMBAZOVA et al. 2009), 'Kreaca' = 'Banati Rizling' (GALBACS et al. 2009), 'Mustos Feher' = 'Mustos' (GALBACS et al. 2009), 'Tamjanika Crna' = 'Muskat Ruza Porecki' (MALETIĆ et al. 1999), 'Žametovka' = 'Schwarzgrobe' (SEFC et al. 2000), 'Zimsko Belo' = 'Karatsoba' (SEFC et al. 2000) = 'Karatsova Naousis' (Greek Vitis Database) = 'Opsimos Lefko' (Greek Vitis Database) and 'Prošip Bijela' = 'Volovnik' (ŠTAJNER et al. 2008) = 'Vela Pergolla' (MALETIĆ et al. 1999; SEFC et al. 2001). P a r e n t a g e a n a l y s i s : Parentage analysis based on 14 microsatellite markers resulted in a panel of potentially interesting parent/offspring trios including Balkan genotypes that were shown to be in either parent or offspring relationship with genotypes from the Vassal collection (Tab. 3). Principal Coordinate Analysis: Finally, the Balkan genotypes that were not uncovered by the meaning of synonymy were further analysed with PCA (Figure). The PCA analysis reflects the relationships among groups from Vassal collection based on the STRUCTURE clustering (A3 - Western and Central Europe genotypes; B3 - Farand Middle-East genotypes and C3 - Balkan and East Europe genotypes) and their distance to the Balkan genotypes referred as D3 group. The PCA grouping shows that D3 Balkan samples are positioned close to the C3 samples that represent Balkan and East Europe genotypes in the study of BACILIERI et al. (2013). Discussion Most of the synonyms identified in this study (Tab. 2) were not reported in the relevant papers published recently (BEŠLIĆ et al. 2012, ZDUNIĆ et al. 2013, MARAŠ et al. 2014, SCHNEIDER et al. 2014). Among twenty-four associations found within two studies, the highest part of the Vassal counterpart are of Balkans origin (67 %) and the rest, except two, are of Italian origin for which BACILIERI et al. (2013) reported high level of admixture as regards to genetic composition of geographic origin. The parentage analysis resulted in identification of parentages for twelve authentic Balkan genotypes having a parent either from our set of samples or from the Vassal collection and to our knowledge they have not yet been described in the literature. All varieties from Vassal have been assigned a geographic origin presented in Tab. 3, where we can see that even when one parent is identified in Vassal, still, most of the time it is of Balkans origin. The only notable exceptions are two parental accessions, one of which is 'Terrano' that is considered of Italian origin, but is widely grown also in Slovenia and Croatia and the second is 'Malvasia del Chianti' being part of a Malvasia group derived from Greece. Another two parental accessions of non-Balkan origin are 'Gouais blanc' = 'Heunisch weiss', one of the oldest varieties in the world, used in several crosses and 'Rosenmuskat' a Muscat used to introduce the muscat flavour. Three out of twelve offspring described in parentage analysis are table grapevines ('Gročanka', 'Krivaja', and 'Šljiva') and their candidate parents are also table grapevines. The same findings were reported by LACOMBE et al. (2013), who stated that comparison of the fruit use of parents for a given progeny showed a majority of crosses usTable 2 List of identical grapevine genotypes based on matching allele sizes for analysed loci Balkan sample INRA sample (LACOMBE et al. 2013) Bela Zgodnja (SVN) Perle de Csaba (#1069) (HUN) Čauš Bel (MKD) Chaouch rose (#1674) (TUR) Drenak (BIH) Rosa menna di vacca (#1662) (ROU) Drenak Beli (SRB) Coarna alba (#749) (ROU) Drenak Crni (SRB) Darkaia noir (#728) (MAR) Elezovka (BIH) Chaouch blanc (#1673) (TUR) Furmint (SRB) Knipperle (#283) (FRA) Gnjet (SVN) Piccola nera (#2387) (ITA) Harslevelu (SRB) Harslevelu (#1609) (HUN) Kratosija (MNE, MKD) Primitivo = Zinfandel (#1277) (ITA) Marburger (SVN) Neuburger (#2172) (AUT) Menigovka (BIH) Italia = Pirovano 65 (#926) (ITA) Muskat Ruza (SRB) Muscat rouge de Madère = Moscato violetto (#576) (ITA) Plavka (BIH) Plavina crna (#1843) (HRV) Prokupac (BIH) Prokupac (#1630) (SCG) Radovaca (MNE) Dattier de Beyrouth = Afuz Ali (#634) (TUR) Refosk (SVN) Terrano (#1293) (ITA) Rezaklija’ (BIH) Razachie rosie (#1887) (ROU) Ruzica (SRB) Kovidinka (#1578) (YUG) Šipon (SVN) Furmint (#25) (HUN) Smederevka (SRB) Dimiat (#1666) (BGR) Srem Zelenika (SRB) Szeremi zöld (#1623) (HUN) Trbljan Beli (SRB) Mostosa = Empibotte bianco (#2054) (ITA) Žilavka (BIH) Zilavka (#1637) (BIH) 70 ŠTAJNER et al. ing parents from the same geographical area and with the same grape use. The main Balkan genitors identified in our analysis are 'Heptakilo' (#743), a traditional table cultivar from Greece and 'Rosa menna di vacca' (#1662), a traditional table cultivar from Romania. These findings are in agreement with the historical view; grapevines arrived in the Balkan Peninsula from Central Asia via the Greek islands and the Romans continued to spread the cultivation of grapevine on the Adriatic islands and on the mainland. During the period of the Ottoman Empire, the production of table grapes increased and was more widespread over the region (SAVIĆ 2003). Several interesting hypotheses have arisen concerning some putative trios; introduced cultivars have often been Table 3 List of full parentages detected for Balkan grapevine accessions. The symbol # refers to the accession number in the Vassal collection (http://bioweb.ensam.inra.fr/collections_vigne/) Offspring First candidate Second candidate Trio loci compared Trio loci mismatching Trio LOD score Dobrogostina (BIH) = Stara Žilavka (BIH) Krkošija (BIH) Rosenmuskat (#3550) (DEU) 14 0 20 Godominka (SRB) Muscat Ottonel (#280) (FRA) Smederevka (SRB) = Dimiat (#1666) (BGR) 14 1 23 Gročanka (SRB) Bela Zgodnja (SVN) = Perle de Csaba (#1069) (HUN) Radovaca (MNE) = Dattier de Beyrouth = Afuz Ali (#634) (TUR) 15 0 30 Krivaja (SRB) Drenak (BIH) = Rosa menna di vacca (#1662) (ROU) Rezaklija (BIH) = Razachie rosie (#1887) (ROU) 14 0 23 Manastirsko Belo (MKD) Drenak (BIH) = Rosa menna di vacca (#1662) (ROU) Heptakilo (#743) (GRC) 14 0 31 Polšakica (SVN) Malvasia del Chianti (#1352) (ITA) Prošip Bijela (BIH) 14 1 27 Prokupac (SRB) Prokupac (BIH) = Prokupac (#1630) (SRB) Refošk (SVN) =’Terrano’ (#1293) (ITA) 14 0 32 Šljiva (BIH) Drenak Beli (SRB) = Coarna alba (#749) (ROU) Heptakilo (#743) (GRC) 14 0 27 Žilavka (BIH) Albaimputotato (#44) (ROU) Dobrogostina (BIH) 14 0 21 Bagrina (SRB) Beli Medenac (SRB) Braghina=Dinkavoros (#1670) (HUN) 13 0 17 Kreaca (SRB) Drenak Beli (SRB) = Coarna alba (#749) (ROU) Plavac Mali (#3144) (HRV) 14 1 14 Prošip (BIH) Drenak Beli (SRB) = Coarna alba (#749) (ROU) Gouaisblanc = Heunisch weiss (#211) (FRA) 14 0 13 Figure: Principal component analysis on joint SSR data A) for non-identified Balkan samples (D3 group) and samples from Vassal collection subdivided in 3 structure groups as in BACILIERI et al. (2013): A3S, Western and Central Europe genotypes, B3S, Farand Middle-East genotypes, and C3S, Balkan and East Europe genotypes) and B) for samples of D3 group graphically displaying their individual distances shown on the same axes as in Figure A; it presents the enlarged picture of Figure A, including names of accessions. Genetic clustering and parentage analysis of Western Balkan grapevines 71 given local names and have eventually been considered native old cultivars. For example 'Žilavka' was considered to be a very old Bosnia and Herzegovina authentic cultivar. The parentage analysis by LACOMBE et al. (2013) showed one of the progenitors of 'Žilavka' to be 'Furmint' (#25) or 'Alba imputotato' (#44) or 'Goher' (#2244). MALETIĆ et al. (1999) reported that DNA profiling has suggested a genetic relationship between 'Žilavka' and 'Teran Bijeli' ('Prosecco') grown in Istria. Our results suggest that 'Žilavka' might be the offspring of an old female Romanian genitor 'Alba imputotato' (#44) and another BIH accession 'Dobrogostina' = 'Stara Žilavka' (Tab. 3). There are no formal records on breeding and selection of 'Žilavka', despite it being a very famous grapevine, particularly in BIH, and having been grown for more than 600 years in the region of Herzegovina. The Bosnian King Tvrtko was mentioned as drinking 'Žilavka' wine in the 14th century (VUKSANOVIĆ and KOVAČINA 1984). Wine made from 'Žilavka' is economically important and very popular in BIH and also in some adjacent regions of Croatia, Macedonia, Montenegro and Serbia, where it is also grown (TOMIĆ et al. 2012). In the field of grapevine studies, there is still an ongoing debate about the origin of the synonymic genotypes: Californian 'Zinfandel', Croatian 'Crljenak Kaštelanski', Montenegrian 'Kratošija' and Italian 'Primitivo' (MALETIĆ et al. 2004; CALÒ et al. 2008; LACOMBE et al. 2007; CIPRIANI et al. 2010). Some data suggest a possible first-degree relationship between the cultivars 'Kratošija' and 'Vranac' (MALETIĆ et al. 2004, CALÒ et al. 2008) and a parent-offspring relationship of 'Kratošija' with 'Plavina' and 'Vranac' has been proposed (MALETIĆ et al. 2004; CALÒ et al. 2008). Others have suggested that 'Plavina' (= 'Plavina crna') could be an offspring of 'Kratošija' and 'Verdeca', a white cultivar grown in southern Italy (LACOMBE et al. 2007, CIPRIANI et al. 2010, LACOMBE et al. 2013). In our analysis, two accessions of 'Kratošija', originating from Montenegro and Macedonia, resulted in identical allelic profiles and were therefore confirmed as synonyms and, on the basis of comparison to genotypes of the Vassal collection, they were also confirmed as synonyms of the cultivar 'Primitivo' = 'Zinfandel' (#1277). In our parentage analysis, 'Vranac' and 'Plavina crna' (#1843) were one of the most likely candidate parent pairs for 'Kratošija' (results not shown) but, in a comparison to the trio proposed by LACOMBE et al. (2013), by which 'Plavina crna' is an offspring of 'Kratošija' (= 'Primitivo' = 'Zinfandel') and 'Verdeca' (= 'Lagorthi'), the latter resulted in a higher LOD score value than the former, with assigned LOD scores of 22.5 and 15.2, respectively. 'Kratošija' is an old Montenegrin grape accession that, according to the literature, existed in these regions before 'Vranac', although 'Vranac' today predominates in Montenegro and is often cultivated together with 'Kratošija' (ULIĆEVIĆ 1966, PEJOVIĆ 1988, MARAŠ et al. 2003, 2004, 2014). MARAŠ et al. (2014) evidenced that ‘Kratošija’ with all its biotypes has been grown in Montenegro for centuries and suggested that this region be considered a likely region for the origin and spreading centre of 'Kratošija'. According to STRUCTURE clustering reported by BACILIERI et al. (2013) cultivars 'Kratošija' and 'Vranac' were considered as admixed, with 61 % and 72 % of its genome belonged to S-3.3 cluster (Balkan and Eastern Europe group), respectively. During the first inventory of Balkan samples, we found that 'Prokupac' obtained from the Sremski Karlovci (Serbia) collection vineyard (where this cultivar is also bred) resulted in a distinct allelic profile as 'Prokupac' from Davis collection, (USA), but they shared 73 % of alleles analysed at 22 loci (ŠTAJNER et al. 2013). The present analysis similarly confirmed that ‘Prokupac’ from Serbia is different from 'Prokupac' (#1630) from the Vassal collection. In contrast, another 'Prokupac' from BIH resulted in a genotype identical to Prokupac from Davis collection and Prokupac from Vassal collection. The parentage analysis revealed that 'Prokupac' from the Sremski Karlovci collection vineyard might be a progeny of "true-totype" 'Prokupac' and 'Terrano', showing high LOD scores (31.6) and no allele mismatch. A possible explanation for this misnomer is that progenies of a particular parent were sometimes treated the same as the parent cultivar due to misleading documentation of events or possible mislabelling. The PCA analysis shows that Balkan samples (D3, Figure A) overlapped with the genotypes from Balkan and Eastern Europe (C3S group). Hence, the genotypes graphically displayed inside the D3 ellipse might be characterized as typically autochthonous Balkan accessions (Figure B). On the other hand, the accessions that are out of ellipse and are classified between two different groups, e.g. 'Blatina' and 'Prosip Bijela' probably are recent crosses between one table and one Western genotype, with one parent in the A3 and the other in the B3 group. Few of the analysed Balkan accessions are shown to be near to either A3 group ('Bena', 'Sipa', 'Refosco') or B3 group ('Manastirsko Belo', 'Krivača Bijela', Figure B), which reflects their admixed origin and corresponds to various breeding activities in the Western Balkan region. However, these non-Balkan accessions represent a minor proportion of the dataset, while about forty (85 %) analysed cultivars are considered true autochthonous Balkan germplasm. Conclusions Parentage analysis and PCA showed to be very valuable tools for identification of relationships. Many of the parentages revealed in this study were previously not known and are of considerable historical interest. The PCA showed that 44 accessions (85 %) are structured within a group of Balkan and Eastern genotypes and only a minor proportion resulted in admixed population assignment (Figure B). However, the identity of approximately forty Balkan accessions remains only partially explained so far. Some of these are presumably old traditional grapevines, with a putative Balkan origin, and are therefore worth exploring for their origin and identity. Acknowledgements The research was funded by the European Commission’s Joint Research Project of the Joint Call of the SEE-ERA.NET 72 ŠTAJNER et al. PLUS, grant no. ERA 155/01 and by the Slovenian Research Agency, grant no. P4-0077. The study was conceived within the framework of FA COST Action FA1003 "East-West Collaboration for Grapevine Diversity Exploration and Mobilization of Adaptive Traits for Breeding". References BACILIERI, R.; LACOMBE, T.; LE CUNFF, L.; DI VECCHI-STARAZ, M.; LAUCOU, V.; GENNA, B.; PÉROS, J. P.; THIS, P.; BOURSIQUOT, J. M.; 2013: Genetic structure in cultivated grapevines is linked to geography and human selection. BMC Plant Biol. 13, 25. BEŠLIĆ, Z.; TODIĆ, S.; KORAĆ, N.; LORENZI, S.; EMANUELLI, F.; GRANDO, M. S.; 2012: Genetic characterization and relationships of traditional grape cultivars from Serbia. 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