Identity and genetic relatedness of Bosnia and Herzegovina grapevine germplasm

Scientia Horticulturae 143 (2012) 122–126

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Identity and genetic relatedness of Bosnia and Herzegovina grapevine germplasm b ˇ ´ Lidija Tomic´ a , Nataˇsa Stajner , Tatjana Jovanovic-Cvetkovi c´ a , Miljan Cvetkovic´ a , Branka Javornik b,∗ a b

University of Banjaluka, Faculty of Agriculture, Bulevar vojvode Petra Bojovi´ca 1A, 78 000 Banjaluka, Bosnia and Herzegovina University of Ljubljana, Biotechnical Faculty, Jamnikarjeva 101, 1000 Ljubljana, Slovenia

a r t i c l e

i n f o

Article history: Received 1 March 2012 Received in revised form 17 May 2012 Accepted 25 May 2012 Keywords: Identity Microsatellite Relatedness Grapevine

a b s t r a c t A set of Bosnia and Herzegovina grapevine cultivars was analyzed by microsatellite markers in order to assess true cultivar identity, genetic relationships and to detect the level of genetic diversity. Survey expeditions resulted in the collection of 51 samples from 8 locations. By amplifying 22 SSR loci, 25 unique genotypes were detected, revealing new synonyms and homonyms. The number of amplified alleles was 172 and the average allele number was 7.82. The average polymorphism of analyzed loci was 71.1% and the most informative loci were VVMD28 (0.867), VChr8b (0.848), ZAG79 (0.830) and VVMD5 (0.806). Parent-offspring relationship analysis resulted in one trio elucidating the parentage of the cultivar Zˇ upljanka. A microsatellite dendrogram was constructed using the unweighted pair group method, in which genotypes clustered into two groups with subgroups, showing an overall genetic similarity of 36.8%. Assessment of true cultivar identity, identification of synonym cultivars, i.e., duplicates, will be used for establishing a grapevine germplasm collection with maximum genetic variability and minimum number of plants. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Bosnia and Herzegovina (BIH) has two wine regions in which grapes and wines are produced, North Bosnia and Herzegovina. An official estimate by the International Organization of Vine and Wine (OIV) is that there were around 4000 ha of vineyards in BIH in the period between 2000 and 2004. Eighty percent of total vineyards are under grapevine cultivars and twenty percent produce table grapes. Although many old varieties have been abandoned in favour of widespread varieties more favoured for the present wine market (Cipriani et al., 2010), wine production in Herzegovina is based on two local cultivars: Zˇ ilavka (white wine) and Blatina (red wine). Zˇ ilavka has been grown in the region of Herzegovina for more than 600 years, being first mentioned in the 14th century (Vuksanovic´ and Kovaˇcina, 1984). In the 19th century, due to the grape quality and, particularly, to its resistance to bunch rot (Botrytis cinerea Pers.), the Austro-Hungarians exploited this cultivar for the pro´ duction of a special dessert wine of the Malaga type (Mijatovic, 1988). In addition to its historic importance and long tradition, Zˇ ilavka is of economic value since it accounts for the majority of total wine production in BIH. Blatina is used for the production of red wines but since it has functional female flowers, its cultivation is limited by the need for a pollinator. Blatina is therefore

∗ Corresponding author. E-mail address: [email protected] (B. Javornik). 0304-4238/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.scienta.2012.05.023

increasingly being replaced by the cultivar Vranac. In vineyards, Zˇ ilavka is often accompanied by the cultivar Krkoˇsija, whilst the cultivar Plavka can be found with Blatina. Overall, the main cultivars for white vine production in BIH used to be Bena, Dobrogostina, Rkatsiteli, Smederevka, Toboluˇsa and Zˇ upljanka whilst Alichante Bouchet, Blatina, Gamay, Merlot and Vranac were the main cultivars for red wine production (Tarailo et al., 1978). There are no exact records of the country’s vineyards today with data about age, health status and accurate evidence of cultivars grown. There are no collection vineyards in BIH. The aim of this study was to establish a microsatellite profile of BIH cultivars in order to assess the genetic diversity, identity of genotypes and their genetic relatedness. Accurate identification of traditional varieties and their conservation could prevent their disappearance and preserve them for future needs. 2. Materials and methods 2.1. Plant material A set of 51 grapevine samples were collected and inventoried during surveys done from 2005 to 2011 (Supplementary Data I). Grapevines were collected from eight vineyards in the Herzegovina region. The collected samples represent old cultivars mainly locally grown or traditional cultivars with a long history of cultivation. Seven reference cultivars (Barbera, Cabernet Sauvignon, Chardonnay, Merlot, Pinot Noir, Sultanine and Touriga National)

L. Tomi´c et al. / Scientia Horticulturae 143 (2012) 122–126

were included in the analysis of microsatellite genotyping, for standardization of the sizes of the obtained microsatellite allele data. Some cultivars from the collection vineyard in Sremski Karlovci, Serbia, which were believed to be the same as BIH cultivars carrying the same name (Blatina, Smederevka, Vranac, Zˇ ilavka and Zˇ upljanka) were included in the analysis for assessing truenessto-type. 2.2. Microsatellite analysis DNA was extracted from young leaves of each sampled grapevine by using the modified CTAB method described by Kump and Javornik (1996). The DNA concentration was measured by fluorimeter and samples were stored in TE buffer at +4 ◦ C. In order to ensure reliable identification of cultivars, the most informative loci were selected, based on previous studies: VVS2 (Thomas and Scott, 1993), VVMD5 and VVMD7 (Bowers et al., 1996), VVMD25, VVMD27, VVMD28 and VVMD32 (Bowers et al., 1999), VrZAG62 and VrZAG79 (Sefc et al., 1999), VChr3a, VChr5a, VChr5b, VChr5c, VChr7b, VChr8a, VChr8b, VChr9a, VChr13a, VChr15a, VChr18a, VChr19a and VChr19b (Cipriani et al., 2008, 2010). The PCR mix prepared in a total volume of 15 ␮l contained 20 ng of genomic DNA, 5× PCR buffer (Promega), 0.2 mM of each of dNTPs (Sigma), 2 mM MgCl2 (Promega), 0.2 ␮m of each primer and 0.5 U of Taq Poly (Promega). The foreword primer was labelled with 6-FAM, VIC, PET or NED fluorescent dye. Touch down PCR was done according to the following thermal scheme: one cycle at 95 ◦ C for 2 min, followed by 10 touchdown cycles at 94 ◦ C for 20 s, 55–1.0 ◦ C/cycle for 20 s and 65 ◦ C for 40 s, followed by 15 cycles at 94 ◦ C for 20 s, 50 ◦ C for 20 s and 65 ◦ C for 40 s and a final step of 1 h at 72 ◦ C. Four by four PCR reactions differing in fluorescent dye were merged together by alliquoting 4 ␮l of each. One ␮l of merged PCR was added to 0.5 ␮l of LIZ 600 size standard and 8.5 ␮l of Hi-Di formamide and separated by capillary electrophoresis using an ABI Prism 3130xl DNA analyzer. The allele sizes were analyzed with Peak Scanner software version 1.0 (Applied Biosystems). The size of each allele was visually inspected in order to prevent effects from potential dropout of long alleles and binning problems. Genotypes showing a single peak in a locus were considered to be homozygous. Data were analyzed with Identity 1.0 software (Wagner and Sefc, 1999) to calculate the probability of identity, synonyms and potential parentage relationships. Data obtained from microsatellite analysis were statistically tested based on the detected allele sizes using the Cervus 3.0 programme (Kalinowski et al., 2007). The following parameters were calculated for each locus: number of alleles at the locus (k), observed heterozygosity (Ho), expected heterozygosity (He), polymorphic information content (PIC) and estimated frequencies of null alleles. Genetic similarity was calculated from band sharing data of each pair of samples using the Jaccard coefficient of similarity. A dendrogram was constructed from the matrix of pairwise distances, using the unweighted pair group method (UPGMA) for clustering in the NTSYS-PC software package version 1.80 (Rohlf, 1993). Statistical support was applied by bootstrap analysis of the binary data set with 100 permutations using WINBOOT software available at http://www.irri.org. Bootstrap values over 50 were considered significant and are presented on the dendrogram. 3. Results and discussion 3.1. Genetic diversity and identity of genotypes A set of 51 BIH grapevine samples from 8 locations were genotyped at 22 SSR loci. The microsatellite profiles of 51 samples are presented by allele sizes in base pairs (Supplementary Data II).

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Table 1 Diversity statistics of analysed loci in terms of the number of alleles – k, observed – Ho and expected – He heterozygosity, polymorphic information content – PIC and frequencies of null alleles – Null freq. Locus VVS2 VVMD5 VVMD7 VVMD25 VVMD27 VVMD28 VVMD32 VrZAG62 VrZAG79 VChr3a VChr5a VChr5b VChr5c VChr7b VChr8a VChr8b VChr9a VChr13a VChr15a VChr18a VChr19a VChr19b Mean

k 11 9 8 6 7 11 7 8 12 8 8 7 7 5 8 12 7 7 5 7 7 5 7.82

Ho

He

PIC

Null freq

0.786 0.778 0.704 0.741 0.741 0.821 0.857 0.926 0.815 0.815 0.857 0.821 0.750 0.750 0.259 0.778 0.679 0.786 0.222 0.643 0.750 0.607

0.776 0.839 0.688 0.698 0.742 0.895 0.806 0.766 0.864 0.799 0.732 0.790 0.769 0.719 0.798 0.878 0.711 0.782 0.456 0.741 0.723 0.680

0.732 0.806 0.635 0.650 0.688 0.867 0.761 0.724 0.830 0.758 0.679 0.740 0.715 0.652 0.757 0.848 0.663 0.737 0.414 0.695 0.675 0.623

−0.0134 +0.0338 −0.0300 −0.0279 −0.075 +0.0362 −0.0418 −0.1202 +0.0200 −0.0225 −0.0907 −0.0291 +0.0064 −0.0235 +0.5032 +0.0473 +0.0406 −0.0090 +0.3446 +0.0668 −0.0343 +0.0392

0.722

0.757

0.711

The analysis of identity of the 51 grapevine samples revealed 25 unique genotypes and 3 near synonyms (with two to twenty-two bp differences in the amplified alleles), which were further used to calculate diversity statistics. Genotypes from pairs of near synonyms were included in the statistical analysis in order not to omit the significance of rare alleles. The number of amplified alleles was 172. Allele length in base pairs varied from 83 to 272, with an average allele size of 175 base pairs. The average allele number per locus was 7.82 (Table 1.), which is within the range of diversity present in, e.g., 19 Bulgarian cultivars (7.86 ± 1.07) (Dzhambazova et al., 2009), 50 Greek cultivars (7.9) (Lefort and Roubelakis-Angelakis, 2001), 11 Romanian cultivars (7.90) (Gheorghe et al., 2010) and 33 Slovenian cultivars ˇ (8.00) (Stajner et al., 2011) but less than 111 Spanish cultivars (9.85) ˜ et al., 2003) or 304 Mediterranean accessions (13.17 ± 3.59) (Ibánez (Laiadi et al., 2009). This inconsistency is due to the increased presence of low frequency alleles in large scale sample sets (Laiadi et al., 2009). The average expected heterozygosity (0.757) was slightly higher than the observed heterozygosity (0.722) (Table 1). This could be explained by a lack of a high degree of human selection, which acts against homozygosity (Sefc et al., 2000). At loci VChr8a and VChr15a, the observed heterozygosity was significantly lower than expected since the number of homozygote loci is high. This could be explained by an over-abundance of null alleles, since their frequencies are positive with the highest values (Table 1). This was the case with the locus VVMD36, at which the presence of null alleles was estimated to be very high (12.2%) in an analysis of Slovenian ˇ cultivars (Stajner et al., 2011). The average polymorphism of analyzed loci was 71.4%, with the most informative loci being VVMD28 (0.867), VChr8b (0.848), ZAG79 (0.830) and VVMD5 (0.806). Microsatellite fingerprinting of 51 samples revealed 25 unique genotypes. Cases of a perfect match were detected in 5 groups of samples with identical SSR profiles at all 22 analyzed loci (Table 2). These perfect matches will be further characterized by ampelographic analysis, in order to confirm their true variety names and identity. Two Kadarun samples that were collected from different locations had the same allelic profile at all loci analyzed, two Radovaˇca samples and eight Zˇ ilavka samples also obtained from different locations, were identical (Supplementary Data II.).

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Table 2 Groups of matching grapevine genotypes with the same allelic profiles at 22 analyzed microsatellite loci. Groups of perfect matches

No.

Kadarun II = Kadarun IV = Surac IV = Podbil I Dalmatinka VI = Kambuˇsa I Mala Blatina I = Proˇsip I Kadarun Bijeli IV = Smederevka II Blatina I = Stara Blatina

1 2 3 4 5

Table 3 Groups of individuals growing in different locations, with the same name but different allelic profiles. Groups of homonyms

No.

Krkoˇsija I = / Krkoˇsija III / Bena III Bena I = / Blatina VIIa and Blatina VIIb Blatina I = / Mala Blatina VIIa and Mala Blatina VIIb Mala Blatina I = / Surac VI Surac IV =

1 2 3 4 5

There were also cases of homonymy (5 groups) amongst samples carrying the same name but growing in different locations (Table 3). The cause of this homonymy is probably mislabelling or misnaming of planting material, since most of those false synonyms were shown to be identical to some other cultivars from the analysed set. We found 5 pairs/groups of identity: Krkoˇsija from location III was shown to have the standard genotype of Zˇ ilavka, Bena from location III was shown to be Smederevka, Blatina from locations VIIa and VIIb are actually the cultivar Plavka, with a different genetic profile than Blatina I at all analyzed loci. Mala Blatina from locations VIIa and VIIb were shown to be the cultivar Vranac, a well known cultivar grown mainly in Montenegro. Surac from location IV had the same profile as Kadarun II, Kadarun IV and Podbil I, whilst Surac VI had a unique profile. According to the Cipriani et al. (2010) conservative criterion, cultivars may be considered synonymous when there is a mismatch at a maximum of two loci out of a total of 32 analysed loci. Plavka from location IV was a perfect synonym of Plavka from location VI and these two were near synonyms of Plavka from location I, with a 2 bp difference at one allele of locus VVS2. Stara Blatina from location VIIa was a perfect synonym of Stara Blatina from location VIIb but showed a 16 bp difference at one allele of locus VChr5b in comparison with Blatina I and Stara Blatina I. A twenty-two bp difference at a locus mismatch was also found between cultivars Crna Proˇsip and Dobrogostina (VChr19a), whilst a two-loci mismatch was detected between Crna Proˇsip and Stara Zˇ ilavka (VChr3a and VChr15a) and Dobrogostina and Stara Zˇ ilavka (VChr15a and VChr19a) (Supplementary Data II.). The groups of near synonyms are likely to be full siblings or a mixture of clones (Cipriani et al., 2010; Lefort and Roubelakis-Angelakis, 2001; Sefc et al., 1998). The cultivars Blatina, Smederevka, Vranac, Zˇ ilavka and Zˇ upljanka, which were compared to cultivars from the collection vineyard Sremski Karlovci for assessing trueness-to-type, perfectly matched at all analyzed loci, thus providing accurate variety identification (data available on request). BIH cultivars were further compared to already genotyped cultivars from neighbouring Croatia (Maletic´ et al., 1999) at five loci (VVS2, VVMD5, VVMD7, VrZAG62 and VrZAG79). Plavka I from BIH was shown to be identical to Plavina from Croatia and Zˇ ilavka from BIH is the same as Zˇ ilavka and Brajdica from Croatia. 3.2. Genetic relatedness A similarity index was calculated from band sharing data of each pair of samples, using the Jaccard coefficient of similarity. The

overall similarity calculated from a matrix of pairwise distances was 36.8%. This was within the range of average similarity of Greece cultivars (34.4%) (Lefort and Roubelakis-Angelakis, 2001) but lower than Croatian (38%) (Maletic´ et al., 1999), Portuguese (38%) (Lopes et al., 1999) and mid-European cultivars (40%) (Sefc et al., 1998). A dendrogram was constructed from the matrix of pairwise distances, using the unweighted pair group method (UPGMA) for clustering. Genotypes clustered on the dendrogram into two main groups, which shared 22% of alleles (Fig. 1). Dobrogostina, Stara Zˇ ilavka and Crna Proˇsip clustered together with Krkoˇsija and Zˇ ilavka as a subgroup in the first group. This subgroup of cultivars is clearly separated on the dendrogram, sharing only 28% of alleles with the rest of the group. Crna Proˇsip, Dobrogostina and Stara Zˇ ilavka are near synonyms, mismatching at two loci, indicating close genetic relatedness and suggesting that they are siblings. Similarly, Lefort and Roubelakis-Angelakis, 2001 suggests that such closely related cultivars may be clones; i.e., one originated from the other. The moderately high bootstrap value support (67%) between the group of near synonyms (Crna Proˇsip, Dobrogostina, Stara Zˇ ilavka) and Zˇ ilavka supports our theory of a parent-offspring relationship, which was also confirmed by parentage analyses (data available on request). Five reference cultivars Cabernet Sauvignon, Chardonnay, Merlot, Touriga National and Pinot Noir classified into the same subgroup of the second group, together with Bena, Radovaˇca and Smederevka. Bijela Proˇsip and Drenak were found to be close on the dendrogram (49%), as well as Bena and Radovaˇca (50%), whilst other cultivars were more distant. Barbera grouped with Menigovka and Dalmatinka, all being table cultivars. Sultanine, Rezaklija and the NPS1 genotype were a distinct outgrouping from the other cultivars. Genotype NPS1 was 86% distinctive from the rest of cultivars, which probably suggests a different or mixed origin of this cultivar. High bootstrap values support the association between some cultivars, especially synonymic ones, but some low values indicate that certain clusters are not well supported by data, which may be a result of the large number of hybrid genotypes in our data set. 3.3. Parentage analysis Identity 1.0 software was used for the analysis of parentoffspring relationships (Wagner and Sefc, 1999). Analyses were done on a set of samples with unique genotypes, together with near synonyms, in order to identify the true parents. The analysis resulted in three trios; however, two of them were false trios, since a parent offspring relationship was shown between near synonyms. The third parent-offspring relationship was revealed for cultivar Zˇ upljanka which was shown to be the offspring of Pinot Noir × Surac VI (Table 4). According to Cindric´ et al. (2000), the cultivar Zˇ upljanka, registered in 1970, is derived from a cross between Pinot Noir and Prokupac. Because of the obtained discrepancies Surac VI was further compared to Prokupac acquired from grapevine collection Sremski Karlovci–Serbia, to identify its trueness-to-type. The genotyping results showed that Prokupac from Sremski Karlovci has unique genotype as Surac VI, which confirmed the parentages published by Cindric´ et al. (2000). Prokupac is described as a native Balkan cultivar belonging to the ecological and geographic group convarietas Pontica, subconvarietas Balcanica (Cindric´ et al., 2000) and has been used in the breeding of several other grapevine cultivars. The most widespread autochthonous cultivar in BIH is Zˇ ilavka. The genotyping results suggest that Zˇ ilavka is in a parent/offspring relationship with three cultivars (Crna Proˇsip, Dobrogostina and Stara Zˇ ilavka), since they share 50% of alleles, exactly one allele at each analyzed locus. This is the first evidence in the evaluation of the origin of Zˇ ilavka, since its parentage still stays unknown. It should be further investigated whether the relationships are the result of spontaneous breeding or if it was planned selection.

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125

61

100 67

100

100 50 100

100

92 100

100 100

100 61 72

100 63

53 63

100

100

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

0.80

0.90

Krkošija_I Dobrogostina_I StaraŽilavka_I CrnaProšip_I Krkošija_III Žilavka_III Žilavka_VIIIb Žilavka_VIIa Žilavka_VIIb Žilavka_II Žilavka_IV Žilavka_I Žilavka_VIIIa ŽutaŽilavka_I Prošip_I MalaBlatina_I NN_VI BijelaProšip_I Drenak_II Plavka_IV Plavka_VI Plavka_I Blatina_VIIb Blatina_VIIa Kambuša_I Dalmatinka_IV Barbera Menigovka_I Blatina_I StaraBlatina_I StaraBlatina_VI StaraBlatina_VI Vranac_I MalaBlatina_VII MalaBlatina_VII Trnjak_I Žlozder_I Podbil_I Kadarun_II Kadarun_IV Surac_IV SuracPlavi_II Župljanka_II Surac_VI Bena_I Radovaèa_II Radovaca_III Merlot Cabernet PinotNoir Chardonnay Touriga Bena_III Smederevka_II KadarunBeli_IV Sultanine Rezaklija_V NPS1_VI

1.00

Coefficient Fig. 1. Consensus phenogram of 100 bootstrap replicates generated from the binary data matrix at 22 microsatellite loci for 58 grapevine genotypes, based on the Jaccard coefficient of similarity.

Table 4 Cumulative likelihood ratio of Zˇ upljanka being the progeny of Pinot Noir (1) and Surac (2), versus alternative parents, including close relatives, combined over 22 SSR loci. Parent combination

(1) × (2)

(1) × X

Rel (2) × (1)

(2) × X

Rel (1) × (2)

With observed allele frequencies With 95% upper confidence limit

2.54e+017 2.00e+009

2.05e+011 1.75e+007

5.55e+003 5.44e+002

1.93e+010 1.82e+006

1.96e+003 1.93e+002

4. Conclusions The grapevine material collected for the genotyping study represents old and traditional BIH cultivars that are still preserved in local vineyards, nurseries or individual farms. Microsatellite analysis at 22 loci is the first attempt to evaluate BIH grapevine diversity at DNA level. Out of 51 collected samples, 25 unique fingerprints were revealed, representing 23 cultivars and 2 unnamed genotypes. Amongst these 23 cultivars 17 were cultivars for production of white wines (Bena, Bijela Proˇsip, Dobrogostina, Krkoˇsija, Radovaˇca, Smederevka (syn. Kadarun Bijeli), Stara Zˇ ilavka, Zˇ ilavka, Zˇ lozder and Zˇ upljanka); 16 are used for production of red wines (Blatina, Crna Proˇsip, Dalmatinka (syn. Kambuˇsa), Kadarun (syn. Podbil), Plavka, Proˇsip (syn. Mala Blatina), Surac, Surac Plavi, Vranac and Trnjak). Drenak, Menigovka and Rezaklija are table grape cultivars. Identity analysis also showed cases of homonymy, which are probably misnaming caused by propagating and planting mistakes.

Assessment of true cultivar identity, identification of synonym cultivars, i.e., duplicates, is an important activity for establishing a grapevine germplasm collection in which maximum genetic variability should be contained in a minimum number of plants. Acknowledgement The research was funded by the European Commission’s Joint Research Project of the Joint Call of the SEE-ERA.NET PLUS, grant No. ERA 155/01 and by the Slovenian Research Agency, grant no. P4-0077. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.scienta.2012.05.023.

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