Original research articles

SSR molecular marker analysis of the grapevine germplasm of Montenegro

Abstract

Aim: Given that the information about the origin, genetic relationships, and diversity of Montenegrin grapevines is still partial, we performed a detailed analysis of the germplasm in this country using simple sequence repeat (SSR) markers. Our main goal was to determine the identity of cultivars unique to Montenegro and those shared with other countries, especially the neighbouring ones.

Methods and results: Seventy samples were collected and 14 genotypes were found. After SSR profile comparison with available molecular databases and literature data, the identity of each genotype was established. Five well-known cultivars were found, the others being minor, lesser-known cultivars.

Conclusion: This research provides an overview of the Montenegrin grapevine assortment. There are cultivars shared with other countries, mainly the neighbouring ones, while others are likely native to Montenegro. The Kratošija population (alias Primitivo, Zinfandel and Crljenak Kaštelanski) has a large number of different names in Montenegro and also a wide morphological variability. Therefore, Montenegro is the best candidate as the origin and spreading point of this cultivar.

Significance and impact of the study: The present study adds information on the identity, origin, diffusion and variability of some grapevine cultivars, allowing us to reconstruct the history and evolution of national and transnational ampelographic assortment of Montenegro.

Introduction

Montenegro is a small country located in the Balkan Peninsula and overlooking the Adriatic Sea. Grapevine growing in Montenegro dates back before the Roman period (Vuksanović, 1977). The climatic, soil and orographic conditions of southern Montenegro define two wine regions: the Montenegro Skadar Lake basin, encompassing the Podgorica, Crmnica, Rijecki, Bjelopavlicki and Katunski sub regions, and the Montenegrin seacoast region, including the Boko-Kotorski, Budvansko-Barski, Ulcinjski and Grahovsko-Nudolski sub regions. Ancient and probably autochthonous cultivars dominate the present grapevine assortment of these two regions, and very heterogeneous populations appear as a consequence of perennial growing. Furthermore, there are numerous local grapevine cultivars lacking heterogeneity in their population, which have been registered as autochthonous even though they were not definitively identified. Most of these cultivars can be recognized using OIV codes, but it is not enough for their reliable identification (Ulićević, 1966; Pejović, 1988; Maraš, 2000).

Current viticulture and wine production in Montenegro is based mainly on presumed autochthonous grapevine cultivars, such as Vranac, Kratošija, Krstač, and Žižak. The dominant cultivars for red wine production are Vranac and Kratošija. Significantly less prevalent is Krstač, used for white wine production, while no commercial wine is produced from Žižak. The presence of Muškaćela and Trojka is significantly lower.

The results of Calò et al. (2008) showed that Kratošija is a synonym of the Californian Zinfandel, the Italian Primitivo and the Croatian Crljenak Kaštelanski, and suggested a first-degree relationship with the Montenegrin Vranac. Contrary to Vranac, which has stable morphological traits and has undergone expansion in the recent years, Kratošija is very ancient and a lot of biotypes have been found within its population, not fully investigated yet. Because of its heterogeneity, Kratošija is less present alone in the vineyards, and is found mostly in combination with Vranac, which dominates in the assortment of red grape cultivars. The heterogeneity of Kratošija has been described by many authors (Stojanović, 1929; Bulić, 1949; Jelaska, 1954; Ulićević, 1966; Avramov, 1988; Pejović, 1988; Burić, 1995; Bozinovik et al., 1998; Maraš, 2000; Maraš et al., 2004). It is well known that the features of an old grapevine cultivar may change with the accumulation and fixation of mutations in the shoot apical meristem, giving rise to many biotypes within the population, even with noticeable morphological or phenological differences. In this context, our work had different purposes. Using simple sequence repeat (SSR) markers, we wanted i) to investigate the identity of almost all grapevine cultivars grown in Montenegro; ii) to determine which cultivars are likely native to Montenegro and which are common to neighbouring countries; iii) to ascertain the identity of presumed Kratošija biotypes collected in the experimental field of the Biotechnical Institute in Ljeskopolje and coming from different Montenegro private vineyards, where they are cultivated under many different names, depending on the area; and iv) to verify the correct identity of Vranac mother vines chosen for clonal selection process.

Materials and methods

1. Plant material

Seventy samples were selected for genotyping during the period from 2006 to 2010. The vines were collected from different viticultural areas of Montenegro (Table 1).

Table 1. Accession list (name, provenance and identity by genotyping).


ID Accession name Provenance (viticultural areas, municipalities) Identity
1 BIKACA Ljeskopolje, Biotechnical Institute, Podgorica Kratošjia
2 BIKACA Kuci, Podgorica Kratošjia
3 BIKACA Doljani, Podgorica Kratošjia
4 BIOKA Brceli, Crmnica, Bar Bioka
5 CRNA LOZA Doljani, Podgorica Crna Loza
6 CRNI KRSTAC Ljeskopolje, Biotechnical Institute, Podgorica Kratošjia
7 CUBRICA Kuci, Podgorica Cubrica
8 KADARUN Herceg Novi Kadarun
9 KRAT. SA DUB. UREZ. Ljeskopolje, Biotechnical Institute, Podgorica Kratošija
10 KRATOSIJA Brceli, Crmnica, Bar Kratošija
11 KRATOŠIJA Cemovsko polje, Podgorica Kratošija
12 KRATOŠIJA Cemovsko polje, Podgorica Kratošija
13 KRATOŠIJA Cemovsko polje, Podgorica Kratošija
14 KRATOŠIJA Crmnica, Bar Kratošija
15 KRATOŠIJA Doljani, Podgorica Kratošija
16 KRATOŠIJA Ljeskopolje, Biotechnical Institute, Podgorica Kratošija
17 KRATOŠIJA Ljeskopolje, Biotechnical Institute, Podgorica Kratošija
18 KRATOSIJA Nikolj Crkva, Cemovsko polje, Podgorica Kratošija
19 KRATOŠIJA SREDNJA Ljeskopolje, Biotechnical Institute, Podgorica Kratošija
20 KRATOŠIJA ILI VRAN Ljeskopolje, Biotechnical Institute, Podgorica Kratošija
21 KRATOŠIJA MALA Ljeskopolje, Biotechnical Institute, Podgorica Kratošija
22 KRATOŠIJA MALA Ovtocic, Crmnica, Bar Kratošija
23 KRSTAC B. Cemovsko polje, Podgorica Krstac
24 KRSTAC B. Cemovsko polje, Podgorica Krstac
25 KRSTAC B. Cemovsko polje, Podgorica Krstac
26 KRSTAC BIJELI Cemovsko polje, Podgorica Krstac
27 LISICINA Brceli, Crmnica, Bar Lisicina
28 LISICINA Godinje, Crmnica, Bar Lisicina
29 LJUTICA Ljeskopolje, Biotechnical Institute, Podgorica Kratošjia
30 MUŠKACELA Cemovsko polje, Podgorica Muscat blanc à petits grains
31 MUŠKACELA Cemovsko polje, Podgorica Muscat blanc à petits grains
32 MUŠKACELA Cemovsko polje, Podgorica Muscat blanc à petits grains
33 MUŠKACELA Nikolj Crkva Cemovsko polje, Podgorica Muscat blanc à petits grains
34 PLAVINA Herceg Novi Plavina
35 RAZAKLIJA Doljani, Podgorica Drenak crven
36 RAZAKLIJA CRNA Kuci, Podgorica Razaklija crna
37 REHULJACA Ljeskopolje, Biotechnical Institute, Podgorica Kratošija
38 SREDNJI VRANAC Ljeskopolje, Biotechnical Institute, Podgorica Kratošija
39 TROJKA Ljeskopolje, Biotechnical Institute, Podgorica Moscato violetto/Muscat rouge de Madere
40 TROJKA Ljeskopolje, Biotechnical Institute, Podgorica Moscato violetto/Muscat rouge de Madere
41 TROJKA Ljeskopolje, Biotechnical Institute, Podgorica Moscato violetto/Muscat rouge de Madere
42 VELJI VRANAC Ljeskopolje, Biotechnical Institute, Podgorica Kratošija
43 VRAN Ljeskopolje, Biotechnical Institute, Podgorica Kratošija
44 VRANAC 9 Ljeskopolje, Biotechnical Institute, Podgorica Kratošija
45 VRANAC Ovtocic, Crmnica, Bar Vranac
46 VRANAC Ovtocic, Crmnica, Bar Vranac
47 VRANAC Ovtocic, Crmnica, Bar Vranac
48 VRANAC Limljani, Crmnica, Bar Vranac
49 VRANAC Brceli, Crmnica, Bar Vranac
50 VRANAC Cemovsko polje, Podgorica Vranac
51 VRANAC Cemovsko polje, Podgorica Vranac
52 VRANAC Cemovsko polje, Podgorica Vranac
53 VRANAC Nikolj Crkva Cemovsko polje, Podgorica Vranac
54 VRANAC Nikolj Crkva Cemovsko polje, Podgorica Vranac
55 VRANAC Nikolj Crkva Cemovsko polje, Podgorica Vranac
56 VRANAC Nikolj Crkva Cemovsko polje, Podgorica Vranac
57 VRANAC Brceli, Crmnica, Bar Vranac
58 VRANAC (clone 2/8) Crmnica, Bar Vranac
59 VRANAC (clone 6/8) Crmnica, Bar Vranac
60 VRANAC NCV 14/39 Cemovsko polje, Podgorica Vranac
61 VRANAC NCV 15/13 Cemovsko polje, Podgorica Vranac
62 VRANAC NCV 62/7 Cemovsko polje, Podgorica Vranac
63 VRANAC NCV 62/13 Cemovsko polje, Podgorica Vranac
64 VRANAC NCV 62/8 Cemovsko polje, Podgorica Vranac
65 VRANCIC Ljeskopolje, Biotechnical Institute, Podgorica Kratošija
66 VRANCINA Ljeskopolje, Biotechnical Institute, Podgorica Kratošija
67 ŽIŽAK Cemovsko polje, Podgorica Žižak
68 ŽIŽAK Cemovsko polje, Podgorica Žižak
69 ŽIŽAK Cemovsko polje, Podgorica Žižak
70 ŽIŽAK Nikolj Crkva, Cemovsko polje, Podgorica Žižak

2. Microsatellite DNA analysis

DNA was extracted from young leaves obtained from cuttings, and genotyping was performed with 11 SSR loci routinely employed at CRA-VIT (Centro di Ricerca per la Viticoltura, Italy) for cultivar identification: VVS2 (Thomas and Scott, 1993); VVMD5, VVMD7, VVMD27 and VVMD28 (Bowers et al., 1996 and 1999); VrZAG62 and VrZAG79 (Sefc et al., 1999); ISV2, ISV3 and ISV4 (Crespan, 2003); and VMCNG4b9 (Welter et al., 2007), as described by Crespan et al. (2006). The varietal identification was achieved by comparing the obtained SSR profiles with available molecular databases and literature data.

3. Statistics on SSR data

To evaluate the discrimination power of the 11 SSRs used in this work, the following parameters were calculated: the polymorphic information content (PIC) and the combined non-exclusion probability for identity of unrelated and full-sib genotypes. SSR data were computed using 1418 unique profiles of Vitis vinifera L. cultivars present in the CRA-VIT molecular database, including the new genotypes found in the present work. CERVUS 3.0.3 software was used for calculations (Marshall et al., 1998; Kalinowski et al., 2007; http://www.fieldgenetics.com). Genotypes showing a single allele were considered as homozygous.

Results and discussion

Fourteen genotypes have been found within the 70 samples (Table 2). Using our set of 11 SSR markers, the mean PIC computed on 1418 unique profiles of Vitis vinifera L. cultivars was 0.8036; the estimated probability of identity to deal with unique genotypes was 3.38E-15 for unrelated genotypes and 9.52E-6 for full sibs. These parameters showed the significant reliability of this set of markers for varietal identification.

Table 2. SSR profiles of grapevine varieties grown in Montenegro (allele lengths are in bp).


VARIETY berry
colour*
use
**
VVS2 VVMD5 VVMD7 VVMD27 VrZAG62 VrZAG79 VVMD28 ISV2
(VMC6E1)
ISV3
(VMC6F1)
ISV4
(VMC6G1)
VMC
NG4B9
BIOKA B W 135 143 226 232 239 249 179 181 187 193 250 258 249 251 137 165 133 139 169 187 166 176
CRNA LOZA N W 139 143 226 232 247 249 179 181 185 199 236 258 261 261 141 141 133 139 177 177 150 164
CUBRICA N W 133 143 236 246 239 249 179 181 187 199 236 258 239 261 141 141 133 139 177 193 164 172
RAZAKLIJA
(Drenak crven)
R T 139 143 232 246 239 247 181 185 185 187 250 258 239 261 141 143 133 145 177 193 150 176
KADARUN N W 143 145 232 236 249 249 179 181 193 199 258 258 249 261 165 165 139 139 177 193 150 150
KRATOŠIJA N W 133 143 226 236 247 249 179 181 199 203 236 258 251 261 141 165 139 139 177 177 150 164
KRSTAC B W 133 139 232 240 239 239 185 185 187 195 250 258 247 261 145 151 133 139 169 177 158 166
LISICINA R W 133 137 238 246 239 239 181 185 187 195 250 250 237 239 141 143 133 139 177 197 158 172
TROJKA (mutant of
Moscato violetto)
B W 133 133 226 228 247 249 179 183 185 203 244 254 249 261 141 161 133 133 169 177 158 158
MUŠKACELA (Muscat
blanc á petits grains)
B W 133 133 228 236 233 249 179 194 185 195 250 254 249 271 141 143 133 139 169 187 158 166
PLAVINA N W 133 143 232 236 239 249 179 189 187 199 236 242 251 261 143 165 139 139 177 177 150 152
RAZAKLIJA CRNA N T 135 139 232 238 247 255 181 185 185 203 250 258 239 247 141 165 139 145 177 187 158 176
VRANAC N W 133 133 226 226 247 249 181 181 193 199 258 258 239 251 151 165 133 139 177 177 164 172
ŽIŽAK B W 143 145 240 240 239 263 179 191 187 193 250 250 251 261 141 159 139 139 183 187 150 152

*B: white, N: black, R: red.
**W: wine, T: table.

The identification work showed that five genotypes match well-known cultivars, namely Kratošija, Muscat blanc à petits grains, Muscat rouge de Madere (alias Moscato violetto), Plavina and Vranac. In particular, the accessions named Kratošija (plus an adjective), Bikača, Ljutica, Rehuljaca, Srednji Vranac, Velji Vranac, Vran, Vrancic, Vrancina and a wrongly named Vranac (id 44 in Table 1) all belong to the Kratošija cultivar, as previously supposed based on ampelographic descriptions (Maraš, 2000). Crni Krstač matches the Kratošija profile and is another clear misnomer. The first remarkable descriptions of Vranac and Kratošija cultivars have been given by Petar Plamenac in the Ampelography of Viala and Vermorel (1910). Later on, many scientists (Stojanović, 1929; Bulić, 1949; Ulićević, 1959 and 1966) state that Vranac is present only in Montenegro. Ulićević (1959) writes the following: "characteristic cultivar of Crmnica from the Skadar Lake viticultural area is Vranac, covering about 40% of the assortment of grape cultivars. As a matter of fact, this is the only viticultural area where it prevails and the only one where it is represented to a greater extent. It is in a proper sense a Crmnica cultivar". Nastev (1967) states that Vranac is a Montenegrin autochthonous grapevine cultivar mostly cultivated in the Skadar lake region (Crmnica), but also on the Montenegrin seacoast. This author declares that Vranac has been spread in the 1950s in Macedonia (experimental field of Butel). Nowadays, it represents the leading cultivar for red wine production in this country (Stajner et al., 2009). Ulićević (1959) states that Kratošija is almost as remarkable as Vranac. In the same paper he writes that Kratošija dominated in Montenegrin vineyards for over 70 years and that it often represented 90% of the assortment. Therefore, Kratošija is the main and probably the oldest Montenegrin grapevine cultivar. Ulićević (1959) argues that the Californian Zinfandel is identical to Kratošija and that Zinfandel likely originates from Montenegro and has been exported by Montenegrin emigrants. This was supported by the work of Calò et al. (2008), confirming that Kratošija has the same genetic profile as Zinfandel. Avramov (1988), Pejović (1988), Cindrić (1994), Cindrić et al. (2000), Burić (1995), Božinovik (1996), Maraš (2000) and Maraš et al. (2004) write that Vranac and Kratošija are autochthonous cultivars from Montenegro and that Kratošija has a very heterogeneous population. The wines of these cultivars are complementary: Kratošija usually provides a higher amount of total acids and sugars, while Vranac is richer in coloured substances. Bulić (1949) gives a description of Kratošija with synonyms (Gartosija, Grakosija, Kratkosija) from the nine municipalities of the Montenegrin seacoast. This cultivar is rarely found in the Dalmatia region, therefore, the author states that it has been possibly introduced to Dalmatia from Montenegro. By 2001, only about twenty living vines of Crljenak Kaštelanski had been found in Dalmatia (Maletić et al., 2009), confirming that this cultivar was not widespread in Dalmatia in the 19th and 20th century (Savić, 2003). Stojanović (1929) writes that the best vineyards in Montenegro are in the Skadar lake region (Crmnica), which is famous for its red wines that can reach a high quality, while the wines produced along the Montenegrin seacoast are similar to those from Dalmatia. This author states that Plavac mali, Dobričić and some others are the main cultivars grown in Dalmatia, whereas he does not mention Kratošija.

Ampelographic descriptions are available for all Kratošija accessions listed in Table 1 and grown at the Experimental Estate of the Biotechnical Institute in Podgorica - Ljeskopolje (Maraš, 2000). Some examples of ampelographic variability in Kratošija accessions are reported in Table 3 and Figure 1. The name of each Kratošija biotype is in connection with some of its particular characteristics. In particular, these biotypes showed a wide range of variability in grape yield, mainly dependent on bud fertility rather than cluster weight. Cluster weight was highly variable among accessions and correlated with bunch morphology; these traits showed to be stable within each biotype (Maraš, 2000). One mother vine was selected per biotype for the sanitary check-up and all showed the presence of viruses. Furthermore, additional investigation of the sanitary status of newly selected vines of Kratošija from all viticultural regions of Montenegro showed a high level of viral infection (Maraš et al., 2014).

Table 3. Examples of ampelographic variability of some Kratošija accessions (Maraš, 2000) adapted to the 2nd Edition of the OIV descriptor list for grape varieties and Vitis species (id number in parenthesis refers to table 1)*.


Accession name OIV CODES
3 4 16 65 68 76 79 84 85 151 202 206 203 204 220 223 225 231 236 241 235
Bikaca (id 1) 3 7 1 7 3 2 9 5 7 3 5 1 3 5 5 2 6 3 1 3 1
Crni krstac (id 6) 3 7 1 7 4 2 7 5 7 3 7 1 5 5 5 2 6 3 1 3 1
Kratošija (id 16) 3 7 1 7 4 2 7 5 7 3 7 1 5 5 5 2 6 3 1 3 1
Kratošija ili Vran (id 20) 3 7 1 7 3 2 9 5 7 3 7 1 5 5 5 2 6 3 1 3 1
Kratošija mala (id 21) 3 7 1 7 4 2 7 5 7 3 5 1 5 9 5 2 6 3 1 3 1
Kratošija sa dub.urez. (id 9) 3 7 1 7 3 2 9 5 9 3 7 1 5 5 5 2 6 3 1 3 1
Kratošija srednja (id 19) 3 7 1 7 4 2 7 5 7 3 5 1 5 5 5 2 6 3 1 3 1
Ljutica (id 29) 3 7 1 7 4 2 7 5 7 3 7 1 5 5 5 2 6 3 1 3 1
Rehuljaca (id 37) 3 7 1 5 3 2 9 5 7 3 7 1 3 3 5 2 6 3 1 3 1
Srednji vranac (id 38) 3 7 1 7 3 2 9 5 7 3 7 1 5 5 5 2 6 3 1 3 1
Velji vranac (id 42) 3 7 1 7 3 2 9 5 7 3 7 1 5 5 5 2 6 3 1 3 1
Vranac 9 (id 44) 3 7 1 7 3 2 9 5 7 3 7 1 5 5 5 2 6 3 1 3 1
Vran (id 43) 3 7 1 7 3 2 9 5 7 3 7 1 5 9 5 2 6 3 1 3 1
Vrancic (id 65) 3 7 1 7 3 2 9 5 9 3 7 1 5 5 5 2 6 3 1 3 1
Vrancina (id 66) 1 7 1 7 3 2 7 5 7 3 7 1 5 5 5 2 6 3 1 3 1

*traits differing in the level of expression compared to the population are in bold.

Figure 1. Examples of cluster variability among Kratošija biotypes (id number in parenthesis refers to table 1).

By putting together different pieces of information from the literature cited, and given the results of our study, we can conclude that Kratošija with all its biotypes has been grown in Montenegro for centuries. Given its presence in all the viticultural regions of this little country, the wide variability of the population, the impressive high number of different names, the close genetic relationship with Vranac, and its current presence (survival) in viticultural areas in Montenegro, we suggest that this area be considered a likely region for the origin and spreading centre of this cultivar. Therefore, the place of origin of this cultivar should be moved further south than the Dalmatian coast, unlike what has been previously suggested by Maletic et al. (2004).

The SSR profile of Muškaćela accessions matches that of Muscat blanc à petits grains. This outcome adds a new synonym to the long list of this very ancient cultivar, largely spread all over the viticultural areas in Europe and recently showed to be the main ancestor of the Muscat family (Cipriani et al., 2010). Trojka accessions have the same profile as Muscat rouge de Madere (alias Moscato violetto), another important member of the Muscat family (Di Vecchi-Staraz et al., 2007; Lacombe et al., 2013), but the berry colour is different (it is white in our analyzed Trojka cultivar). Muscat rouge de Madere is a progeny of Muscat blanc à petits grains and Sciaccarello (Lacombe et al., 2013) and has little importance. Supposed to be native to Portugal, it does not have any agronomic relevance. Actually, it is not included in any official list of grapevine cultivars authorized for cultivation in 24 national catalogues (Lacombe et al., 2011). It is considered a grape for enthusiasts (Galet, 2000) and is grown mostly in germplasm repositories. That is why it was a surprise to find it among Montenegrin cultivated grapevines. Plavina is a progeny of Kratošija and Verdeca (Cipriani et al., 2010) and is an important cultivar in the sub region of North Dalmatia. All mother plants chosen for clonal selection of Vranac (NCV 62/13; NCV 14/39; NCV 15/13; NCV 62/7; NCV 62/8) showed to be correctly identified.

Although in literature Bioka is considered as a synonym of Krstač in Crmnica (Ulićević, 1959), SSR data showed that it has a different molecular profile. Therefore, Bioka is another cultivar. We have discovered that Bioka shares the same genotype as the Italian Francavidda, a cultivar registered in the Italian Catalogue and grown in Apulia, the Italian region facing Montenegro on the other side of the Adriatic Sea (http://catalogoviti.politicheagricole.it). Moreover, Bioka has the same SSR profile as the Croatian Zlatarica Vrgorska (Zdunić et al., 2013). Therefore, these three cultivars represent a new case of previously unknown synonyms grown in different countries facing the shores of the Adriatic Sea (Dalmatia and Apulia).

Comparing our data with the European Vitis database (http://www.eu-vitis.de/index.php), we found one match at all seven common SSR loci between the Montenegrin Kadarun and a Kadarun accession from Croatia (HRV041-SA#11-95) lacking the true-to-type validation. In the Vitis International Variety Catalogue (VIVC, http://www.vivc.de), Kadarun is reported as a Turkish cultivar.

The red berry Razaklija accession from the Skadar Lake region (Doljani), a known synonym of the Serbian Drenak crven, matches the SSR profile of Crven Drenok (Vitis-WBC, Western-Balkans Vitis Database, http://vitis.atcglabs.com). Table grapes in Montenegro only accounts for 5%, of which 95% is the Razaklija cultivar (Drenak crven) from both the Skadar Lake and the seacoast region (Ulićević, 1959). Many authors from Yugoslavia consider that Razaklija originates from Asia Minor (Ulićević, 1966). It is not known how and when it arrived in Montenegro, Macedonia and other countries and how it spread.

Crna Loza, Čubrica, Krstač, Lisičina, Razaklija crna and Žižak show unique SSR profiles. Crna Loza was considered as a Kratošija synonym, but our analysis shows a different SSR profile for this cultivar. Based on SSR allele sharing at all analyzed loci, Crna Loza could be another offspring of Kratošija, but further research should be done to confirm this hypothesis.

Ulićević (1959) states that Čubrica is a cultivar for red wine production and is represented in very small percentage in the vineyards of the Podgorica sub region (Doljani and Kuci). The sample of Čubrica from Kuci has a DNA profile confirming that it is an independent cultivar.

Ulićević (1959) writes that in the Skadar Lake region, in the sub region of Podgorica (Beri and Doljani), Krstač was dominant among the white grapevine cultivars used for white wine production, produced in small amount in that period. In the sub region of Podgorica, it represents only 10% of the vineyard area, mostly in Beri and Doljani, and very little in the Ljesanska district. Krstač is believed to be autochthonous of Montenegro and probably originated in Beri (near Podgorica); its name comes from the look and shape of the bunch that resembles a cross (Viala and Vermorel, 1910; Vujović, 1956). Žižak (or Žižak bijeli after Bulić, 1949) is considered another autochthonous cultivar of Montenegro and its origin is unknown. Individual specimens can be found nearby Podgorica, but it is mostly grown on the Montenegrin seacoast (Boko-Kotorski sub region), where it is used for dessert wine (Ulićević, 1959).

About Lisičina, Stojanović (1929) reports the use of this cultivar for white wine production in Montenegro. Bulić (1949) and Ulićević (1959) found Lisičina (synonyms are Lisica, Ružica, Sjemerava, Sjeruša) in Montenegro around Bar, Ulcinj, Podgorica and Virpazar (Crmnica). The VIVC catalogue reports that Lisičina is a synonym of the Croatian Lisica. According to Nastev (1967), Lisičina is a wrong synonym of the Plovdina (Pamid) cultivar. Accordingly, the SSR profile of our Lisičina sample is different from that of the Bulgarian Pamid (Hvarleva et al., 2004; Dzhambazova et al., 2009).

Razaklija crna (black), having a dark blue coloured skin, was found only in Kuci (sub region of Podgorica) in old vineyards and under this name. It has to be noted that this name indicates a table grape cultivar originated by crossing Razaklija Rossa and Vranac (performed by Ulićević M. at the Biotechnical Institute in Podgorica). Based on SSR allele sharing at all analyzed loci, Razaklija crna could really be a progeny of Drenak Crven, but our SSR data rule out Vranac. Further data are required to validate this partial pedigree.

Conclusion

Seventy accessions belonging to the Montenegrin grapevine germplasm were analyzed using SSR markers. Fourteen genotypes have been found, three of them being native to Montenegro (i.e., Vranac, Krstač and Žižak). Kratošija has been recently recognized as an international cultivar, being a synonym of Italian Primitivo, Californian Zinfandel and Croatian Crljenak Kaštelanski, whose origin has been widely investigated. Given the large number of different names used in Montenegro for Kratošija and the wide morphological variability of Kratošija biotypes, we propose here, for the first time, Montenegro as the best candidate country for the origin and spreading centre of this group of synonyms. Crna Loza, Čubrica, Lisičina and Razaklija crna are cultivars likely native to Montenegro. Muškaćela and Trojka, considered autochthonous of Montenegro, are clearly of different origin. We discovered that Montenegrin Bioka, Italian Francavidda and Croatian Zlatarica Vrgorska represent a new group of synonyms.

Overall, our genotyping study gave us the opportunity to check the available information based on ampelographic descriptions and comparisons, providing a more stringent and sometimes new perspective on the origins and relatedness of grapevine cultivars in Montenegro.

Further analyses should be done in order to clarify the genetic relationships among Montenegrin grapevine cultivars as well as the degree of relatedness and origin of commercially important Montenegrin cultivars.

The sharing of common cultivars among neighbouring countries was expected as a natural consequence of easily vegetative propagation of grapevine, but only recently we have started to get accurate information on their identity and the present research contributes to shed some light on this topic.


Acknowledgments: This research was supported by the following projects: "Variability of Kratošija cultivar in Montenegrin viticultural regions" and "Clonal selection of Kratošija cultivar" funded by the Ministry of Science of Montenegro; "Identification and characterization of Montenegrin autochthonous grapevine cultivars" and "Clonal selection of autochthonous grapevine cultivars" funded by Company 13 jul Plantaze and the Ministry of Agriculture of Montenegro; and "IDENTIVIT/ASER" funded by the Italian Ministry of Agricultural, Alimentary and Forestry Policies. Special thanks to Dordjije Rajkovic for useful suggestions and support and to Angelo Costacurta for initiating us to cultivar characterization and identification.

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Authors


Vesna Maraš

Affiliation : Department of development, 13 jul Plantaže, P. Radomira Ivanovica 2, Podgorica, Montenegro


Vladan Bozovic

Affiliation : Department of development, 13 jul Plantaže, P. Radomira Ivanovica 2, Podgorica, Montenegro


Sabrina Giannetto

Affiliation : Consiglio per la Ricerca e la sperimentazione in Agricoltura - Centro di Ricerca per la Viticoltura (CRA-VIT), viale XXVIII Aprile 26, 31015 Conegliano (Treviso), Italy


Manna Crespan

Affiliation : Consiglio per la Ricerca e la sperimentazione in Agricoltura - Centro di Ricerca per la Viticoltura (CRA-VIT), viale XXVIII Aprile 26, 31015 Conegliano (Treviso), Italy

manna.crespan@entecra.it

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