Ampelographic and Genetic Characterization of Montenegrin Grapevine Varieties

Montenegro is a small country in Balkan Peninsula with very long tradition of grapevine growing and wine making that originate from the pre-Roman period. Dominant place in Montenegrin viticulture belongs to autochthonous grapevine varieties Vranac, Kratosija, and Krstac, while in minor part, the other varieties are presented. Among many literature sources, the oldest historical document that pointed out the importance of autochthonous varieties is The Medieval Statute of Budva from fifteenth century. In order to better present Montenegrin germplasm, this research provides an overview of literature, ampelographic, and genetic analysis on autochthonous and domesticated varieties. Achieved results showed an important breeding history of grapevine and a large number of unique DNA profiles. Montenegro has the richness of grapevine diversity that can significantly enrich the diversity of vines in Europe.


Introduction
Montenegro is a small country placed in the Balkan Peninsula with one part overlooking the Adriatic Sea, right across the Italian region Puglia. A long tradition of grapevine growing in Montenegro is very well known and it dates back before the Roman period [1]. On the Montenegrin territory, a large number of tombstones with grapevine and wine motives which originated in ancient era were found. Found decorations were in the vine form and were directly related to the Dionysus god cult [2]. Numerous archeological sites and found objects that originated from the Illyrian period indicate that the wine was much appreciated and was quite used as the beverage. In the middle century, vine growing and winemaking were well developed in Montenegro and one of the oldest written documents that point out the importance of grapevine cultivation and importance of autochthonous varieties on the territory of today's Montenegro is the Medieval Budva's Statute from fifteenth century. After all, more organized work started during the reign of the King Nikola Petrović , who introduced the grape-growing and winemaking regulations. Within the grape varieties that have a long tradition of growing in Montenegro, the dominant place belongs to autochthonous grape varieties Vranac and Kratošija that are used for making red wines, and for production of white wines, the Krstač was the dominant one [3,4], while in a minor part, the other varieties were also presented. A major turning point in the development of Montenegrin viticulture was the realization of the project Ćemovsko polje since 1977-1982, during which 1500 ha of vineyards were planted and modern wine cellar with 2 million liters capacity was established. However, as autochthonous grape varieties were in that time the most important, they also now constitute the viticulture and winemaking sector of Montenegro. In fact, Vranac grape variety represents more than 70% of total production and promotes Montenegro as an important wine country. In order to better present the germplasm of grapevine varieties in Montenegro, beside literary research, ampelographic and genetic analysis of autochthonous and domesticated grapevine varieties was done in order of their secure identification.

Materials and methods
Multi-year research included work on autochthonous and domesticated grapevine varieties in Montenegro. A detailed review of available literature and writing of earlier and contemporary authors regarding autochthonous and domesticated grapevine varieties was done. For a better understanding, we did also ampelographic and genetic analysis of selected varieties. Analysis included 188 samples of old representative vines aged between 50 and 300 years that are grown in affirmed vineyards in Montenegro. These studies also included 17 biotypes of the Kratošija variety ( Figure 2) that were collected in 1987 in an experimental field in Ljeskopolje-Podgorica.

Literature survey
The first mention of Montenegrin grapevine varieties was in fifteenth century [14], and later they were studied and described by many authors. Early mentioning of autochthonous Montenegrin grapevine variety was done by M. Plamenac [3]. He stated that in Montenegrin grape growing region Crmnica, Kratošija, Vranac, Krsmač, Sjerovina, Lisica, and Muskacelica varieties were grown. But the first more significant description of varieties Vranac and Kratošija was given by P. Plamenac [15]. All authors from the former Yugoslavia [16][17][18][19][20][21][22][23][24][25][26][27][28][29] reported Vranac and Kratošija as Montenegrin autochthonous grapevine varieties. Moreover, they stated that Vranac and Kratošija were grown only in Montenegro. From Montenegro, these varieties were spread to Macedonia [20] and Dalmatia [17] and to other countries in the former Yugoslavia. The Macedonian professor Nastev [20] states that Vranac is a Montenegrin autochthonous grapevine variety mostly cultivated in the Skadar lake region (Crmnica), but also in the Montenegrin seacoast. This author declares that Vranac has been transferred in the 1950s in Macedonia (experimental field Butel), from where it has been spread out through the former Yugoslavia. Montenegrin academic Ulicevic [18,19] states that Vranac is a characteristic variety of vine growing area Crmnica in famous vine growing region Skadar lake, which occupies about 40% of the assortment. According to the same author, this is the only vine growing area where this variety is dominant and the growing area was not wider than 30 km.
The earliest reference of the Montenegrin variety Kratošija is reported in the Budva's Medieval Statute [14] in fifteenth century (1426-1431). In particular, it mentioned the "Kratošija's vineyards" indicating the importance of the Kratošija variety in that time in Budva (Montenegro). The Dalmatian ampelographer Bulić [17] described Kratošija (also considering the synonyms Gartošija, Grakošija, and Kratkošija) from nine municipalities of the Montenegrin cost (Budva, Grbalj, Luštica, Krtole, Kotor, Paštrovići, Prčanj, Tivat, and Herceg Novi). Moreover, the author stated that this variety was rarely found in the Dalmatia region where it was likely spread over from Montenegro. For the Kratošija variety, Ulicevic [18,19] states that, it is strongly dominated in all plantations older than 60-70 years in that period and made 90% of the assortment in other regions. According to Ulicevic [18], the growing area of Kratošija was between 100 and 150 km through Montenegro and that is the main and probably the oldest Montenegrin variety.
Krstač was dominant among the white grapevine varieties used for white wine production. Its name comes from the look and shape of the bunch that resembles a cross [15,31]. Ulicevic [18] wrote that Krstač was believed to be autochthonous of Montenegro and probably originated from Beri (near Podgorica) with a growing area of 40-50 km. The same author stated synonyms for Krstač, in Doljani it was called Krstača bijela, in Vražegrmci Bijeli Krstač and in Beri Bijela vinogradarska.
Beside the most important grapevine varieties for viticulture in Montenegro Vranac, Kratošija, and Krstač there are also some literature data about minor grapevine varieties.
Žižak or Žižak bijeli [17] is considered another autochthonous variety of Montenegro and its origin is unknown. Individual vines can be found nearby Podgorica, but it is mostly grown on the Montenegrin seacoast (Boko-Kotorski sub region). Ulićević [18] also described Žižak as an important variety from which, in some places in Boka, are produced dessert wines called Prošek.
Ulicevic [18] states that Čubrica is used for red wine production and is represented in very small percentage in the vineyards of the Podgorica sub region (Doljani and Kuči). According to the author, vineyards in Doljani are quite old and none of the contemporary people, neither then nor now, did not know when these vineyards were planted.
Literature data about Muškaćelica were given by M. Plamenac [3], and he stated that it is the little grown white grapevine variety that has very strong smell while the variety Sjerovina is russet grapevine variety that has round berries.
The first mention of Lisica was in Grlica [3], where the author reported that this variety is grown in Crmnica. About Lisičina, Stojanović [16] reports the use of this cultivar for white wine production in Montenegro. Bulić [17] and Ulićević [18] wrote that Lisičina (synonyms are Lisica, Ružica, Sjemerava, and Sjeruša) was grown in Montenegro around Bar, Ulcinj, Podgorica, and Virpazar (Crmnica).
Zadrimka was a major variety grown in the Ulcinj viticultural region in the late 1800s until phylloxera, and later World War II devastated the vineyards and almost drove the cultivar to extinction [19].
According to Ulićević [18], large areas under vineyards were in Bokokotorski subregion. Kadarun was dominated, while Kratošija and Vranac were less represented.
Ulićević [18] states that the Razaklija cultivar from both the Skadar Lake and the seacoast region constituted 95% of total table grape production in Montenegro. Many authors from Yugoslavia consider that Razaklija originates from Asia Minor [19]. It is not known how and when it arrived in Montenegro, Macedonia, and other countries and how it was spread.

Ampelographic descriptions
Ampelographic descriptors for certain varieties (Figure 1) already existed and through these researches data with some descriptions of additional varieties were fulfilled. Ampelographic descriptions of researched varieties, except Trojka, are given in Table 1.
Ampelographic descriptions are available for all Kratošija accessions listed in Table 2 and grown at the Experimental Estate in Podgorica-Lješkopolje [27]. The name of each Kratošija biotype is in connection with some of its particular characteristics. Cluster weight was highly variable among accessions and correlated with morphology and these traits showed to be stable within each biotype [27].

Genetic identification
The varietal identification was achieved by comparing the obtained SSR profiles with available molecular databases and literature data. The work on the genetic identification of Montenegrin autochthonous varieties began in collaboration with Istituto sperimentale per la viticoltura-Susegana-Conegliano-TV. The analysis confirmed that Vranac, Krstač, and Žižak have an original DNA profile. Kratošija, an old Montenegrin grape variety, has an identical genetic profile as the Zinfandel from California, Primitivo from Italy, and Crljenak kaštelanski from Croatia [32]. The same authors also suggest a probable first degree relationship between Vranac and Kratošija. Research on Montenegrin grapevine diversity continued and further analysis of 70 samples revealed 14 different genotypes. The results showed already identified genotypes: Vranac, Kratošija, Krstač, and Žižak and 10 new identified genotypes [33]. In Table 3, SSR markers of identified varieties are presented. Also, all 17 biotypes of Kratošija were confirmed to have the same genetic profile as Zinfandel/Primitivo [33]. According to Maras et al. [33] Table 1.
Ampelographic descriptions of Kratošija accessions [27].  Out of the six remaining samples, three accessions were found to be misnomers, one coincides with a previously identified variety in another country, and two accessions showed the original SSR profile which did not match any of the known varieties [37]. Accession of Japudžak from Montenegro is identical to the Turkish variety Yapıncak [38]. As a result of this project, in 2012, Montenegro presented and included its autochthonous and domesticated grapevine varieties in the EU Vitis database (Vranac, Kratošija, Čubrica, Krstač, Žižak, Japudžak, Sijer, Lisičina, Zadrimka, and Kovačka bijela). To preserve grapevine germplasm, the National collection of identified varieties was planted in Ćemovsko polje.

Conclusions
According to available literature and obtained results of ampelographic and genetic identification, Montenegro has a very long tradition of grapevine growing and very rich grapevine germplasm. There are varieties whose identification was done, but there are a lot of varieties with unknown origin and identity. Ampelographic description of 18 identified varieties as well as of 17 Kratošija biotypes was done and presented. During multiple years of research, genetic identification of 188 samples was carried out and the results revealed the original DNA profile for Vranac, Krstač, Žižak, Crna Loza, Čubrica, Lisičina, Razaklija crna, Kovačka bijela, Zadrimka, and Sijer. Kratošija, and every of its 17 biotypes, have the same DNA profile as Italian Primitivo, Californian Zinfandel and Croatian Crljenak Kaštelanski. Muškaćela is Moscato bianco while Trojka is Moscato violeto. Montenegrin Bioka is the same as Italian Francavidda and Croatian Zlatarica Vrgorska. The variety Razaklija is the same as Drenak crveni. For Japudžak the same SSR profile as for Turkish Yampincak is discovered.
Research and work on autochthonous and domesticated grapevine varieties in Montenegro are of great importance for the viticulture and winemaking sector. Having in mind this and very interesting results achieved, it was necessary to continue with investigation of Montenegrin grapevine germplasm. Further research with partners from the Institute for Vine and Wine in La Rioja will be done with the aim of analyzing a large number of samples across Montenegro, and then determining its origin and genetic relationships (pedigree analysis).
the Joint Call Secretariat (JCS) at the German Aerospace Center (DLR) within SEERA NET 91/01 project: "Preservation and establishment of true-to-type and virus free material of endangered grapevine cultivars in Croatia and Montenegro". The author gratefully acknowledges all partners for the participation and collaboration.

Conflict of interest
I declare I have no affiliations with or involvement in any organization or entity with any financial interest (such as honoraria; educational grants; participation in speakers' bureaus; membership, employment, consultancies, stock ownership, or other equity interest; and expert testimony or patent-licensing arrangements), or nonfinancial interest (such as personal or professional relationships, affiliations, knowledge, or beliefs) in the subject matter or materials discussed in this manuscript.