Ampelographic description of research grapevine varieties using 26 OIV codes .
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 Among many literature sources, the oldest historical document that pointed out the importance of autochthonous varieties is The Medieval Statute of Budva from fifteenth century.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.
- autochthonous varieties
- grapevine diversity
- genetic characterization
- ampelographic characterization
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 . 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 . 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 (1860–1918), 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.
2. 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.
2.1 Ampelographic description
Ampelographic analysis, that is, a method of describing characteristics of grapevine varieties, was done with codes—a descriptor prescribed by O.I.V. (Office International de la Vigne et du Vin)—International Wine and Wine Office . Observations were made on young shoots (OIV-003 and -004), young leaves (OIV-051 and -053), mature leaves (OIV-067, 068, 070, 076, 079, 080, 084, and 087), flowers (OIV-151), shoots (OIV-155), bunches (OIV-202, 204, 206, and 208), berries (OIV-220, 223, 225, 235, and 236), and, when possible, on must quality (OIV-505, 506, and 508). Ampelographic description also was done for 17 Kratošija biotypes (with following OIV codes: 003, 004, 016, 065, 068, 076, 079, 084, 085,151,202, 203, 204, 206, 220, 223, 225, 231, 235, 236, and 241).
2.2 Genetic analysis
For genetic analysis, DNA was extracted from young leaves. In the first phase of research, genotyping was performed with 11 SSR loci for variety identification: VVS2 ; VVMD5, VVMD7, VVMD27 and VVMD28 [7, 8]; VrZAG62 and VrZAG79 ; ISV2, ISV3 and ISV4 ; and VMCNG4b9 , as described by Ref. .
During the second phase of research, genotyping was done with nine microsatellite loci: VVS2 ; VVMD5, VVMD7, VVMD25, VVMD27, VVMD28, and VVMD32 ; and ssrZAG62 and ssrZAG79  as proposed by the GrapeGen06 consortium and by the European Vitis Database .
3. Results and discussion
3.1 Literature survey
The first mention of Montenegrin grapevine varieties was in fifteenth century , and later they were studied and described by many authors. Early mentioning of autochthonous Montenegrin grapevine variety was done by M. Plamenac . 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 . 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  and Dalmatia  and to other countries in the former Yugoslavia. The Macedonian professor Nastev  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  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  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 , the growing area of Kratošija was between 100 and 150 km through Montenegro and that is the main and probably the oldest Montenegrin variety.
In addition to very long Kratošija growing in Montenegro, there is also a huge heterogeneity of its population and it was described by many authors. M. Plamenac  for the first time mentioned biotypes of Kratošija and described some kind of Kratošija whose clusters are not compacted, but loose and it was called Reavica. Authors [16, 17, 19, 23, 24, 25, 30, 27, 28] also described different Kratošija’s biotypes. Ulicevic  mentioned three types of Kratošija: Obična Kratošija, Slaborodna Kratošija, and Rehuljava Kratošija. Bozinovik et al.  stated that Kratošija has a high number of biotypes and described three of them (Kratošija standardna, Kratošija rehuljava, and Kratošija neoplodjena). The variability of the Kratošija population in Montenegro was also studied by Pejovic  and Maras . Ampelographic analyses  were done on 17 biotypes of Kratošija, which are known under different names in viticultural areas in Montenegro: Velja Kratošija, Velji Vran, Crni Krstač, Vrančina, Bikača, Vran, Srednja Kratošija, Kratošija or Vran, Srednji Vranac, Velji Vranac, Vrančić, Ljutica, Kratošija, Čestozglavica, Kratošija mala, Kratošija sa dubokim urezima, and Rehuljača.
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  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  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  also described Žižak as an important variety from which, in some places in Boka, are produced dessert wines called Prošek.
Ulicevic  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 , 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 , where the author reported that this variety is grown in Crmnica. About Lisičina, Stojanović  reports the use of this cultivar for white wine production in Montenegro. Bulić  and Ulićević  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 .
According to Ulićević , large areas under vineyards were in Bokokotorski subregion. Kadarun was dominated, while Kratošija and Vranac were less represented.
Ulićević  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 . It is not known how and when it arrived in Montenegro, Macedonia, and other countries and how it was spread.
3.2 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.
|Young shoot: intensity of anthocyanin coloration on prostrate hairs of the shoot tip||Young shoot: density of prostrate hairs on the shoot tip||Young leaf: color of upper side of blade (fourth leaf)||Young leaf: density of prostrate hairs between main veins on lower side of blade (fourth leaf)||Mature leaf: shape of blade||Mature leaf: number of lobes||Mature leaf: area of anthocyanin coloration of main veins on the upper side of blade||Mature leaf: shape of teeth||Mature leaf: degree of opening/ overlapping of petiole sinus||Mature leaf: shape of base of petiole sinus||Mature leaf: density of prostrate hairs between main veins on lower side of blade||Mature leaf: density of erect hairs on main veins on lower side of blade||Flower: sexual organs||Shoot: fertility of basal buds||Bunch: length (peduncle excluded)||Bunch: density||Bunch: length of peduncle of primary bunch||Bunch: shape||Berry: length||Berry: shape||Berry: color of skin||Berry: firmness of flesh||Berry: particular flavor||Sugar content of must (Oe)||Total acidity of must||Must -specific pH|
Ampelographic descriptions are available for all Kratošija accessions listed in Table 2 and grown at the Experimental Estate in Podgorica-Lješkopolje . 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 .
|Young shoot: intensity of anthocyanin coloration on prostrate hairs of the shoot tip||Young shoot: density of prostrate hairs on the shoot tip||Shoot: attitude (before tying)||Mature leaf: size of blade||Mature leaf: number of lobes||Mature leaf: shape of teeth||Mature leaf: degree of opening/ overlapping of petiole sinus||Mature leaf: density of prostrate hairs on main veins on the lower side of blade||Mature leaf: density of erect hairs on main veins on the lower side of blade||Flower: sexual organs||Bunch: length (peduncle excluded)||Bunch: width||Bunch: density||Bunch: length of peduncle of primary bunch||Berry: length||Berry: shape||Berry: color of skin||Berry: intensity of flash anthocyanin coloration||Berry: firmness of flesh||Berry: particular flavor||Berry: formation of seeds|
|Kratošija ili Vran||3||7||1||7||3||2||7||5||7||3||7||5||7||1||5||3||6||3||3||1||3|
|Kratošija sa dubokim urezom||3||7||1||7||3-4||2||7||5||9||3||7||5||7||1||5||3||6||3||3||1||3|
3.3 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 . 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 . 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 . According to Maras et al.  the variety Muškaćela is identical to a variety Muscat bianco–Muscat a petits grains. Trojka accession has the same profile as Muscat rouge de Madere (alias Moscato violetto), another important member of the Muscat family. Based on analysis, it can be concluded that Plavina is a progeny of Kratošija. Bioka shares the same genotype as the Italian Francavidda and Croatian Zlatarica Vrgorska. Comparing data with the European Vitis database in the Vitis International Variety Catalog  Kadarun is Reported as a Turkish cultivar. The red berry Razaklija accession that was analyzed matches the SSR profile of Crven Drenok . Crna Loza, Čubrica, Lisičina, and Razaklija crna show unique SSR profiles. Crna Loza was considered as a Kratošija synonym, but analysis shows a different SSR profile for this variety. Based on SSR allele sharing at all analyzed loci, Razaklija crna could really be a progeny of Drenak Crven . Bearing in mind the importance of grapevine germplasm in Montenegro, the research was continued through two international projects SEEDNet and SEE.ERA NET. As result of SEEDNET project, from 16 considered samples, 6 different genotypes were identified . The identified varieties are Vranac, Kratošija, Krstač, Čubrica, Lisičina, and Razaklija. The variety Razaklija has the identical SSR profile to Drenok crveni from Macedonia.Within SEE.ERaNet project on various viticulture areas, from different vine growing regions, 96 samples of vines were selected and marked for identification. From these samples, 15 different genotypes were revealed some already known (Vranac, Kratošija, Krstač, Žižak, Čubrica, and Lisičina) and some with original SSR profile (Kovačka bijela, Sijer, and Zadrimka). 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 . Accession of Japudžak from Montenegro is identical to the Turkish variety Yapıncak . 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.
|Variety||VVS2||VVMD5||VVMD7||VVMD27||VrZAG62||VrZAG79||VVMD28||ISV2 (VNC671)||ISV3 (VMC6F1)||ISV4 (VMC6G1)||VMC NG4B9|
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).
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 the company 13 jul Plantaze and the Ministry of Agriculture of Montenegro. Part of this work was financially supported by the Swedish International Development Agency (SIDA) within SEEDNET project: “Identification, characterization and conservation of old and autochthonous vine varieties in Eastern European countries” and by the respective national funding bodies and the European Commission represented by 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.