Population genetic analysis in old Montenegrin vineyards reveals ancient ways currently active to generate diversity in Vitis vinifera

Abstract

Global viticulture has evolved following market trends, causing loss of cultivar diversity and traditional practices. In Montenegro, modern viticulture co-exists with a traditional viticulture that still maintains ancient practices and exploits local cultivars. As a result, this region provides a unique opportunity to explore processes increasing genetic diversity. To evaluate the diversity of Montenegrin grapevines and the processes involved in their diversification, we collected and analyzed 419 samples in situ across the country (cultivated plants from old orchards and vines growing in the wild), and 57 local varieties preserved in a grapevine collection. We obtained 144 different genetic profiles, more than 100 corresponding to cultivated grapevines, representing a surprising diversity for one of the smallest European countries. Part of this high diversity reflects historical records indicating multiple and intense introduction events from diverse viticultural regions at different times. Another important gene pool includes many autochthonous varieties, some on the edge of extinction, linked in a complex parentage network where two varieties (Razaklija and Kratošija) played a leading role on the generation of indigenous varieties. Finally, analyses of genetic structure unveiled several putative proto-varieties, likely representing the first steps involved in the generation of new cultivars or even secondary domestication events.

Figure 1

Location of Montenegro (MNE) in the Western Balkans (a) and sampling sites of cultivated and wild grapevines in Montenegro (b). In (b), cultivated and wild vines are shown as blue and orange circles, respectively. Main Montenegrin cities are shown as grey dots. Maps were generated using MapChart (https://mapchart.net) and Tableau v. 10.3.

Figure 2

Population structure analysis of 131 non-redundant grapevine genotypes found in Montenegro. In (a), an unweighted neighbor-joining (UwNJ) radiation tree showing the relationship between wild vines (orange dots) and cultivated vines (blue dots) is shown. In (b), a principal coordinate analysis (PCoA) with both vine types in the same colors is shown. The % variance explained by the PCoA1 and PCoA2 is indicated in the plot axes. Both (a,b) plots were obtained from a dissimilarity matrix calculated in DARwin using 194 SNPs. In (c), STRUCTURE analysis revealing the existence of two major genetic groups, mainly corresponding to varieties sampled as cultivated and wild respectively. Every non-redundant genotype is shown as a vertical line, with color segment lengths proportional to their inferred ancestry to Str1.1 and Str1.2, (shown in green and pink, respectively). The optimal number of genetic groups (2) was set considering the ΔK criterion. Considering a critical ancestry coefficient of q ≥ 0.50, 87 and 44 genotypes were assigned to Str1.1 and Str1.2, respectively.

Figure 3

Population structure analysis of 91 non-redundant cultivated grapevine genotypes found in Montenegro. In (a), STRUCTURE analysis revealed the existence of two major genetic groups (Str2A.1 and Str2A.2). Every non-redundant genotype is shown as a vertical line, with color segment lengths proportional to their inferred ancestry to Str2A.1 (green) and Str2A.2 (purple). The optimal number of genetic groups (K = 2) was set considering the ΔK criterion. Considering a critical ancestry coefficient of q ≥ 0.70, 31 and 23 genotypes were assigned to Str2A.1 and Str2A.2, respectively (37 genotypes were considered as admixed). In (b), a principal coordinate analysis (PCoA) obtained from a dissimilarity matrix calculated in DARwin from genetic data (194 SNPs) from the non-redundant genotypes is shown. The variance explained by the PCoA1 and PCoA2 is indicated as %. In (c), the unweighted neighbor-joining (UwNJ) radiation tree obtained for the same dataset by means of DARwin is shown. In (b,c), genotypes assigned to Str2A.1 and Str2A.2 are shown as green and purple dots, respectively (admixed genotypes are shown in grey).

Figure 4

First-order genetic relationships (trios and duos) detected for cultivated and wild grape varieties sampled in Montenegro. Chlorotypes (A, B, C or D) are indicated with different colors, according to the inserted code. If white, no information on chlorotype was available. Unidentified and unique genotypes in the ICVV-SNP database are shown in boxes with broken borders. These genetic relationships were obtained with the likelihood-based method implemented in Cervus v.3.0 for parentage analysis, on the basis of SNP genetic data.