Biochemical Response and Gene Expression to Water Deficit of Croatian Grapevine Cultivars (Vitis vinifera L.) and a Specimen of Vitis sylvestris.

The biochemical response and gene expression in different grapevine cultivars to water deficit are still not well understood. In this study, we investigated the performance of four traditional Croatian Vitis vinifera L. cultivars ('Plavac mali crni', 'Istrian Malvasia', 'Grasevina', and 'Tribidrag'), and one wild (Vitis vinifera subsp. sylvestris) genotype exposed to water deficit (WD) for nine days under semi-controlled conditions in the greenhouse. Sampling for biochemical and gene expression analyses was performed at days six and nine from the beginning of WD treatment. The WD affected the accumulation of metabolites with a significant increase in abscisic acid (ABA), salicylic acid (SA), and proline in the leaves of the stressed genotypes when the WD continued for nine days. Lipid peroxidation (MDA) was not significantly different from that of the control plants after six days of WD, whereas it was significantly lower (297.40 nmol/g dw) in the stressed plants after nine days. The cultivar 'Istrian Malvasia' responded rapidly to the WD and showed the highest and earliest increase in ABA levels (1.16 ng mg-1 dw, i.e., 3.4-fold increase compared to control). 'Grasevina' differed significantly from the other genotypes in SA content at both time points analyzed (six and nine days, 47.26 and 49.63 ng mg-1 dw, respectively). Proline level increased significantly under WD (up to 5-fold at day nine), and proline variation was not genotype driven. The expression of aquaporin genes (TIP2;1 and PIP2;1) was down-regulated in all genotypes, coinciding with the accumulation of ABA. The gene NCED1 (9-cis-epoxycarotenoid dioxygenase) related to ABA was up-regulated in all genotypes under stress conditions and served as a reliable marker of drought stress. This work suggests that the stress response in metabolite synthesis and accumulation is complex, treatment- and genotype-dependent.

Microstructure of Croatian Wild Grapevine (Vitis vinifera subsp. sylvestris Gmel Hegi) Pollen Grains Revealed by Scanning Electron Microscopy.

Wild grapevine (Vitis vinifera subsp. sylvestris Gmel Hegi) is dioecious with male and female plants, whereas domesticated grapevine is mostly hermaphrodite with self-fertile hermaphrodite flowers. The pollen morphology of wild grapevine has been poorly studied. There is no detailed palynological study of V. sylvestris in Croatia and neighboring countries. Here, scanning electron microscopy (SEM) was used to analyze the pollen of V. sylvestris from male and female individuals growing at two natural sites in Croatia. The selective APT3 marker was used to confirm the flower phenotype with the genetic background. SEM analysis showed that the pollen grains of V. sylvestris were isopolar and radially symmetrical, with foveolate perforated ornamentation, regardless of the flower type of the individuals. All male flowers were 3-colporate and prolate in shape, whereas female individuals varied from subprolate to spheroidal and had inaperturate pollen grains. Pollen shape, dimensions and exine ornamentation proved very informative, and here we address the most polymorphic traits in the analyzed V. sylvestris individuals. Principal component analysis (PCA) and clustering based on pollen morphology variables clearly differentiated individuals by their flower type, and no grouping specific to population was observed, pointing to the conserved pollen structure of V. sylvestris. The results indicate the need to continue the palynological study of V. sylvestris and serve as a good phenotypic basis for functional genetic studies on genes involved in pollen morphology and function.

Identification of powdery mildew resistance in wild grapevine (Vitis vinifera subsp. sylvestris Gmel Hegi) from Croatia and Bosnia and Herzegovina.

Wild grapevine (Vitis vinifera subsp. sylvestris) is widely recognized as an important source of resistance or tolerance genes for diseases and environmental stresses. Recent studies revealed partial resistance to powdery mildew (Erysiphe necator, PM) in V. sylvestris from Central Asia. Here, we report resistance to PM of V. sylvestris collected from different regions of Croatia and in seedling populations established from in situ V. sylvestris accessions. Ninety-one in situ individuals and 67 V. sylvestris seedlings were evaluated for PM resistance according to OIV 455 descriptor. Three SSR markers (SC47-18, SC8-071-0014, and UDV-124) linked to PM resistance locus Ren1 were used to decipher allelic structure. Nine seedlings showed resistance in in vivo evaluations while leaf disk assays revealed three PM-resistant accessions. One V. vinifera cultivar used as a control for PM evaluations also showed high phenotypic resistance. Based on the presence of one or two resistance alleles that are linked to the Ren1 locus, 32 resistant seedlings and 41 resistant in situ genotypes were identified in the investigated set. Eight seedlings showed consistent phenotypic PM resistance, of which seven carried one or two alleles at the tested markers. This study provides the first evidence of PM resistance present within the eastern Adriatic V. sylvestris germplasm.

Early Defoliation Techniques Enhance Yield Components, Grape and Wine Composition of cv. Trnjak (Vitis vinifera L.) in Dalmatian Hinterland Wine Region.

Defoliation and cluster thinning are of practical importance in a control of the grapevine source-sink balance, cluster architecture, microclimate and berry composition. Nevertheless, their effectiveness on wine composition is unexplored. In this work, the impacts of preflowering (T1), after berry set (T2), and veraison defoliation (T3) and cluster thinning (T4), on yield components, grape and wine composition of cv. Trnjak are given. Implemented techniques significantly reduced yield and affected grape and wine components in comparison to untreated control (C). Despite lowest number of clusters ensured by cluster thinning, defoliation at veraison had lowest yield. Defoliations improved cluster architecture parameters. Highest berry per se was in preflowering T1 and lowest at veraison T3 defoliation. Berries of T1 had lowest sugar content (19.47 °Brix) while T3 had highest (22.3 °Brix), and the reverse is seen in total acidity highest in T1 (6.12 g/L) and lowest in T3 (5.01 g/L). Wines of early defoliations (T1 and T2) had lowest alcohol and highest anthocyanin concentration. Both techniques applied at veraison produced wines with lower anthocyanins and flavonols than those obtained without any intervention (C). In conclusion, the early defoliations (T1 and T2) improve yield and wine composition of cv. Trnjak in the Mediterranean region of Croatia.

Genetic Structure and Relationships among Wild and Cultivated Grapevines from Central Europe and Part of the Western Balkan Peninsula.

The genetic diversity and relationship between wild (Vitis vinifera L. subsp. sylvestris (Gmel.) Hegi and cultivated (V. vinifera L. subsp. vinifera) grapevine in the western Balkan region and Central Europe have not been studied together previously, although this area has a rich viticultural past. Here, we studied wild grapevine populations sampled from their natural habitats in several countries of the western Balkan region and Central Europe. Their genetic diversity and structure were compared to cultivars that are traditionally in use in this region. A sample set of 243 accessions was genotyped at 20 nuclear microsatellite loci, including 167 sylvestris and 76 diverse vinifera cultivars. The genetic diversity of the wild grapevines was lower than that of cultivars by all genetic parameters. Both hierarchical and nonhierarchical clustering methods differentiated two main groups, indicating clear separation between wild and cultivated vines but also revealed clear gene flow between the cultivated and wild gene pools through overlaps and admixed ancestry values in the graphs. There was greater affinity to the wild grapes in Central European cultivars than in Balkan cultivars. Fine arrangement of the structure among cultivated grapevines showed differentiation among Central European and Balkan cultivars. These results confirm the divergence of wild grapes from vinifera and highlight the "crossroad" role of the western Balkan peninsula in the broader context of European viticulture.

Assessment of wild grapevine (Vitis vinifera ssp. sylvestris) chlorotypes and accompanying woody species in the Eastern Adriatic region.

The Eastern Adriatic region, encompassing Croatia and Bosnia and Herzegovina, is considered an important area of natural populations of wild grapevines (Vitis vinifera ssp. sylvestris). The wild grapevine arises in the Eastern Adriatic region in a contact zone of the EU-Mediterranean and the sub-Mediterranean characterized by typical karst relief. This study focuses on the chloroplast DNA (cpDNA) analysis of wild grapevines and the biodiversity of accompanying woody species to better understand the genetic variation of the sylvestris populations of the Eastern Adriatic region and to investigate how this variation fits within today's wild grapevine distribution in the European continent. The allelic variation at nine cpDNA microsatellite loci of wild individuals was used to characterize haplotype diversity in 53 individuals from four population sites. All individuals were grouped into two chlorotypes: A and D, D being the rare haplotype among wild populations on the European continent. In total, 52 woody plant species were identified. However, the studied vegetation structures have been affected by permanent human pressure on natural resources and the preservation status of the collection sites. Based on our results, we conclude that the investigated areas were probably shelter zones for wild grapevine preservation during the unfavorable glaciation era.