Wild grapes of Armenia: unexplored source of genetic diversity and disease resistance.

The present study is the first in-depth research evaluating the genetic diversity and potential resistance of Armenian wild grapes utilizing DNA-based markers to understand the genetic signature of this unexplored germplasm. In the proposed research, five geographical regions with known viticultural history were explored. A total of 148 unique wild genotypes were collected and included in the study with 48 wild individuals previously collected as seed. A total of 24 nSSR markers were utilized to establish a fingerprint database to infer information on the population genetic diversity and structure. Three nSSR markers linked to the Ren1 locus were analyzed to identify potential resistance against powdery mildew. According to molecular fingerprinting data, the Armenian V. sylvestris gene pool conserves a high genetic diversity, displaying 292 different alleles with 12.167 allele per loci. The clustering analyses and diversity parameters supported eight genetic groups with 5.6% admixed proportion. The study of genetic polymorphism at the Ren1 locus revealed that 28 wild genotypes carried three R-alleles and 34 wild genotypes carried two R-alleles associated with PM resistance among analyzed 107 wild individuals. This gene pool richness represents an immense reservoir of under-explored genetic diversity and breeding potential. Therefore, continued survey and research efforts are crucial for the conservation, sustainable management, and utilization of Armenian wild grape resources in the face of emerging challenges in viticulture.

Dual domestications and origin of traits in grapevine evolution.

We elucidate grapevine evolution and domestication histories with 3525 cultivated and wild accessions worldwide. In the Pleistocene, harsh climate drove the separation of wild grape ecotypes caused by continuous habitat fragmentation. Then, domestication occurred concurrently about 11,000 years ago in Western Asia and the Caucasus to yield table and wine grapevines. The Western Asia domesticates dispersed into Europe with early farmers, introgressed with ancient wild western ecotypes, and subsequently diversified along human migration trails into muscat and unique western wine grape ancestries by the late Neolithic. Analyses of domestication traits also reveal new insights into selection for berry palatability, hermaphroditism, muscat flavor, and berry skin color. These data demonstrate the role of the grapevines in the early inception of agriculture across Eurasia.

Comparative Study on Grape Berry Anthocyanins of Various Teinturier Varieties.

The red-fleshed grape cultivars, called teinturier or dyer grapes, contain anthocyanins in both the skin and flesh. These phenolic compounds exhibit excellent coloring ability, and as antioxidants, they are important bioactive compounds in food crops. In this work, anthocyanin patterns of grape berries of fifteen teinturier varieties collected from the gene bank located at Pécs in the southwest of Hungary were compared. Anthocyanin profiles of numerous varieties originating from Hungary such as 'Bíborkadarka', 'Kármin', 'Kurucvér', and 'Turán' are reported for the first time. Anthocyanins extracted separately from the skin and juice were analyzed using high-performance liquid chromatography coupled with a photodiode array detector. For the identification of compounds, high-resolution orbitrap mass spectrometry was used. All in all, twenty-one anthocyanins were identified and quantified. We found that anthocyanin patterns differed significantly in the skin and juice for all investigated cultivars. For Vitis vinifera varieties, the predominant anthocyanin in the skin was malvidin-3-O-glucoside, while the main pigment in the juice was peonidin-3-O-glucoside. For the first time, a significant amount of diglucosides was detected in two Vitis Vinifera cultivars with a direct relationship. In general, the pigment composition of the skin was much more complex than that of the juice. The comparative study with presented patterns gives valuable and beneficial information from a chemotaxonomical point of view. Our results also help to choose the appropriate teinturier varieties with the desired anthocyanins for food coloring or winemaking purposes.

Tannin phenotyping of the Vitaceae reveals a phylogenetic linkage of epigallocatechin in berries and leaves.

BACKGROUND AND AIMS: Condensed tannins, responsible for berry and wine astringency, may have been selected during grapevine domestication. This work examines the phylogenetic distribution of condensed tannins throughout the Vitaceae phylogenetic tree. METHODS: Green berries and mature leaves of representative true-to-type members of the Vitaceae were collected before 'véraison', freeze-dried and pulverized, and condensed tannins were measured following depolymerization by nucleophilic addition of 2-mercaptoethanol to the C4 of the flavan-3-ol units in an organic acidic medium. Reaction products were separated and quantified by ultrahigh pressure liquid chromatography/diode array detection/mass spectrometry. KEY RESULTS AND CONCLUSIONS: The original ability to incorporate epigallocatechin (EGC) into grapevine condensed tannins was lost independently in both the American and Eurasian/Asian branches of the Vitaceae, with exceptional cases of reversion to the ancestral EGC phenotype. This is particularly true in the genus Vitis, where we now find two radically distinct groups differing with respect to EGC content. While Vitis species from Asia are void of EGC, 50 % of the New World Vitis harbour EGC. Interestingly, the presence of EGC is tightly coupled with the degree of leaf margin serration. Noticeably, the rare Asian EGC-forming species are phylogenetically close to Vitis vinifera, the only remnant representative of Vitis in Eurasia. Both the wild ancestral V. vinifera subsp. sylvestris as well as the domesticated V. vinifera subsp. sativa can accumulate EGC and activate galloylation biosynthesis that compete for photoassimilates and reductive power.

A 69 kbp Deletion at the Berry Color Locus Is Responsible for Berry Color Recovery in Vitis vinifera L. Cultivar 'Riesling Rot'.

'Riesling Weiss' is a white grapevine variety famous worldwide for fruity wines with higher acidity. Hardly known is 'Riesling Rot', a red-berried variant of 'Riesling Weiss' that disappeared from commercial cultivation but has increased in awareness in the last decades. The question arises of which variant, white or red, is the original and, consequently, which cultivar is the true ancestor. Sequencing the berry color locus of 'Riesling Rot' revealed a new VvmybA gene variant in one of the two haplophases called VvmybA3/1RR. The allele displays homologous recombination of VvmybA3 and VvmybA1 with a deletion of about 69 kbp between both genes that restores VvmybA1 transcripts. Furthermore, analysis of 'Riesling Weiss', 'Riesling Rot', and the ancestor 'Heunisch Weiss' along chromosome 2 using SSR (simple sequence repeat) markers elucidated that the haplophase of 'Riesling Weiss' was inherited from the white-berried parent variety 'Heunisch Weiss'. Since no color mutants of 'Heunisch Weiss' are described that could have served as allele donors, we concluded that, in contrast to the public opinion, 'Riesling Rot' resulted from a mutational event in 'Riesling Weiss' and not vice versa.

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.

PhenoApp: A mobile tool for plant phenotyping to record field and greenhouse observations.

With the ongoing cost decrease of genotyping and sequencing technologies, accurate and fast phenotyping remains the bottleneck in the utilizing of plant genetic resources for breeding and breeding research. Although cost-efficient high-throughput phenotyping platforms are emerging for specific traits and/or species, manual phenotyping is still widely used and is a time- and money-consuming step. Approaches that improve data recording, processing or handling are pivotal steps towards the efficient use of genetic resources and are demanded by the research community. Therefore, we developed PhenoApp, an open-source Android app for tablets and smartphones to facilitate the digital recording of phenotypical data in the field and in greenhouses. It is a versatile tool that offers the possibility to fully customize the descriptors/scales for any possible scenario, also in accordance with international information standards such as MIAPPE (Minimum Information About a Plant Phenotyping Experiment) and FAIR (Findable, Accessible, Interoperable, and Reusable) data principles. Furthermore, PhenoApp enables the use of pre-integrated ready-to-use BBCH (Biologische Bundesanstalt für Land- und Forstwirtschaft, Bundessortenamt und CHemische Industrie) scales for apple, cereals, grapevine, maize, potato, rapeseed and rice. Additional BBCH scales can easily be added. The simple and adaptable structure of input and output files enables an easy data handling by either spreadsheet software or even the integration in the workflow of laboratory information management systems (LIMS). PhenoApp is therefore a decisive contribution to increase efficiency of digital data acquisition in genebank management but also contributes to breeding and breeding research by accelerating the labour intensive and time-consuming acquisition of phenotyping data.

Genetic Diversity of Armenian Grapevine (Vitis vinifera L.) Germplasm: Molecular Characterization and Parentage Analysis.

Armenia is an important country of origin of cultivated Vitis vinifera subsp. vinifera and wild Vitis vinifera subsp. sylvestris and has played a key role in the long history of grape cultivation in the Southern Caucasus. The existence of immense grapevine biodiversity in a small territory is strongly linked with unique relief and diverse climate conditions assembled with millennium-lasting cultural and historical context. In the present in-depth study using 25 nSSR markers, 492 samples collected in old vineyards, home gardens, and private collections were genotyped. For verification of cultivar identity, the symbiotic approach combining genotypic and phenotypic characterization for each genotype was carried out. The study provided 221 unique varieties, including 5 mutants, from which 66 were widely grown, neglected or minor autochthonous grapevine varieties, 49 turned out to be new bred cultivars created within the national breeding programs mainly during Soviet Era and 34 were non-Armenian varieties with different countries of origin. No references and corresponding genetic profiles existed for 67 genotypes. Parentage analysis was performed inferring 62 trios with 53 out of them having not been previously reported and 185 half-kinships. Instability of grapevine cultivars was detected, showing allelic variants, with three and in rare cases four alleles at one loci. Obtained results have great importance and revealed that Armenia conserved an extensive grape genetic diversity despite geographical isolation and low material exchange. This gene pool richness represents a huge reservoir of under-explored genetic diversity.

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.

Color Intensity of the Red-Fleshed Berry Phenotype of Vitis vinifera Teinturier Grapes Varies Due to a 408 bp Duplication in the Promoter of VvmybA1.

Grapevine (Vitis vinifera) teinturier cultivars are characterized by their typical reddish leaves and red-fleshed berries due to ectopic anthocyanin formation. Wines of these varieties have economic importance as they can be used for blending to enhance the color of red wines. The unique and heritable mutation has been known for a long time but the underlying genetic mechanism still is not yet understood. Here we describe the association of the red-fleshed berry phenotype with a 408 bp repetitive DNA element in the promoter of the VvmybA1 gene (grapevine color enhancer, GCE). Three different clones of 'Teinturier' were discovered with two, three and five allelic GCE repeats (MybA1t2, MybA1t3 and MybA1t5). All three clones are periclinal chimeras; these clones share the same L1 layer, but have distinct L2 layers with different quantities of GCE repeats. Quantitative real time PCR and HPLC analysis of leaf and berry samples showed that the GCE repeat number strongly correlates with an increase of the expression of VvmybA1 itself and the VvUFGT gene regulated by it and the anthocyanin content. A model is proposed based on autoregulation of VvmybA1t to explain the red phenotype which is similar to that of red-fleshed apples. This study presents results about the generation and modes of action of three MybA1t alleles responsible for the red-fleshed berry phenotype of teinturier grapevines.

Unraveling the genetic origin of 'Glera', 'Ribolla Gialla' and other autochthonous grapevine varieties from Friuli Venezia Giulia (northeastern Italy).

'Glera' and 'Ribolla Gialla' are the most economically relevant local grapevine cultivars of Friuli Venezia Giulia region (north-eastern Italy). 'Glera' is used to produce the world-renowned Prosecco wine. 'Ribolla Gialla' cultivation is constantly increasing due to the strong demand for sparkling wine and is the most important variety in Brda (Slovenia). Knowledge of local varieties history in terms of migration and pedigree relationships has scientific and marketing appeal. Following prospections, genotyping and ampelographic characterization of minor germplasm in Friuli Venezia Giulia, a further research was developed to understand the parentage relationships among the grapevine varieties grown in this region. An integrated strategy was followed combining the analysis of nuclear and chloroplast microsatellites with the Vitis 18k SNP chip. Two main recurrent parents were found, which can be regarded as "founders": 'Vulpea', an Austrian variety parent-offspring related with at least ten Friuli Venezia Giulia cultivars, among them 'Glera', and 'Refosco Nostrano', first degree related with other six Friuli Venezia Giulia varieties. 'Ribolla Gialla' was shown to be another member of the impressively long list of offspring derived from the prolific 'Heunisch Weiss'. Combining molecular markers and historical references was a high-performance strategy for retracing and adjusting the history of cultivars.

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.

Extended diversity analysis of cultivated grapevine Vitis vinifera with 10K genome-wide SNPs.

Grapevine is a very important crop species that is mainly cultivated worldwide for fruits, wine and juice. Identification of the genetic bases of performance traits through association mapping studies requires a precise knowledge of the available diversity and how this diversity is structured and varies across the whole genome. An 18k SNP genotyping array was evaluated on a panel of Vitis vinifera cultivars and we obtained a data set with no missing values for a total of 10207 SNPs and 783 different genotypes. The average inter-SNP spacing was ~47 kbp, the mean minor allele frequency (MAF) was 0.23 and the genetic diversity in the sample was high (He = 0.32). Fourteen SNPs, chosen from those with the highest MAF values, were sufficient to identify each genotype in the sample. Parentage analysis revealed 118 full parentages and 490 parent-offspring duos, thus confirming the close pedigree relationships within the cultivated grapevine. Structure analyses also confirmed the main divisions due to an eastern-western gradient and human usage (table vs. wine). Using a multivariate approach, we refined the structure and identified a total of eight clusters. Both the genetic diversity (He, 0.26-0.32) and linkage disequilibrium (LD, 28.8-58.2 kbp) varied between clusters. Despite the short span LD, we also identified some non-recombining haplotype blocks that may complicate association mapping. Finally, we performed a genome-wide association study that confirmed previous works and also identified new regions for important performance traits such as acidity. Taken together, all the results contribute to a better knowledge of the genetics of the cultivated grapevine.