Insight into grapevine (Vitis vinifera) genetic resources from Caucasus using an integrative approach

European grapevine, Vitis vinifera L., is one of the most important tree crops worldwide for both economic value and cultivated area. The social-economic significance of Vitis vinifera led to the development of new cultivation techniques in order to produce hybrids characterized by the resistance to diseases like downy and powdery mildews typical of american and asian vitis and at the same time characterized by the quality of European grapevines. During the last years, a major sensitivity to environmental issues along with market demand led the research towards the crops genetic improvement and the greater attention over the biodiversity conservation. The advent of new technologies and the improvement of crop farming and winemaking made the wine growing activity very profitable. Indeed, traditional methods of breeding, which include the employment of rootstocks for the resistant hybrids production, together with the advent of new techniques such as marker assisted selection (MAS) and the chance to isolate gene of agronomic interest throughout experimental populations (QTL mapping) or natural populations (association mapping, GWAS) represent, to date, the best method to produce and study grapevine. Recently, a major attention was turned to the exploitation of cultivated and wild genetic resources from Caucasus, primary center of grapevine domestication, characterized by a high genetic diversity. The present research project was focused on the characterization of a previously unexplored Caucasian germplasm collection composed by 25 accession of Vitis vinifera, both cultivated and wild, with a low susceptibility to downy mildew based on Toffolatti et al., 2016 [1] evaluations. In order to exclude any possible introgression of resistant traits from American and Asian species, a screening with markers linked to the major Rpv loci has been performed, apparently confirming the absence of introgressions. A first phylogenetic analysis, performed with 21 microsatellites, allowed to compare these accessions with a collection of cultivars of various geographic origin previously described (Vv1, Vv2, Vv3, Vv4) together with other Georgian cultivars and some rootstocks classifying them in V. vinifera clade. Enlarging the databases with European, Caucasian, Israelian, American and Asian accessions, a second classification allowed to place these genotypes within the Georgia region, characterized by a strong genetic diversity, a high number of alleles and private alleles. PCoA analysis and STRUCTURE analysis identified a certain amount of admixture between wild and cultivated grapevine species. In order to deepen these results, a new phylogenetic analysis based on plastid markers described in literature has been performed to classify the entire collection on the base of chlorotype. In agreement with obtained data, a few Georgian cultivars, particularly interesting for their low susceptibility to downy mildew, showed a high homozygosity and a chlorotype similar to the ones of European sylvestris, suggesting ancestral features typical of hybrids between wild and cultivated varieties. Among these ancestral grapevine accessions a Georgian cultivar, known as ‘Mgalobshivili N.’, was mainly deepened for its low susceptibility to downy mildew (Toffolatti et al., 2016) [1]. In order to study this tolerance, a genetic map derived from a segregating mapping population, composed by 154 offspring and obtained by selfing, was constructed. Despite the high homozygosity, all the 19 chromosomes have been reproduced, using 177 molecular markers (SSRs and SNPs). The obtained map has been integrated with phenotypic data to perform a preliminary QTL analysis. The population, maintained in pots, has been evaluated after artificial infections with P. viticola during different years: 2013, 2014, 2015 (may and july) and 2016. However, plants behavior has not been reproducible in the various seasons and it should be evaluated in conditions of greater stability on adult population. QTL analysis detected three QTLs in 2014 on chromosome 1, 7 and 11 and a minor QTL in 2016 corresponding to Rpv11 locus. A fine mapping has been performed on these chromosomes in order to restrict the region of interest. Within these regions, genes mainly involved in ethylene pathway have been identified. Moreover, an integrative approach based on the study of gene expression has been started. Two genotypes of the offspring, which showed a contrast behavior to P. viticola infection among the years have been selected and, together with the parental line, have been micropropagated, starting from internodes. In the time frame of seven months seedlings, grown enough to carry out a gene expression experiment, have been obtained. In order to test the efficiency of inoculum and the response to infection of each genotype, a few phenotypic evaluation on leaf discs, indicating the differences among the rates of sporulation, were carried out. Subsequently, the same inoculum was tested on the different biological replicas of the whole plants and various samplings were performed at four time points post infection (0hpi, 8hpi, 24hpi and 48hpi). If the phenotypic evaluation on leaf discs defined a clear differentiation among genotypes behaviors, the ones performed on the entire plant highlighted significant differences among biological replicas which influenced also the further gene expression. Once obtained reverse transcribed cDNAs, samples were tested with candidate genes, chosen both on the base of detected QTL regions and literature reports mainly based resistance traits studied in wild species, given the ancestral features of the cultivar. Each experiment was carried out in qRT-PCR and statistical test like ANOVA and Tukey confirmed the significance levels. Results revealed, in the most resistant accession, an HR response associated to a considerable induction of genes involved in ethylene pathway. Moreover the high expression of MYB and VvWRKY transcription factors marked out an immune response linked with the production of phytoallexins and secondary metabolites. In conclusion, this research work allowed to characterize a V. vinifera germplasm collection with a low susceptibility to downy mildew and originated from Georgia, the cradle of grapevine domestication, composed by a few cultivar, phylogenetically very similar to European sylvestris varieties. This suggested the probable “hybrid” nature of these accessions marked out by ancestral features. Further analyses on ‘Mgalobshivili N.’ suggested that this interesting phenotype (Toffolatti et al., 2016) [1] could be controlled by genes which induce production of phytoallexins therefore strictly associated to a basal immunity system influenced by ethylene production. This work allowed to stress the presence of interesting resilience features in cultivated grapes which maintained ancestral traits and which, presumably, assumed a central role in the domestication process, improving the plant’s defense process from the pathogen.