Discovery and genotyping of DNA sequence variations in grape.

The common grapevine, Vitis vinifera subsp. sativa L. (2n=6x=38) is one of the major fruit crops worldwide in terms of economic value and cultivated area. In the panorama of Italian viticulture and oenology, Campania region is characterized by a rich biodiversity, which includes a distinctive number of very ancient varieties (more than one hundred years old). This biodiversity needs to be preserved to protect producers and consumers from frauds, and on the counterpart is an essential genetic resource for breeding. The recent sequencing of grape genome represents an essential step forward for structural and functional genomics studies. In this context, one major objective of this research was to investigate the molecular basis of Campania grape germplasm. Microsatellite and retrotransposon-based markers were used in a collection of 62 grapevines. Homonymies and synonymies were found, pointing out that molecular evaluations can provide further insights into genetic structure and differentiation of Vitis germplasm accumulated during centuries of cultivation and selection. Indeed, Procidana and Coglionara are grapes cultivated and sampled on the island of Ischia and we classified them as synonymous. In our effort, we were able to identify 19 grape-specific alleles, which represent useful tools for many purposes such as traceability, typicity preservation and varietal identification. Within the collection analyzed, Aglianico del Taburno is a model for astringency and the strong resveratrol content confers high positive nutriceutical properties to its grapes and wines. For this reason, a transcriptomic analysis of key genes of the phenyl-propanoid pathway was carried out in different tissues of the berry during fruit maturation. This part of the study was correlated with the spectrophotometric assay of total phenols, flavans, anthocyanins and tannins detected in skin and berry. Anthocyanin analysis revealed the highest amount of these molecules in ripening berry skin and the lowest in seed. Correlating the transcriptomic and chemical data, the key-genes able to control production of poly-phenolic compounds in berry tissues were determined. Poly-phenolic compounds are key molecules of grapevine defense and in particular of PTI (PAMP-Triggered Immunity). Indeed, they are produced as defense compounds after pathogen recognition. Nowadays, much has been written about defense mechanisms and chitin perception in Arabidopsis, but little is known in grapevine. Considering powdery mildew as the most destructive disease of grapevines world-wide, an intense structural and functional study of PTI-involved grape genes was conducted. Grapevines are highly resistant to many powdery mildews of other species, called non-adapted (e.g. E. cichoracearum). Using Arabidopsis as model species, we identified and functionally studied the grape genes involved in the perception of chitin in the epidermal cells apoplast, as LysM-RLK, and in the intracellular signal transduction. Our findings suggest that three candidates, VvLYK1, VvLYK2 and VvLYK3, can play a different role in chitin signaling in V. vinifera. We demonstrated that a total resistance to powdery mildew was re-established in the A. thaliana cerk1 mutant only by VvLYK1, but not by VvLYK2 and VvLYK3. Further experiments also suggested an inactive VvLYK2 gene and an active but unknown function of VvLYK3. Regarding the signal transduction pathways, we found that VvMAPK3 and VvMAPK6 are not involved in the biotic response, since they both did not show expression differences in powdery mildew infection and chitin treatment time courses. The transcriptomic data generated in this study allowed us to identify VvWRKY24 as the positive regulatory factor of grape defense to powdery mildew, but excluded VvWRKY16. Indeed, VvWRKY24 had the strongest up-regulation in response to powdery mildew infection and chitin treatment. To better understand the possible interactions between two or more genes and regulatory mechanisms, an over-expression and silencing transformation was conducted. The transformations gave rise to few positive embryos after ten months from co-cultivation with Agrobacterium. This result is strictly correlated with the known recalcitrance of grapevine to transformation. As far as we know, this represents the first complete structural and functional genomics study in grapevine aimed to the identification of the major genes involved in chitin perception and signal transduction.