SNP genotyping as powerful tool to discriminate clones of Vitis vinifera

Grapevine (Vitis vinifera L.) is one of the major fruit crop worldwide with thousands of cultivated varieties. Among the important Italian cultivars, 'Nebbiolo' is characterized by a wide genetic variability expressed by a high number of clones (i.e. vegetatively propagated lines of selected mother plants) registered in the National Catalogue. However, little morphological differences exist between clones and often it is very difficult to visually distinguish the different clones, and the Simple Sequence Repeats (SSRs) extensively used as markers, showed low or no success in discriminating clones. The aim of this on-going study is to identify a set of single nucleotide polymorphisms (SNPs) able to discriminate the registered 'Nebbiolo' clones, setting up an effective and rapid SNP genotyping protocol based on the TaqMan® probes technology. Three 'Nebbiolo' clones were sequenced by Illumina technology (CVT 71, CVT 185 and CVT 423) and processed reads were mapped against the PN40024 reference genome. SNPs were first detected using the GATK-UnifiedGenotyper tool with default parameters followed by filtering and by a calibration step. SNPs were validated as true positive by Sanger sequencing, and nine of the 11 SNPs confirmed to date were successfully used to perform TaqMan® genotyping assays in genomic DNA from 37 'Nebbiolo' clones. Combining results obtained from each single SNP assay, we classified the 'Nebbiolo' clones in six main groups linked to the geographical origin of accessions. Furthermore, these SNPs were tested in other grapevine cultivars, and interestingly in some case, these markers are polymorphic also in clones of 'Brachetto' and 'Nebbiolo rosè'. This work is in progress and the methodology will be applied in the vine supply chain to ensure a traceability from grape nurseries up to wine industry, providing a product of sure origin to the consumers. This research was funded by Fondazione Cassa di Risparmio di Cuneo: "Nebbiolo Genomics"; Fondazione Cassa di Risparmio di Torino: "Track W/Vine"; and Regione Piemonte: "MicroGenVine".