High resolution melting analysis of microsatellite markers applied to grapevine varietal fingerprinting throughout the wine production chain

Abstract

The accurate varietal identification is an essential requirement for every process involved in the exploitation of grapevine resources and derived products. The advancements achieved during the last years allowed the simultaneous analysis of multiple molecular markers capable of identifying grapevine varieties. Despite the establishment of a recommended set of nine microsatellite (SSR) markers for this purpose, their effective application with DNA extracted from must and wine samples remains a challenging task. This work aimed to develop High Resolution Melting (HRM) assays based on SSR markers applicable for grapevine varietal identification using leaf, must and wine samples. The grapevine varieties used were Cabernet Sauvignon, Touriga Franca, Touriga Nacional and Rufete. A total of 12 SSR markers were used to screen the varieties: nine markers recommended by the OIV (VVMD5, VVMD7, VVMD25, VVMD27, VVMD28, VVMD32, VVS2, VrZAG62 and VrZAG79) and three markers selected based on their repeat motif and length (VvIv35, VChr5c and VChr9a). The results from multiplex PCR amplification of DNA from wine samples revealed that these three markers performed better than the nine established SSR markers. HRM assays were developed targeting markers VvIv35, VChr5c and VChr9a, successfully discriminating the varietal composition in must DNA samples. Promising results were obtained using wine DNA, where assay HRM-VChr9a proved to have the highest discriminant power. The HRM-SSR assays need to be applied in a larger number of varieties, to explore its suitability for grapevine fingerprinting applications throughout the wine chain. Overall, the proposed small SSR makers can be more suitable for wine DNA analysis. The HRM-SSR approach presented here provides fast results, allowing the complete discrimination of varietal composition in must DNA. It also shows to be a promising tool to discriminate the varieties using wine DNA, a task usually hampered by the inherent complexity of wine samples.