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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 s

Precise identification is crucial to study and for the valorisation of ancient grapevine varieties, with many synonyms and misidentifications hampering this task. SSR-Multiplex PCR and SNP genotyping combined with HRM were used, to identify 19 varieties present in an ancient Portuguese vineyard. The combination of the methodologies allowed the identification of 18 grapevines, from which 10 were correctly identified by ampelography and the remaining were misidentified. SSR and SNP data retrieved for one sample did not match any profile publicly available, however pedigree analysis suggests it is a new variety. One sample identified as Cerceal Branco presented an unexpected red berry. A sample identified as Malvasia Preta/Preta Roxa also presented a berry colour non coincident with its molecular identification. This suggests they might represent new somatic variants of existent varieties. Three HRM assays were performed to test its robustness in comparison to SSR/SNP results. Molecular identification was successful, revealing possible new variants. HRM revealed to be useful in identifying varieties by melting curve profile comparison against reference material. The results provide a better knowledge of a valuable pool of genetic patrimony: ancient varieties that represent a piece of history and heritage from an important winegrowing region.

Wine authenticity assessment throughout different winemaking steps is a topic of interest for consumers and producers. The correct identification of grapevine varieties is a main goal of different authenticity strategies. PCR amplification and capillary electrophoresis of microsatellite markers is the standard DNA-based method for varietal identification. Although successfully applied to vines, it reveals to be unreliable when applied to DNA extracted from must and wine, mainly due to the degradation and contamination of the DNA present in these matrices. High Resolution Melting has been proposed as a PCR-based alternative for this purpose, but it could benefit in targeting fragments of small size, when analysing must or wine DNA samples. This study aimed to discriminate five grapevine varieties using DNA extracted from samples with different complexity, using HRM analysis of three SNP markers1 identified in fragments of approximately 100 bp. We used Cabernet Sauvignon as the international reference and four Portuguese varieties - Touriga Franca and Touriga Nacional, two grapevine varieties commonly used in Portugal, and Donzelinho Tinto and Rufete, two less utilized autochthonous varieties. HRM assays were performed using gDNA extracted from leaf, monovarietal must and monovarietal wine samples. Using the melting profiles obtained from leaf gDNA as reference, varietal discrimination was remarkably achieved in must gDNA samples from all varieties in study. In relation to wine gDNA, Cabernet Sauvignon and Rufete samples showed to be the most troublesome in giving reproducible results. The varieties were clustered into different groups according to their genotype, in line with the data from the ICVV-SNP database. Considering the sensitivity and precision of this methodology, our results show that this approach is promising to analyse samples collected throughout the wine production chain.