Grapevine cultivars are vegetatively propagated to maintain their varietal characteristics. However, long periods of cultivar multiplication result in the accumulation of spontaneous somatic mutations that can differ among clonal lines. Here, we explored this intravarietal genetic diversity to trace back the origin and dissemination history of ‘Tempranillo Tinto’, the third most cultivated wine grape variety worldwide. A stringent somatic variant calling over whole-genome resequencing data of 35 ‘Tempranillo Tinto’ grapevines from seven Iberian winemaking regions revealed 158 somatic single nucleotide variants (SNVs) shared by some of the plants. Among them, 56 highly informative SNVs were used to custom-design a high-throughput intravarietal genotyping assay, which was validated and used to analyze 185 vines representing a broader geographic distribution. Phylogenetic analyses revealed three major clonal lineages in ‘Tempranillo Tinto’ that grouped the samples according to their geographic origin. By inferring the ancestral SNV alleles in ‘Tempranillo Tinto’ from whole-genome resequencing data of its parents, we determined the Ebro River Valley in Northeast Spain as the most likely birthplace of the cultivar. Derived alleles revealed one major historical human-mediated westward dissemination route from this original site towards the winemaking regions following the Duero River Valley and then, to the South in Portugal. Clonal lineages also revealed the polyphyletic nature of somatic variant traits of interest for grape and wine quality production under climate change conditions. Our findings elucidate the origin and historical dispersal of ‘Tempranillo Tinto’ and underscore genomic strategies for advancing clonal improvement to ensure the sustainability of valuable traditional grapevine cultivars.

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