Grapevine Variety Determination from Herbarium and Archeological Specimens

Genotyping old grapevine samples, in particular cultivar identification via microsatellite profiling, is not yet routine. Success depends on several factors, the age of the investigated material being the most obvious. In addition, the amount and integrity of DNA depends on specimen storage/preservation history. Moreover, contamination and fragmentation processes make the isolated DNA a difficult template for PCR amplification. Three old grapevine specimens were investigated in this study. Two derived from a 90-year-old Croatian grapevine herbarium collection: cv. 'Tribidrag' and cv. 'Brzamin'. Both are indigenous Croatian grapevine cultivars not present in contemporary germplasm collections. The third was a 2000-year-old archeological underwater grapevine specimen. Several different approaches for DNA isolation were tested. A commercial plant DNA isolation kit was the best choice in terms of avoiding contamination with contemporary grapevine DNA. Initial attempts to PCR-amplify standard grapevine microsatellite loci from isolated DNA did not work satisfactory. Therefore, a whole genome amplification (WGA) kit was applied to overcome this problem. The WGA performed well with the cv. 'Tribidrag' herbarium sample and the 2000-year-old archeological specimen. While the amplified, cloned and sequenced DNA from the archeological specimen was from origins other than grapevine, indicating contamination of the specimen, clones deriving from the cv. 'Tribidrag' corresponded to different grapevine chromosomes. Therefore, we used the cv. 'Tribidrag' DNA for successful amplification of the VVS2 microsatellite locus by PCR, thereby demonstrating the proof of principle. Eventually we genotyped the cv. 'Tribidrag' specimen at six standard microsatellite loci. The SSR profile obtained was identical to that of 'Zinfandel'/'Primitivo'/'Crljenak kastelanski'. These results are in line with the previously proposed Croatian origin of 'Zinfandel'. This time we identified the name 'Tribidrag' as the most ancient name for cv. 'Zinfandel', which has been cultivated in Croatia, according to several historical documents, since the beginning of the 15th century.