engleski

Grapevine variety determination from herbarium and archeological specimens

Genotyping old grapevine samples, in particular variety identification via microsatellite profiling, is not yet a routine. The success depends on several factors, the age of the investigated material being the most obvious one. In addition, the amount and integrity of DNA depends on specimen storage/preservation history. Moreover, contamination and fragmentation processes make the isolated ancient DNA (aDNA) a difficult template for PCR amplification. Three old grapevine specimens were investigated in this study. Two specimens derived from a 90-years old Croatian grapevine herbarium collection: cv. Tribidrag and cv. Brzamin. Both are Croatian indigenous grapevine varieties not present in contemporary germplasm collections anymore. The third was a 2000 years old archeological underwater grapevine specimen (woody parts). Several different approaches of DNA isolation were tested. Eventually, the use of a commercial plant DNA isolation kit showed to be the best choice in terms of avoiding contamination with contemporary grapevine DNA. The initial attempts to PCR-amplify standard grapevine microsatellite loci from isolated aDNA 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 years 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, six clones deriving from the cv. Tribidrag corresponded to six different grapevine chromosomes. Therefore, we used the cv. Tribidrag multiplied aDNA 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 obtained SSR profile 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 Zinfandel, which has been cultivated in Croatia, according to several historical documents, since the beginning of 15th century.

aDNA; Crljenak kastelanski (CK); whole genome amplification; Tribidrag; Zinfandel

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