engleski

Grepevine variety determination from herbarium and arheological specimens

Genotyping old grapevine samples, in particular variety identification via microsatellite profiling is still far from being a routine. The successful identification 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 conditions of samples storing. Contamination and fragmentation processes make the isolated ancient DNA (aDNA) a rather difficult template for PCR amplification. Three old grapevine samples were investigated in this study. Two samples were taken from approximately 90-years old herbarium of cv. Tribidrag, a Croatian autochthonous grapevine variety not anymore present in the germplasm collections and one was approximately 2000 years old underwater archeological grapevine specimen (woody parts). Several different approaches of aDNA isolation were tested. However, the use of a commercial plant DNA isolation kit showed to be the best choice in terms of yield and prevention of possible contamination with temporary grapevine DNA. The initial attempts to amplify standard grapevine microsatellite loci by standard PCR protocol failed, most likely due to low copy number of template DNA. Therefore, the whole genome amplification (WGA) kit was successfully applied to overcome this problem. The WGA approach worked in case of one herbarium sample and the archeological specimen. DNA fragments ranging between 100 and 500 bp were cloned and sequenced. While the isolated DNA from the underwater archeological sample was proved to be from different origins (not from grapevine), indicating contamination of the specimen, the six sequenced clones from the herbarium specimen corresponded to six different grapevine chromosomes. Therefore, we used the WGA-processed herbarium sample and managed to PCR-amplify the VVS2 locus, thereby demonstrating the proof of principle. Eventually, we successfully genotyped the same template using six standard microsatellite loci (VVS2, VVMD5, VVMD7, VVMD27, VvZAG62 and VvZAG79). The obtained SSR profile was identical to Zinfandel/Primitivo/Crljenak Kastelanski, except for the VVS2 locus which was homozygous (141/141) instead of heterozygous (131/141). In the light of these results we hypothesize on the origin of Zinfandel as an old Croatian variety under the historical name Tribidrag, which was being used in documents dating as late as 15th century.

aDNA; grapevine; SSR

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