conference schedule and program with abstracts - Horticulture ...

P-61
Grepevine variety determination from herbarium and arheological specimens
N. Malenica 1 , S. Simon 2 , E. Maletic 3 , I. Pejic* 2
1
University of Zagreb, Faculty of Science, Department of Molecular Biology, Zagreb, Croatia; 2
University of Zagreb, Faculty of Agriculture, Department of Plant Breeding, Genetics and
Biometrics, Zagreb, Croatia; 3 University of Zagreb, Faculty of Agriculture, Department of
Viticulture and Enology, Zagreb, Croatia
*Corresponding author: ipejic@agr.hr
Genotyping old grapevine samples, in particular variety identification via microsatellite profiling
is still far from being 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
DNA 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 15 th
century.
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