ISHS Acta Horticulturae 546: International Symposium on Molecular Markers for Characterizing Genotypes and Identifying Cultivars in Horticulture
ROUTINE EXTRACTION OF DNA FROM GRAPEVINE (Vitis ssp.) CANES AND ROOTS FOR VARIETY IDENTIFICATION BY RAPD-PCR |
| Authors: | T. Wolf, C. Ortlieb, K. Eimert, R. Ries |
| Keywords: | differentiation, DNA, mycorrhiza, roostock, variety, Vitis |
| Abstract:
Distinguishing grapevine varieties from wooden canes is particularly difficult. Traditional ampelographic methods, based on morphological characters of leaves and shoot tips, cannot be applied. On the other hand, DNA extraction from wooden canes for analysis with methods like RAPD-PCR, is difficult because of the presence of polyphenols and polysaccharides. Therefore, it is not usually used on a routine basis. Another problem is the variety determination of grafted rootstocks, where only roots can be sampled. A particular problem in this regard is the presence of VA mycorrhiza in most roots, which can yield additional bands. The aim of this study was to develop a routine technique for DNA extraction from roots and wooden canes of grapevines. DNA yield and the reproducibility of various extraction methods were compared. A modified method, described by Lodhi et al. (1994) proved to be most reliable and easy to use. The amount of DNA obtained was up to 230µg/g from wooden canes, with a purity of 2.0 to 1.8. Pure grapevine DNA could also be extracted from roots by the same method, if the roots were surface sterilised before extraction. The described method provides a simple, reliable protocol for the routine extraction of DNA. Over 50 samples per day could be processed by this method. DNA extracted in this way was further used for molecular genetic studies: 36 rootstock varieties can be routinely distinguished by RAPD-PCR with a single primer. | |
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