The RAPD analysis of several cultivars of grapevine (Vitis viniferaL.) and their clones

Abstract

Nine identification RAPD markers (MORAVCOVÁ et al. 2003) were used to distinguish 24 clones and grapevine cultivars. No polymorphism was detected among all the tested clones of Chardonnay, Pinot gris and Zweigeltrebe from Polešovice. Pinot noir, Pinot gris, Pinot blanc and Pinot Meunier were indistinguishable within clones, they also showed the identical RAPD profile within cultivars (except discussed sample No. 26). On the other hand, Auxerrois as a relative to cultivars of Pinot group showed unique patterns and may be classified as a different cultivar. Some irregularities within the cultivars of Pinot family from Oblekovice were also found, several of them gave different results from those expected: Pinot blanc sample 26 has the RAPD profile typical of Chardonnay. A new abnormal RAPD pattern as a marker of typical Chardonnay and Pinot profiles was observed in two cases. While RAPD banding patterns could not distinguish between the known clones, they were useful for distinguishing between phenotypically similar cultivars and for assessing the origins of cultivars thought to have originated as sports. Table 2.) DNA amplificationThe RAPD amplification was carried out in 25 µl of reaction mix containing 20 ng DNA, deionized sterile water, 0.5 U of Dy-Nazyme TM II DNA polymerase (Finnzymes), 1x Buffer for Dy-Nazyme TM II DNA polymerase (10 mM Tris HCl, pH 8.8; 1.5 mM MgCl2; 150 mM KCl and 0.1% Triton X-100), 0.2 mM of each dNTP (Promega), 0.4 µM of primer in length of 10 nucleotides (Operon technologies).DNA was denatured with one cycle of 94°C for 3 min, followed by 45 amplification cycles of 94°C for 1 min for denaturation, 36°C for 1 min for annealing of primers, 72°C for 1 min 30 s for extension. The final step of the program consisted in 72°C for 9 min for finishing the synthesis of all PCR products.Electrophoresis was performed in 1.5% of TAE agarose gels, stained with ethidium bromide. 100 bp DNA ladder (Biolabs, New England) was used as a molecular marker for approximation of the mass of DNA. RESULTS AND DISCUSSION Distinguishing of clones by RAPDOnly the intense bands that showed repeatable patterns in all cases were scored. All the used primers gave one or several quality and strong bands that are used for cultivar identification (MORAVCOVÁ 2004). Usually it was the combination of two or three primers discriminating the tested cultivars ( Table 2). The combination of primers can determine some varieties, but it is not able to discriminate between their clones. The RAPD pat- terns typical of the tested varieties were obtained in all cases (Fig. 4). The inability to separate clones may be attributable to the rarity of genetic changes responsible for clonal identity which would make the detection of these changes highly improbable (YE et al. 1998). RAPD study of Pinot familyPinot family and relative cultivars: Pinot noir, Pinot gris, Pinot blanc, Chardonnay, Pinot Meunier and Auxerrois from Oblekovice were the object of analysis.Our data support the opinion that the cultivars from the Pinot fam...