Identification Of Cold Resistance And RAPD Markers Linked To Cold Resistance Genes In Chinese Wild Vitis Species

The cold resistance of 151 materials which included 38 accessions of 10 Chinese Wild Vitis species, 5 cultivars of V. vinifera, 2 accessions of V. riparia, 1 cultivar of V. vinifera×V. Labrusca, 51 hybrids of V. vinifera×Chinese Wild Vitis species or hybrids and 54 hybrids of self crosses was studied by Leakage of Electrolytes, Water-lost Ratio and Bud Bursting in the winter and evaluated by subordinative function. And the molecular marker linked to cold resistance genes in the Chinese Wild Vitis was studied by Random Amplified Polymorphism DNA (RAPD) .The results are as following: 1.Subordinative levels of the V. amurensis of Chinese Wild Vitis and the V. riparia were more than 0.7,belonging to the most hardiness species. That of the V. yeshanensis, the V. adstricta and the V. piaseikii was between 0.5 and 0.7,they were more cold hardiness. While the hardiness species are the V. davidii , the V. davidii var. cyanocarpa (Gang) Sarg, the V. romanetii, the V. quinquanguliros Rehd., the V. qinlingensis, and the V. bashanica, whose Subordinative levels are 0.4~0.5. And Subordinative levels of the V. vinifera was less than 0.4, belonging to cold-susceptibility species. The hybrids Beichun (V. vinifera×V.amurensis) and Kyoho(V. vinifera×V. labrusca) were 0.572 and 0.541 respectively, and the cold resistance of a majority of interspecies progenies were between their parents, but a few of them were beyond their parents, and progenies of the two self crossings were also segregated from 0.005~0.525, 0.360~0.147. 2. The RAPD marker S241-691 linked to cold resistance gene of Chinese Wild Vitis was acquired by taking the hybridizations of Red Globe×Beichun as materials. and the marker S241-691 was further tested in other hybrids(Muscat Hamburg×Heilongjiang Seedling, Self crossing of Red Globe and Beichun), species(other species of Chinese Wild Vitis, V. vinifera, V. reparia). The results showed that S241-691 was universal in the Vitis. 3. The specific DNA fragment was retrieved from agarose gel and ligated into the pGEMR –T Easy Vector System, and then transformed into E. coli DH5-α. Sequencing of positive clones was carried out and the result showed that the S241-691 was 691bp. This provided a DNA evidence for Molecular marker-assisted Selection (MAS), obtaining Sequence Characterized Amplified Region (SCAR) marker, and cloning of the cold resistance gene through gene walking. 4. The restriction-sites of the sequences of S241 were analyzed by software 'Wingene231'. 52 restriction enzymes with recognition sites equal or longer than 6 bases had cutting sites on this sequences: EcoRI had no cutting sites, but HindIII had 2 cutting sites at 357bp and 521bp on this sequence. 5. ORFs(Open Reader Flames) of the sequences of markers linked to cold resistance gene were found in GenBank. There was an ORF in the sequence of S241-691 (from 317 to 448), length 132bp, coding 44 amino acids. The sequence of this amino acid was detected by BLASTp, but the expect value was insignificant, the practical function of this ORF needs to be tested further.