| Grape is one of the most important economic crops cultivated widely in the world, germplasm resources and variation type is extremely rich. In the long-term process of natural selection and artificial cultivation, the genetic background of the grape variety become complicated. To breed high quality grape varieties, it is necessary to analyse the genetic diversity and genetic basis of the variety. In this paper, 137 grape germplasm resources collected from grape germplasmresources garden in Shanshan and Turpan as experimental materials, using ISSR molecular markers to analyze the genetic diversity. Our aim is to identificate and classificate the grape germplasm resources and homonym or synonyms varieties by using effective molecular marker technology, and to provide guidance for cross breeding and grape industry healthy and sustainable development. The main results are as follows:1. Formulation of CTAB extraction buffer was improved, the CTAB concentration was increased up to 3%, PVP40 concentrations up to 4%, and β-mercaptoethanol concentration up to 2%, while other ingredients unchanged. This study shows that the genomic DNA extracted from grape leaves by the improved CTAB buffer in high quality and purity could satisfy the requirement of ISSR analysis, and compared with the traditional extraction method, this modified method can effectively reduce the contamination of phenolic compound, polysaccharide and other compounds during DNA extraction process.2. In this study the ISSR-PCR reaction system was optimized by single-factor test, including dNTPs, Taq DNA polymerase, DNA templates and primers, finally, orthogonal design was applied for optimize the ISSR-PCR amplification system. The optimized ISSR-PCR amplification system contains 0.225 mmol/L dNTPs, 1.0 U TaqDNA polymerase, 1.0 μmol/L primer, 30 ng of template DNA in total 20 μL of reaction solution. Amplification using this ISST-PCR reaction system could obtain clear and stable bands, which could be used for analyzing grape genetic diversity and relationships of germplasm resources.3. The suitable amplification polymorphic primers for analysis of grape germplasm genetic diversity were screened. 17 out of 50 random primers were selected for the amplification reaction, and showed highly efficient and polymorphic, then used for ISSR analysis of 137 grape materials. A total of 166 bands were obtained by amplification by the 17 polymorphic primers, among which 109 bands were found to be polymorphic, polymorphism rate reached 65.66 percent, the average amplified bands of each ISSR primers was 9.76, among which average polymorphic bands was 6.41.4. We analyzed the genetic diversity and genetic relationships of 137 grape germplasm. Clustering results showed that the similarity coefficient ranged from 0.22 to 1.00. 137 grape varieties geneticly divided into four groups when the similarity coefficient was 0.60. The first group included 127 V.vinifera Land five varieties of hybrids of V. vinifera and V. Labrusca. The second group contains four hybrids of V. vinifera and V. Labrusca. The third group contains only one V.vinifera L.. The fourth group has only one Vitis labrusca L.. Most of the clustering analysis in this study are consistent with the traditional family pedigree, most of the V.vinifera L., hybrids of V. vinifera and V. Labrusca, Vitis labrusca L. were clustered into same group respectively, but still a few of the clustering results are differente from the pedigree to a certain extent. Among them, Fu Lieao, Riesling, Gernischt, Deng Wasi Roses and other grape varieties clustered together which are resistant to biotic or abiotic stress, can be used as resistance breeding material. Horse Milk, White Racha, Centennial Seedless and other high yield grape varieties clustered together, can be used as high-yield grape breeding material.5. Study results show that Xinjiang local grape originated from V.vinifera L.. The Xinjiang local grape varieties clustered with V.vinifera L. populations in the same group, suggested the Xinjiang local grape varieties are not native origin, but spread from outside Xinjiang, gradually evolved from V.vinifera L..Grape varieties we used for analysis showed significantly different genetic diversity, our results could provide reference for germplasm resources cultivation, preservation and utilization of Xinjiang. |
