Study On Genetic Diversity And Construction Of Core Collections About Vitis Amurensis Rupr.germplasm Resources By SSR Markers

In Vitis genus, Vitis amurensis Rupr. is one of the important wild fruit germplasm resources, which has the strongest cold hardiness and stronger resistance to many diseases, so it is a very useful resource in grape breeding. Vitis. germplasm resources are very rich, including 70 species,29 varieties in China. The early study about V. amurensis focused on its cultivation, but the research in molecular aspects started later, which seriously affected the utilization of V. amurensis germplasm resources and the innovative exploration. In recent years, as the study on cultivation physiology of V. amurensis has advanced stepwise and the research methods on molecular have developed rapidly, some research results on molecular level about V. amurensis germplasm resources have been reported, but the reports about genetic diversity by SSR molecular markers on the resources are rare and the experimental materials used are relatively less. In this paper, the SSR molecular marker technique was employed in the analysis of genetic diversity and construction of core collections about V. amurensis germplasm resources, and the research results clarified the phylogenetic relationships and taxonomic status within the species, and displayed the preliminary core collections of V. amurensis germplasm resources. It provided the molecular basis for the further usage of V. amurensis germplasm resources in grape breeding and provided the scientific basis for the conservation, evaluation and identification of V. amurensis resources. The main results are as follows:1. The SSR reaction system about V. amurensis was optimized.19 pairs of SSR primers were screened out and their appropriate annealing temperatures were established. The leaves of Zuoshaner (V. amurensis Rupr.) were used as the experimental material in the system optimization, and the main factors of SSR reaction system were optimized. Effects of template concentration, primer concentration, dNTP concentration, TaqDNA polymerase on the amplification were studied in this paper. The results showed that appropriate concentration and amount of each component in 20μl SSR system were:10×PCR buffer 2μl,3μmol/L primer 2μl, 30ng/μl-1 template DNA 1μl, 1mmol/L dNTP 4μl,2.5U/μL-1 TaqDNA polymerase 0.4μl, ddH2O 10.6μl.By this SSR system,3 primers that were screened out from 50 primers amplified the clear and reproducible bands from 5 accessions of V. amurensis, which confirmed the applicability and stability of the system.19 pairs of SSR efficient primers were selected from 256 primer pairs, including Vmc9a2.1,VVMD31,VVMD19,UDV-017,UDV-025,UDV-033,UDV-041,UDV-048,UDV-050,UDV-054,UDV-059,UDV-060,UDV-067,UDV-088,UDV-134,VMC6f1,VVS29,VMC7b1,VMC7b3. About the appropriate annealing temperature of each primer, Vmc9a2.1 was 66℃, UDV-134 51℃, VMC6f1 59℃, VVS29 58℃, VMC7bl and VMC7b3 were 61℃, and the others were 56℃.2. The genetic diversity of 360 accessions of V. amurensis germplasm resources were studied and analyzed.18 primers were selected from 256 primers for SSR amplification on V. amurensis germplasm resources. The amplified bands were clear and the polymorphism was very high. A single primer amplified 4-13 bands, and the average was 9.44. Amplified length about DNA fragments was between 150-1000bp, and the majority was 200-750bp.18 primers totally amplified 170 bands, in which 167 bands were polymorphic and the polymorphic percentage was 98.2%. Of 360 V. amurensis resources, the average allele frequency (pi) was 0.106169, the effective number of alleles (Ae) was 5.240862, the Shannon's diversity index (Ⅰ) was 1.778051. In all 360 resources, some cultivars or strains produced 5 unique bands, which accounted for 2.95%. The analysis of genetic diversity among populations showed that:allele frequency (pi) and Shannon's diversity index (I) of the resources from Liaoning province were highest, followed by Jilin-originated resources, and in Heilongjiang-originated resources they were lowest. The effective number of alleles (Ae) of population from Jilin province was highest, and the number was 5.33388; the Ae of population from Heilongjiang province was followed by, and the number was 5.076471; the Ae of population from Liaoning province was lowest, and the number was 5.03173. In Nei-Li genetic distance, the cluster analysis was conducted according to SSR fingerprints from 360 accessions of V. amurensis germplasm resources, and it was found that the genetic similarity coefficient about the whole resources was 0.56 to 0.99 and the average value was 0.775. At the point of genetic similarity coefficient 0.66,360 accessions of V. amurensis resources could be divided into 6 groups; and in the sixth group, at the point of the genetic similarity coefficient 0.72, the resources could be further divided into 2 groups in addition to small part of resources. 3. The construction of core collections was finished about 360 accessions of V. amurensis germplasm resources. Using the 18 SSR primers which could amplify clear and stable bands, the analysis of genetic diversity was carried out and completed to the 360 V. amurensis germplasm resources, and cascading compression method was used to choose the core collections. And using 5 groups with different number of resources, the number of alleles, allele frequencies, effective number of alleles, Nei's genetic diversity and Shannon's information index and other parameters were compared.79 accessions of V. amurensis germplasm resources were selected as the core collections finally, and the sampling ratio of core collections was 21.9% of the initial resources. With DPS software, T test was conducted to the effective number of alleles, Nei's genetic diversity and Shannon's information index of the initial resources and the core collection resources respectively. The number of core collections retained 21.9% of the initial samples, and the retention rate of alleles, effective number of alleles, Nei's genetic diversity, Shannon's information index were 94.1%,106.3%, 101.9%,102.2% respectively. The core collections had a good representative of the initial germplasm resources. The core collections not only contained the wild and cultivated varieties, but also contained some resources with special characters, such as bisexual flower varieties, female plants and male plants. The core collections had very good representative, heterogeneity and diversity.4. The genetic relationship between V. amurensis and other grape resources was analyzed. The genome polymorphism analysis was conducted in the 20 materials by SSR markers, and 19 primers were screened out from 256 primers and were used to SSR amplification. Amplified bands were clear and the polymorphism was rich. Total of 144 bands were amplified and all of them were polymorphic (the percentage of polymorphism was 100%). A single primer amplified 3-12 bands, and the average was 7.58. Amplified product length was between 150-1000bp, and the majority DNA fragments were 200-750bp. According to the results of SSR amplification, the analysis of Jaccard similarity coefficient was conducted with NTSYSpc2.10e software. The genetic similarity coefficient of 20 grape resources was 0.63 to 0.90, and the average was 0.765. By UPGMA cluster analysis,20 resources could be divided into 4 major groups at the point of genetic similarity coefficient 0.665. Category 1 included European grapes, American hybrids and Euro-American hybrids. Category 2 included V. davidii Foex, V. quinquangularis Rehd., V. piasezkii Maxim., V. hancockii Hance, V. thunbergii Sieb.&.Zucc and V. pseudoreticulata W. T. Wang, all of which grew in southern China. Category 3 contained the hybrids of V. amurensis×V. vinifera. Category 4 constituted of V. amurensis. The results showed that the genetic relationship among V. amuensis with V. vinifera, Euro-American hybrids and American hybrids had far genetic distance, but the relationship among V. vinifera, Euro-American hybrids and American hybrids had the close distance. The relationship between V. amuensis and the other grapes growing in southern China also had far genetic distance. At the point of genetic similarity coefficient 0.630, V. amuensis and the grapes growing in southern China clustered together. In all 6 accessions of grapes growing in southern China, V. pseudoreticulata had relatively far genetic relationship with the other grapes. The distance of genetic relationship between V. amuensis-European hybrids and V. amuensis or European grapes related with the selected multiple-generation backcrossing parents when the hybrid was bred.