Screening Of Tea Genomic-SSR Molecular Markers Based On The Tea Genome Information And Preliminary Application

SSR molecuar markers possess incomparable superiority than other molecuar markers. Development of SSR primers must be based on the information of known nucleic acid sequences, so the reported tea SSR primers were almost EST-SSRs, which were oringined from coding regions in the tea genome. However, EST-SSR markers were discovered to provide lower polymorphism than the genomic-SSR (gSSR) markers developed based on the genome information. Up till now, the gSSRs in Camellia sinensis have been rarly reported. This study was aimed at developing tea gSSR primers based on the genomic information of C. sinensis obtained from previous sequencing work by our laboratoty, and establishing a SSR molecular marker system, so as to accelerate research of genetic diversity, build genetic map, phylogenetic system and molecular marker assisted breeding in tea plant. The main results of this research were as follows:1. The method for extraction of genomic DNAs in tea plant were appropriately modified based on previously reported methods, which can be applied to isolated DNA samples from a variety of fresh tea leaf samples with high quality.2. A number of factors in SSR-PCR reaction system were optimized so as to set up an amplificaton system suitable for simultaneous amplification of multiple samples and multiple pairs of primers. In additon, optimization the degree of crosslinking of the non-native polyacrylamide gel were also performed in order to obtain the best separation conditions. It showed that the best degree of crosslinking of the polyacrylamide gel was5.0%.3. A total of228pairs of SSR primers were designed based on the genomic information of C. sinensis obtained from our previous study. Among these SSR primers,86primers were successfully amplified, and the SSR fragment size of different SSR locus ranged from150to400bp. Their PCR products were visualized by8%non-native polyacrylamide alleles gel electrophoresis.27pairs of SSR primers with higher polymorphism were screening from the polymorphic alleles visualized from their SSR-PCR products. The results revealed that the27primers could be used for SSR polymorphism analysis.4. The27pairs of primers were used for genetic diversity analysis of the24samples which included wild ancient tea plants in Yunnan Province, the natural hybrid groups of wild ancient tea plants and modern tea cultivars. A total of95alleles and158kinds of genotypes were detected. The average number of DNA alleles and genotypes detected by each primer was2to6and3to11, respectively. The average number of alleles, genotypes and PIC value were3.52,5.85and0.4549, respectively. The Nei index was between0.05and0.72, and the average Nei index was0.51. Shannon information index (I) fluctuated from0.11to1.54, and the average Shannon information index was0.91.The observed heterozygosity(Ho) was between0.10and0.52, and the average Ho was0.2875. The expected heterozygosity(He) was0.05to0.78, and the average number was0.52. Genetic similarity coefficients were between0.51and0.85.The results revealed that the tea resources and species studied in this paper showed abundant genetic diversity on the DNA molecular level.5. UPGMA cluster result demonstrated that when the similarity coefficient was0.58, all the cultivars and wild ancient tea plants resources were clustered to2independent groups, which was in accordance with the species evolution law. This result indicated that the27pairs of genomic-SSR primers initially developed by our laboratory were reliable in the analysis of genetic diversity.