The ISSR And RAPD Analysis On Genetic Diversity And Phylogenetic Relationship Of C.Sinensis Cv.Gulin-niupicha Germplasm

The Subsect.Theas is one of the pristine genera in the Camellia, the Theaceae in many parts of the world, of which the overwhelming majority was situated in China. The southwest China which is resourceful remarkably, turn out the center of distribution of Theas. The source area at which C. sinensis cv. gulin-niupicha growed is located in district Jiaozigou region, Gulin County, Luzhou City, which is situated on the edge of Sichuan and Guizhou province, and contiguous to Yunnan province. It is distributed mainly over N28°0.4′in latitude and E105°48.2′in longitude, and800-1200meters in altitude. Through identifying the Morphologic characterization, The tea showed abundant morphological diversity, for example, arbusele or small arborescence in tree form, opening or half opening in tree posture, middle leaf in lamina size, elliptical or long elliptical in leaf shape, dark green in leaf color,et.al.In this study, two molecular makers (ISSR and RAPD) were emoloyed to analyze the total95accessions with76C.sinensis cv. gulin-niupicha,11C.sinensis cv. huangjing-dashucha and8materials taken from cultivated species derived from Sichuan, Chongqing and Guizhou. We have come to conclusions as follows:1. The optimal annealing temperatures for ISSR primers were found by screening primers tested and optimizing the ISSR reaction system. The annealing temperatures were decided by the bases of each primer, while were handly related to the Tm value. The selected range of temperature,48-62℃, would be effective for using ISSR on C.sinensis cv. gulin-niupicha.2.18ISSR primers screened from60primers, which amplified a total of160clear-cut bands, were tested on the95materials. The percentage of polymorphic bands was91.88%and the length of fragments vary ranged from200bp to4500bp. mostly from500bp to2000bp. 3.10primers which amplified64clear-cut bands which have56polymorphic loci, were used to the95materials. The percentage of polymorphic bands was87.5%and the length of fragments vary ranged from200to4500bp, mostly from500to3000bp.4. According to ISSR and RAPD markers, coefficient of genetic similarity for the95materials in the study have been computed completely.95materials can be divided into2groups which were in accord with that of the systematic of species. Divisions of subsets in some groups were related to the genetic structure difference of C.sinensis cv. gulin-niupicha and C.sinensis cv. huangjing-dashucha.5. Mantel’test method on RAPD, ISSR and SPAR(ISSR+RAPD) produced correlation coefficients constructed dendrograms using the UPGMA method based on SM coefficients, and proved the SPAR to be the most reliable one. Furthermore, the correlation between ISSR and RAPD based on SM coefficients showed moderate relation (r=0.501), and based on cophenetic value showed high(r=0.77), which constructed that ISSRs and RAPDs could be confirmed and sustained each other.