| Tea plant [Camellia sinensis (L.) O. Kuntze] originated from China, it had been spread to many countries and became the important economic plant and popular healthy beverage plant of twenty first century in the world. The initiatory researches about molecular marker technology and genetic diversity of tea plant had made some achievements in the middle of ninety decades of twenty century, but it was drop behind the other economic plants. Although the RAPD marker was used frequently, but the RFLP, SSR, AFLP markers only were used rarely. It is very difficult to construct genetic map of tea plant due to being multi-year growth, highly heterogenous and xenial plant. The special PCR marker technology, such as PCR-RFLP, is based on the information of nucleotide sequence, however, the information of nucleotide sequence in tea plant is very little. Therefore, the application of special PCR maker in genetics research of tea plant was restricted. To develop general molecular marker into sequencing the DNA base and finding the polymorphism of base sequence is the necessary trend of modern molecular colony genetics research. However, the new analysis methods about gene mutation and SNP were mainly applied in the medicine domain, and the reports revealing gene mutation and SNP in agriculture field were infrequent.In this study, the AFU ISSR, PCR-SSCP technology which were used to analyze tea plant genome DNA were established firstly, then the genetic relationship of tea germplasms was researched by using the established methods, and the genetic map in tea plant was constructed by adopting "double-pseudo-testcross strategy" which had been used in molecular map construction of perennial fruit tree and forest, the rare F1 population utilized to construct molecular map originated from Qi Men No.4 × Chao An Da Wu Ye. The SNP in polyphenol oxidase (PPO) gene of tea plant was studied in the part two of this thesis by combination of PCR-SSCP analysis and sequencing method, the primers were designed according to the cloned sequence. The polymorphism of SNP inducing restriction site mutation was analyzed further with the PCR-RFLP method, to arm at the relationship between SNP and processing property or heredity background of different tea cultivars. The main results were as follows:1. In order to isolate high-quality genomic DNA from tea plant leaves enriching polyphenols and polysaccharides, four methods of DNA extracting were reformed, including CTAB general method, CTAB subarea method, SDS general method, SDS subarea method, then the effect of four modified methods on DNA extracting were compared, results showed that the OD260/OD280 of the DNA samples obtained with these four modified methods were betweenl.8~1.92, the molecular weight of DNAs were more than 21Kb, DNAs were digested completely with restriction endonucleases and amplified with polymerase chain reaction (PCR). The high-quality genomic DNAs were obtained with any modified method. To preserve tea fresh leaves which obtained from distant place as the material of DNA extraction, three methods of -20, -70 and dehydrated desiccation with silica gel were designed to preserve the fresh leaves, and use fresh leaves as control, the quality of DNA was inspected, results showed that quality of DNA extracted from the leaves of the three preservation methods was the same as fresh leaves (control), so, the silica gel dehydrated desiccation preservation is a good method, which can be utilized when the material of tea leaves for DNA extraction from tea plant is sampled from distant place.2. The AFLP analysis system with highly resolution response and reproduction was established by adopting the silver staining technology instead of isotopes. Fingerprinting of some cultivars was made with this AFLP silver staining protocol, the fingerprinting polymorphism of different cultivars with the same primer or the same cultivar with different primers was quite abundant. It was showed that the AFLP analysis technology is a high performance method to probe genetic mutation of tea... |
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