| Tea is an important economic crop with a very long cultivation history and wide rangedistribution. The genetic resource of tea plant was very abundant. However, the geneticresearches in tea plant were droped behind comparing with other important economicplants. Especially, the application study about SSR molecular marker technology was veryrare. This study had focused on the development of SSR primers in terms of the Genbankdatabase and the establishment of SSR molecular marker system. Furthermore, theoptimized SSR technology was used to analyze the genetic diversity of tea germplasms.The main results were as follows:The genomic DNAs in tea plant were extracted by the modified method, and theconcentration of DNA extracts were quantitated by Lambda DNA standard, which lead toa new method was established for estimating the concentration of genomic DNA in teaplant. The new method was firstly brought forward and applied in this study.A number of factors were studied to set up the optimizing SSR reaction system. And theoptimized technology was: each 20μL amplification reaction solution comprised 1×Buffer,1.5～2.5 mmol/L MgCl2, 0.2 mmol/L dNTPs, 0.25μmol/L of each primer, 0.75U Taq DNApolymerase, 30 ng template DNA. Amplification program was: 5 min at 95℃; then 35cycles that consisted of 60 s at 95℃, 45 s at the best annealing temperature of each primer,60 s at 72℃; followed by 10 min extension at 72℃. This system had well reproducibilityand stability proved by time after time amplification practice.The EST sequences were collected from the Genbank datebase, and 33 pairs of SSRprimers were firstly designed by Primer 3 software. And another 17 pairs of SSR primerswere selected in terms of the literatures. Then, a total of 50 pairs SSR primer weresynthesized for amplifying the genomic DNA. Out of a total of 50 primer pairs screened,37 primers were successfully amplified and the amplification primers accounted for 74％.These 37 pairs of primer were crossed with a set of tea cultivars, and their PCR products were visualized by 8％native polyacrylamide gel electrophoresis. The polymorphicalleles were visualized from the PCR products amplified by 34 pairs of primer out of 37.The number of bands per primer pair ranged from 1 to 11 and the SSR fragment size ofdifferent SSR locus ranged from 150 to 350 bp.16 pairs of primer out of 34 were choosed to cross the total of 43 accessions and the SSRfragments were visualized by fluorescence capillary electrophoresis. A total of 142polymorphic alleles were detected. Based on the SSR results, the genetic distance andsimilarity coefficient were calculated using the Nei & Lei's coefficient method by DPSsoftware. The results showed that the gene differentiation was very remarkable amongcultivars because the genetic distance coefficient among 43 accessions ranged from 0.059to 0.820. Based on the genetic distance and the UPGMA cluster, all the 43 accessions wereclustered to 7 groups at the average genetic distance level. Majority closely relatedcultivars according to pedigree were grouped together. SSR marker was a sort of excellentmethod to find the genetic relationship among tea germplasms.With native polyacrylamide gel electrophoresis and fluorescence capillary electrophoresisto visualize the SSR fragments, the SSR fingerprinting of 43 cultivars at a group of SSRloci had generated. The method of study SSR in this research would facilitate theestablishing of SSR genotype database in tea germplasms.This research would facilitate the development of EST-SSR based on the database and layout a foundation for further utilization of SSR marker, such as used for varietyidentification, breeder's right protection, origin and evolvement study, genetic mapconstruction, and marker-assisted breeding. |
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