Studies On The Development Of EST-SSR Markers And Its Application In Tea Plant

Based on ESTs of tea plant existing in the public database, ESR-SSR marker was developed after mining and evaluating SSRs in them for the first time. As well as the application in resource evaluation and transferability to other plant species of these EST-SSR markers were discussed and explored.The frequency and characteristics of SSR were analyzed and clarified in tea pant ESTs. 1989 ESTs of tea plant were downloaded from the public database of NCBI and some of them redundant or with low quality were removed, finally resulting in 1589 non-redundant ESTs with total length of 734.54 kb. Totally 281 SSRs distributed in 246 ESTs were mined out, accounting for 17.68% of the non-redundant ESTs. The average length of the EST-SSRs searched is 33.06 bp and the average distance of distribution is 1/2.16 kb. The dinucleotide repeat is the dominant type with repeat motif AG/CT being the most common. These results provide a base for the development and future application of EST-SSR markers in tea plant.The EST-SSR marker was developed in tea plant. By comparing effects of different concentration of the Vc, PVP and P-mercaptoethanol adding to extraction buffer, CTAB method was improved firstly for extraction of genomic DNA with high quality from tea plant. It was observed that addition of 0.1% Vc in extraction buffer could effecteively improve the purity and quantity. Then 16 primer pairs for EST-SSRs were designed. After testing on the annealing temperature and the concentration of primers, dNTP and MgCl₂, a suitable PCR system was established. Under the condition of reaction system developed, the primers designed were screened against genomic DNA of Longjing 43 from which most ESTs were derived, and 13 primer pairs showed the amplification, accounting for 81.3% of total primers. Then the primers showing amplification were subjected to PCR for DNAs from 10 tea plant varieties and 7 primer sets showed polymorphisms, accounting for 53.8% of primers available. Results prove it is an effective and feasible approach to develop SSR markers based on ESTs in tea plant.The application of EST-SSR marker developed above in resource evaluation and variety identification was investigated. Forty-two tea varieties were analyzed by using 16 SSR primer sets designed in this study, and 13 of them produced clear bands and 10 showed polymorphism, accounting for 76.9%. The PIC value for each polymorphic primer set varied from 0.522 to 0.866, with a average about 0.73. Totally 84 genotypes and 74 alleles were detected in all varieties tested by 10 polymorphic markers, with the range from 4 to 12 andfrom 3 to 10 for each polymorphic primer set, respectively. The genetic distance among 42 tea varieties varied from 0.074 to 0.667, averagely 0.363, suggesting that the materials used in the experiment possess a broad genetic variation. Based on the similarity coefficient about 0.55, all the tea varieties tested could be classified into 3 groups and most of them were included in the first group. The results show that the EST-SSR marker is very effective in evaluation of tea germplasms.The transferability of tea plant EST-SSR markers to other plant species was explored, such as rice, sorghum and cabbage. 16 pairs of EST-SSR primer were tested and 8 of them showed the amplification in all the three species. The percentage of available primers is 62.5%, 75%, 68.8% and the polymorphic primers is 60.0%, 50.0%, 36.4% of available primers in rice, sorghum and cabbage, respectively. The results reveal that tea plant EST-SSR marker is transferable in some degree to distant plant species.