| Simple sequence repeats (SSR) is a new type of DNA fingerprint, with 1-6 bases as the basic unit of tandem repeat sequences. SSR markers have co-dominant, good polymorphism and characteristics such as good repeatability, stable and reliable, which are incomparably superior to other molecular markers. In recent years, there are lots of reports of SSR markers of tea plants(Camellia sinensis (L.) O. Kuntze) and mainly of EST-SSR which is developed based on expressed sequence tags. Compared with gSSR, its shortcoming is of lower polymorphism and hard to evenly cover the whole genome when apply to the construction of genetic map. Therefore, it is urgent to speed the development and application of gSSR of tea plant. Based on the DNA sequence information of Tieguanyin through the whole genome survey, the study has predicted the gSSR and its flanking sequence. We also constructed a system of developing SSR on the base of 4300 DNA analysis system and applied it on the analysis of genetic diversity of some Anhui tea strains and on the detection of gSSR genetic characteristics on the offspring of hybridization and backcross 1 generation. The main results of this research were as follows:(1) Based on the result of genome survey of Tieguanyin, we got 45310 SSR through the biological information prediction. Among them, dinucleotide was the most common repeat motif with a frequency of 75.29%, its most repeat bases is AT. Followed by 17.55% trinucleotide, TTA is its most repeat bases.(2) Single factor experiment and L9 (34) orthogonal design test that carried out on the main parameters affecting tea SSR-PCR system were used to optimize the system, we set up a suitable SSR-PCR reaction system for 4300 DNA analysis system. In addition to, we proved that self-made polyacrylamide gel solution (acry:bis is 29:1, concentration of 6.5%) can be alternative to the gel solution provided by 4300 DNA analysis system.(3) We selected 99 gSSR from the data pool for SSR-PCR amplification,63of them were successfully amplified. Polymorphism analysis was carried out on the 22 gSSR by using 18 Anhui tea plants,4 national varieties of other province and Shubeizhong of Japan. Results show that a total of 95 allelic loci and 158 genotypes were detected; Range of each pair of the primers’allelic and genotype can be detected were 2-6 and 2-12, respectively; The average number of alleles, of genotype and PIC values was 4.1,6.9 and 0.53. These results indicate that the detected gSSRs have rich polymorphic. Meanwhile we also analyzed the genetic diversity of the 23 tea plants. Results show that observed heterozygosity (Ho) was from 0.05 to 0.87, with an average of 0.45; Expected heterozygosity (He) from 0.13 to 0.77, with an average of 0.6; Changes between Nei index (H) in the range of 0.13 to 0.75, with a mean of 0.58; Shannon Information Index (Ⅰ) ranges from 0.25 to 1.49, mean of 1.1; The genetic similarity coefficient were between 0.55 and 0.78. All of these results indicate that the detected tea samples have abundant genetic diversity in DNA molecular level.(4) UPGMA cluster result demonstrated that when the similarity coefficient was 0.65, the tested 23 tea plants resources were clustered into 4 independent classes. The first and second class only included YukanglO and Zhuerzhong, respectively; the third class included Huangshanzhong, Yangshulin, Tieguanyin, Fuding Dabaicha; the remaining 17 strains were classified into the forth. When the similarity coefficient was 0.67, the plants of the forth class could be clustered into 3 subclasses. The first included Shuchazao, Xianyuzao, Huangguanyin, Zhuyezhong in Keemun and Anhuil; the second only included Hengmeizhong; the remaining 11 strains were classified into the third subclass.(5) Amplifying individuals of cross and backcross 1 generation of tea plant by 8 pairs gSSR primers, we found that in cross and backcross generation 1, co-dominant individuals respectively accounted for 72.75% and 60.53%, individuals with only father’s alleles for 10.19% and 18.42%, individuals with non-parents alleles for 1.75% and 11.21%. As for TGS13 and TGS25, the individuals with father’s alleles separately accounted for 40.63% and 52.63% cin cross and backcross 1 generation. |
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