Screening Of Hubei Snails (oncomelania Hupensis) Microsatellite Dna Library To Establish And Polymorphic Loci

1. The GoalAs the only intermediate host snail of Schistosoma japonicum in mainland China, the Oncomelania hupensis is very important to disseminate the schistosomiasis. It mainly distributed in the Yangtze valley and the south of the Yangtze valley, including Jiangsu, Zhejiang, Anhui, Jiangxi, Fujian, Shanghai, Hunan, Hubei, Guangdong, Guangxi, Yunnan, Sichuan.Because Oncomelania hupensis is widespread and in the influence of the geographical isolation, there is very obvious variation between the different areas population of mainland China, then evolving many different sub-species; And in the same condition, there's significant and different susceptivity to the Schistosoma japonicum. Therefore, the analysis about the genetics diversity of Oncomelania hupensis both has the theoretically value in biology, and has the useful value in medicine. In the study, I choosed the SSR technology to build the Oncomelania hupensis SSR sequence library, and in the foundation, choosed the SSR sequences that had the better polymorphism to analysis more than 200 samples in 30 fields of 6 provinces.2. The MethodChoosed the wild Oncomelania hupensis strain which was in the Haokou, Qianjiang, Hubei to build the Oncomelania hupensis SSR sequences library: Used the phenol-chloroform method to withdraw the whole geneome of Oncomelania hupensis , then cutting the geneome by a kind of incision enzyme called Sau3AI, and recycling the fragment which was between 200-900 bp, after connecting the SAUL attachment, using the PCR technology to concentrate, screening the SSR sequences and using ultra filters to concertrate by the 10 widowed nucleotide probes that was marked by the biotin and unifies with the Avidin. We obtained the majority SSR sequences after 2 repeats.In established SSR DNA sequences library, selected the sequenced which had the obvious the the selection redundant characteristic and the suitable length, in the foundation of the sequences, designing and synthesis the primers. Using PCR technology to concertrate, and 5% polyacrylamide gel electrophoresis and dyeing by the silver, and in the same time, screening the polymorphism locus by the gene scanning technique. Six sequences that had the better polymorphism were selected to genetics diversity analysis about Hunan, Hubei, Jiangxi, Anhui, Jiangsu and the Zhejiang 6 province more than 200 samples.3. The Result(1) 292 sequences were obtained, included the 266 SSR DNA sequences. There's 12 identical sequeces in the 266 sequences. And all the sequences were above 200 bp. Compares the computation criterion of the SSR sequences, totally obtained effective SSR DNA sequence altogether 254 sequences, in the 254 sequeces, 66 sequences were the perfect repetitive sequences, accounted for 25.98%, 94 sequences were the imperfect repetitive sequences , accounted for 37.01%, 94 sequences were the compound repetitive sequences, accounted for 37.01%.(2) Sixty-seven typical SSR sequences were selected in the Oncomelania hupensis library. And designed the primers in the amphisequences, there's 67 pairs primers grand total, in the last, synthesized 20 pairs primers for amplification. In the 20 paries primers, there's 4 pairs primers that had presented the non-specificity sequences. In the other primers, there's 14 (70%) polymorphism sites after preliminary screening. And in the 14 sites, 7 sites each 30 samples has completed to marked by fluorescence and to gene scanning, in the results, there's 6 sites thar was better. In the 6 sites, the observed heterozygosities(Ho) and the Polymorplism Information Content(PIC) of the primer P84 was lower, it's 0.1667 and 0.1813. The others Ho and PIC were 0.36-0.8929 and 0.8437-0.9289, which had the better polymorphism.(3) By gene scanning the 219 samples of Hunan, Hubei, Anhui, Jiangxi, Jiangsu, Zhejiang, Sichuan's of the 6 SSR polymorphism sites. The result output by the Excel tabular form, and I obtained altogether 2628 SSR DNA sites data. The number of the mean number of allele/locus (MNA) is 34.17, and the average con-population heredity heterozygosities was 0.869, and the average endo-population heredity heterozygosities was 0.023. Checking by the Hardy-Weinberg balance, the primers T5-13, T6-27, P82 were at the state of equilibrium, the primers T5-11 and T4-22 were at the non-equilibrium state in the populations of Hubei and Jiangsu. But P84 was at the non-equilibrium state in the populations of Anhui, Jiangxi, Jiangsu and Zhejiang.4. The Conclusion(1) In the established SSR DNA library, the double nucleotides duplication sequences were the most of the sequences (73.62%), and the three nucleotide duplication sequences were the second, and the multi-nucleotide duplication sequences were rarest; The double nucleotide nucleotides duplication sequences is most commonnly by (CA)_n and (GT)_n, the number of duplication may reach 98 times.(2) Screening by marked by the fluorescence and gene scanning the 30 samples of obtained 6 sites.The alleles number of the different sites was different, from 5 to 22, and the average was 16.7; Among the total, the PIC number of P84 was less than 0.25, It's the lowest polymorphism in the 6 SSR sites, and in the other sites, the PIC number were more than 0.5, there's better polymorphism.(3) In the 6 SSR sites, the P84, T5-11, and T4-22 were more or less non-equilibrium. In the total population, I obtained 205 alleles. The average alleles were 15.33 in the different sites; Allele distribution in different populations was not obvious central tendency.Showed by the heredity analysis of the endopopulation, the average Ho, He and PIC of the all sites were 0.637, 0.811, and 0.777. Tthe polymorphism is obvious. And synthesizing all the indicatrix, the Hubei population was the highest mutation; but the Jiangsu population was the lowest.In the analysis about the hereditary between the populations, the Jiangsu and Jiangxi populations had the high mutation, Anhui and Hunan populations had the low mutation. The community of the gene exchange is not high, so the heterozygosity is high; however, between the populations the mutation is lower, the mutation maily comes from the individual of the populations.