SNP Analysis Of Partial Mitochondrial DNA Of Different Pelodiscus Sinensis Strains And Germplasm Identification

In this study, partial sequences of mitochondrial DNA from four strains (Taihu Lake strain, Taiwan strain,Yellow River strain and Japanese strain) of Chinese soft-shelled turtle (Pelodiscus sinensis) were amplified andsequenced. The genetic diversity parameters were calculated and a phylogenetic tree was constructed. Also, thestrain specific SNPs of each strain were detected and classified. Specific SNP genotyping was performed usingPCR-RFLP and high-resolution melting (HRM), thus to identify different strains of P. sinensis. Specific results areas follows.(1) Genetic diversity and phylogenetic relationship analysisAbout1607bp of full-length16S rRNA gene from the four P. sinensis strains (20individuals per strain) weresequenced and analyzed. The genetic diversity parameters showed that the Taihu Lake strain displayed the highesthaplotype genetic diversity (0.863) followed by the Taiwan strain (0.484), Yellow River strain (0.195) andJapanese strain (0.100). A total of18haplotypes were detected including ten in the Taihu Lake stain, three in theTaiwan strain, three in the Yellow River strain and two in the Japanese strain, and no haplotype was sharedbetween any two strains. It indicated that it is feasible to identify different P. sinensis strains by mitochondrialDNA. Phylogenetic MP tree and genetic distance analyses showed similar results that the Yellow River and TaihuLake strain reflected a relatively closer relationship to the Japanese and Taiwan strain, respectively. It indicatedthat the Japanese strain possibly originated in the Yellow River population.(2) Seeking of strain specific SNPsAbout13000bp of mitochondrial DNA of the four P. sinensis strains (15individual per strain) was sequenced andanalyzed. A total of83strain specific SNPs were detected, including18in Taihu Lake strain,22in Taiwan strain,24in Yellow River strain and19in Japanese strain. Three strain-specific enzyme restriction sites were foundamong these SNPs.(3) SNP genotyping and germplasm identificationThree SNPs in restriction sites were tested by PCR-RFLP, and this method could distinguish these four P. sinensisstrains. Fifteen pairs of primers were designed and could be successfully used for SNP genotyping by HRManalysis. Among these fifteen pairs of primers, one could be used for the identification of Taihu Lake strain, twofor Taiwan strain, three for Yellow River strain, one for Japanese strain, three simultaneously for Taihu Lake andTaiwan strain, one simultaneously for Yellow River and Japanese strain, and four could divide the four P. sinensisstrains into two groups. So, the method of SNP genotyping can be used for the identification of these four P.sinensis strains effectively.