Mitochondrial DNA Sequence Analysis Of Portunus Trituberculatus And Charybdis Japonica And Identification Of Single Nucleotide Polymorphisms Of MIH In Portunus Trituberculatus

Genetic structure of Portunus trituberculatus and Charybdis japonica from different geographical groups was analyzed using multianalysis and DNA Sequencing technique. And Single nucleotide polymorphisms(SNP) in MIH gene were studie in Portunus trituberculatus. The main results were shown as follows:Fragments from two mitochondrial genes of Portunus trituberculatus sampled from three sites(beihaidao of Japan, the east coast of South Korean and huichang in china) were amplified and sequenced .523bp 16SrRNA,and 658bp COI fragments were obtained. Variable sites,nucleotide diversity,average number of nucleotide diferences and average number of nucleotide substitutions per site were calculated to analyze and compared the sequence diference and genetic diversity level among different populations.Results showed that levels of genetics diversity of Chinese wild population were low. The molecular phylogenetic tree constructed with NJ method using software MEGA 4. 0 based on 16S rRNA was not consistent with that based on COI gene. The main difference is the classification of Charybdis.The phylogenetic tree based on16SrRNA gene sequences showed that Portunidae are lustered into two branches: different haplotypes of Portunus trituberculatus together first,and then with Portunus pelagicus and Portunus sayi; the three kinds of Scylla together clusted first,and then clusted with Charybdis japonica.These findings of the study can be used for the protection and utilization of Portunus trituberculatus resources of China,519 base sequence of the 16SrRNA gene and 658 base sequence of the cytochrome oxidase 1 gene were amplified and sequenced in specimens of Charybdis japonica from Laizhou Bay and Jiaozhou Bay.The analysis of the homology sequences of 16S and COI gene indicated that Little sequence variation was observed within species.A total of 7 and 8 nucleotide site were detected variable defining 7 and 7 haplotypes in 16S and COI gene,respectively. Variable sites,nucleotide diversity,average number of nucleotide differences and average number of nucleotide substitutions per site were calculated to analyze and compared. Rich levels of nucleotide diversities were detected in two Charybdis japonica wild population. The molecular phylogenetic trees were constructed with NJ and UPGMA method using software MEGA 4. 0 based on 16S rRNA and COI gene. The phylogenetic tree based on 16SrRNA gene sequences showed that Portunidae are clustered into two branches: different haplotypes of Charybdis japonica together first,and then with the three kinds of Scylla;and three crabs of Portunus and two of Callinectes clustered into the other branche; While the phylogenetic tree based on COI gene sequences showed that different haplotypes of Charybdis japonica together first,and then with three crabs of Charybdis; Portunus trituberculatus and Portunus pelagicus of Portunus and two of Callinectes clustered into the other branche. The results are consistent with classical taxonomy.SNPs of MIH gene were detected in Portunus trituberculatus by using PCR-SSCP technique. According to the three exons, we designed seven pairs of specific primers, M1 primer amplification products with a polymorphism SSCP electrophoresis, and the polymorphic bands were cloned and sequenced, contrasting with the sequencing peak map, we found one SNP site, that was the G212A, nucleotide changes led to changes in arginineâ†'glutamine, for the molt-inhibiting hormone biological activity, it is worth further study.