Analysis Of The COI Gene Fragment Of The Perna Viridis And The Complete Mitochondrial Genome Of The Crassostrea Hongkongensis

It is known that doubly uniparental inheritance (DUI) mitochondrial DNA are present in the species M. trossulus, M. edulis,M.galloprovincialis, M.californianus, Geukensia demissa, R. philippinarum, Pyganodon grandis, P. fragilis and Fusconaia flava. In this study, we examined whether the phenomenon of double uniparental inheritance exist in mussel Perna viridis, a species in the same family with M. galloprovincialis. We also analyzed the phylogenetic relationship of the three species from the same genus (P. viridis, P. canaliculus and P. perna) based on partial mitochondrial COI gene data.Partial mt COI gene sequences from the gonads (male and female) and somatic tissue of Perna viridis were separately amplified using the universal primers COIL 1490/COIH 2198. A 661bp nucleotide sequence of COI gene was obtained. It is found that this sequence was identical among different tissues of male individual, and low divergence (0.31%) was detected between male and female sequences. Only one haplotype was found in both female and male sequences. No doubly uniparental inheritance (DUI) was detected. Together with available COI sequences of other Perna species from GenBank, NJ tree, MP tree and Bayesian inference (BI) tree were constructed respectively with PAUP and MrBayes, and phylogenetic relationships among them were analyzed accordingly. Results showed that the sequence divergence among these 3 species was consistent with the estimation of time of differentiation.The oyster Crassostrea Hongkongensis, belongs to Mollusca-Bivalvia-Pterioida- Ostreidae-Crassostrea, is one of the most important cultured oysters along the South China Sea. Ten pairs of genus-specific primers were designed in this study based on analysis of the complete mitochondrial DNA sequences of Pacific oyster (Crassostrea gigas, NC₀01276) and eastern oyster (Crassostrea virginica, AY905542). Ten large fragments were amplified by long PCR accordingly. Through primer walking sequencing and sequnce assembly, the complete mitochondrial sequence of 16,475bp was obtained. The genome contains 22 transfer RNA genes (tRNA), 2 ribosomal RNA genes (rRNA), 12 protein-coding genes, 1 dominating noncoding region, and 1562 bp noncoding inter-genic sequences. In comparison with other mitochondrial genome of Crassostrea species, no protein-coding gene rearrangement was found in C. hongkongensis.The nucleotide compositions were calculated for the complete sequence, protein-coding genes, tRNA genes, rRNA genes, Intergenic nucleotide and dominating noncoding region, respectively. The AT content of dominating noncoding region is the highest, with the value of 77.8%, and that of Intergenic nucleotide was 72.1%. The AT content of complete sequence, protein-coding genes, tRNA genes and rRNA genes are 65.4%, 64.6%, 64.2% and 60.5%, respectively. As for the criterion of L-strand, GC-skew=-0.207, AT-skew=0.134.All of the genes are encoded in H-strand. Most of the protein-coding genes are separated by tRNA genes or spacer with size range from 1bp to 294bp in length, except for tRNA-His and ND4 genes which have 1bp overlapped.In protein-coding genes, only two type of start codon are found: ATG and ATA. The start codon exist at the CytB gene in Crassostrea hongkongensis is CTA, which are CTA and TTA in the Pacific oyster and eastern oyster respectively. The start codon CTA and TTA both code for the amino acid Leu. Two type of stop codon are found, they are TAA and T(only ND4L gene for T). In tRNA genes, anticodon loop and the amino acid arm are the most conserved regions among species.Evolutionary rate of different genes are compared. Results show that NDAH dehydrogenase subunits (ND1-ND6) and Cyt B genes are the fastest evolved genes, ATP6 subunits gene take second place, and then followed by Cytochrome c oxidase subunits gene and then are the tRNA genes. Evolutionary rate in small and large subunits rRNA genes (12s rRNA and 16s rRNA) are the lowest. In Cytochrome c oxidase subunits, COIII has a higher evolutionary rate than the other two.In NDAH dehydrogenase subunits, evolutionary rate are higher in ND6 and ND2.Phylogenetic relationship among three oyster species Crassostrea hongkongensis, C. gigas, C. virginica and M. edulis (Mytiloida: Mytilidae) and Artemia franciscana (Pectinoida: Pectinidae) are analyszed. Ruditapes philippinarum (Heteroconchia: Veneridae) is used as out-group. Every single gene, genes encode the same protein group, all protein-coding genes, all rRNA genes, all tRNA genes and all genes as one fragment are used respectively to construct NJ phylogenetic tree, MP phylogenetic tree, ML phylogenetic tree and BI phylogenetic tree by the program Paup and MrBayes. Most of the results show that C .hongkongensis is close to C. gigas, and is different from C. virginica. When single gene is used, the bootstrap supporting rate is low. When genes are used together, the bootstrap supporting rate is up to 100%. This result indicates that genes used together can complement the defect of each other's, hence it is more resolvable in phylogenetic research. Therefore it is evident that mitochondrial genome can be used to research relationship among far-related species .Pairise distances among the three oysters calculated with COI gene and 16s rRNA gene sequence are consistent with the results from others..Pairwise distance is also estimated with different gene sequences. The values between species calculated with rRNA genes and tRNA genes are low. But it is very high when calculated by ATP subunit genes and NADH subunit genes.When different genes are combined, species with distance can be seen as different genera. When all genes are combined, the pairwise distances are ranged between 0.15 and 0.368.