Comparative Analysis On Some Genes Of Mitochondrial DNA And Their Molecular Identification Markers In Three Species Of Soft-turtles

Some genes of mitochondrial DNA of Pelodiscus sinensis , Pelodiscus axenaria and Palea steindachneri were cloned and sequenced by using molecular technology of mitochondrial DNA. Based on the comparative analysis on their sequence data, we confirmed Pelodiscus axenaria is a new species in Pelodiscus from the molecular level and got their molecular identification markers in three species of soft-turtles. The main results were summarized as follows:(1) The primers for Pelodiscus sinensis and Pelodiscus axenaria were designed and 562 base pairs of mitochondrial DNA 12S rRNA gene were amplified and sequenced. The results showed that the length of their sequence is the same, the A, T, G, C contents are similar. Based on their sequence data, we found there are 13 different nucleotide sites (percentage divergence is 2. 31%) between Pelodiscus sinensis and Pelodiscus axenaria by using the software of ClustalW, whereas the average number of different nucleotide sites is 3 (percentage divergence is 0.53%) in Pelodiscus sinensis and 2 (percentage divergence is 0.36%) in Pelodiscus axenaria. The nucleotide percentage divergence is obvious between species, so it suggests Pelodiscus axenaria is a new species in Pelodiscus. Restriction endonuclease analysis based on sequence data of this DNA fragment revealed the presence of polymorphic sites for Msp I endonuclease, there is one site for Msp I endonuclease in Pelodiscus axenaria, but none in Pelodiscus sinensis. The restriction profiles obtained by agarose gel electrophoresis when amplicons were cut with Msp I enzyme allowed the unequivocal identification between Pelodiscus sinensis and Pelodiscus axenaria.(2) The primers for 12S rRNA and 16S rRNA genes of Pelodiscus sin ensis, Pelodiscus axenaria and Palea steindachneri were designed and the complete sequences of the two rRNA genes were amplified and sequenced. Based on their sequence data of 12S rRNA gene, we can know the nucleotide percentage divergence is 4. 0% between Pelodiscus sinensis and Pelodiscus axenaria, 11.0% between Pelodiscus sinensis and Palea steindachneri, 10.8% between Pelodiscus axenaria and Palea steindachneri, whereas the nucleotide percentage divergence is 4. 0% between Pelodiscus sinensis and Pelodiscus axenaria, 13. 2% between Pelodiscus sinensis and Palea steindachneri, 12.8% between Pelodiscus axenaria and Palea steindachneri when the sequence data of 16S rRNA gene is analysised. The phylogenetic relationship of 12S rRNA and 16S rRNA genes of four species of soft-turtles was respectively described. All of the molecular phylogenetic trees indicated that Pelodiscus axenaria is more closely related with Pelodiscus sinensis than the other two species of soft-turtles and supported Pelodiscus axenaria is a new species in Pelodiscus.( 3 ) The primers for Pelodiscus sinensis , Pelodiscus axenaria and Palea steindachneri were designed and 1140 base pairs of mitochondrial DNA cytochrome b gene were amplified and sequenced. The results showed that the length of their sequence is the same, A> C G^ T contents are similar. Based on their sequence data, we can know the nucleotide percentage divergence is 7. 7% between Pelodiscus sinensis and Pelodiscus axenaria, 15. 0% between Pelodiscus sinensis and Palea steindachneri, 15. 9% between Pelodiscus axenaria and Palea steindachneri, whereas the individual nucleotide percentage divergence is 2. 37% in Pelodiscus sinensis , 0. 88% in Pelodiscus axenaria and 0. 18% in Palea steindachneri. The nucleotide percentage...