DNA Barcoding And Molecular Phylogeny Of Several Amphibians In Hunan Province

Mitochondrial is an important organelle existing in most of eucaryote cells.Vertebrate mtDNA is a closed circular double strand DNA,which is self-duplicate in a strict maternal inheritance way and doesn't recombinate in multigenerational transmission process.MtDNA has been extensiveiy used in vertebrate population genetics and evolutionary genetics,which provides an ideal research materials for resolving phylogeny on molecular level. The study is based on the mtDNA COI gene (Mitochondrial cytochrome coxidase subunit I), aimimg to classify the amphibians in Hunan Province and reseach the related Phylogenetic problems.The COI gene in eleven species of amphibians was amplified by PCR, and there are about 650 base pairs sequenced. The pairwise genetic similarities and differences among sequences of COI gene in 11 ampibia species were calculated by using MEGA4.1.The results showed that:(1) The content of A+T was 56%and The content of G+C was 44%. The content of A+T is obviously more than G+C. As regards the point of codon usage frequency, the use frequency of G as the first base codon was the lowest, while the third highest frequency was T, which showed the use of bases and codons had certain partiality.(2) The pairwise genetic similarities were between 55.4%and 98.6%and the sequence differences of COI gene between 1.5%and 33.7%.The pairwise genetic similarities between Rana limnocharis and Rana boulengeri of the COI gene was higher than that among other species and Bufo gargarizans and Microhyla pulchra was lower than that among other species.The sequence differences of COI gene between Andrias davitianus and Rana guentheri were bigger than that among other species and Rana limnocharis and Rana boulengeri was the smallest.(3) The topology of trees illustrated that the three species of Ranidae,Rhacophridae and Microhylidae formed the monophylyletic group while the Bufonidae's three belong to the same branch. (4) Comparing the collected COI gene sequences with the species record in Genbank, we have found that they are basically consistent with each other. But there are also differences in the larger sequence, which is probably due to the different subspecies, individual difference, geographical isolation and so on.On the other hand, in the same species the COI genes could be different among different subspecies, which showed the better resolution capability in the subspecies.The analysis revealed above was of efficient validation to identify these species in amphibian specialists, and it's a useful tool for reflecting the genetic relationship among them. The conclusion is the same with traditional methods of species taxonomy.