Mitochondrial Genome Comparison Of N. Sinensis And P. Striata And Phylogenetic Study Of The Frog Subfamily

There are a lot of researches about the mitochondrial genome of Anura. The mitochondrial genomes of over 200 species in Anura have been sequenced. However, the mitochondrial genomes of only 21 species in the suborder Mesobatrachia of Anura were reported. The mitochondrial genomes of the genus Scutiger, most species of which are endangered species, Mesobatrachia have never been reported. The mitochondrial genomes of two species, Scutiger ningshanensis and Scutiger liupanensis, are reported in the present study. The phylogenetic relationship among the species that their mitochondrial genomes were reported are also analysised in this study.The primer-walking and genomic-walking were used to sequencing the genome in this research, and the sequences were authenticated, assembled, annotated and analyzed by Bioinformatics method. Combined with 21 species mitochondrial genome of Mesobatrachia deposited in GenBank database, PCGs, rRNAs and rRNAs & PCGs were built to analyze phylogenetic relationship of Mesobatrachia by Maximum Parsimony, Maximum Likehood and Bayesian Inference. The results were showed as follows:1. The content and arrangement of two toads'mitochondrial genomeThe obtained mitochondrial genome sequences length of S. ningshanensis and S. liupanensis are 16799 bp and 16890 bp respectively. Containing 13 protein-coding genes (ATP6, ATP8, ND1-6, ND4L, COX1-3, CYTB),2 ribosomal RNA genes(12S-rRNA,16S-rRNA),19 transfer RNA genes(tRNAIle, tRNAGln, tRNAMet were not sequenced through) and a D-loop region in same order and orientation as the mitochondrial genome sequences of vertebrates. The consistency sites of two species accounted for 87.6% by comparison.The obtained mitochondrial genome sequences arranged densely and all contained overlap regions and spacer regions.S. ningshanensis contains 8 overlap regions spanning 34 bp and 9 spacer regions spanning 17 bp. S. liupanensis contains 14 overlap regions spanning 87 bp and 12 spacer regions spanning 63 bp. All 2 new mitochondrial genomes have a high A+T content, A+T content is greater than the G+C content.10 protein-coding genes have typical ATG initiation codon, except ATP8, COX1, ND6 genes of S. ningshanensis and S. liupanensis, which are tentatively designated by the GTG codon. Meanwhile, there are two kinds of termination codon, complete termination codon and incomplete termination codon. The obtained mitochondrial gene sequences of two species encoding 19 tRNA genes, which distributed between rRNA and PCGs. There are 37and 44 mismatched base pairs in secondary structure of tRNA genes and the main mismatchs were U-G. The D-loop of two species was between tRNAPro and tRNAphe. The position and direction of tRNATrp was different in various species indicated the genome sequence were rearranged. The position of rRNA was fixed relatively and located on the H-strand.2. Analysis of mitochondrial gene in Mesobatrachia.Analysing the nucleotide composition of all mitochondrial genome sequence in Mesobatrachia, we found that all species showed obvious AT skew and the content of the bases G were the lowest. The composition of the bases content were A>T>OG on H-strand and A>C>T>G on L-strand, it lead to AT/GC-skew change. PCGs were extremely dependent on the choice of base composition, this study found the characteristics of protein-coding genes that the content of T were greater than A. This result led to the synonymous codon terminated with A or T were more than other synonymous codon.3. Analysis of phylogenetic relationship in Mesobatrachia.Phylogenetic relationship was reconstructed using the Maximum Parsimony (MP), Maximum Likehood (ML) and Bayesian Inference (BI) methods on the combined dataset of PCGs, rRNAs and rRNAs & PCGs. The results showed that Mesobatrachia was not an effective taxonomic category, it constituted with two branches and composed Archaeobatrachia with Leiopelma and Bombina. Pipidae and Rhinophrynidae were relatively original and had a near relationship. Megophryidae, Pelobatidae and Pelodytidae were relatively evolutionary, together they comprised other main branch of Mesobatrachia. S. ningshanensis and S. liupanensis as the represent species of Scutiger got together for a branch, it proved that the two species were belonged to Scutiger boulengeri group.