Red Tail Paracobitis Mitochondrial Genome Sequencing And Phylogenetic Analysis Of The Carp Suborder

In this study, the complete mitochondrial genome (mitogenome) sequence of Paracobitis variegatus was sequenced using the strategy of sub-PCR after long-PCR amplification and annotated in detail. These sequences would enriched the database of mitochondrial genome sequences. We also use parts of five families of cyprinoidei mitochondrial genes which were published in NCBI to infer the phylogenetic relationships among these families and subfamilies. The conclusions were mainly summarized as follows:1. Mitochondrial genome of Paracobitis variegatus is a closed-circular molecule. This mtDNA is 16571bp in length, include 37 genes and a 923bp D-loop control region as found in other vertebrates mitochondrial genomes. The genomic structure is arranged densely, besides D-loop control region there is only 63bp intergenic spacer, and 33bp overlap between genes. Genes arrangment and transcription direction are consistent with Crossostoma lacustre.2. The nucleotide composition in P. variegatus mtDNA shows A+T bias. The overall A+T content of is 55.6%, and it is 66.4% in D-loop control region.3.13 protein-coding genes started with ATG start codons, except CO I gene is putative GTG as start codon. The 9 of 13 protein-coding genes terminated with the typical TAA or TAG, while CO II, COIII and Cyt b were infered to have an incomplete termination codon T, and ND4 gene terminated with an incomplete termination codon T.4. All P. variegatus tRNA genes formed typical clover-leaf structure except for tRNA-SerAGN, with which DHU arm could not form stable stem-loop structure, while only form a loop. There are unmatched base pairs occur in all the secondary structures of P. variegatus mt tRNAs, and majority of them are G-U pairs.5.The secondary structures of P. variegatus mt 12S rRNA and 16S rRNA are predicted. The secondary structure of 12S rRNA include 43 stem-loop structures and the secondary structure of 16S rRNA include 6 domains and 53 stem-loop structures. Their secondary structures are conservative.6. There are 1 terminal associated sequence named TAS1,3 central conserved sequence blocks named CSB-F, CSB-E and CSB-D,2 conserved sequence blocks named CSB-2, CSB-3, a conservative terminal combined sequence named TAS and it's complementary sequence in the control region of P. variegatus. Among them, central conserved sequence blocks CSB-F, CSB-D and conserved sequence blocks CSB-2 are conservative. 7. Based on the three data sets and three phylogenetic analysis, we can conclude that:1) Cobitinae and Nemacheilinae have more closely relationships than Botiinae within Cobitidae relatively. And there are more closely relationships among Homalopteridae, Cobitinae and Nemacheilinae than the relationships among Botiinae, Cobitinae and Nemacheilinae.2) Catostomidae and Cyprinidae have closely relationship, and both are more ancestral groups within Cyprinoidei, while Homalopteridae and Cobitidae belong to advanced groups.8. The phylogenetic relationships among each families within Cyprinoidei were analysed using phylogenomic techniques, and the reslt is Catostomidae+Cyprinidaeâ†'Homalopteridae+Cobitidae. The phylogenetic relationships among subfamilies within Cobitidae is Botiinaeâ†'Nemacheilinaeâ†'Cobitinae.9. The mitogenome data is proved to be an effective tool in resolving deep relationships within the superfamily.