Determination Of Mitochondrial Genome And Molecular Evolution Analysis Of Three Species

We determined the complete mitochondrial genome sequences of Schizopygop-sis younghusbandi, Schizothorax oconnori and Schizothorax waltoni (Cyprinidae: Schizothoracinae), and annotated in detail. These sequences would have enriched the database of mitochondrial genome sequences. We also used parts of63species of Cy-priniformes mitochondrial genome which were published in GenBank to infer the phylogenetic relationships among these families and subfamilies and estimate diver-gence time. Second, an adaptive evolutionary analysis was performed by PAML. Fi-nally, in order to understand the liver gene expression of S. younghusbandi and select genes against hypoxia or extreme cold, we constructed a liver cDNA library and chose the clones to sequence and analysis the ESTs through bio informatics methods. The main conclusions were as follows:1) We determined the whole mitochondrial genome sequence for the S. young-husbandi, S. oconnori and S. waltoni. These mtDNA were closed-circular molecule and16,593bp,16,567bp、16,589bp in length, include13protein coding genes,2rRNA,22tRNA, D-Loop control region respectively; Gene arrangment and transcrip-tion direction were consistent with other Cyprinidae mitochondrial genomes.2) The overall A+T%content was56%in S. younghusbandi mtDNA. While the overall A+T%content was54.8%in S. oconnori and S. waltoni. The nucleotide com-position in three schizothoracine fish mtDNA showed A+T bias.3) On start codons of protein coding genes, except COX1gene was putative GTG as start codon, the other genes were ATG as start codon. On termination codons, COX1, COX3, ND4and CYTB were inferred to have the incomplete termination co-don T or TA, the other PCGs were terminated with the typical TAA or TAG in S. younghusbandi; ATP6, COX2, ND2, ND4, ND5and CYTB were inferred to have the incomplete termination codon T or TA, the other PCGs were terminated with the typ-ical TAA or TAG in S. oconnori and S. waltoni.4) The srRNA and lrRNA genes in three schizothoracine fish mitogenome were conservative; All three schizothoracine fish tRNA genes formed typical clover-leaf structure; D-Loop control region were933bp,935bp and935bp in length respectively. In this study, we found that the D-Loop control region of three schizothoracine fish generally had the2terminal associated sequences named TAS, and3central con-served sequence blocks named CSB-F, CSB-E, CSB-D and3conserved sequence blocks named CSB-1, CSB-2, CSB-3.5) Phylogenetic analyses of the two datasets based on rRNA&PCGs and PCGs expressed that the results were as follows:(1) The subfamily schizothoracinae was considered to have a close relationship with the subfamily cyprininae and barbinae; primitive stage schizothoracinae and highly specialized stage schizothoracinae formed a monophyletic clade with strong support and they were sister clades;(2) the esti-mated divergence time of the subfamily schizothoracinae was about51.7Mya-67.5Mya. Geological research showed that this period was when Indo-Asia continental collision and Qinghai-Tiber Plateau formation happened, and the results further con-firmed that Qinghai-Tiber Plateau was a center of origin and divergence of the subfa-mily schizothoracinae; and (3) an adaptive evolutionary analysis was performed on the rRNAs genes and PCGs genes (not ND6) of63the family of the family Cyprini-dae by PAML software. The results from branch model indicated that the ancestor branch of S. younghusbandi was under the positive selection with ω>1, and11the positive selection sites of six genes were detected under the branch-site model.6) The original cDNA library was acquired with the reorganization frequency of98%and storage capacity1.86×106×6=1.12×107CFU. To check the length of insert fragments,96positive colones were randomly selected to do PCR, the results showed that the mean length of insert fragments were about1200bp.7) The531single clones were randomly selected to be sequenced from5’-end, and we obtained506high-quality expressed sequence tags. These506ESTs were clustered through BioEdit software and total of198unigenes, and highly Abundance genes were chosen to be done the bio informatics analysis.