Wheat Fish Pseudorasbora Parva Mitochondrial Genome Sequencing And Analysis

The mitochondrial DNA with the properties of high polymorphism,rapid evolutionary rate,maternal interitance, makes it as a good model for research in the field of gene structure,function,transcription and expression. Currently, complete mitochondrial genome sequences were available in Genbank for 1790 metazoan species which included 1250 Vertebrate,625 Osteichthyes,98 Cypriniforme,69 Cyprinidae,12 Gobioninae.In the research, the complete mitochondrial genome of Pseudorasbora parva belonged to Gobioninae was sequenced, assembled, annotated and analyzed in detail with the methods of long-PCR and sub-PCR. The new mitochondrial genome combined with other Cyprinidae fishes deposited in Genbank were used to reconstructed Cyprinidae phylogeny and explored the phylogenetic utility of different combined data sets. Conclusions were as follows:1. The Pseudorasbora parva mtgenome is 16600 bp long. It encodes 13 protein genes:COⅠ, COⅡ, COⅢ, ATP6 and ATP8; ND1, ND2, ND3, ND4, ND4L, ND5, ND6 and Cytb; 2 rRNA genes: srRNA and 1rRNA; 22tRNA genes and a D-loop control region.2. The nucleotide composition of Pseudorasbora parva mtgenome shows some A/T bias. The A+T content of the complete sequence of Pseudorasbora parva is up to 58.9%. The A+T content of D-loop control region is up to 66.2%, which is significantly higher than the complete sequence and protein coding gene sequences of mtDNA.3. Among 13 protein genes, Most protein genes use the typical start codon ATG, except CO I gene using special start codon GTG; there are two kinds of stop codons, one is the complete, including TAA (ND2, COI, ATP6, ND4, ND5, ND6) and TAG (ND1, ATP8); the other is incomplete, including TA (ND3, ND4) and T (COⅡ, COⅢ, Cytb).4. All 22 tRNA genes of Pseudorasbora parva mitochondrial genome have a typical clover leaf structure. Some unmatched base pairs occur in the Pseudorasbora parva tRNAs, and overwhelming majority of them are G-U pairs.5. The complete secondary structure of srRNA and 1rRNA genes in Pseudorasbora parva mitogenome are predicted. Compared with published secondary structure of srRNA in Gobiomorphus australis and 1rRNA in Bos Taurus mitogenome. the secondary structure of srRNA is much conserved, the differences are mainly in stem of 17,19 to 20 and loop of 26 and 42; the secondary structure of 1rRNA differences are mainly appeared in two terminal regions.6. The D-loop control region is located in the conserved position between tRNAPro and tRNAPhe. The length is 931 bp, the A+T content is 66.2%, which is much less of the third site of protein coding genes (69.3%). In this study, we found that the D-loop control region of Pseudorasbora parva generally has the 2 terminal associated sequences named TAS1 and TAS2; 2 central conserved sequence blocks named CSB-F and CSB-D; 3 conserved sequence blocks named CSB-1, CSB-2, CSB-3. The TAS1 can fold a potential secondary stem-loop structure, the same structure is observed in other Cyprinidae.7. Phylogenetic analysis from the 3 datasets based on rRNAs data set expressed that the Rasborins are located in the most basal cyprinid group, the monophyly of the Rasborinae was not supported. The remaining cyprinids were clustered into two lineages, Barbine and Leuciscine. Within Barbine, the clade comprised Cyprininae,Schizothoracinae and Barbinae is monophyletic group.In Leuciscine, the close monophyletic group relationships existed between North American phoxinins and Eurasian leuciscins, also in Gobioninae, East Asian endmic cyprinid taxa such as Cultrins, Xenocyprinins, Rasborins, Ctenopharyngodon idellus and Mylopharyngodon piceus, were closely related and recognized as a monophyletic group. The position of Tinca tinca which possibly belonged to the Leuciscine formed a Tincinae. The relationship among these subfamilies were hypothesized to be the following:(Rasborinae, ((Cyprininae, (Schizothoracinae, Barbinae)), (Tincinae, ((Xenocypridinae, Cultrinae), (Gobioninae, Leuciscinae))))). Pseudorasbora parva and Pseudorasbora pumilia are closely relationship by high bootstrap value.8. The result based on 13 protein genes and combined data sets was hypothesized to be the following:((Cyprininae, (Schizothoracinae, Barbinae)), (Rasborinae, ((Xenocypridinae, Cultrinae), (Tincinae, (Gobioninae, Leuciscinae))))). The monophyletic group of Rasborinae and Leuciscinae was not supported as well. The controversial Tinca tinca formed a Tincinae. Pseudorasbora parva and Pseudorasbora pumilia are closely relationship, and the interspecific difference is much little according to the research of morphological classification and molecular evolution.