Study On Mitochondrial Genome Analysis And Phylogeny Of Four Species In Melanotaeniidae

Melanotaeniidae belongs to the order Atheriniformes,and is mainly distributed in Australia.Their body length vary in the range of 6-18 cm,and has high degree of variability in color.So it’s also named as "beauty" or "rainbow fish" in China.There is no domestic species of Melanotaeniidae,all of which are imported from the origin inhabitat,but there are few molecular(mtDNA)data about them,causing difficulties for their introduction and conservation.Melanotaeniidae has obvious geographical distribution characteristics and is considered to be an important biomaterial of biogeography in Australia.So,it has important phylogenetic significance.In this study,the method of PCR and cloning sequencing were applied to determine the mitochondrial genome sequence of four Melanotaeniidae species,then were stitched,annotated and analyzed by bioinformatics software.We selected 12 Mitochondrial genome sequences of Atheriniformes species and a Mitochondrial genome sequence of Oryzias latipes as the outgroup,used the adjacency method(NJ),maximum likelihood method(ML)and Bayesian method(BI)to explore the phylogenetic relationship among Melanotaeniidae species.On the basis of phylogenetic results combined with geographical distribution,we explored the phylogenetic relationship between the Melanotaenia praecox with the genus Melanotaenia,the genus Glossolepis and the genus Chilatherina.The main results are as follows:A.Mitochondrial genome sequencing and structure analysis of 4 Melanotaeniidae speciesThe measured mtDNA lengths of the four species were 16,539bp(Glossolepis incisus),16,493bp(Melanotaenia boesemani),16,536bp(Melanotaenia praecox)and 16,530bp(Melanotaenia australis).The length of the difference is mainly caused by the large difference in the noncoding region.The complete mitochondrial genome include 13 protein-coding genes,22 transfer RNAs,two ribosomal RNAs genes and 2 main noncoding region(the light-strand replication origin and control region).Except that ND6 and 8 tRNA genes are encoded in the light chain(L-strand)and the rest are encoded in heavy chain(H-strand).Normally only CO Ⅰ genes using GTG as initiation codon,whereas the ATP6 gene of the Glossolepis incisus was also used GTG as the initiation codon.The AT base content of total mtDNA was higher than GC,and the third locus of protein codon was shown to have more severe low G bias.In the 22 tRNAs,only the tRNA-Ser(UGA)gene was deleted from the DHU arm,the other tRNA genes could form a stable clover type secondary structure.The light-strand replication origin(OL)of four kinds of fish can form a stable stem-ring structure.In the four fish control areas,we find terminal associated sequences(ETAS),central conserved sequence block(CSB-B,CSB-E),conserved sequence block(CSB-1,CSB-2,CSB-3).By comparison,it is found that the variation of the ETAS and central conserved sequence block are greatest.Mitochondrial genome composition and arrangement of 4 fishes are unanimous,the tRNA secondary structure is also similar,rRNA and tRNA mutation sites accounted for 7.3% and 5.6%.These show the conservative nature of the mitochondrial genome of the Melanotaeniidae in the evolutionary process.Protein genes and non-coding regions bases are very different and have rich variation.the mutation sites accounted for 19.5% and 20.3%,respectively.These reflect that Melanotaeniidae species have a lot of genetic diversity.B.Phylogenetic study of Melanotaeniidae based on mitochondrial genomeWe selected 12 Mitochondrial genome sequence of Atheriniformes species and a Mitochondrial genome sequence of Oryzias latipes as out group,used the adjacency method(NJ),maximum likelihood method(ML)and Bayesian method(BI)to build phylogenetic trees.The results show that the three methods have been consistent with the topological structure.firstly,Melanotaeniidae and Atherinidae was clustered into single groups,then they poly as a single group with Isonidae,Atherinopsidae is located at the outermost layer of the topology.Seven species of the Melanotaeniidae form a very obvious monophyletic group.M.praecox do not get together with other Melanotaenia fish,but cluster into sister group with G.incisus.Through their geographical distribution analysis,we believe that M.praecox is far from genus Melanotaenia in genetic relationships and geographical relationships,and is closer to genus Chilatherina and genus Glossolepis.The results will provide a basis for the re-division of the classification of M.praecox.