Determination Of Mitochondrial Genome Sequence Of Five Species Of Chrysosporium And The Phylogenetic Development

Pyraloidea is the second largest groups of the Lepidoptera, the vast majority of which are destructive pest in agriculture, forestry and storage, especially the pests of borer class. A large area of forestry pests can cause serious economic losses, so how to prevent and treat insect infestation has always been an important topic in the research of scholars. However, with the development of molecular techniques, the study of mitochondrial genome in the Pyraloidea began to increase. But it is mainly concentrated in the mitochondrial genome of individual insect. There are few research in the characteristics of Pyraloidea and the phylogenetic relationship to conduct a comprehensive analysis in depth.In this study, the complete mitochondrial genome of Hypsopygia regina, Pseudargyria interruptella, Evergestis junctalis, Tyspanodes striata and Maruca vitrata had been determined by long amplification polymerase chain reaction (LA-PCR) technique and primer walking method. And then the five new mitochondrial genomes of Pyraloidea were sequenced, annotated and analyzed in details. Combined with the 20 species mitochondrial genome of Pyraloidea has been released in GenBank datebase, used the sequence of the protein-coding genes (PCGs) and the Drosophila yakuba as the dataset and the outer group, respectively. The methods of Maximum likelihood (ML) and Bayesian Inference (BI) were applied to reconstructe and analyse the phylodenetic relationship of these Pyraloidea insects. The main results were showed as follow:1. The complete mitogenome sequences of five Pyraloidea insects (H. regina, P. interruptella, E. junctalis, T. striata and M. vitrata) were 15,212 bp,15,231bp,15,438 bp,15,255 bp and 15,200 bp in size, respectively. So far the mitochondrial genome length of M. vitrata measured in this study is the shortest complete sequence in Pyraloidea. The composition and structure of the mitochondrial genomes measured in the study are consistent with Pyraloidea ones which had been reported. It is closed circular double-stranded DNA molecules, including 13 protein-coding genes (PCGs),22 tRNA genes,2 rRNA genes and an A+T-rich region. The location of the tRNAMet gene in Pyraloidea is different with the primordial groups in Lepidoptera and tRNAIle-tRNAGln-tRNAMet could be replaced by tRNAMet-tRNAIle-tRNAGln.2. Except the non-coding A+T-rich region in whole mitochondrial genome of E. junctalis, there are two longer non-coding intergenic spacers, located between tRNAAla and tRNArg (114 bp), ND3 and tRNAAla (62 bp), which are inserted in series repeat; Additionally, one overlapping sequence involving a total of 88 bp between tRNAPr0 and ND6.3. The base composition of the mitochondrial genome in the five Pyraloidea insects have significant AT bias, H. regina with a lower AT content of 78.65% and T. striata with the hightest content of 81.38%, M. vitrata, P. interruptella and E. junctalis with an AT content of 80.86%,79.40% and 81.08%, respectively.4. In the PCGs of the five Pyraloidea, except the COI gene start with CGA, the remaining 12 genes use the ATN as the initiation; for termination codon usage, COâ…¡ gene stop with the single T in all five mitochondrial genomes, ND5 gene of the P. interruptella use the incomplete codon TA as termination codon, and then the rest of all the PCGs stop with the complete codon TAN.5. All tRNA genes in the five species can be folded into the typical cloverleaf structure with the exception of tRNASer(AGN), in which the dihydrouridine (DHU) arm simply forms a loop. In the secondary structure of the tRNA, there are a certain number of base composition mismatches including U-U and G-U. And the type of G-U is the main existence.6. The A+T-rich region in the five Pyraloidea with a high AT content, and the AT contents of H. regina, P. interruptella, E. junctalis, T. striata and M.vitrata are 94.86%, 90.99%,94.30%,94.51% and 93.62%, respectively. While there are some related conserved elements such as ATTTA, ATAG, poly-T and varying lengths of microsatel-lites in the A+T-rich region of the five moths.7. The PCGs codon usage of the 25 kinds of Pyraloidea moth present the high frequency of the UUA(L), AUU(L), UUU(F) and AUA(M) with an average count of 465.4,415.5,344.5 and 256.5, respectively. Meanwhile the PCGs amino acid usage based on the codon usage, the order from high to low is leucine and isoleucine, phenylalanine, methionine.8. For the phylogeny of the Pyraloidea, the methods of ML and BI are used to re-construct the phylogenetic tree, and they have the same overall topology. Meanwhile, the results showed that they are consistented with the traditional classification, two phylogenetic tree are very well supported the monophyletic of Crambidae and Pyralid-ae, as well as support for the 11 subfamilies (4 in Pyralidae, and 7 in Crambidae) monophyly in the system tree, the relative position of the performance of each subfamily in ML is:((((Phycitinae+Pyralinae)+Epipaschiinae)+Galleriinae)+ ((((Crambinae+Scopariinae)+Schoenobiinae)+(Acentropinae+Evergestinae))+ (Spilomelinae+Pyraustinae))).