Measurement And Analysis Of Mitochondrial Genome Sequences Of Three Species Of Chinese Tiger And

Lepidoptera is the the second largest families in the insecta, including butterflies and moth. Lepidoptera have so huge taxonomic diversity that the traditional morphological classification is difficult to give an accurate classification and definition. With the development of molecular biology and the computer technology application, the DNA data more and more used in the studies of Lepidoptera. As of october2013, the complete mitogenome sequences of Lepidoptera have been reached to99species in the GenBank database, which include the butterfly50and moth49. Relative to the species-richness of Lepidoptera insects, the existing information of Lepidoptera is very limited.In order to enrich the lepidopteran data of mitochondrial genome, and go further into phylogenetic relationships of superfamilies in Lepidoptera. The Sub-PCR technique and primer walking were used to finish the whole genome amplification in this research. Mitochondrial DNA (mtDNA) of three (Ningshan(Luehdorfia choui Shou et Yuan), Zhouzhi, Huayin) population of Luehdorfia chinensis (Leech,1893) and Tyspanodes hypsalis (Warren,1891) were sequenced, assemblyed, annotated and analyzed in details. Combined with the99species complete mitochondrial genome of Lepidoptera deposited in GenBank database and4new sequenced complete mitochondrial genome, the phylogenetic relationship were reconstructed by using the phylogeny methods of Maximum parsimony, Maximum likelihood and Bayesian, to reconstructed the phylogenetic relationship of lepidopteran insects. The results were showed as follows:1. The content and arrangement of four lepidopteran insects mitochondrial genomeThe mitochondrial genome size of the three (Ningshan, Zhouzhi, Huayin) population of I. chinensis and T. hypsalis are15,580bp,15,580bp,16028bp and15329bp, respectively. Containing13protein-coding genes (PCGs),2ribosomal RNA genes (rRNAs),22transfer RNA genes (tRNAs) and a non-coding A+T-rich region in same order and orientation as the complete sequenced mitochondrial genomes of the other lepidopteran insects.The orientation and arrangement of genes are identical to the completely sequenced lepidopteran tDNA. The determined mitochondrial genome sequences of the three population of L. chinensis and T. hypsalis are all contain overlap regions and intergenic spacers. The Huayin population contains8overlap regions spinning18bp and18intergenic spacers spanning193bp. The Zhouzhi population contains9overlap regions spinning28bp, and18intergenic spacers spanning193bp. And, there are9overlap regions spinning28bp, and18intergenic spacers spanning192bp in the Ningshan population. Genes overlap in T. hypsalis mitochondrial was9of26bp, intergenic of its was14of180bp.The three (Huayin, Zhouzhi, Ningshan) population of L. chinensis and T. hypsalis complete mitochondrial genome have obvious bias with AT, and the A+T content of mitochondrial genomes (J strand) are81.90%,81.40%,81.60%and81.42%, respectively. Meanwhile, tRNAs gene, rRNAs gene, PCGs and A+T-rich region have biased toward adenine and thymine.Most protein-coding genes have typical ATN initiation codon, except the COI gene of the three population of L. chinensis and T. hypsalis, which is tentatively designated by the CGA codon as observed in other lepidopterans. There were two kinds of termination codon, complete termination codon (TAG and TAA) and imcomplete termination codon (TA and T). Most protein genes use the typical and complete termination codon:TAA, except the COI and COII gene in the three population of L. chinensis, which use the incomplete termination codon:T. The T. hypsalis complete mitochondrial genome, most protein-coding genes have typical TAN termination codon, but the COII and ND5gene respectively use imcomplete termination codon T and TA.Except tRNASer(AGN) gene, all of the tRNAs gene of the three population of L. chinensis and T. hypsalis have a typical clever-leaf structures. There are some unmatched base pairs in all22tRNAs secondary structure in the three population of L. chinensis and T. hypsalis, and the main unmatched base pairs were G-U.The A+T-rich region in the three (Huayin, Zhouzhi, Ningshan) population of L. chinensis and T. hypsalis are1095bp,646bp,648bp and350bp respectively in length, with the AT content up to96.35%,97.37%,97.76%and95.43%. And the three population of L. chinensis contain some typical structures of other lepidopteran mitogenomes, for example, microsatellite-like repeating sequences (AT)n elements, poly-A and poly-T structures etc.2. The content and arrangement of mitochondrial genome in LepidopteraThe orientation and arrangement of97sequenced lepidopteran insects mitogenomes are identical to the three population of L. chinensis and T. hypsalis. But the placement of tRNAMet is different from the ancestral gene order of insects, and the tRNA cluster of tRNAne-tRNAGln-tRNAMet in ancestral insects has turned into the arrangement of tRNAMet-tRNAIle-tRNAGln.3. Phylogenetic analyses of LepidopteraPhylogenetic relationship of Lepidoptera were reconstructed using the maximum likelihood (ML), the bayesian inference (BI) and the maximum parsimony (MP) methods based on the combined dataset of nucleotide sequences of13protein coding genes, rRNA genes (srRNA and lrRNA) and PCGs&rRNAs, build9phylogenetic trees. The results showed that analyses based on the combined dataset of PCGs are trustworthier compared with the combined dataset of rRNAs and PCGs&rRNAs. The conclusions were drawn as follow:1) Hesperoidea is a branch of Papilionoideaa, however, this is inconsistent with the traditional classification, which classify Papilionoidea and Hesperoidea as a sister group.2) Noctuoidea, Bombycoidea and Geometroidea, these three superfamilies have a closer relationship.3) The three population of L. chinensis were got together in all9phylogenetic trees constructured by three methods, which do not supported the opinion that L. choui should be a new species. But more likely support that the population attributed to the Ningshan population of L. chinensis.