Sequencing And Analysis Of The Full Mitochondrial Genome Of Six Species Of The Subfamily

In this study,the complete mitochondrial genomes(mitogenome)of six Spilomelinae species which contain Conogethes punctiferalis(Guenee,1854),Diaphania perspectalis(Walker,1859),Palpita nigropunctalis(Bremer,1864),Pleuroptya inferior(Hampson,1898),Pycnarmon lactiferalis(Walker,1859)and Pycnarmon pantherata(Butler,1878)were sequenced by PCR with primer walking method and analyzed them in comparative genomics and phylogenetic relationship.The main results showed as follows:1.The mitogenome sequences of the C.punctiferalis,D.perspectalis,P.nigropunctalis,P.inferior,P.lactiferalis and P.pantherata were 15325 bp,15249 bp,15226 bp,15348bp,15219 bp and 15545 bp in length.Furthermore,the complete mitochondrial genome of P.pantherata is the largest genome compared with published Pyraloidea mitogenome,which is caused by the varying length of the control region.The composition and arrangement of six Spilomelinae species mitogenomes were consistent with other sequenced Pyraloidea,including 13 protein-coding genes(PCGs),22 transfer RNA(tRNA),2 ribosomal RNA(rRNA)and a variable A+T rich region.2.Among thirty-seven genes,twenty-three genes(nine PCGs and fourteen tRNA)were coded on the majority strand(J-strand),and the rest fourteen genes(four PCGs,two rRNA and eight tRNA)were located on the minority strand(N-strand).The gene order and orientation were the same as what in other sequenced pyraloidea species,in an order of tRNAMet-tRNAIle-RNAGln between the A+T-rich region and ND2,which differs from the species Thitarodes renzhiensis and Ahamus yunnanensis in Hepialoidea with the ancestral gene arrangement of tRNAIle-tRNAGln-tRNAMet.This indicated that the mitogenome arrangement of the lepidopteran insects may have evolved independently after splitting from its stem lineage.3.There were a few non-coding regions named intergenic spacer in the complete mitochondrial genomes of six Spilomelinae species with the exception of control region,in which the largest one occured between tRNAGln and ND2 gene.Additionally,there were a few overlapping nucleotides ranged in sizes from 1 to 8 bp in the six Spilomelinae species mitogenome.The longest overlapping is 8 bp(AAGCCTTA)between tRNATrp and tRNACys,and the lengths of another major overlapping regions is 7 bp(ATGATAA)between the ATP8 and ATP6.4.The mitogenomes of the six Spilomelinae species were biased towards AT.The A+T nucleotide compositions of the whole mitogenome of C.punctiferalis,D.perspectalis,P.nigropunctalis,P.inferior,P.lactiferalis and P.pantherata were as follows:80.52%,81.01%,81.03%,81.49%,81.68%,81.42%,respectively.Significantly higher G+C content.The AT skewness was not only reflected in base composition but also in codon usage.5.The start codon and stop codon usages of 13 PCGs in six Spilomelinae species were analysed.Twelve of the 13 PCGs started with typical ATN codons,but the COI began with CGA.For stop codon,TAA was used in all the PCGs,while the COI,COII and ND5 existed incomplete stop codon T or TA.6.The length of 22 tRNAs in the six Spilomelinae(C.punctiferalis,D.perspectalis,P.nigropunctalis,P.inferior,P.lactiferalis and P.pantherata)ranged from 62 to 74 bp.All tRNAs had the typical cloverleaf secondary structure except for tRNASer(AGN)which lacks the DHU stem.Furthermore,there were some number of unmatched base pairs in tRNAs secondary structure,the number of unmatched base pairs are 24 pairs,25 pairs,23 pairs,24 pairs,24 pairs and 27 pairs,respectively.As other lepidoptera insects,the rRNA genes(16S rRNA and 12S rRNA)genes located between the tRNALeu(CUN)and A+T-rich region were separated by tRNAVal.7.The A+T-rich region of C.punctiferalis,D.perspectalis,P.nigropunctalis,P.inferior,P.lactiferalis and P.pantherata was located between the 12S rRNA and tRNAMet,and their lengths were 354 bp,339 bp,366 bp,327 bp,339 bp and 506 bp,respectively.A+T-rich region displayed the higher A+T content than 37 genes in the mitogenome.In addition,there are some conserved sequences,poly-T and poly-A structures,as well as different tandem repeats in the A+T-rich regions.8.Phylogenetic analysis was performed based on the 13 PCGs of the thirty-six sequenced Pyraloidea species mitogenomes using BI and ML methods.Both the BI and ML trees are similar topologies.The tree was divided into two clades named Pyralidae and Crambidae.Within Crambidae it is composed of two main clades,one clade contains Crambinae,Nymphulinae,Schoenobiinae and Evergestistinae,the other clade includes Pyraustinae and Spilomelinae.The phylogenetic relationships among the two families were analysed using BI as follows:((((Epipaschiinae+Pyralinae)+Phycitinae)+Galleriinae)+(((Crambinae+Schoenobiinae)+(Nymphulinae+Evergestistinae))+(Pyraus tinae+Spilomelinae))).