Determination And Analysis Of Mitochondrial Genomes Of Six Stored-grain Beetles

The Coleoptera is the most diverse of all organismal lineages with more than 390,000 kinds,accounting for about one-third of the world’s known total number of insects,larvae and adults of many species are seriously harmful to animal husbandry,agroforestry and storage industry.It is known as beetles,belonging to class Insecta,Pterygota,Holometabolism,and composed of four suborders Archostemata,Myxophaga,Adephaga and Polyphaga.The beetle is the most important insect in the stored grain pests.The stored grain beetle is a kind of insect which has a normal life and reproduction in the dry stored grain,currentlywe use respiratory agents such as phosphine to control it.Phosphine mainly act on mitochondrial respiratory chain,many genes encoding by mitochondrial genome are directly involved in respiratory process.As a result of the characteristics of easy obtained,length and genetic constitution relatively stable,rapid evolution rate and maternal inheritance,mtDNA has been widely used as molecular markers and in the fields of insect phylogeny,genetic variation and differentiation of populations.Nowadays,44 whole sequences and 76 nearly complete sequence of Coleoptera mitochondrial genome are available in the GenBank database.However,there are only two complete mitochondrial genome sequences which come from stored grain beetle.In this study,we successfully sequenced mitochondrial genomes of Tribolium confusum,Tribolium audax,Sitophilus oryzae,Oryzaephilus surinamensis,Callosobruchus chinensis and Lasioderma serricorne by PCR amplification.The new mitochondrial genomes were annotated with reference to the mitochondrial genome informations of other Polyphaga beetles in the NCBI database.Combining with other Polyphaga mitochondrial genomes in the database,protein-coding genes have been completely compared,and phylogeny of Polyphaga has been reconstructed.The main conclusions drawn from this study were listed below:1.The obtained sequences length of T.confusum,T.audax,S.oryzae,O.surinamensis,C.chinensis and L.serricorne mitochondrial genomes are 15,813 bp,15,925 bp,14,274 bp,14,288 bp,16,148 bp and 14,241 bp respectively.The sequenced mitochondrial genome structures expect the C.chinensis are consistent with those of other Polyphaga insects.In the typical gene order of Invertebrates,a tRNA-gene cluster between A+T rich region and ND2 gene includes tRNAIle(I),tRNAGln(Q),and tRNAMet(M)genes in this order(IQM).However,in the mitogenome of the C.chinensis,the tRNAGln gene was rearranged between the tRNASer(UCN)and the ND1 genes.2.All protein-coding genes of Polyphaga mitochondrial genomes in this study have an obvious AT skew,especially in the third codon positions,whose A+T content can be as high as 78%.All mitochondrial genome have intergenic region and overlapping region,and a conserved motif(TACTA)in intergenic region between the tRNASer(UCN)and ND1 genes can be found in all six species.In addition,one conserved motif(ATGATAR)can be found in the overlapping sequence between ATP8 and ATP6 genes;the other one(YTATCAT)can be also found in both ND4 and ND4L genes.3.The functional RNA of the six species included tRNAs and rRNAs.Nearly all of the tRNAs could form the classical clover leaf structure,while tRNASer(AGN)was excluded for its DHU arm was lost.There were many mismatches in tRNAs secondary structure,and in the presence of mismatches,G-U mismatch was the main type.Most of the anticodons of the tRNAs were identical and completely conserved,while the tRNALys of S.oryzae and C.chinensis used the atypical UUU as their anticodon.4.All protein-coding genes of 41 kinds of Polyphaga beetles mitochondrial genomes in this study have an obvious AT skew.In terms of single gene,all PCGs distributed in J-strand generally have a slight skew of bases C and T,while each of the other four PCGs coded by N-strand has an obvious skew of base G and T.Namely,AT skew and GC skew of mitochondrial protein-coding genes is related to the coding strand.The four amino acids(Leu,Ser,Ile and Phe)are having a high using frequency in protein-coding genes of Polyphaga beetle mitochondrial.The statistics of start codon use of the mitochondrial protein-coding genes in Polyphaga beetles show that most of the genes uesd ATG as their start codon,while ATA,ATT and ATC also occupy a certain proportion.5.The phylogeny of Polyphaga was reconstructed using ML and BI method based on all 13 protein-coding genes.The monophyly of Chrysomeloidae,Curculionoidea and Tribolium were supported in the analysis.At the same time,the fact of sister-group relationship of the Chrysomeloidae and Curculionoidea was well confirmed.Topology of the resulting phylogenetic tree showed that O.surinamensis was at the basal to the Tenebrionidae,the L.serricorne and the Chaetosoma scaritides of Cleroidae clustered into one branch.And we speculated that it might be because of these two beetles were the only one sample representative of its family level.