Complete Mitochondrial Genome, RDNA ITS-1 Sequence Evidenced Population Differentiation And Phylogeography Of Pieris Rapae Linnaeus

The Pieris rapae Linnaeus is a worldwide distributed insect species which is harmful to a lot of species of vegetable plants．This important economic speciesWas not endemic in China and imported tO our countW about one or two hundreds of years ago and now distributed in most areas of our country However．up tO now．the higher level taxonomic and phylogenetic studies about the butterflies arelimited and remains many controversial issues．in addition．the molecular evolution and comparative genomics researches about the butterflies on the genomic levelhave not been conducted．In this paper,the Pieris rapae．as one representative spcies of Pieridae．its mitochondrial genome sequence was determined by longPCR and primer—walking techniques．meanwhile its mitchondrial genome waspreliminary analyzed here compared with those of other rep—resentative butterflyspecies dertermined,furthermore．the phylogentic trees based on the 1 3 proteinencoding sequecnce of their genome were reconstructed to clarifv theirphylogenetic relationships as well as the phylogenetic position of the Pieridae．Onthe other hand．though there exists a lot of reports about the biological charactersand the measures of biological conrtol of this pest．the sudies about its molecularidentification．population genetic structure and phylogeography are remarkedly rare．In this paper,we determined and compared the nuclear interval transcribed spacers1(ITS一1)sequences of Pieris rapae Linnaeus from 1 6 different localities of Chipa．and using Pieris canidia as the outgroup,we reconstructed the phylogenetic treesby means of neighbor-joining and minimum evolution analyses．aiming to clarifytheir biOgeOgraphical divergence pattem．The details and the main results of thisstudy are listed as following:1．The obtained genome sequence is 1 5 1 57bp in size．containing 1 3protein—encoding genes,2 ribosomal RNA and 22 transfer RNA genes．All the 37genes are conserved in the same orientations as observed in those of other bu~erfly species determined．1 1 1 96 bp of the mtDNA are coding for proteins．1 474 bp fortRNAs,1 3 1 9 bp for rRNA large subunit(1rRNA)．and 764 bp for rRNA small subunit(srRNA)．The A+T—rich region is 393 bp in size．The genes overlapping sequences are 59 bp long in total and are spreading over 10 locations(1-35 bp at each site)．In addition．a total of 1 20 bp intergenic spacer sequences are scattered in1 3 regions at the lengths of 1 to 46 bp,of which the largest(46 bp)is located between the tRNA and tRNA0 genes．2．The nucleotide composition．orientation and gene order of Pieris rapae iS almost same to that of other Lepidoptera．The protein·encoding gene(PCG)ATPase8．ATPase6．ND4L and ND6 genes．which are next to each other at their 3'end in turn,all had the sequences potential to form a hairpin structure．suggestingthe importance of such a structure for precise cleavage of the mRNA of mature PCGs．The putative start codon for COI gene appears to be a tetranucleotide TTA A A G instead of the typical ATN appearing in other 1 2 PCGs．The TAA andTAG codons are used as stop codons in all the protein coding genes except COI,which use the imcomplete stop codon T Total 28 mismatches appear in all the 22tRNAs．and all the 22 tRNAs form typical secondary structures except tRNA0。。(AGN)．which formed a simple loop(7nt)on DHU arm with the absence of thetypical clover leaf structure．2．The NJ and MP phylogenetic trees of eleven representative butterflies basedon the complete 1 3 protein encoding gene sequence data were reconstructed usingthe neighborI_oining(NJ)and the maximum parsimony(IvlP)methods．The resultsshowed that the Papilionidae and Pamassiidae constitute a large lineage．whereas．the Pieridae．which is commonly considered as the close relatives of Papilionidae．constitute another lineage with the Lycaenidae．Nymphalidae and Acraeidae．3．The results of the sequence variaiton analyses showed that the sizes of theITS一1 of Pieris rapae range from 337 to 458 nucleotides in length．AIigned withthe ClustalW software,the total 347 sites,which contain 28 1 conserved．63variable,1 6 parsimonious informative．were obtained．Meanwhile．for the wholepopulation of this study．the nucleotide diversity Pi and the theta(per site)Eta werecaculated to be 0．014 and 0．041．respectively．Morever,total 48 haplotypes wereidentified and the haplotypes diversity O．989 was obtained by the software DNAsp． Additionally,the fixation index fFST)0．351(P