Sequencing, Annotation And Analysis For Mitochondrial Genomes Of Three Booklice

Booklice (Psocoptera:Liposcelididae) are pests of stored grain products. They pose a considerable economic threat to global food security and safety. To date, only one booklouse, Liposcelis bostrychophila, has been sequenced for complete mitochondrial (mt) genome. Unlike most insect, the mt genome of L. bostrychophila has two circular chromosomes. Meanwhile,5parasitic lice in Phthiraptera also have more than one mt chromosome, but their mt genomes are different from that of L. bostrychophila. The relationship between Psocoptera and Phthiraptera has not been reached an agreement. Different results have been aquired by phylogenetic analysis based on several partial genes and the traditional morphology. In this study, two methods were used to build phylogenetic tree based on mt genome sequences. To further understand whether the fragmented mt genome occurred in other booklice, and the relationship between the genus Liposcelis and lice, more sequence of booklice mt genomes should be sequenced. Meanwhile, the sequence information can enrich mt genome database and provide materials for comparative genomic study.In this study, Long-PCR was used to amplify the mt genomes of L. decolor, L. entomophila and L. paeta. Subsequently, the characteristics of four mt genomes were analyzed by bioinformatic approaches. Then, two methods (ML and BI) were used to infer phylogenetic relationships between Psocoptera and Phthiraptera based on mt genome sequences. The main results are as follows:1The mitochondrial genome of L. decolorThe mt genome of L. decolor is one typical circular chromosome, in size of14,405bp (GenBank Acc No:JX870621). The size and the circular organization of this mt chromosome were confirmed by two long, overlapping PCR amplicons. The amplification fragments are9.1kb (dl-d2) and5.5kb in size (d3-d4), with a33bp overlap in cox3and a92bp overlap in rrnS. The nucleotide composition of the strand with19genes is:A:40.49%(5,832), T:34.74%(5,005), C:12.14%(1,749) and G:12.63%(1,819), with a73.25%A+T content. This mt genome contains37genes that are typically found in animal mt genomes (13PCGs,22tRNAs and2rRNAs),19genes were transcribed on a strand, and the other18genes on the complementary strand. However, the arrangement of these genes in L. decolor differs substantially from that observed in L. bostrychophila and other insects. These two booklice just share a gene block, atp8-atp6.2The mitochondrial genome of L. entomophilaThe mt genome of L. entomophila is different from L. decolor, and consists of two smaller chromosomes like that of L. bostrychophila. The two chromosomes were named Le chromosome Ⅰ (GenBank Acc No:KF649223) and Ⅱ (GenBank Acc No: KF649224). They are assembled by four long overlap fragments:E1-E2(5.6kb) and E3-E4(6.4kb), E5-E6(10.2kb) and E7-E8(3.4kb) and in size of11,599bp and12,675bp.28of the37genes were found in almost all animals and15pseudogenes were annotated for the two chromosomes. However, there are massive non coding sequences in this mt genome. Coding sequence, pseudogenes sequence and non coding sequences account for52.76%,10.53%and36.72%of the entire length of L. entomophila mt genome. Le chromosome Ⅰ just consists of11protein coding genes (atp6, cob, coxl-cox3and nadl-nad6) and three pseudogenes. Le chromosome Ⅱ encodes two ribosomal RNA genes (rrnL and rrnS),14transfer RNA genes(trnD, trnF, trnI, trnK, trnLl, trnL2, trnM, trnP, trnQ, trnR, trnSl, trnS2, trnT and trnY) and atp8, additionally, with12pseudogenes. Although37typical genes were found in animal mt genome, nad4L and8tRNA genes have not been identified in the mt genome of L. entomophila. The A+T content of this mt genome is78.64%, which is much higher than that of other booklice. Meanwhile, L. entomophila has a unique mt gene arrangement and share no gene boundary or gene block with L. bostrychophila or L. decolor.3The mitochondrial genome of L. paetaLike L. entomophila and L. bostrychophila, the mt genome of L. paeta consists of two chromosomes. The two chromosomes were named as Lp chromosome Ⅰ (GenBank Acc No:KF649226) and Ⅱ (GenBankAccNo:KF649225), in size of11,500bp and10,322bp. Each of them is assembled by two long overlap fragments:P1-P2(4.9kb) and P3-P4(7.7kb), P5-P6(1.9kb) and P7-P8(8.9kb), respectively.29genes,8pseudogenes and a lot of non coding sequences have been found in the mt genome of L. paeta. They account for59.40%,11.31%and29.29%of the entire length of the mt genome, respectively. Lp chromosome Ⅰ consists of10protein coding genes (atp6, atp8, cob, coxl-cox3, nadl, nad.2, nad4and nad5), rrnL and three tRNA genes (trnS2, trnQ and trnH), and Lp chromosome Ⅱ consists of3protein coding genes (nad3, nad4L and nad6), rrnS and11transfer RNA genes (trnA, trnC, trnF, trnG, trnK, trnL2, trnM, trnR, trnT, trnV and trnY).8of22typical tRNA genes of animal are not found in this mt genome. For psedogenes, just PrrnL-1locates in mt chromosome Ⅰ, and the others locate in mt chromosome Ⅱ. The A+T content of this mt genome is75.69%, slightly lower than that of L. entomophila. L. paeta shared3mt gene boundaries with L. bostrychophila:atp8-atp6, cox3-coxl and nad4-nadl.4Comparative analyses of four booklice mitochondrial genomesFour mt genomes of booklice are compared and several differences among them are identified.1) The four booklice have different mt genomes stuctures, one typical mt chromosome for L. decolor and two small chromosomes for each of the other three.2) The mt genomes of L. entomophila and L. paeta are in size of24,274bp and21,822bp, much larger the mt genomes of L. bostrychophila (16,463bp) and L. decolor (14,405bp).3) Booklice mt genomes vary greatly in composition. L. decolor just contains37genes, which were typical in insect mt genome, however, the three other booklice contain several psedogenes and long non coding sequences additionally.4) Different distributions of mt gene, psedogene and non coding sequence exist in the three fragmented mt genomes. The two mt chromosomes of L. bostrychophila contain nearly equivalent amounts of mt genes, psedogenes and long non coding squences. For L. entomophila and L. paeta, mt genes are gathered in one chromosome, whereas psedogenes and non coding sequences in the other chromosome.5) There is a great variation among A+T content in four booklice mt genome, and the highest is78.64%(L. entomophila) and the lowest is68.63%(L. bostrychophila).6) Four booklice have different mt gene arrangements and share no gene block. Only L. bostrychophila and L. paeta share three gene blocks:atp8-atp6, cox3-coxl and nad4-nadl.5Phylogenetic analysesTwo methods (ML and BI) are used to identify the phylogenetic relationship among17species from Psocoptera and Phthiraptera according to the mt genome sequences. These phylogenetic trees show that the genus Liposcelis clustered into the same branch. The close relationships among the four booklice are strongly supported in the analyses. L. decolor (group B) is split off from the others, then, L. entomophila (group A) is separated from the remainders. Furthermore, these four booklice from Liposcelis formed a sister clade to all lice (order Phthiraptera). It proved that Phthiraptera is a monophyletic group.