| Objective: To conduct sequencing of and annotation to the mitochondrial genome of the Lepidoglyphus destructor of Glycyphagidae and Chortoglyphus arcuatus of Chortoglyphidae,with an attempt to define the phylogenetic status of the two species of Acariformes on the mitochondrial genome level basis.Methods: DNeasy Blood & Tissue Kit was used to extract the total genomic DNA from L.destructor and C.arcuatus.The complete mitochondrial sequence of L.destructor was initially amplified using the common primers into the target fragments of COX1,CYTB,rrn S and ND4-ND5 genes.Then the amplified products were cut,recovered,cloned and purified,and underwent first-generation sequencing through contract with Sangon Bioengineering（Shanghai）Co.,Ltd.Specific primers were designed based on the sequencing results for amplification until splicing into a ring.DNA was also extracted from C.arcuatus,and directly subjected to second-generation sequencing by Sangon Bioengineering Co.LTD.Mitochondrial genomes of both L.destructor and C.arcuatus were preliminarily annotated using MITOS Webserver,and compared according to mitochondrial genomes of Acaroid mites available in Gen Bank（NC023778,NC024637,NC026079,NC038207）.Then the mitochondrial genome was mapped using OGDRAW,and manually corrected using bioinformatics software Geneious.The secondary structure of the t RNA gene（with a parameter of 05-invertebrate）was predicted via MITOS Webserver,and the unpredicted t RNA was compared manually with the annotated Acaroid mite mitochondrial gene.Finally 22 t RNA genes were obtained.r RNA models for the lines of L.destructor and C.arcuatus were constructed based on the previously reported mitochondrial r RNA model of Tyrophagus longior.Stem loop structure at the control region was speculated in compliance with the Mofld web tool,and the tandem repeat sequences at the control region were detected using Tandem Repeats Finder.Phylognetic tree was constructed as maximum likelihood（ML）and Bayesian inference（BI）according to the mitochondrion in 13 protein genes in the 37 species of Acariformes released in Gen Bank and 2 species of Parasitiformes used as an out-group,together with the protein gene of Carpoglyphus lactis,L.destructor and C.arcuatus of protein gene（a total of 42 species）,respectively to construct phylogenetic tree.Results: The length of mitochondrial gene was 14 641 bp and 14 865 bp respectively for L.destructor（Gen Bank accession no.: MT248117）and C.arcuatus（Gen Bank accession no.: MT248116）.Both species included 13 protein-coding genes,22 t RNA genes,two r RNA genes and a control region.The sequence of L.destructor and C.arcuatus genes was consistent with that of Aleuroglyphus ovatus,Caloglyphus berlesei,Dermatophagides farina and Rhizoglyphus robini,whereas t RNA genes of L.destructor[（54.18±2.72）bp] and C.arcuatus [（53.59±4.66）bp] were shorter,and generally failed to be folded into typical clover structures except for the trn K gene from L.destructor and the trn N and trn Y genes from C.arcuatus.The rrn S and rrn L gene were 661 bp and 1 020 bp,and 660 bp and 1 019 bp,respectively for L.destructor and C.arcuatus.In addition,the rrn S and rrn L genes of the two species of mites were located in the J-strand and between the trn K and trn W genes,yet blocked by the trn V gene.Both L.destructor and C.arcuatus contained a control region as well as two stable stem-ring secondary structures and a tandem repeating unit that could be regarded as microsatellite elements.The phylogeny of main taxa in Acariformes was analyzed by ML and BI.The results showed that the phylogenetic trees constructed by the two methods resulted in similar topological structure.Phylogenetic analysis showed that C.arcuatus and the two species of Histiostoma,Histiostoma blomquisti and Histiostoma feroniarum,were clustered into one branch,indicating that the family Chortoglyphidae was closely related to the family Histiostomatidae,while Glycyphagidae was fallen into a single branch.Conclusion: We successfully sequenced and annotated the mitochondrial genomes of L.destructor and C.arcuatus,which is also the first annotation of mite genes in Glycyphagidae and Chortoglyphidae,and complement to the mitochondrial genomes of Acarid mites,the reservoir of Astigmata,and even Acari.Our findings may provide a reference for genetic identification of these mites.Importantly,C.arcuatus is identified to be closely related to the Histiostomatidae mites through phylogenetic analysis of the mitochondrial genomes in the 38 species of Acariformes mites,yet Glycyphagidae,Carpoglyphidae and Acaridae as a single branch. |
