| Mitochondrion, an important organelle existing in eukaryotic cells, is correlated with energy metabolism, apoptosis, aging, disease and oxidative phosphorylation. The growth and proliferation of mitochondrion are controlled by nuclear genome and its own genome, so it is called semi-autonomous organelle. Mitochondrial genomes have been widely used in phylogenetic studies and comparative and evolutionary genomics of insects, and mitochondrial genomes are ideal molecular markers of population genetics and molecular evolution. All of these are because of its matrilineal inheritance, lacking of extensive recombination, conservative gene structure and composition, low mutation rate and faster evolution than nuclear genome. Diptera is one of the largest families among the insecta with described species widely distributed throughout the world. It included kinds of mode insects and medical pests, and it is important for research of the control of many infectious diseases.In order to enrich the Dipteran mitochondrial genome data, and go further into studies of the mitoehondrial genomes in Dipteran insects, we analyzed the structural characteristics, base composition and conserved regions of dipteran mitochondrial genome based on known26whole-length mitochondrial genome sequences of Diptera using comparative genomics and bioinformatics methods, and designed a set of universal primers for mitochondrial genome sequencing of Diptera. We successfully got the complete mitochondrial genome of Delia antiqua using these primers, and annotate the complete mitochondrial genome of the D. antiqua.Some conclusions drawn from the study as follows:①The mitochondrial genomes of Diptera are quite conservative with37coding genes (13protein-coding genes,22tRNA genes and two rRNA coding genes)and a non-coding region (AT District) with various length. The base composition on these genomes is not balanced with AT content significantly higher than G and C.We identified11conservative regions based on the nucleotide and amino acid sequence conservation analysis of whole genome, and designed26pairs of universal primers for mitochondial genome sequencing of Diptera with each pair of primers anchored to identified conservative regions. The application of this set of primers to the Delia antiqua mitochondrial genome sequencing showed that the set of primers is efficient and operable.②The D. antiqua mitochondrial genome is a double-stranded circular molecule with16141bp in length. Gene numbers and organization is similar to that of the typical insect mitochondrial genomes. It contains37genes and a non-coding region.14intergenic spacers were found ranging in size from2-26bp, the longest intergenic spacer was located between tRNAArg and tRNAAsn. On the other hand, there were12gene overlaps in the mitochondrial genome of D. antiqua and involved a total of43bp, the longest overlap was8bp and appear between tRNATrp and tRNACys.③The nucleotide composition of the mitochondrial genome of D. antiqua showed obvious bias towards A and T. The A+T content of the whole genome was78.5%, the A+T content of isolated PCGs, tRNAs, rRNAs and control region were all above70%, the control region has the highest A+T content (93.7%). The skew statistics of the whole genome showed that the whole mitochondrial genome of D. antiqua were CG-skewed distinctly and AT-skewed.④All the13protein-coding genes use ATN as initiation codon except COI gene, which was started with ATCA. Most protein-coding genes have complete termination codons. only COⅡ and ND5have the incomplete termination codon T. The bias is reflected in the codon usage and amino acid composition. The protein-coding genes of D. antiqua mitochondrial genome preferred to use codon UUA (Leu) and Leucine.⑤All the tRNAs have the typical clover-leaf structure, except tRNASer (AG which does not contain the dihydrouridine (DHU) arm. and there are22mismatches with G-U and U-U in the tRNAs. The locations of the16S rRNA and12S rRNA genes were at N-strand. and the16S rRNA gene is1330bp long, and the12S rRNA is784bp long. Their A+T content were82.26%and78.32%respectively. The control region is1266bp in length, and includes three kinds of special regions—poly-T stretches, a (TA)n stretch and several G(A)□T structures.⑥Finally, nucleotide sequences of13protein-coding genes are used in a phylogenetic study. Both maximum likelihood and Bayesian inference analyses highly support that the Delia antiqua has a nearer relationship with Calliphoridae.This work is the first report about structural characteristics, base composition and conserved regions of dipteran mitochondrial genome, and designed a set of universal primers for mitochondrial genome sequencing of Diptera. The application of this set of primers to the D. antiqua mitochondrial genome sequencing showed that the set of primers is efficient and operable, and have got the complete mitochondrial genome of the D. antiqua. We enrich the Dipteran mitochondrial genome data and provide reference and basis for further research of Diptera mitochondrial genomes. |
