Classification Technique Research Of DNA Barcode In Dermestidae

Dermestidae is a kind of important stored pests, polyphagous, strongly fertile and cause serious damage, belongs to Dermestoidea, Coleoptera. It is widely distributed around the world, including more than 1000 known species, about 100 species in China. Correct identification of species is the basis of pest control, the morphological classification of Dermestidae insect focus on adults, and similar morphology in larva is difficult to identify. Because of high requirements on classification knowledge and experience of professional, the identification of species is spiny in practical work. In recent years, the springing up of molecular biological technology provides new approaches for rapid identification of insect species. But the molecular biological research of Dermestidae insect is still at the preliminary stage, concentrate on Trogoderma, and lack of systematic molecular phylogenetic relationship between related genera in Dermestidae.It is the first time to use DNA barcode technology to study on classification of genetic barcode for stored pest of Dermestidae. The research discussed the experiment of DNA extraction and PCR amplification system, constructed the molecular phylogenetic relationship in Dermestidae, and established DNA barcode database initially, in order to make use of DNA barcode technology to realize rapid identification of pests in Dermestidae. Research contents and results are as follows:1. Morphological classification research in Dermestidae, analyzed characteristics of main species, recorded all specimens with dorsal, ventral and lateral pictures for aided identification.2. Optimize DNA extraction method, explore the effect of different pretreatments for it. After preprocessing, it reduced the chain scission of genomic DNA to some extent, make it easier to amplificate long segments, also benefit amplification of different locus. Of which 9%Na Cl for 3h is best in DNA quality and PCR amplification results with magnetic method.3. Establish suitable PCR amplification system. Reaction system with ABS and increasing Mg2+ can improve the efficiency of PCR amplification and the quality of electrophoretic bands. The research designed 4 pairs of peripheral nested primers, of which A5/A6 showed best in amplification results for the optimal annealing temperature of 49℃. 5 pairs of nested primers combination both could be applied to amplification of a low concentration DNA.4. Sequence analysis of COⅠ(cytochrome C oxidase subunitⅠ)gene. The obtained COⅠ gene sequences of Dermestidae include the front-end 357 bp and back-end 609 bp fragments, adjustment and alignment respectively, computed conserved sites, variable site, parsimony information site, nucleotide composition, nucleotide pairfrequency, codon usage and so on with MEGA. Tested the phylogenetic signal of data set, confirmed it as non-random sequences, which can provide guarantee for the construction of phylogenetic tree.5. Phylogenetic studies. With two methods of maximum parsimony and maximum likelihood, analyzed nucleotide and amino acid sequences of two ends of COⅠgene groups, constructed phylogenetic trees respectively. The back-end fragments of Dermstidae contain more genetic informations, less divergence of phylogenetic trees, higher consistent with morphological classification, so more suitable for phylogenetic research of Dermestidae.6. Preliminary construction of DNA barcode. Evaluated COⅠgene sequences of Dermestidae from 3 parts of genetic distances between species, NJ phylogenetic tree and barcoding gap. Results showed that the segment completely accords with the inspection standards of effectiveness as DNA barcode, can achieve the purpose of identification. We initially established DNA barcode database of 3 genera in Dermestidae with abtained gene.