Identification Of Necrophagous Flies Species In Some Regions Of China By Molecular Systematics Approaches

Identification of necrophagous flies species is an important step to use insects for forensic analysis, and it plays a key role in busting a crime. The traditional method of identifyingn necrophagous flies species is mainly based on morphological features. However, in the circumstances of the incomplete specimens and early growth periods, it is difficult to identify types by morphological methods. Researchers have been looking for effective classification methods to resolve this problem. With rapid development of molecular biology techniques, especially the application of sequencing technology in the past few years, the research of insect molecular systematics has gone further, and application of molecular marker techniques in necrophagous flies classification and identification is a hot spot of forensic entomology. Currently, the main methods are restriction fragment length polymorphism (RFLP), random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), simple sequence repeat (SSR), inter simple sequence repeat DNA (ISSR), and DNA sequence analysis. ISSR molecular marker technology has the advantages of good repeatability, high stability, and rich polymorphisms, and it can provide more information on the genome. In this study using ISSR marker technology, we analyzed common necrophagous flies in Guangzhou, Shenzhen, Yangjiang,Nanjing, Changchun, Yichang, Beijing, Wuhan, Chengdu and other areas to find specific fragments of the species and provide molecular taxonomy evidence for identification of necrophagous flies. In this study a total of 22 primers were designed, and eight specific primers were selected for necrophagous flies species identification. Fly samples in the above-mentioned areas were amplified by PCR method, and they produced 882 clear stable fragments, of which 692 fragments were polymorphic, and polymorphism percentage was 78.5%; each necrophagous fly was amplified to the specific fragments; the results of cluster analysis results were the same as the results of morphological classification. The results showed that the Musca domestica, Chrysomyia megacephala, Lucilia sericata, Boethcherisca peregrina and Helicophagella melanura in different cities and regions of China have different Band, and the houseflies of different regions show geographical and genetic polymorphism Band. ISSR marker method could distinguish houseflies' intraspecies genotype and genetic differences; it also could distinguish several necrophagous flies interspecific and intraspecies genetic polymorphism. ISSR marker method is a simple, stable and reliable molecular marker, and it can be used as an effective method of necrophagous flies identification. This study identified five kinds of necrophagous flies of different regions in China using eight kinds of primers and found species-specific fragments. In order to further conversion to five necrophagous flies species specificity markers(SCAR) cloning, sequencing, and taking identification necrophagous flies SCAR gene fragments, it established the basis of the work. Compared with the ISSR markers, SCARs(Sequence Charactered Amplified Region)have weak sensitivity, high specificity and reproducibility. This study, based on the housefly ISSR-PCR reaction in different areas, identified species-specific bands. It turned this fragment into SCAR fragment to look for houseflies specific Marker, and provide effective tools for rapid and accurate Musca domestica identification. using ISSR06 primers amplified specificity fragment from the cultivated houseflies genome, purified gene SCAR , and constructed pMD 18-T-SCAR recombinant plasmid. According to the results of sequencing, we got houseflies SCAR full-length gene sequences. Specific primers were designed by Premier 5.0 software. Using different parts of Musca domestica genome as a template and specific primers to amplify the Musca domestica species specificity band, the fragments size is consistent with the expected . This study found that the Musca domestica a new gene, which is species specificity fragment. We have cloned the gene, sequence length of which is 500 bp, and find a new method for the rapid and accurate Musca domestica identification. In recent years, DNA sequence analysis methods has been widely used in animals and plants classification. These methods provide the new ideas and new ways for the forensic insects classification and identification . Eukaryotic ribosomal DNA (rDNA) is a multi-copy gene family. Each copy is composed with external transcribed spacers, 18S gene, internal transcribed spacers (ITS), 5.8S gene, 28 S gene and gene spacer. 18S gene, 5.8S gene, 28S gene sequence have high conservation and slow rate of evolution. ITS 1 and ITS2 do not join mature ribosomes, so they suffer small selection pressure, and have the faster evolution, significant differences between species and also a kind of individual differences. They could provide rich variable sites and information sites. In this study, we using the SSCP and gene sequence analysis analyzed Beijing, Jilin, Changchun, Chengdu, Nanjing, Guangzhou, Jingzhou and cultivated housefly ITS2. The aim is to explore the housefly ITS2 gene sequence intraspecies differences and the feasibility as intraspecies molecular identification markers and to provide molecular basis for necrophagous flies species identification. Methods: extract Musca domestica genome and construction pMD 18-T-ITS2 recombinant plasmid. The masc recombinant plasmid was sequenced, and then different regions of the housefly ITS2 gene carried out SSCP and gene sequence analysis. Using PAUP (4.0b10) Software UPGMA, NJ, MP phylogenetic tree was constructed. The results showed that: ITS2 sequence base of Musca domestica in different regions has AT deflection characteristics; sequence base variation mode has conversion, transversion and shortcoming, and transversion rate is higher than the conversion rate; Musca domestica population of different regions exist rich genetic polymorphism , shows the differences of intraspecies the gene sequence; NJ tree and MP tree have equal topology, and it has got high value. Conclusions: Using PCR-SSCP and rDNA-ITS2 gene sequence analysis analyzes the Musca domestica population in some regions of China, and reveals that there are some differences in Musca domestica genome sequences of different regions of our country , but nowadays no literature report.