Research And Demonstration Application Of DNA Barcode Testing Technology In Medium Rats Identification

ObjectivesThis research take the intercepted rats in the frontier ports and the rats captured in regions to explore the accuracy of the DNA barcode testing technology and the general pattern, the identification of DNA barcode technology used in sibling species, the DNA barcode technology used for identification of decay rats intercepted in the frontier port ocean liner, the feasibility of the DNA barcode technology tracing the geographical origin of intercepted media.MethodsThis research take the320rats as object, captured or intercepted in guizhou, sino-russian border bolshoy ussuriysky island and shandong huangdao port of entry and exit, which belong to2families,5genera and12species. The genomic DNA was extracted. The COI gene was amplified and sequenced. Partial sequences were uploaded to NCBI database and BOLD systems. The multiple sequence alignment was then carried out. The Intraspecific genetic distance and interspecific genetic distance were calculated with MEGA5.0based on Kimura double parameter model. The NJ and MP tree was ultimately carried out. The sequence clustering and phylogenetic analysis was executed.ResultsAmong the three species captured in Maolan region, Guizhou province, the intraspecific genetic distance in Rattus niviventer, Rattus flavipectus and Mus pahari respectively is0.008,0.001and0.002. The average interspecific genetic distance between Rattus niviventer and Rattus flavipectus, Rattus niviventer and Mus pahari, Rattus flavipectus and Mus pahari is respectively0,142,0.185and0.210. There is a similar topology between NJ and MP tree. All the individuals belonging to the same species form a monosystem. Node approval rating is100%.The41COI gene sequences of rodents sample captured in Bolshoy Ussuriysky island area was carried out in the NCBI database for BLAST, and results show that the similarity is in99%to100%. The COI sequences of two sibling species in Bolshoy Ussuriysky, Clethrionomys rufocanus Sundevall and Clethrionomys rutilus Pallas, Microtus fortis Buchner and Microtus maximowiczii Schrenck, were analyzed. The results show that the genetic distance among the same species is very small,0.003～0.008, with an average of0.005; Interspecific difference is very big,0.108-0.346, with an average of0.258. The average interspecific genetic distance is51times of intraspecific genetic distance. NJ and MP tree shows that different species of mice are in different branches. Every single branch corresponds to a specific species, node approval rating of100%. On the branch length, branch of interspecific is greatly longer than intraspecific branch.The COI gene sequences of intercepted rats in Huangdao ports is carried out in the NCBI database for BLAST, and the results show that similarity99%-100%of the sequences are mice Mus musculus. A preliminary inference is that the decayed rat intercepted in Huangdao Port is Mus musculus. The COI sequence similarity, between the intercepted rat in Huangdao and10mice COI sequences from Guyana, Mexico, Canada, the United States and China, is from97%to100%. The10mice COI sequences was classified as high ratings uniparental branch, the average genetic distance was0.017. Rattus norvegicus from different region was classified as the other, the genetic distance between the two branches is24%. In intraspecific genetic distance and phylogenetic analysis, we also found that intercepted mice in Huangdao ports and the mice from different regions belong to a single monosystem, but also showed a small difference. The intercepted mice in Huangdao Ports is closely related with the mice from Guyana and Mexico. The difference in distance characteristics showed obvious regionality.ConclusionsOur research indicated that DNA barcode can not only correct errors among morphology-based diagnoses, but also identify allied species. DNA barcode technology can identify intercepted decay mice from frontier ports, and make a preliminary judgment on the geographical origins of intercepted mice in frontier ports. DNA barcoding based on COI gene can provide convenient and accurate identification of rats.