Research And Application Of DNA Barcoding On Quarantine Moth And Termites (Coptotermes Spp.)

The accurate identification of pest species was an important part in the inspection and quarantine work.At present,the identification of plant quarantine insects was mainly morphological method,but there were some limitations in the traditional morphological identification.For example,the early morphological and incomplete insects of the insects were difficult to be identified,the results were subjective,the identification period was longer,in some cases,they seriously affected the accuracy of quarantine work and the detection rate of the epidemic.Moths,belonging to Lepidoptera,often cause huge economic losses to food,cotton,vegetables,oil,herbs and other crops and storage in their larvae.At the same time they also constituted a great threat of our agriculture and forestry production.In recent years,with the rapid development of international trade,imported goods,especially fruits,dried fruits,nursery stock and other species increased rapidly,the national inspection and quarantine port intercepted a large number of moth larvae,pupae and adults.The threat to our agricultural production and forest ecological environment was increasing.As an important group of quarantine pests,Coptotermes showed a rapid increase trend in imported logs especially in the past 10 years.The phytosanitary department of the port of China has intercepted the Coptotermes species from the imported timber records account for more than one third of the total number of termites.However,due to morphological convergence,genital degeneration,identification of morphological characteristics can be done very little,the genus was more chaotic in classification,species identification was difficult,human subjective classification,synonyms and the same name foreign body phenomenon was common.The difficulty of classification and identification of Coptotermes is recognized by the world classification experts.DNA Barcoding has been the main focuses of biological taxonomic research in decade years.The development of this technique breaks the limitation of traditional morphological identification and provides a new method for molecular identification of harmful organisms.It has been widely used in the molecular identification of species,found new species or hidden species and phylogeny research.This study attempted to use this technique to identify quarantine moth and termites.The main research results were as follows:1.We used the mitochondrial CO?gene of Hyphantria cunea,Spilosoma menthastri,Spilosoma niveus,Stilpnotia candida,Amsacta lactinea,Creatonotus transiens,Spilarctia subcarnea,Spilosoma album and Spilarctia album as template and screened the specific DNA sequence of H.cunea,and designed a MGB probe for rapid identification of H.cunea and got specific amplification by real-time PCR method.We got comparative analysis by DNA barcode.The results showed that the DNA barcode of H.cunea and other allied species were unique for each species.In other words,the barcode which have better ability to recognize H.cunea could be used for the species identification.The results of this study provide a new technical of monitoring and treatment of the pest,which were means for the quarantine and identification of H.cunea.2.Using the common CO? primers could basically amplify all the moths CO? gene fragments.Sequencing of the 658 bp fragments obtained by the database Genbank and BOLD published DNA sequence alignment analysis,could effectively distinguish between Cryptophlebia leucotreta,Epichoristodes acerbella Waller,Grapholitha molesta,Cydia pomonella,Thaumatotibiabatrachopa,Mussidia pectinicornella,Helicoverpa zeaBoddie and ITS related species.3.In order to reveal the genetic differences between European and Asian,the CO? gene sequences of Lymantria disparsamples were determined,which from the United States,Japan,Russia,Mongolia and China of different geographical origins.According to DNASTAR software,the Lymantria dispar CO? gene was 1531 bp in length,and there were 14 stable base loci in American and Asian populations.There was only a change between base and no transversion,insertion or deletion.There was no difference in the amino acid sequence of the single base difference,and the difference of single base at 406 bp resulted in the difference of amino acid at this site.The Upgm phylogenetic tree constructed using the CO? gene sequence shows that the European type(American population)and the Asian type are clearly divided into two part,and the Japanese population and other Asian populations are distant and independent.Based on the single base difference of 406 bp,the MGB probe was designed and the allele genotyping method was used to distinguish the European and Asian chrysanthemum samples.This study provided a scientific basis for the study of genetic differentiation of Gypsy moth and proposed a new method for distinguishing different geographical populations of the Lymantria dispar.4.In order to explore the applicability of DNA barcoding technology in Lepidoptera identification of apple orchard,the mitochondrial cytochrome oxidase I(CO?)of Lepidoptera from seven apple orchards in Shanxi was amplified and analyzed by universal primers of DNA barcodes.Gene fragments,based on the amplified CO? sequence to calculate interspecies and intraspecific genetic distance,and to build NJ tree(Neighbor-Joining tree)and ME tree(Minimum-Evolution tree).The results showed that the collected Lepidoptera species belonged to 9 species of insects belonging to 7 genera.The interspecific and interspecific genetic distance analysis based on p-distance model showed that the interspecific genetic distance was 8.4%-69.4 %,And the genetic distance between species was 0-0.6%.There was no overlap between the maximum genetic distance between species and the minimum genetic distance between species.Meanwhile,NJ and ME trees were constructed based on interspecies and intraspecific genetic distance.In the system tree are in the same evolutionary branch;therefore,the DNA barcoding identification method can distinguish between these apple orchid different Lepidoptera pests.5.Based on sequence and phylogenetic analysis,we designed specific primers and probes for CO? and ITS sequences in the species to optimize the detection system and conditions,which more conserved and interspecific..Based on real-time PCR we established molecular detection and identification technology system of Coptotermes,C.curvignahus,C.travians and C.sjostedti.In this paper,the method of real-time PCR detection has the characteristics of fast,accurate,specific and reliable,and it is suitable for the detection of different grade samples of C.formosanus.Sensitivity experiments showed that the real-time PCR method could detect 1pg/?l concentration of the sample,which provided a new method for the rapid and accurate diagnosis of quarantinous Coptotermes.