DNA Barcoding For Identification Of Common Whitefly Species In China

Whiteflies (Hemiptera:Aleyrodidae) are species-rich order, which can cause huge losses to the crops. In recent years, the invasion events occur frequently, the Bemisa tabaci MED cryptic specie with highly invasive had spread to most area of our China, the Paraleyrodes pseudonaranjae Martin and Aleurodicus dispersus Russell invaded to China in succession, which have high risk to the Chinese mainland. Whitefly species with small body can spread with flowers nursery stock and other trade activities easily. It is difficult in their morphological identification. In this study, we clearified the effectiveness of3’-and5’-end sequences of the COI gene in identification of common whitefly species and application of DNA barcoding technology for species identification of common whiteflies. We do research on the mitochondrial cytochrome c oxidase subunit I (COI) genes in whitefly species. We constructed the whitefly DNA barcoding identification system, which have significant influence in identifying whitefly accurately and quickly and preventing their transmission and spread, in further maintaining the credit of our import and export and ensuring the safety of agriculture industry. The specific results of the research as follows.(1) Effectiveness of3’-and5’-end sequences of the COI gene in identification of common whitefly species. We amplified the different ends of mtDNA COI gene with the universal primers using16common whitefly species. Subsequently we sequenced and aligned the sequences, counted the intra-and inter-species genetic distance, constructed the phylogenetic tree using Neighbor-Joining method (NJ). The results indicated that the identification based on the phylogenetic tree was consistent with those based on morphological characteristics for the16whitefly species when the5’-end fragments were analyzed. However, when the3’-end fragments were analyzed the identification based on the phylogenetic tree was not consistent with those based on morphological characteristics in the case of Pealius mori Takahashi and B. tabaci (Gennadius) Asia Ⅱ1. The average genetic distance between species was193.1(0.2897vs.0.0015) and402.4(0.2817vs.0.0007) times higher than the corresponding within species values for the5’-end and3’-end fragments, respectively. There was no overlap between inter-and intra-species genetic distances when the5’-end fragments were analyzed; but overlap was observed between P. mori and B. tabaci Asia Ⅱ1when the3’-end fragments were analyzed. The DNA barcoding based on the5’-end fragment of the partial COI gene can provide a rapid and accurate identification of these whitefly species.(2) Application of DNA barcoding technology for species identification of common whitefly. In this study, we amplified the5’-end of mt DNA COI gene with the universal primers using27whitefly species and sequenced the PCR production. We aligned and analyzed the sequences, counted the intra-and inter-species genetic distance, constructed the phylogenetic tree using Neighbor-Joining method (NJ) and Maximum Likelihood method (ML). The results showed that average genetic distance between species was0.3094(0.5105vs0.1036), which was162.8times higher than the corresponding within species values, and the average genetic distance within species was0.0019(0.0075vs0.0000). All of the genetic distance between species was above2%, and there was no overlap between the inter-species distance and the intra-species distance. There was some difference in revealing the relationship of B. tabaci cryptic species from the phylogenetic tree constructed by different method, but the topological structure was alike.(3) The establishment of whitefly barcoding system. The database mainly includes the specimen database, the knowledge database and barcoding sequence database. The specimen database and the knowledge database recorded27whitefly species belong to15genera, involved the content such as specimen information, collecting locality and the pictures of collecting and microscopy. Barcoding sequence database recorded4464sequences (22genera,62species), in which4185sequences (19genera,47species) came from the NCBI database and279sequences (15genera,27species) from this research, the database enlarged the number of species and sequences. This database with simple web interface and easy to be operated makes uesers identify unkown whiteflies accurately and quickly.The research on effectiveness of3’-and5’-end sequences of the COI gene in identification of common whitefly species contain many3’-end sequences, which make up the deficit of the existing database limited by the number of species. In this study, we establish the whitefly barcoding system, which has the function to detect and identify the whitefly pests, and provide the technical support to the port inspection and quarantine and whitefly pest remote monitoring...