Studies On Classification And Identification Of Grapholita Based On DNA Barcoding

Some species of Grapholita (Lepidoptera:Tortricidae) are important fruit pests, which are harm seriously in north China. Because they are all feeding on fruits in their larval stages, the growth of plant and the quality of fruits are reduced seriously. And the preventions are difficult, they caused great losses of agriculture and forestry production, so the study of these has very important economic significance. In order to control oriental fruit moth, we have to classify these pests, just like Grapholita. At present, the morphological characteristics identification is the common method, however, identification of morphological identification is difficult for relative species and it need professionals, so a rapid, accurate and automatic tools of classification identification which can be used in global is urgently needed. With the widely used of molecular biology techniques in insect classification, DNA Barcoding provided a new method to classify Grapholita spp., which is rapidly and accurately.In these studies, we treat some species of Grapholita as the study object, which are composed of Grapholita molesta, Grapholita dimorpha, Grapholita funebrana and Grapholita delineana. The different gender, different stages, different regions of these species' partly mitochondrial COI gene were sequenced, and then we combined the results with the COI sequences of Grapholita which download from GenBank to construct Phylogenetic tree of Grapholita based on NJ and MP. In addition, some individuals of ribosomal internal transcribed spacer ITS2 partial sequences were studied, the results are as follows:1. Four species of Grapholita were proved to be Grapholita molesta, Grapholita dimorpha, Grapholita funebrana and Grapholita delineana respectively through traditional morphological identification. COI and ITS2 sequences amplified of these species with different regions are as follows: Grapholita molesta include 35 COI sequences and 13 ITS2 sequences. Grapholita dimorpha include 1 COI sequence and 1 ITS2 sequence; Grapholita funebrana include 5 COI sequences and 4 ITS2 sequences; Grapholita delineana include 14 COI sequences and 4 ITS2 sequence; 55 COI sequences and 22 ITS2 sequences were obtained from Grapholita. COI and ITS2 gene sequences we obtain as a label of barcode, have riched the nucleotide identification database, which will providing a convenient for the study of other scholars.2. Though the COI gene sequence analysis of different species of Grapholita under different gender, different stages and different regions, we found that COI gene sequences have no differences among gender, stages, regions (except Grapholita funebrana), and different species have themselves'fixed nucleic acid site.3. Large number of samples for genetic studies of mitochondrial COI of four Grapholita species and outgroup genera Cydia pomonella found that after taking into account geographical factors and related species, combined inter-intraspecific genetic distance analysis between species, and the use of Neighbor-joining (NJ) and maximum parsimony (MP) analysis method building tree, the genetic relationship with the genus of Grapholita delineana and the outgroup of Cydia pomonella is more closer, manifest that molecular identification of COI sequence alone as DNA barcoding marker can classifiy Grapholita genes accurately.4. With the nuclear ITS2 gene study of four kinds large samples of Grapholita spp., after taking into account geographical factors and related species, combined with inter-intra specific genetic distance analysis and NJ, MP analysis method building tree, we discovery that ITS2 sequence is a suitable accessory molecular marker of Lepidoptera.5. Use the COI and ITS2 gene to explore genetic relationship between Grapholita spp.:the genetic relationship between Grapholita molesta and Grapholita dimorpha is closest, followed is between Grapholita funebrana, the genetic relationship between Grapholita molesta and Grapholita delineana and is farthest.