| The species of Aphelinidae are the important natural enemies of whiteflies, aphids and scale insects. Many of them play an important role in biological control of pests. Currently, it is a more difficult task for taxonomy identification only by morphological characteristics. Developing a new method to identify species more effectively is urgently needed. This study aimed to make molecular identification analysis of common species of Aphelinidae by three molecular markers including 28S rDNA D2 expansion region,5’-end and 3’-end of mt DNA CO1 gene. The analysis method is DNA barcoding technology. In recent two decades, the research on the insect phylogeny and systematics based on the complete mitochondrial DNA sequence data is becoming more and more important. The mitochondrial gene of Encarsia formosa were sequenced using PCR technology. The results are as follows:1. Seventeen species belonging to four genera of Aphelinidae were identified using DNA barcoding technology based on CO1 gene. Specific primers for CO1 gene amplification were designed because some species cannot be amplified using general primers(LCO1490/hco2198), and the DNA sequences amplified by specific primers met the demands of standard DNA barcode of BOLD database. Twelve DNA sequences were obtained while eleven of them were not included in BOLD systems.2. Based on the sequencing of 28S rDNA D2 expansion region of 29 species, the interspecific average genetic distance was 355times higher than the intraspecific average genetic distance, but intraspecific genetic distances of 25 species among them is 0. The sequences of 28s rDNA were highly conserved within these species. The interspecific genetic distance between Coccophagus havaiiensis and Coccophagus japonicus is 0, so as Encarsia brimblecombe and Encarsia normarki. This molecular maker cannot distinguish the closely related species completely, but it is effective among different genus and species groups.3. Based on the identification on aphelinids using 5’-end and 3’-end of mt DNA CO1 gene, the results showed that base composition and base substitution of these two markers were not distinctly different; A+T content was about 75%, showing significant T+A bias; the average variances of abasic sites were similar; the ratio of abasic transition to transversion was the same(R=0.36); each aphelinid formed a separate branch according to the cluster analysis of the phylogenetic tree.The interspecific genetic distance was 30.8 times of the intraspecific genetic distance of sequence of 3’-end, while 46.2 times of sequence of 5’-end. Both 5’-end and 3’-end of mt DNA CO1 gene are effective molecular markers to identify species of Aphelinidae while sequence of 5’-end is better.4. Seven gene fragments of Encarsia formosa,16S, CO1, CO2, CO3 and Cyt b were amplified successfully using 7 pairs of universal primers. These results laid the foundation for future research on the whole mitochondrial genome. |
