Molecular Phylogenetic Study And Molecular Identification In Five Species Of Fruit Flies(Diptera: Tephritidae)

Fruit fly is one of the familes of Diptera, belong to Bactrocera and Tephritidae,make a serious damage on production of fruits and vegetables. Nowadays, Fruit flyare regarded as a kind of serious pests by 64 contries and areas. Many governmentsadopts a series of serious quarantine methods to avoid the introucation of fruit fliesand pay more attention to their classification and identification.In this study, five species of fruit flies are researched. By methods of RAPD,ISSR and partial mtDNA cytb sequences analyzed, studies on the molecularphylogenetic and molecular identification of fruit flies were carried out. The resultsare follows:1. Genomic DNA in fruit flies (stored in alcohol)was extracted by six methods,such as CTAB and SDS. We analysed the concentration and quality of DNA extractedby different methods. The results showed that the best method is hydroxybenzene/chlorine2. An optimal PCR system for RAPD in fruit flies has been found: in 25μLreaction solution, contained 80ng DNA template, 2.0mM Mg²⁺, 0.2mM dNTP, 0.5μMprimers, 1U Taq polymerase.The amplification program was devised: 94℃for 3min;denaturing at 94℃for 45sec, primer annealing at 36℃for 1min, extension at 72℃for 2min, 40cyclesm, at last extension at 72℃for 10min.Then ten primers werescreened from 40 random primers and amplified 316 bands(from 100bp to 2500bp).The phylogenetic tree were constructed based on DICE genetic distance. In thephylogenetic tree, B.scutellata,B.cucurbitae and B.tau clustered together, then theyclustered with B.dorsalis into one group, and clustered with B.minax at last. All theseresults suggested that RAPD is suitable for analysis of phylogenetic relationshipsamong fruit flies.3. It is the first time that fruit flies was reaserched by ISSR. We has been foundan optimal PCR system for ISSR in fruit flies. Then eight primers were screened from40 ISSR primers. The phylogenetic tree of the five fruit flies were constructed withmethod of UPGMA, and identified them by amplified bands. All these resultssuggested that ISSR is suitable for analysis the phylogenetic relationships andidentification among fruit flies.4. The mtDNA Cytb genes of 5 fruitfly species, including Bactrocera dorsalis,B.cucurbitae, B.correcta, B.tau, B.scutellataand and B.minax were coloned andsequenced. The sequence data revealed consderble variation in 128 nucleotide sites among the sequences analyzed, then get 20 sites to identify different species. Amolecular phylogenetic tree was constructed by using neighbor joining (NJ) andUnweighted Pair Group Method with Arithmetic mean (UPGMA) methodologies.TAlltwo phylogenetic analysis generated similar affinities, the congeners B. cucurbitae andB.tau clustered with B.scutellata, then clustered with B.dorsalis, at last clusteredB.minax.This text has carried on research to the molecular phylogenetic and molecularidentification of fruit flies by three different molecular markers. At the same time,these methods could be consulted in the identification of other fruit flies. So, the studycould constrcucted the fundmention for further research.