| The Chinese citrus fly, Bactrocera (Tetracus) minax (Enderlein)(Diptera:Tephritidae) is a kind of quarantine pest of citrus fruit, which has been reported in several Asian countries including China, India, Bhutan, Nepal and so on. The statistics of relevant departments indicate that B. minax occurs mainly in seven provinces in China, and has caused huge losses to Chinese citrus production. Therefore, it is of great significance to understand the genetic structure and to analyze the diffusion path of B. minax for the formulation of prevention and control measures. Through amplifying a mitochondrial DNA (mtDNA) gene fragments and four microsatellite loci of B. minax, this research used the parameter analysis of genetic diversity, phylogenetic analysis and phylogeography analysis to systematically and profoundly compare the18B. minax populations in different areas of China mainland for the first time. The main conclusions was as follows:1. Selection of molecular markersIn the selection of molecular markers for the mitochondrial gene, we chose mitochondrial COX1, CYTB, ND4and ND5gene of B. minax as the primary targets according to the previous studies. Then, the selections were carried out in consideration of6kinds of genetic diversity indexes, namely percentage of A+T (%), numbers of polymorphism loci, numbers of parsimony information sites, numbers of singleton sites, numbers of haploid types and diversity of nucleic acid, as well as the principle of choosing the gene fragments greater than500bp after removing the front and back primers. Among them,6genetic diversity indexes of four mitochondrial genes were: COX1,56.40,56,30,26,7,0.0092; CYTB,61.74,40,23,17,5,0.0147; ND4,65.50,64,42,22,11,0.0116; ND5,66.20,36,22,14,9,0.0102. The results indicated that the ND4gene was with the highest rate of evolution among the four mitochondrial genes of B. minax, therefore we chose ND4gene fragments as the mitochondria molecular marker for B. minax.2. Selection of the microsatellite markersIn order to screen effectively suitable microsatellite primers for the genetic structure research of B. minax, we firstly selected33microsatellite primers with high polymorphism from congeneric species of B. minax, i.e., B. dorsalis, B. papayae, B. cucurbita and B. cacuminata, and the PCR amplification products were screened using agarose electrophoresis firstly and using6%denaturing polyacrylamide gel electrophoresis secondly. After that, we analyzed the allele frequency, polymorphism information and effective allele numbers. Finally, we got6pairs of microsatellite primers which showed higher polymorphism in B. minax, of which were respectively loci Bp125(PIC=0.1546), BcuB5.2(PIC=0.4105), BcuF1.6(PIC=0.3086), Bd47(PIC=0.0886), Bcac5.10(PIC=0.2745) and Bcac6.12(PIC=0.1118). At last, according to limitations of the multiple PCR (Difference of two target gene fragments should be over30bp, and the pair of primers could not form hairpin structure or dimers), we chose the4pairs of primers (Bp125BcuF1.6Bcac5.10and Bcac6.12) as the microsatellite markers for B. minax.3. Genetic structure and genetic diversity of B. minaxThis experiment has amplified ND4gene sequences of359individuals in18B. minax populations and4microsatellite loci information of478individuals. We found93variation sites and91haplotypes in ND4gene; while in the four microsatellite loci, we detected respectively7,14,9and8alleles, and alleles in various populations ranged from19to30with an average of23.06. The results showed that the185. minax species were with highly polymorphic sites and haplotypes, and the high degree of the alleles heterozygosity also demonstrated the higher genetic diversity of B. minax.The SAMOVA analysis results of ND4sequences and microsatellite datas of B. minax (The genetic variation of18B. minax populations was high with38.01%) proved that the genetic structure of the B. minax was weak. At the same time, AMOVA analysis results on genetic coefficient of FST in18B. minax populations showed that there were obvious genetic differentiation among B. minax populations, but the genetic coefficient values of microsatellite datas were samller than those in ND4sequences, the reason for which may be that the number of B. minax homozygotes was relatively large and that there existed some ineffective alleles on the microsatellite loci.4. Analysis of the dispersal centre and the diffusion path for B. minax in Chinese mainlandCombining the genetic differentiation coefficient, the phylogenetic tree, and gene flow analysis results of ND4sequences and microsatellite datas in18B. minax populations, we speculated the dispersal center and the main diffusion path of B. minax in Chinese mainland and made the following deductions: (1) By comparing genetic differentiation coefficients and gene flow values among the adjacent B. minax populations, especially the five populations in Chongqing, we thought that B. minax might have a "homesick" behavior, that is, it would not spread from the settlement to the mountains around.(2) The analysis results of phylogenetic tree structure and gene flow values (the asymmetry between immigration and emigration rates) for18B. minax populations showed that the South China group was in the middle of the phylogenetic tree, of which the emigration rate was greater than its immigration rate (22.785and1.175, respectively), and that its genetic differentiation coefficients with other groups were pretty small. Therefore, we believed that the SC group should be the dispersal center of B. minax in Chinese mainland.(3) At the same time, we found that the Shiyan population of Central China group was in the special position of18B. minax populations. The genetic differentiation coefficient of Shiyan population with other populations was smaller, and the gene flow values showed that the Shiyan population belonged to mainly emigration type, so we duduced that Shiyan should be the "bridgehead" for B. minax in Chinese mainland.In conclusion, we speculated that B. minax began to spread out from Southern China, through Shiyan of Central China which acted as the "transfer station", spread to elsewhere of Central and Western China, especially to the Southwest regions.5. The origin of B. minax in ChongqingAs one of the main locality of B. minax, we analyzed the diffusion model and the genetic relationship among populations of B. minax in Chongqing. The five populations of Chongqing (Wulong, Zhongxian, Wanzhou, Yunyang and Wushan populations), had almost no shared haplotypes, and the results of Structure analysis showed that the Zhongxian population should be newly recorded in recent years which was introduced from Southern China, and that the population genetic structures of Wulong, Wanzhou and Wushan populations were similar to Jingzhou’s in Central China, and that the genetic structure of Yunyang population was similar to that of Shiyan and Yichang populations. Therefore, we though that B. minax in Chongqing was introduced from Southern China, Jingzhou and Shiyan of Central China. |
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