Molecular Identification And Genetic Structure Of Frankliniella Occidentalis(Pergande) Based On Multiple Gene Barcoding,mtDNA CO?,CO? And RDNA ITS2 Markers

The western flower thrips,Frankliniella occidentalis?Pergande??Thysanoptera:Thripidae?is a worldwide agricultural pest and quarantine object,causing huge threaten to vegetables and ornamental crops in China.Population genetic diversity and genetic structure in a species can reflect its genetic feature of gene constitution and distribution pattern on large spatial and temporal scales.Population genetics research,which can explore origin information and differentiation of species or population,provide theoretical basis for analyzing evolutionary potential and environmental adaptability as well as to make integrated prevention and control measures accurately.In the present study,two mitochondrial genes?cytochrome oxidase I,CO?and cytochrome oxidase II,CO??and one nuclear ribosomal gene?internal transcribed spacer,ITS2?were selected to evaluate the effectiveness in identification of glasshouse strain and lupin strain of F.occidentalis,and hope to establish the molecular identification system.Then,from the population genetics point of view,we examine the influence of geographic variation,host plants as well as uncultivated land and related cultivated land host plants on the genetic diversity and genetic structure of F.occidentalis populations.The main results are as follows:?1?Identification of F.occidentalis based on multiple gene barcoding method.In the present study,the mtDNA CO?,CO?and rDNA ITS2 were selected as molecular marker to evaluate the effectiveness in identification of glasshouse strain and lupin strain of F.occidentalis.Samples of F.occidentalis and its relative species F.intonsa were collected from 21 different sites in China.The results indicated that the mtDNA CO?,CO?and rDNA ITS2 genes can efficiently distinguish F.occidentalis and F.intonsa.But for the two strains of F.occidentalis,the average genetic distance between interstrain based on mtDNA CO?and CO?sequences were 0.035 and 0.036 respectively.Genetic distance of interstrain was 12.9 and 11.3 times respectively than that of intrastrain,and no overlap of interstrain and intrastrain genetic distance was observed.Each of the phylogenetic trees had two clear divergent clades corresponding to glasshouse strain and lupin strain of F.occidentalis.Thus,the mtDNA CO?and CO?can provide reliable and accurate identification of the glasshouse strain and lupin strain of F.occidentalis.While the rDNA ITS2 showed that the glasshouse strain and lupin strain of F.occidentalis were just two kinds of haplotype with fewer variable sites,and the average genetic distance between interstrain was 0.006.The genetic distance of interstrain was just 1.5 times than that of intrastrain.Although there was no overlap between interstrain and intrastrain genetic distances,phylogenetic tree indicated that glasshouse strain and lupin strain of F.occidentalis could not generate clear divergent clades.Thus,identification based on ITS2 gene might be inaccurate.The result provides technology support for accurate identification of different strain of F.occidentalis and conducting biological experiment in laboratory.?2?Genetic diversity and structure among different F.occidentalis geographical populations in China.We used the mtDNA CO?,CO?and rDNA ITS2 to assess whether the geographic variation affect the genetic diversity and genetic structure of F.occidentalis population.Samples were collected at21 different sites covering most invasion areas in China.The results indicated that F.occidentalis populations had lower genetic diversity totally,and the invasion process of F.occidentalis had experienced the"genetic bottlenecks"and might be in accumulating genetic variation and generating adaptive evolution currently.Both neutrality tests and mismatch distribution analysis implied there might not be an obvious population expansion in recent period of history,and F.occidentalis population was at demographic dynamic equilibrium.The genetic structure analysis based on the sequences of mtDNA CO?and CO?indicated that there was mild genetic differentiation among different pairwise geographic populations,while rDNA ITS2 suggested no obvious genetic differentiation among geographic populations.This discrepancy could be due to inconsistent rate of evolution between mitochondrial gene and nuclear gene.In addition,we divided the 21 populations into four groups?southwest region,northwest region,northeast region,northern region?according to geographical and climatic feature of sample sites so that we can perform analysis of molecular variance?AMOVA?among different area in China.Both mtDNA CO?,CO?and rDNA ITS2 showed most of the genetic variation was within populations,and less variation was detected among groups or among populations within groups,and no obvious genetic differentiation among different area.So,it is reasonable to regard F.occidentalis population did not showed any geographical pattern in China.Frequency of haplotype distribution and phylogenetic relationship also shared the similar result.?3?Genetic diversity among five different host plant populations of F.occidentalis?Pergande?in north-western China.The mtDNA CO?and CO?were selected to assess whether the host plants affect the genetic diversity and genetic structure of F.occidentalis populations.Samples were collected from five common host plant populations including Capsicum annuum,Solanum melongena,Althaea rosea,Rosa chinensis and Canna indica at 9 localities in north western China.The results indicated that populations collected from Capsicum annuum and Solanum melongena had a lower genetic diversity,while populations collected from Althaea rosea,Rosa chinensis and Canna indica had the relatively high genetic diversity.Fixation index?Fst?and gene flow?Nm?suggested that the high level of gene flow and no obvious genetic differentiation among host populations of F.occidentalis.Analysis of molecular variance?AMOVA?results showed most of the genetic variation was within populations.Frequency of haplotype distribution and phylogenetic relationship indicated that glasshouse strain of F.occidentalis occured in all host plants,and lupin strain of F.occidentalis preferred to happen on perennial host plants.?4?The genetic diversity and genetic structure of F.occidentalis between cultivated and uncultivated host populations was assessed by mtDNA CO?gene.Samples were collected from ten host plant populations including Capsicum annuum,Solanum melongena,Cucurbita pepo,Lycopersicon esculentum,Solanum nigrum,Datura stramonium,Lycium barbarum,Pharhiris nil qianniuhua,Convolvulus arvensis and Solanum melongena in ZhangYe region.The results indicated that host populations of F.occidentalis which were collected from cultivated field including Capsicum annuum,Solanum melongena,Cucurbita pepo and Lycopersicon esculentum had a lower numbers of haplotype,haplotype diversity and nucleotide diversity,while host populations of F.occidentalis which were collected from uncultivated field including Solanum nigrum,Datura stramonium,Lycium barbarum,Pharhiris nil qianniuhua,Convolvulus arvensis and Solanum melongena had the relatively high genetic diversity.Fixation index?Fst?and gene flow?Nm?suggested high level of gene flow and no obvious genetic differentiation among host populations of F.occidentalis.Analysis of molecular variance?AMOVA?results showed most of the genetic variation was within populations,and less variation was detected among groups or among populations within groups.Fixation index among groups(F_CT)was0.05439?P<0.05?and fixation index within populations(F_ST)was 0.07809?P<0.05?,indicated that there was a certain level of genetic differentiation between cultivated and uncultivated host populations as well as within populations.Frequency of haplotype distribution and phylogenetic relationship indicated that cultivated host populations consist of the glasshouse strain of F.occidentalis,and uncultivated host populations consist of both glasshouse strain and lupin strain of F.occidentalis.