Genetic Diversity In Bt-resistance Strains Of Ostrinia Furnacalis (Guenée)

Asian corn borer?ACB?,Ostrinia furnacalis?Guenée?,is an important pest of maize.Although the commercial cultivation of transgenic insect-resistant maize has not permitted by now in China,domestic and international research and development of Cry1Ab,Cry1Ac,Cry1Ie,Cry1Ah,Cry1F and other Bt?Bacillus thuringiensis?insecticidal toxin transgenic corns expressing Cry1Ab,Cry1Ac,Cry1Ie,Cry1Ah,Cry1F,etc,toxins developed by domestic and international Agriindustries and Institutions have provide a potential technology reserve for the efficient control of ACB.In the past two decades,the commercialization of Bt corn for protecting agaist lepidopteran pests such as the European corn borer,Ostrinia nubilalis?Hubner?,has been widely used in more than 20 countries in North America,Western Europe,South America,and the Philippines.However,evolution of resistance has been reported in target pests driven by wide spread Bt corn.Accurate and effective resistance testing methods will provide scientific basis for the effectiveness of resistance monitoring and resistance management strategies.In this study,MISA?MicroSAtellite?software was used to search in the Asian corn borer transcriptome dataobtained from high-throughput sequencing.Totally we analyzed 61,622 EST sequences and identified 3,467 SSR loci,The SSR frequency of distribution and occurrence are 4.93%and 5.63%respectively?one SSR locus in 15,162 bp?.In the Asian corn borer transcriptome,the major repeat type of SSR was trinucleotide,accounting for 51.37%of the total number of SSRs,followed by single nucleotides,23.45%,and dinucleotides 14.25%.The number of tetra-and penta-nucleotide repeats was very small,accounting for 5.62%,3.06%,and 2.25%of the total.By designing and screening,we obtained 3,316 pairs of specific primers and 150 pairs were selected for PCR amplification,and 51 pairs of them produced amplification bands of expected sizes.Through polymorphism detection,20 high polymorphic primers were obtained among susceptible ACB population?ACB-BtS?and 5 Bt-resistant populations including ACB-AbR,ACB-AcR,ACB-AhR,ACB-FR and ACB-IeR who are selected by Cry1Ab,Cry1Ac,Cry1Ah,Cry1F and Cry1Ie via diet incoperation.Genetic polymorphisms of a susceptible and 5 Bt toxins selected strains of ACB were detected using 20 pairs of polymorphic primers screened.One hundred and twenty six alleles were detected among 180 individuals from six strains.The average heterozygosity and expected heterozygosity were0.4892 and 0.6183 respectively.The average Nei's gene diversity index of the population was 0.5376,and Shannon's index was 1.0015.It indicated that the ACB population was rich in genetic diversity.There was significant genetic differentiation among the strains.The average genetic differentiation coefficient(F_ST)between the strains was 0.1959 in the ACB,which revealed 80.4%of genetic differentiation from intra-strains and 19.6%was from inter-strains.UPGMA phylogenetic tree was established based on genetic distance and showed similarity of six populations,i.e.,high similarity between ACB-AbR and ACB-AcR,and high similarity between ACB-FR and ACB-IeR.The pattern of genetic variation similarity among the strains is similar to that of resistance and cross-resistance patterns to each Bt toxins reported previously.In addition,the genetic variation among the strains was analyzed using mitochondrial cytochrome C oxidase subunit I?CO I?gene.A total of 14 haplotypes were identified in 144 individuals from 6strains,and they shared one haplotype.The haplotype specific was the most in the ACB-BtS strain.The molecular variation analysis?AMOVA?showed that the genetic variation accounted for 48.0%in intra-strains,and 52.0%in inter-strain,which showed significant genetic differentiation among the groups.The UPGMA phylogenetic tree was constructed based on the genetic distance between populations.It was shown that the six population clusters were divided into two major groups,of which the ACB-BtS was self-contained and the other strains were clustered into one.In conclusion,the Bt susceptible and resistant strains of the ACB could be identified via mitochondrial cytochrome C oxidase subunit I?CO I?gene,but it could not distinguish of which resistant strains was.The identified SSR loci can be used as molecular detection methods for identifying different Bt toxin resistant strains,which could be used to establish a molecular technique for detecting the resistance in the field.