Study On Minning Of Genes Associated With The Resistance To Acanthoscelides Obtectus(Say) And Resistance Mechanism In Common Bean

Phaseolus vulgaris L.is one of important edible legumes,and the seeds are severely damaged by Acanthoscelides obtectus Say during storage,which affects its quality and commercial value.It is a safe,economical and effective method to control the damage of A.obtectus by screening the germplasm resources of bruchid resistance and excavating the resistant genes.In this research,the linkage analysis and genome-wide association analysis were used to excavate resistant genes and study the resistant genes,which laid a foundation for further revealing the molecular mechanism of bruchid resistance and utilization of resistant genes.1.Genetic analysis of resistance to A.obtectusThe black kidney bean displays resistance to A.obtectus.The recombinant inbred lines（RILs）were constructed from a cross between the black kidney bean and a high susceptible cultivar,Longyundou3.The bruchid resistance of the F₁,F_2:3population and RILs were identified.Genetic analysis showed that the resistance to A.obtectus was a quantitative trait controlled by recessive polygenes.Further analysis of the relationship between bruchid resistance and biochemical substances in seeds,seeds size,seeds coat color showed that the percentage of damaged seeds and the number of perforations were significantly negatively correlated withα-amylase inhibitors.Both in F₂population and natural population,the percentage of damaged seeds and the number of perforations were significantly positively correlated with 100-seeds weight,seed length and seed width.Black seeds exhibited a lowest percentage of damaged seeds and least number of perforations.2.Construction of a high-density genetic linkage map and fine mapping of resistant genesThe genetic linkage map with the high density of markers was constructed by whole-genome resequencing using 157 lines of F₆population.The total map distance was 1,283.68c M,containing2,234,769 SNP distributed in 3,106 bin,and the average distance between bins was 0.61c M.A QTL was detected between Bin1565 and Bin1566 markers on chromosome 6,which was associated with both PDS and NP.The LOD value was 5.49 and 4.31,and explained 16.10%and16.37%of the phenotypic variation,respectively.New markers were developed for the candidate regions,and the resistant locus was further fine-mapped to the 122.3 Kb interval between SSR markers I6-4 and I6-16.This region contained five genes.Phvul.006G003700 encodes a bifunctionalα-amylase/protease-inhibited protein and may be a potential candidate gene for bruchid resistance.Sequencing analysis of the candidate gene identified an 5bp insertion-deletion in promoter of gene Phvul.006G003700 between resistant and susceptible parent,located in the TATA box.Expression analysis of the candidate gene revealed Phvul.006G003700 of the resistant parent was markedly higher than that in susceptible parent both in the seeds and leaves.3.Genome-wide association analysis to excavate resistant genes for A.obtectusBy using improved indoor artificial inoculation method,a total of 659 germplasms were identified for resistance to A.obtectus in two years.Among them,four germplasms showed stable resistance both in two years,with the percentage of damaged seeds less than 10%.Genome-wide association analysis was conducted based on 3,767,432 SNP and phenotypic data of the percentage of damaged seeds and the number of perforations from 625 germplasms.A total of 32 significant SNPs were associated with the resistance to A.obtectus.And 460 genes were contained in all loci,including 20 genes encoding bifunctional inhibitors,phytagglutinin,peroxidase and so on,which are related to the defense of bruchids.These results laid a foundation for molecular breeding and selection of insect resistant resources in common bean.