Cloning And Functional Analysis Of Seedborne Fusarium Verticillioides Resistance Gene In Maize

Internal fungal contamination in cereal grains may affect plant growth and result in health concerns for humans and animals.Fusarium verticillioides（F.verticillioides）is seed-borne fungus that can systemically infect maize.However,few efforts have been devoted to genetic studies on maize resistance to seedborne F.verticillioides.In this study,we developed a disease evaluation method to identify the resistance to seedborne F.verticillioides in maize.Two RIL populations were used to identify major QTLs for seedborne F.verticillioides resistance in multiple environments,and genome-wide association analysis was conducted to identify QTLs for seedborne F.verticillioides resistance.The candidate gene Zm WAX2 was identified by GWAS analysis,and another candidate gene,Zm MAPKKK18,was identified by QTL mapping and association analysis of two RIL populations.Finally,the regulatory mechanism of the candidate gene Zm WAX2 on the resistance of seedborne F.verticillioides in maize was explored,and the function of the candidate gene Zm MAPKKK18 was verified.The main results are as follows:1.We developed a disease evaluation method to identify resistance to seedborne F.verticillioides in maize.2.A total of 121 inbred lines were evaluated using the evaluation method to identify the resistance to seedborne F.verticillioides in maize,and 17 inbred lines were selected for their strong resistance to seedborne F.verticillioides.These lines are CNW133,CML172,CNW035,CNW058,CML141,CNW054,CML171,CML300,CNW134,CNW120,CML140,CNW051,CML40,CNW028,CNW031,CML480 and CNW026,respectively.3.Linkage mapping was developed in the BT × Xi502 RIL population and QTL anlaysis was tested in three environments.A total of 13 QTLs were identified on chromosomes 1,3,4,8 and 10.At the same time,linkage mapping was developed in the N6 × CML170 RIL population for resistance to seedborne F.verticillioides.Eleven QTLs were detected using combined analysis of two environments and six replicates,explaining 6.99 % of the maximum phenotypic variation.The interval of chr3（3₂05367812-3₂08620990）located by RIL of N6 × CML170 consistent with that of chr3（3₁99466935-3₂11327406）located by RIL of BT × Xi502,which may be a stable QTL.We name it QTL q ISFR3.4.Association analysis of 170 materials for resistance to seedborne F.verticillioides identified 21 significant SNPs（p< 1×10-4）,with each independent site contributing9-13% of phenotypic variation.Among the 21 significant SNPs loci,S5₉0344973 was the most significant,which was located on chromosome 5 with a P value of 1.09×10-6.The corresponding gene,GRMZM2G083526,which was taken as a candidate gene.In addition,the linkage analysis results of the two RIL populations combined with association analysis identified that a significant SNP site,Chr3.S₂05394278,was located in the QTL q ISFR3 region.The gene corresponding to this significant SNP encodes MAPKKK for GRMZM2G404078,named Zm MAPKKK18.This gene was used as another candidate functional gene.5.We identified a gene of Zm WAX2 associated with quantitative resistant variations to F.verticillioides through a genome-wide association study in maize.A lack of Zm WAX2 compromises maize resistance to F.verticillioides-caused seed rot,seedling blight,and stalk rot by reducing cuticular wax deposition,while the transgenic plants overexpressing Zm WAX2 show significantly increased immunity to F.verticillioides.A natural occurrence of two 7-bp deletions within the promoter increases Zm WAX2 transcription,thus enhancing maize resistance to F.verticillioides.Upon Fusarium stalk rot,Zm WAX2 greatly promotes the yield and grain quality of maize.Our studies demonstrate that Zm WAX2 confers a broadspectrum resistance to F.verticillioides-caused diseases and can serve as an important gene target for the development of F.verticillioides-resistant maize varieties.6.Functional markers of ZmMAPKKK18 were developed,and functional variation sites were found.The near-isogenic lines of Zm MAPKKK18 were constructed based on functional variation.The reliability of Zm MAPKKK18 as a candidate gene was proved by the identifiying the resistance of Zm MAPKKK18 to F.verticillioides through near-isogenic lines.The phenotype of Zm MAPKKK18 was preliminarily identified,and it was concluded that Zm MAPKKK18 positively regulated the resistance to seedborne F.verticillioides in maize.