Screening And Identification Of Fusarium Head Blight Resistance Gene By T-DNA Insertion Mutants In Brachypodium Distachyon

Fusarium head blight is a fungal disease caused by Fusarium graminearum,which seriously threatens wheat yield and safety.However,because of the complexity of the wheat genome,difficulties in transformation,long lifecycle,and lack of suitable model plants,the mechanisms of wheat scab make slow progress.Brachypodium Distachyon as a new model plant in temperate grasses,the genome exhibits a high degree of sequence similarity and conserved gene order with wheat.The T-DNA mutant collections have provided invaluable resources for functional genomics studies.Brachypodium distachyon 21(Bd21)was previously proved to interact with F.graminearum and Our group have explored and confirmed that Brachypodium genotypes can be applied to the analysis of type II resistance to Fusarium head blight.The purpose of this study was to explore the resistance gene of Fusarium graminearum and analysis of signal pathway related to disease resistance by T-DNA insertion mutants of Bd21,and carrying out re-sequencing of the whole plant genome.The specific results are as follows:1.In this study,6000 lines of T-DNA insertion mutants of Brachypodium Distachyon were used to screen F.graminearum resistance genes.Combined with the previous laboratory research results,we finally determined that the temperature of 23℃ and relative humidity of 60%-65%,Fusarium graminearum mycelium can grow fully on isolated leaves without spreading too quickly and affecting phenotypic screening,we create successfully a highthroughput in vitro leaf screening system.2.145 mutant materials were finally identified through high-throughput in vitro leaves and single flower infusion,and then these 145 materials were resequenced.After quality control and filtering of the sequencing results,the effective sequences obtained were compared to the wheat reference genome,SiwssProt protein database,and Top hit was selected as the result of homology annotation.Homologous annotation was performed on all candidate genes and the extracted intergenic region sequences.Finally,a total of 142 wheat homologous genes were obtained.3.In order to obtain comprehensive gene annotation information and provide clues for the research on the mechanism of resistance to scab,we used ClusterProfiler R package(v3.6.3)for GO enrichment and KEGG enrichment analysis of candidate genes,and we analyzed their functions and pathways at the same time.GO annotation results indicate that these genes are mainly classified into 77 functional categories.Most of them are involved in the response plant to damage,fungi,abscisic acid signals,and alcohols.They are mainly located on the cytoplasmic membrane or various biological membranes,and function as transmembrane carriers and protein kinases.4.We further performed KEGG analysis on these genes and classified them into 16 metabolic pathways.Some genes are located in the amino acid biosynthesis and metabolic pathways,such as tryptophan,cysteine,arginine,and proline,which can regulate plant cell wall synthesis and protein cross-linking,thereby affecting the plant’s physical barrier against pathogens.Some genes affect the metabolism of coumarate,ubiquinone,and other terpenoids,which can affect the growth of mycelia,and some can affect the production of plant hormones,which can regulate the resistance to FHB.