The Functional Verification Of TaCWI-B1 On Wheat Fusarium Crown Rot And Sharp Eyespot And Analysis Of Grain Metabolome

Wheat fusarium crown rot(FCR)and sharp eyespot(SE)are common soil-borne disease in many regions.Due to straw-returning and high density planting methods,wheat FCR and SE have become severe threats to wheat production and quality in china.Until now,only a few wheat genotype show moderate resistance to these fungal disease,and the genetic determinants of wheat resistance to these devastating disease are poorly understood.Plant metabolites are fundamental for plant growth and development,however,the analysis of the genetic structure of wheat metabolomics has not been fully studied.In this study,genome wide association analysis(GWAS),quantitative traits loci(QTL),Chinese spring database,wheat660 K chips,transcriptome sequencing,re-sequencing and widely targeted metabolome methods were used to analysis the wheat FCR,SE and grain metabolites,the main results were listed as following:1)A annotated as cell wall invertase gene(Ta CWI-B1)were founded to associated with wheat FCR by muti-methods(transcriptome sequencing,re-sequencing,expression level,virus induced gene silencing).haplotype analysis of Ta CWI-B1 in 446 wheat accessions shows that Ta CWI-B1_Hap1(Ta CWI-B1)are resistant to wheat FCR but Ta CWI-B1_Hap2/3 are susceptible to it.Ta CWI-B1_Hap1 was further verified its resistance to FCR in two RIL populations.The Ta CWI-B1 mutants and overexpression lines confirmed that Ta CWI-B1 could positive regulate wheat FCR resistance.2)Histological and cell wall composition analysis of the Ta CWI-B1 mutants and overexpression lines.Results showed compared with their wild type,Ta CWI-B1-overexpression lines exhibited a thicker cell wall,a higher pectin and cellulose content,whereas the mutants exhibited a thinner cell wall,a lower pectin and cellulose content.Y2 H and LUC experiment shows that Ta CWI-B1 can interact with Ta GAL(alpha-galactosidase).q RT-PCR indicated that Ta GAL expression level can be depressed by Ta CWI-B1.Investigation the FCR resistance of Ta GAL mutant indicated that Ta GAL can negatively modulated wheat FCR resistance.3)To verify whether Ta CWI-B1 conferred a resistance to wheat SE,the resistance of all test accessions was investigated.Results showed that wheat cultivars with Ta CWI-B1_Hap1 showed significantly stronger SE resistance than that with Ta CWI-B1_Hap2 in 446 test wheat accessions and Ta CWI-B1_Hap3 in two bi-parental populations.Ta CWI-B1-overexpression lines exhibited significantly increased SE resistance whereas the Ta CWI-B1 mutants exhibited significantly decreased SE resistance compared with their wild types.Ta GAL mutants showed significantly increased SE resistance compared with wild-type.4)349 wheat accessions from Huanghuai Valley and 138 F10 recombined inbred lines were used for metabolome analysis by using liquid chromatography tandem mass spectrometry(LC-MS/MS)-based widely targeted metabolome method.Total 947 distinct metabolites in the mature kernels were detected,of these metabolites features,159 were structurally identified by using authentic standards,while 302 were putatively annotated.The 947 metabolites were used for GWAS and QTL analysis,a total of 33,566 significant SNP(P<0.0001)were detected from m GWAS analysis.Total 3804 QTL(LOD≥2.5)were mapped from the RIL population based on m QTL analysis.Combined with m GWAS and m QTL,there are total 94 annotated metabolites had been both mapped in the same interval.Further analysis showed that Traes CS7A03G0128100 was significant associated with 11 metabolites.5)Total 33 traits were used for phenotype genome wide association analysis(p GWAS),22,099 significant SNP(P< 0.0001)were identified.Combined with m GWAS,p GWAS and m QTL,1799 SNP can be found to be co-detected.Further analysis showed that Traes CS4A02G343700 was significant with GA4 and roundness.Mutants,transgenic wheat,gene editing and other ways will be used to verify its functions.In conclusion,Ta CWI was cloned and associated with resistance against Fusarium crown rot and sharp eyespot in common wheat,and an underlying molecular mechanism was proposed,in which Ta CWI conferring thickened cell wall counteracts the effects of Ta GAL,an alpha-galactosidase gene associated with susceptibility to the two diseases.Besides,our study constructed a larger number of metabolic related sites for the study of wheat metabolomics,and also provides insights into genetic and biochemical bases of wheat metabolome and can be used as a powerful complementary tool to classical phenotype traits mapping for wheat improvement.