Genome-wide Association Analysis And Candidate Gene Mining Of Wheat Sharp Eyespot

Wheat(Triticum aestivum L.)is one of the most important three grain crops in China,providing one-fifth of humanity’s energy and protein.Wheat sharp eyespot,powdery mildew,scab and rust are the main diseases in wheat production in China,which seriously affect the yield and quality of wheat.Among them,wheat sharp eyespot,mainly caused by Rhizoctonia cerealis,is a very destructive wheat disease,generally causing 5 %-40 % yield loss,and even no grain harvest when the disease is serious.Breeding and application of resistant varieties is the safest,effective and green way to control wheat sharp eyespot.In this study,243 wheat varieties(lines)cultivated in the Huang-Huai wheat region in recent years were selected.Genome-wide association analysis was used to excavate wheat sharp eyespot resistance genes.Combined with transcriptome data,gene expression analysis and gene annotation,a candidate gene was identified.Furthermore,virus-induced gene silencing(VIGS)and Kronos mutants were used to confirm that the gene was positively regulating sharp eyespot resistance.Subsequently,an overexpression vector of the gene was constructed and transformed into wheat to verify the function of the gene.The main results of this study are as follows:1.In this study,the highly virulent race R0301 was used as the inoculation pathogen,the disease index(DI)and Relative resistance Index(RRI)were used as evaluation indexes,and the highly susceptible variety Yumai 49 was used as control.The resistance of 243 wheat varieties(lines)to sharp eyespot was identified and evaluated.The results showed that the variation of resistance DI of all tested materials ranged from 26.67 to 81.48,the correlation coefficient between repeats ranged from 0.59 to 0.89,and the variation coefficient ranged from 15.7% to 17.8%.The results showed that the identification conditions were suitable,the incidence was sufficient,and the population phenotypic variation was wide.According to the relative disease resistance index analysis,215 materials with RDI value less than 0.4 were identified,accounting for 88.5% of all identified materials.Among them,82 cases(33.7%)with RDI less than 0.2,such as Yunong 169 and Hangmai 8,etc.No material with RDI value greater than 0.8 was identified,but the RDI value of wheat variety Yongfengnong 2 was 0.61,indicating relatively high resistance.The results showed that most wheat varieties in Huang-Huai wheat region had poor sheath blight resistance,and the protection situation was serious,so breeding of sheath blight resistance was in urgent need.2.Genome-wide association analysis was conducted for sharp eyespot resistance of 243 wheat samples based on 660 K SNP microarray.A total of 560 SNPS were found,mainly distributed on chromosomes 1D,2B,3B,5B,7Aand 7B,explaining phenotypic variation in 2.2-12.1%range.A total of 37 significant SNPS were detected at the same time,mainly distributed on chromosome 7B(652.9-653.8 Mb)and chromosome 3B(786.3-786.9 Mb),and one multiple SNP was detected on chromosome 2B(7.1-8.5%),which could explain the phenotypic variation.3.Based on the attenuation distance,650.8-656.8 Mb near the Lead SNP marker on chromosome 7B was identified as the candidate region.The SNP in the segment was haplotype analyzed,and two blocks were obtained,which were divided into 6 haplotypes.The mean disease index of haplotype 3 and 5 was 48.87 and 53.86,which was significantly lower than that of other haplotypes.4.A total of 45 annotated genes were identified in candidate regions based on the Genome database of The Chinese spring and AK58.In combination with the transcriptome before and after the induction of bacterial resistance material,and the previous BSR-seq transcriptome,it was found that 9 genes were strongly induced by Bacterial resistance material.According to the analysis of Gene annotation,Induced expression and stem base expression,one candidate gene was selected and silenced by VIGS technology.The results showed that when Gene27 was silenced,the disease index of plants increased significantly and the disease resistance decreased,suggesting that Gene27 may play an important role in sharp eyespot resistance.Annotation results showed that the protein encoded by this gene has a BAG domain as well as PTZ00121 and MDN1 domains,phylogenetic tree analysis showed that this gene had a closer homology with Zm BAG6 gene in maize.named Ta BAG6.Kronos mutant of Ta BAG6 was screened for sharp eyespot resistance identification,and the average disease index of the mutant was significantly higher than that of the wild type.It was further confirmed that Ta BAG6 plays an important role in regulating wheat sharp eyespot resistance.5.The gene was sequenced and 113 polymorphic loci were found in the natural variety of Ta BAG6.Five SNPS with wide variation were selected for gene haplotype analysis and four haplotypes were formed.The average disease index of Hap4 was significantly lower than that of Hap1 and Hap2,indicating that Hap4 was an excellent resistance haplotype.A pair of codominant markers were developed based on variable clipping and insertion deletions in polymorphic loci.The disease index of Ta BAG6_G1(with splice acceptor)was significantly lower than that of Ta BAG6_A0(with no splice acceptor).6.The overexpression vector of Ta BAG6 gene has been constructed and successfully transferred into the recipient plant Fielder.At present,the T0 generation is in the grain filling stage.