Functional Analysis Of TaRLK-B And Genome-wide Association Study Of Fusarium Head Blight Resistance In Wheat

Fasurium Head Blight(FHB)is a fungal disease mainly caused by Fusariurn graminearum infecting the heads of wheat during flowering period,causing wheat ear rot and ear blight,reducing wheat yield and quality.The accumulated mycotoxins such as DON(deoxynivalenol)are harmful materials threatening causes human and livestock health.In wheat FHB epidemic areas,the utilization of FHB-resistant wheat varieties is the most efficient and economic approach for disease control.Wangshuibai is a landrace originally from Liyang county,Jiangsu province.Wangshuibai is considered as one of the best FHB resistance resources.Fhb1,originally mapped on chromosome 3BS in Wangshubai and another FHB resistant variety Sumai 3,is the most stable and best resistance locus for FHB resistance.Cloning FHB resistance genes,elucidating the resistance mechanism of Wangshuibai is of great significance for the utilization of Wangshuibai in wheat breeding for FHB resistance.The phenotyping of wheat varieties collected from different areas,together with high-throughput genotyping by SNP array will not only evaluate the genetic effects of known resistance genes,but also allow the identification of new ressiatnce loci with different origin.Genome wide association study(GWAS)has been widely used in wheat for different research purposes.We have collected rich wheat varieties from different areas,and the discovery of new FHB resistance loci will provide new gene resources for wheat breeding.The main results obtained are as follows:1.Functional analysis TaRLK-B gene in FHB resistance by generating transgenic wheatIn previous RNA-seq experiment,the expression of a receptor protein kinase gene TaRLK-B was significantly different in Wangshuibai and its deletion mutant NAUH117(about 78Mb of the Fhb1 region deleted).TaRLK-B is located within the Fhb1 region,and knockdown TaRLK-B by VIGS significantly compromised FHB ressitance of Wangshuibai.We generated transgenic wheat lines by overexpressing TaRLK-B in receptor variety Fielder via Agrobacterium-mediated transgenic approach.In total,6 positive transgenic plants(ZT1～ZT-6)at T0 generation positive were identified.Their derived T1 lines were analyzed by PCR for transgene segregation.The segregation ratios of ZT-1,ZT-3 and ZT-4 were 3:1,indicating their transgenes were all single copy,those of ZT-2 and ZT-6 were 15:1,indicating their transgenes were all two copies.The only 31.25%were positive in ZT-5 derived T1 lines,indicating the transgenewas genetically unstable.The transgene expression and FHB resistance were analyzed using the ZT-6 derived lines.In average,TaRLK-B expression were 5.35 times higher in T1 transgenic plants than in that of Wangshuibai.Accordingly,average diseased spikelet rate of T1 transgenic plants was lower(14%)than Fielder(31.75%).Aaverage TaRLK-B expression in T2 transgenic plants was 4.84 times higher than that in Wangshuibai,and average diseased spikelet rate in T2 transgenic plants was lower(11.4%)than Fielder(17.9%).We further inoculated the Fusarium graminearum(Eg.)isolate F0609 into coleoptile of wheat seedlings to test the FHB resistance of T3 transgenic plants.The FHB resistance rate of transgenic line was significantly higher(80%)than that of Fielder(13%).By inoculating a GFP-marked F.g.isolate CF0915-4,it was found that the intensity of GFP signals of the inoculated florets in T3 transgenic lines was significantly less than that in Fielder at 3-6 d after F.g.inoculation.The above results reveal that the resistance of TaRLK-B transgenic plants to FHB is significantly higher than that over-expression of TaRLK-B significantly enhanced FHB resistance.However,the relationship of transgene copy number,transgene expression level and FHB resistance need to be further studied.2.Two different haplotypes of gene TaRLK-B may be related to FHB resistanceIn previous research,two haplotypes were present in FHB resistant and susceptible wheat varieties.Compared with the coding sequence(CDS)of TaRLK-B in Aikang58 and Alondra’s(FHB susceptible),CDS of TaRLK-B in Wangshuibai and Chinese spring(FHB resistant)lost a base G(1132bp).This change results in a frameshift and premature termination in Wangshuibai.We defined the premature terminated haplotype in resistant varieties as "G-".and those complete CDS TaRLK-B haplotype in susceptible varieties as“G+".TaRLK-B sequences were searched and compared using the released genome sequences of hexaploid wheat and tetraploid wheat.All TaRLKs in diploid species(A,S,D)or subgenomes A-,and D-in tetraploid and hexaploidy wheat are“G+" type.Both haplotypes TaRLK-B are found in wild emmer and durum wheat(Svevo and Kronos),and G-haplotype TaRLK-B in tetraploid and hexaploidy wheat are complete identical.We speculate that the G+is an ancient haplotype,G-haplotype is a new mutation occurred in the tetraploid level,introduced into hexaploid wheat(AABBDD)by the cross of tetraploid wheat(G-)and hexaploid wheat(G+).A SNP marker was developed based on the deletion/insertion of the the two haplotypes of TaRLK-B.A Wangshuibai×Alondra’s RIL2:8 population was used for association analysis using the new developed marker and seven markers,including Fhb1HRC gene-based marker and other six Fhb1-linkage markers.The results showed that the group consisting of RIL lines with G-haplotype exhibited better FHB resistance than group consisting of RIL lines with G+haplotype.The expression of two TaRLK-B haplotypes were significantly different.How about the regulation mechanism?Taking the advantage of the MNase-seq data of Chinese spring(Kindly provided by Professor Zhang Wenli,NAU),we compared the upstream TaRLK-B gene sequences and expression patterns in Chinese Spring(G-),Wangshuibai(G-),Aikang 58(G+)and Alondra’s(G+).The G-and G+haplotypes of TaRLK-B were significantly different in their promoter and 5’UTR regions.Total length of G-type is 9315bp,containing a 8,172bp super-long 5’UTR.There is a 7840bp intron in the G-type’s 5’ UTR,and the upstream of this intron is 230bp and its downstream is 102bp.The upstream of the 5’UTR is predicted to be a promoter region,which is an MNase hypersensitive site.However,for TaRLK-B G+type,its 5’UTR region only contains 730bp homologous sequence(similarity 99.32%)of the C-terminal of the above mentioned 7840bp intron and its downstream 102bp sequence.The G+type does not have the 5’ UTR upstream promoter sequence of Wangshuibai G-type TaRLK-B.At 0h,24h,and 48h after F.g inoculation,the TaRLK-B expression in Wangshuibai was up-regulated,while the TaRLK-B expression in Aikang 58 was hardly detected.It is speculated that the 5’UTR difference,especially in the promoter region,led to significant different gene expression patterns of the two TaRLK-B haplotypes.3.Genome-wide association study of to FHB resistance using a natural wheat variety populationA natural population consist of 369 wheat varieties from five different regions(Southern China,Northern China,Europe,Africa,and Asian coutries except China)were evaluated for FHB resistance,and varieties from Southern China had better resitance performance.The population was genotyped for association analysis using molecular markers for the three cloned genes(TaPFT,TaHRC and TaRLK-B)in the Fhb1 region.Those varieties(12 varieties)carrying all three genes had best FHB resistance(PSS was 10.19%).For varieties carrying two genes,the combination of TaHRC and TaRLK-B(PSS was 14.69%)were more resistant;For varieties carrying single gene,TaHRC(PSS was 15.12%)were the best.Overall,FHB resistance of varieties having none of the three genes was similar as the susceptible control Alondra’s(PSS was 24.15%).These suggest that the analyzed three genes all contribute to FHB resistance,with TaHRC has the best effect.The association of the TaRLK-B expression and FHB resistance was analyzed using 110 representative varieties in the natural population.It was found that varieties with higher FHB resistance had higher TaRLK-B expression levels.This further verified the function of TaRLK-B in FHB resistance,and the TaRLK-B expression level may associate with its function.A total of 268 Chinese varieties and landraces from the natural population was used for genome-wide association study(GWAS).The population were genotyped using 55K SNP array.A total of 50 SNPs were found to be significantly associated with FHB resistance in 2 environments.Ten QTLs were mapped on chromosomes 1B,3B,5D,6A,7B and 7D.Among them two were the most significant,one located in 760.7-771.3Mb region of 3BL and another in 607.6-609.3Mb region of 7DL.The predicted candidate genes for 3B QTL region include:zinc finger proteins,leucine-rich repeat family proteins and endo-β-1,3glucanase;predicted candidate genes predicted for the 7D QTL include:transcription Factors,E3 ubiquitin protein ligase,NBS-LRR gene and protein kinase family.