Mapping,cloning And Functional Verification Of Wheat Leaf Rust Resistance Gene

Leaf rust,caused the fungal pathogen by Puccinia triticina(Pt),is one of the major and dangerous diseases of wheat worldwide.The epidemic of leaf rust has caused serious yield and economic losses in major wheat-producing regions,whihch seriously threatened the food security of various regions.The economical and effective method to control wheat leaf rust is the application of resistant varieties.The screening of leaf rust resistance resources and the discovery of leaf rust resistant loci are the keys to breeding disease-resistant varieties.Based on this purpose,in this study,large-scale leaf rust resistance identification was carried out at adult plant stage on different types of wheat varieties collected in the previous stage.Some varieties were selected to detect the resistance-related loci of wheat leaf rust by genome-wide association analysis(GWAS)and QTL mapping of the recombinant inbred line(RIL)population.The leaf rust resistance genes were screened by combing GWAS and QTL mapping,and the function of the candidate gene was verified.The main results are as follows:1.In this study,1660 wheat varieties were identified for resistance to leaf rust at adult stage for two years and the average MDS of all varieties was 46.92%.The average MDS of 219 varieties with foreign background was 63.47%,and there were more moderately susceptible types.The average MDS of 224 landrace was 19.92%,and there were more high-resistance varieties.The average MDS of 753 historical wheat varieties/lines was 46.47%,and most of them showed medium resistance or susceptible to leaf rust.The average MDS of 464 modern varieties was 52.87%,and there were more medium-sensitivity varieties.At the same time,a batch of varieties with high resistance or near immunity to leaf rust were screened out,which could be used as resistance resources in breeding.According to the variety sources and characteristics,406 varieties were selected to construct two association populations for subsequent GWAS analysis.2.GWAS analysis was performed on the association population I composed of wheat varieties in different periods in China,using the wheat 90 K SNP assay.A total of397 significant SNPs were detected in 10 environments over 3 years,distributed on 21 chromosomes,explaining 6.67%-37.42% of the phenotypic variation.There were 85 significant SNPs detected in at least 2 environments,distributed on 19 other chromosomes except 2D and 7B,among which,fourteen SNPs were located on chromosome 2B and the lead SNP could explain 19.46%-26.58% of the phenotypic variation.3.GWAS analysis was performed on the association population II composed of wheat varieties breeded in Yellow and Huai wheat region in recent years,using the wheat 660 K SNP assay.A total of 1031 significant SNPs were detected in 14 environments over 4 years,distributed on 21 chromosomes,explaining 6.78%-37.06%of the phenotypic variation.There were 608 SNPs with multi-environmental significance,of which 2B were the most,mainly distributed in two segments of 133.01-142.47 Mb and 154.85-168.62 Mb.The results of haplotype analysis showed that the significant SNPs on 2A,5A and 6A each formed one block,the significant SNPs on 2B formed three blocks,and all blocks contained two haplotypes.The leaf rust resistance differences among different haplotypes reached significant levels.4.QTL mapping was performed on the F7 generation RIL population constructed by Xianyang Dasui and Xinmai 208,using wheat 660 K SNP assay.There were 3 multienvironmentally stable QTLs detected,of which,QLr.henau-7D had the greatest effect,explaining 5.27%-34.70% of the phenotypic variation,but this QTL contained the cloned wheat leaf rust resistance gene Lr34.QLr.henau-2B.1 and QLr.henau-6D explained 8.29%-10.65% and 7.33%-9.67% of the phenotypic variation,respectively.5.QTL mapping was performed on the F7 generation RIL population constructed by Xinmai 26 and Zhoumai 22,using wheat 660 K SNP assay.There were 3 multienvironmentally stable QTLs detected,among them,QLr.henau-2B.2 could be detected in all environments,could explain 6.09%-15.49% of phenotypic variation,and was located at 140.78-157.97 Mb on 2B,co-localizing with the GWAS results.6.Combined analysis of GWAS results and QTL mapping results revealed a colocalization interval of 154.85-157.97 Mb on 2B.The results of haplotype analysis showed that there were 2 blocks in this interval,covering 84.12 Kb and 282.58 Kb respectively,and both contained 3 annotated genes.According to the lead SNP position and gene annotation,two candidate genes,TaCNL and TaCN,were screened,both encoding NBS-LRR-like proteins.7.The candidate genes TaCNL and TaCN were sequenced and found that their sequence polymorphisms were significantly associated with resistance to leaf rust.The SNP at 1684 bp of TaCNL gene was a key locus affecting leaf rust resistance.There was a frameshift mutation at 215 bp of TaCN gene.The varieties without mutation presented a susceptible phenotype(TaCN-S),and after frameshift mutation,protein translation was terminated prematurely,showing a resistant phenotype(TaCN-R).The protein sequence analysis showed that TaCNL contains three domains of Rx-CC_like,NB-ARC and LRR,TaCN-S contains two domains of Rx-CC and NB-ARC,and TaCNR only has a partial sequence of CC domain.8.The expression levels of the two candidate genes in different tissues of wheat were analyzed and the results showed that TaCNL was highly expressed in leaves,which may play an important role in regulating leaf rust resistance.The resistant sequence of TaCNL was overexpressed in the wheat variety Fielder by Agrobacteriummediated transgenic technology and it was found that the positive plants had significantly increased leaf rust resistance at the adult stage in the field compared with the wild type.9.Based on previous findings that adjacent NLR protein pairs could interact and co-regulate plant disease resistance,in this study,the strong interaction between TaCNL and TaCN-R was confirmed by luciferase complementation imaging assay,in vitro pull-down assay and bimolecular fluorescence complementation(Bi FC)technology,and this interaction could be achieved through the N-terminal CC domain.The results of protein co-localization showed that TaCNL was mainly expressed in the nucleus,and TaCN-R was expressed in both the nucleus and the cell membrane.The Bi FC results showed that the interaction between TaCNL and TaCN-R occurred on the cell membrane,indicating that TaCN-R may alter the subcellular localization of TaCNL.Further analysis found that TaCN-R only had half of the CC domain of TaCNS,but there were 5 amino acid differences,which was different from previous studies on the direct interaction of NLR proteins,and may reveal a new mechanism of plant immune regulation mediated by NLR proteins.In conclusion,this study carried out large-scale leaf rust resistance screening of different types of wheat germplasm resources,some varieties were selected for GWAS based on variety characteristics and population structure,and multiple important genetic loci were located.The biparental segregating populations were further constructed to cloned the candidate genes located on chromosome 2B and it was found that TaCNL was an important wheat leaf rust resistance gene.At the same time,it was found that there was a strong interaction between TaCN-R and TaCNL,which may affect wheat leaf rust resistance through the latter.Therefore,the screened resistant varieties,located resistance loci and discovered resistance genes in this study can provide important genetic and material resources for wheat leaf rust resistance breeding in China,and it could also provide an important theoretical basis for further analysis of the resistance mechanism of wheat leaf rust.