QTL Mapping Analysis Of Wheat Stripe Rust Resistance And Disease Resistance Expression Analysis Of Wheat ABC And SNARE Families

Wheat stripe rust and powdery mildew can lead to substantial yield losses and even crop failures during pandemics.The discovery of Yr61(yellow rust)initially occurred in the winter wheat variety Pindong 34,which exhibits immunity or high resistance to various physiological strains of wheat rust.Pindong 34,a valuable germplasm resource,possesses additional characteristics such as large seed size and high yield.MY11847 and Warran,which are foreign germplasm resources,have undergone extensive field testing over many years and demonstrate moderate resistance to the mixed species CYR32/CYR33.To delve into the mechanisms of stripe rust resistance,clone genes associated with this resistance,and address several challenges in breeding for resistance,this study utilizes a genetic population consisting of Pindong 34,MY11847,Warran,and Shaanmai 159 for an indepth investigation.In this study,we employed Pindong 34 as the parent and crossed it with MY11847,Warran,and Shaanmai 159 to generate three RIL populations,thus forming a Nested Association Mapping(NAM)population of 1297 lines.Through simplified genome sequencing,we developed the population using high-quality SNP markers,which were then merged to obtain bin markers and establish a genetic linkage map.Genome-wide association analysis was conducted using the MLM method,and JICIM/RQTL analysis was performed through linkage mapping employing SNP markers.The RIL populations of Shaanmai 159 and Pindong 34 were subjected to stripe rust treatment at the seedling stage.Subsequently,samples were taken at four time points,and the populations were divided into disease resistance pools and susceptible pools for transcriptome sequencing,SNP marker identification,and acquisition of QTL intervals.Differential gene expression,GSEA,and WGCNA analyses were conducted on the transcriptome sequencing data to gain initial insights into the regulatory mechanism of seedling wheat-Pst interactions.Interestingly,the findings suggest that ABC transporter protein and SNARE protein might play crucial roles in the disease resistance process.Bioinformatics methods were employed to perform genome-wide identification of the ABC transporter protein family and SNARE protein family,and these families were subjected to biotransformation analysis and qPCR analysis.Additionally,disease resistance functions of the SNARE protein family members,TaSYP137 and TaVAMP723,were further examined.The study yielded the following results:1.The three RIL populations collectively harbored 164991,227202,and 159164high-quality SNPs,respectively.Combining these SNPs resulted in a large population with50554 SNP markers,which served as the foundation for generating bin markers to construct a genetic linkage map.The MP population encompassed 8268 bin markers distributed across 21 clusters,spanning a total length of 2757.59 cM.The average distance between markers was 0.34.Similarly,the WP population comprised 9626 bin markers covering 21 clusters,with a total length of 2944.84 cM and an average marker distance of 0.32.The MWSP population consisted of 6822 bin markers distributed among 21 clusters,spanning a total length of 1687.2 cM.The average marker distance in this population was 0.26.2.This paper identified a total of nine QTL loci associated with stripe rust resistance.The QTL intervals(Mb)were as follows: 2B:647.1-701.1;4A:729.9-744.5;4D:416.4-446.8;5B:525.4-547.3;7B:427.3-427.7;490.6-496.9;556.4-576.4;619.4-624.4;and 7D:37.1-71.3.Notably,the interval on chromosome 7D consistently appeared across all populations,exhibiting a negative additive effect and potentially representing the dominant QTL interval.3.Transcriptomic profiling unveiled the enrichment of photosynthesis-related biological processes and signaling pathways in the Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)analyses of differentially expressed genes at0-1 days post inoculation(dpi).Furthermore,the disease-resistant population exhibited the presence of the SNARE protein pathway in Gene Set Enrichment Analysis(GSEA).At 1-2dpi,the disease-resistant population demonstrated an abundance of disease-resistant processes,including phenylalanine,isoquinoline alkaloid synthesis,aromatic compound catabolism,curcumin synthesis,ubiquitinone,and terpenoid quinone synthesis.Conversely,susceptible monocots primarily exhibited enrichment in GSEA for photosynthesis-related pathways and processes.By 20 dpi,the susceptible population displayed a substantial enrichment of secondary metabolites associated with disease resistance,cell wall-related genes,and activated ABC transporter proteins.WGCNA analysis identified SNARE-related pathways,fatty acid-related pathways,and other crucial genes associated with stripe rust resistance.4.In wheat,a total of 463 ABC genes were identified using wheat genome sequence and annotation information.Phylogenetic tree analysis revealed the division of the ABC family into nine evolutionary branches and eight subfamilies.The TaABC genes exhibited an average of 11.93 exons.Notably,the promoter region of this gene family contained a high number of cis-acting elements,such as light-associated elements,Me JA-associated elements,ABA-associated elements,and MYB-associated elements.Region duplication emerged as the primary cause of TaABC gene amplification.The Ka/Ks analysis indicated that the majority of TaABC genes underwent purifying selection.Co-lineage analysis of TaABC with other species demonstrated the high conservation of ABC genes throughout evolution.RNA-seq and qPCR data exhibited the induction of expression in most TaABC genes by powdery mildew or stripe rust bacteria.The expression patterns of different genes displayed significant variability,highlighting the diversity in gene expression.5 Using wheat genome sequence and annotation information,a total of 173 SNARE genes were identified in the wheat genome and classified into 21 classes within five subfamilies.Phylogenetic tree analysis revealed the distribution of most SNAREs across the21 classes.Gene structure analysis showcased substantial differences among these 21 classes,although genes within the same class exhibited similar structures.Except for the first group,the number of homologous groups remained consistent.The promoter region of the group contained numerous W-box,MYB,and disease-associated cis-acting elements.The qPCR-based analysis demonstrated similar expression patterns among genes belonging to the same subfamily of SNARE in a given wheat variety.At 6 hours post-infection(hpi)after powdery mildew infestation staining,notable differences in expression patterns were observed among the same genes in resistant and susceptible varieties.6 Significant observations were made 48 hours after the injection of SNARE-GFP fusion protein into wheat that had been inoculated with powdery mildew.At 72 hours post-inoculation(hpi),disease-resistant samples exhibited notably longer mycelium lengths in both TaSYP137 and TaVAMP723 compared to the control.Similarly,at 7 dpi,disease-resistant samples displayed more severe development in TaSYP137 and TaVAMP723 compared to the control.Notably,silencing TaSYP137 and TaVAMP723 in susceptible samples resulted in an improvement in plant resistance against powdery mildew.Additionally,yeast two-hybrid experiments demonstrated the in vitro interaction between TaSYP137 and TaVAMP723.