Identification Of Adult Resistant Candidate Genes To Stripe Rust In Yunnan Wheat Landraces Based On GWAS And Rna-Seq

Wheat stripe rust is a fungal disease caused by Puccinia striiformis f.sp.tritici(Pst).China is the largest epidemic area of wheat stripe rust in the world which is the largest area of disease occurrence,suffered most severe which about 10% of yield loss annually.Breeding and planting disease-resistant varieties is the most economical,environmentally friendly and efficient means to control diseases and ensure high and stable yields of wheat.However,broadening the basis of resistance sources and mining disease resistance genes are the first priority in cultivaring disease-resistant varieties.In this study,wheat landraces in Yunnan were used to evaluate stripe rust resistance of adult plants at multiple locations over years,and genome-wide association analysis(GWAS)and weighted co-expression network analysis(WGCNA)were conducted to identify and screen the loci associated with adult stage stripe rust resistance in local wheat varieties in Yunnan.The results of this study are as follows:(1)Stripe rust resistance was evaluated in 335 wheats at the adult plant for 4 years(2018-2022).The results showed that the stripe rust infection type(IT)of all experimental materials ranged from 0 to 9,with an average IT of 3.10,and there were significant differences in the infection type of stripe rust between varieties.The Pearson correlation coefficients between stripe rust infection types in different environments ranged from(0.33 to 0.69)and the mean correlation was(0.54),indicating that the data of stripe rust infection types of the population in different environments were relatively consistent.Considering the results of resistance identification in the six environments,181 out of 335 wheat germplasm showed resistance at the adult plant(IT ≤ 6)under six environments,and 60 of181 had stable resistance at the adult plant(IT ≤ 3).(2)Through genotyping by sequencing(GBS),rust resistance loci were mined at the whole genome level.Tassel’s general linear model(GLM)and mixed linear model(MLM)were used to perform genome-wide association analysis for stripe rust resistance in the materials.180 SNP markers significantly associated with wheat stripe rust resistance were detected and screened on 21 chromosomes.These SNP loci significantly associated with resistance were mainly distributed on the B genome,with a total of 101 loci,of which the highest number was 23 on chromosome 3B while the lowest number was only 16 on the D genome,of which no significant SNP loci were obtained on chromosome 5D and only one significant SNP locus was obtained on both chromosomes 3D and 4D;and 28 of the 180 SNP loci significantly associated with resistance were associated twice or more in six different environments.(3)Stripe rust infestation experiments were conducted on disease-resistant variety Y0337 and disease-susceptible variety Y0402,and transcriptome sequencing(RNA-Seq)and analysis were performed on the leaves at 0h,24 h,48h,72 h,120h,and 168 h after inoculation with the stripe rust fungus CYR32,and a total of 42,490 differentially expressed genes were obtained.The changes in differential genes were mainly concentrated at 24 hpi and 48 hpi after inoculation,GO and KEGG enrichment analysis suggested that a large number of genes were enriched for molecular functions and metabolic pathways related to the resistance response in these two periods.The expression patterns of differentially expressed genes were further categorized in different periods,and combined with data of two sample groups for analysis.Finally 6343 genes were screened that were differentially expressed in both groups of samples and consistent with the expression trends of wheat resistance response.(4)The above 15 candidate genes were further identified using WGCNA analysis,and finally five disease resistance candidate genes were screened,including wheat ribonuclease gene Traes CS1B02G170200,flavonol 3 sulfotransferase gene Traes CS2D02G181000,F-box proteins Traes CS4B02G117200 and Traes CS6A02G189300,and cell wall receptor kinase gene Traes CS3A02G122300.The results of the gene interaction network and the expression pattern after stripe rust infection demonstrated that the functions of these five candidate genes are highly correlated with the defense regulatory mechanism of disease resistance response.In this study,wheat landraces in Yunnan were used to evaluate stripe rust resistance of adult plant at multiple locations over years,which the disease-resistant germplasm screened out are going to provide a material basis for disease resistance breeding;Using GWAS and RNA-Seq analysis and gene function prediction,five candidate disease resistance genes were finally identified,providing a theoretical basis for further functional validation of disease resistance genes and analysis of disease resistance mechanisms at the adult stage.