Association mapping with a diverse population of Puccinia graminis f. sp. tritici identified avirulence loci interacting with the barley Rpg1 stem rust resistance gene.

BACKGROUND: Wheat stem rust, caused by Puccinia graminis f. sp. tritici (Pgt), is an important disease of barley and wheat. A diverse sexual Pgt population from the Pacific Northwest (PNW) region of the US contains a high proportion of individuals with virulence on the barley stem rust resistance (R) gene, Rpg1. However, the evolutionary mechanisms of this virulence on Rpg1 are mysterious considering that Rpg1 had not been deployed in the region and the gene had remained remarkably durable in the Midwestern US and prairie provinces of Canada. METHODS AND RESULTS: To identify AvrRpg1 effectors, genome wide association studies (GWAS) were performed using 113 Pgt isolates collected from the PNW (n = 89 isolates) and Midwest (n = 24 isolates) regions of the US. Disease phenotype data were generated on two barley lines Morex and the Golden Promise transgenic (H228.2c) that carry the Rpg1 gene. Genotype data was generated by whole genome sequencing (WGS) of 96 isolates (PNW = 89 isolates and Midwest = 7 isolates) and RNA sequencing (RNAseq) data from 17 Midwestern isolates. Utilizing ~1.2 million SNPs generated from WGS and phenotype data (n = 96 isolates) on the transgenic line H228.2c, 53 marker trait associations (MTAs) were identified. Utilizing ~140 K common SNPs generated from combined analysis of WGS and RNAseq data, two significant MTAs were identified using the cv Morex phenotyping data. The 55 MTAs defined two distinct avirulence loci, on supercontig 2.30 and supercontig 2.11 of the Pgt reference genome of Pgt isolate CRL 75-36-700-3. The major avirulence locus designated AvrRpg1A was identified with the GWAS using both barley lines and was delimited to a 35 kb interval on supercontig 2.30 containing four candidate genes (PGTG_10878, PGTG_10884, PGTG_10885, and PGTG_10886). The minor avirulence locus designated AvrRpg1B identified with cv Morex contained a single candidate gene (PGTG_05433). AvrRpg1A haplotype analysis provided strong evidence that a dominant avirulence gene underlies the locus. CONCLUSIONS: The association analysis identified strong candidate AvrRpg1 genes. Further analysis to validate the AvrRpg1 genes will fill knowledge gaps in our understanding of rust effector biology and the evolution and mechanism/s of Pgt virulence on Rpg1.

Identification of Candidate Avirulence and Virulence Genes Corresponding to Stem Rust (Puccinia graminis f. sp. tritici) Resistance Genes in Wheat.

Stem rust, caused by the biotrophic fungal pathogen Puccinia graminis f. sp. tritici (Pgt), is an important disease of wheat. However, the majority of Pgt virulence/avirulence loci and underlying genes remain uncharacterized due to the constraints of developing bi-parental populations with this obligate biotroph. Genome-wide association studies (GWAS) using a sexual Pgt population mainly collected from the Pacific Northwestern United States were used to identify candidate virulence/avirulence effector genes corresponding to the six wheat Sr genes: Sr5, Sr21, Sr8a, Sr17, Sr9a, and Sr9d. The Pgt isolates were genotyped using whole-genome shotgun sequencing that identified approximately 1.2 million single nucleotide polymorphisms (SNPs) and were phenotyped at the seedling stage on six Sr gene differential lines. Association mapping analyses identified 17 Pgt loci associated with virulence or avirulence phenotypes on six Pgt resistance genes. Among these loci, 16 interacted with a specific Sr gene, indicating Sr-gene specific interactions. However, one avirulence locus interacted with two separate Sr genes (Sr9a and Sr17), suggesting two distinct Sr genes identifying a single avirulence effector. A total of 24 unique effector gene candidates were identified, and haplotype analysis suggests that within this population, AvrSr5, AvrSr21, AvrSr8a, AvrSr17, and AvrSr9a are dominant avirulence genes, while avrSr9d is a dominant virulence gene. The putative effector genes will be fundamental for future effector gene cloning efforts, allowing for further understanding of rust effector biology and the mechanisms underlying virulence evolution in Pgt with respect to race-specific R-genes. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.