Robert Saxon Brueggeman

Identifying and genetically characterizing new sources of resistance in barley (Hordeum vulgare) effective against the virulent wheat stem rust (Puccinia graminis f. sp. tritici [Pgt]) population in the Pacific Northwest (PNW) is critical. Isolates from this population, including Pgt isolate Lsp21, were virulent on barley stem rust resistance (R) genes Rpg1, Rpg2, Rpg3, rpg4, Rpg5, and rpg8. Notably, 10% of the Pgt isolates from the population were virulent on barley line Q21861, which contains Rpg1 and rpg4/5 stacked together. Virulence on these 2 broad and effective stem rust R-genes/loci, when combined, is unprecedented Pgt virulence on barley. To discover novel resistance, 277 wild barley (H. vulgare subsp. spontaneum) accessions from the Wild Barley Diversity Collection (WBDC) were screened with Pgt isolate Lsp21. Twelve percent showed moderate resistance, with WBDC-94 and WBDC-238 from Jordan exhibiting exceptional resistance, likely conferred by Rpg7, previously reported in both. To genetically characterize resistance in the WBDC, a genome-wide association study was conducted using disease reactions to Lsp21 and 37,338 genotyping-by-sequencing SNPs. Twelve resistance-associated loci were identified on chromosomes 1H, 2H, 3H, 5H, 6H, and 7H. Rpg7 was not detected due to its low allele frequency in the panel. Importantly, 7 novel resistance loci, WQRpg-2H01, WQRpg-2H02, WQRpg-3H01, WQRpg-5H01, WQRpg-5H03, WQRpg-7H02, and WQRpg-7H03, were discovered. These new sources of resistance can be integrated into cultivated barley, and the associated SNPs will aid in tracking resistance loci in prebreeding lines, enhancing breeding efforts against the virulent PNW Pgt population.

To exploit allelic variation in Hordeum vulgare subsp. spontaneum, the Wild Barley Diversity Collection was subjected to paired-end Illumina sequencing at ∼9 × depth and evaluated for several agronomic traits. We discovered 240.2 million single nucleotide polymorphisms (SNPs) after alignment to the Morex V3 assembly and 24.4 million short (1 to 50 bp) insertions and deletions. A genome-wide association study of lemma color identified one marker-trait association (MTA) on chromosome 1H close to HvBlp, the cloned gene controlling black lemma. Four MTAs were identified for seedling stem rust resistance, including 2 novel loci on chromosomes 1H and 6H and one co-locating to the complex RMRL1-RMRL2 locus on 5H. The whole-genome sequence data described herein will facilitate the identification and utilization of new alleles for barley improvement.

Wheat stem rust, caused by the obligate biotrophic fungal pathogen Puccinia graminis f. sp. tritici ( Pgt), is an important disease of barley and wheat worldwide. Alarmingly, the Pacific Northwest contains a highly virulent Pgt population on barley. This population includes the Pgt isolate Lsp21, which is virulent on the barley stem rust resistance genes Rpg1, Rpg2, Rpg3, rpg4, Rpg5, and rpg8. The virulence on barley lines containing Rpg1 and the rpg4/ Rpg5-mediated resistance locus, when stacked together, is a Pgt virulence profile on barley not previously reported. Thus, this population contains the most virulent Pgt isolates on barley resistance genes characterized worldwide. The Elliot line (PI 592261) was identified from the world barley core collection as containing effective seedling resistance to Pgt isolate Lsp21. To genetically characterize the resistance present in Elliot, 129 recombinant inbred lines were developed by advancing a population from the cross Elliot (resistant) × Palmer (susceptible) to the F₆ generation. The population was phenotyped with Pgt isolate Lsp21 at the seedling stage and genotyped with the Illumina 50K bead express single-nucleotide polymorphism chip, resulting in 7,284 high-quality single-nucleotide polymorphism markers. Two significant resistance quantitative trait loci (QTLs) (EP Rpg_4H-1 and EP Rpg_5H-1) contributed by Elliot were identified on chromosomes 4H and 5H, respectively. The major QTL, EP Rpg_4H-1, is novel, whereas EP Rpg_5H-1 localized to a region approximately 9 Mbp distal of the rpg4/ Rpg5-mediated resistance locus within a region of the barley genome that contains previously identified stem rust resistance loci. These QTLs should be useful in developing barley cultivars with resistance to the virulent Pacific Northwest Pgt population.

To exploit allelic variation in Hordeum vulgare subsp. spontaneum , the Wild Barley Diversity Collection was evaluated for several agronomic traits and subjected to paired-end Illumina sequencing at ∼9X depth, generating 109.5 million single nucleotide polymorphisms after alignment to the Morex V3 assembly. A genome-wide association study of lemma color identified one marker-trait association (MTA) on chromosome 1HL close to HvBlp , the cloned gene controlling black lemma. Four MTAs were identified for stem rust resistance: one co-locating to the complex RMRL1-RMRL2 locus on 5HL, and three novel loci on 1HS, 1HL, and 5HL. Six MTAs for days to heading (DTH) on vernalized plants were identified on all chromosomes except 1H and 6H. Two MTAs for DTH on non-vernalized plants were identified on chromosomes 1HL and 2HS. All MTAs for DTH were novel. The whole genome sequence data described herein will facilitate the identification and utilization of new alleles for barley improvement.

Barley net form net blotch (NFNB) is a destructive foliar disease caused by Pyrenophora teres f. teres. Barley line CIho5791, which harbors the broadly effective chromosome 6H resistance gene Rpt5, displays dominant resistance to P. teres f. teres. To genetically characterize P. teres f. teres avirulence/virulence on the barley line CIho5791, we generated a P. teres f. teres mapping population using a cross between the Moroccan CIho5791-virulent isolate MorSM40-3 and the avirulent reference isolate 0-1. Full genome sequences were generated for 103 progenies. Saturated chromosome-level genetic maps were generated, and quantitative trait locus (QTL) mapping identified two major QTL associated with P. teres f. teres avirulence/virulence on CIho5791. The most significant QTL mapped to chromosome (Ch) 1, where the virulent allele was contributed by MorSM40-3. A second QTL mapped to Ch8; however, this virulent allele was contributed by the avirulent parent 0-1. The Ch1 and Ch8 loci accounted for 27 and 15% of the disease variation, respectively, and the avirulent allele at the Ch1 locus was epistatic over the virulent allele at the Ch8 locus. As a validation, we used a natural P. teres f. teres population in a genome-wide association study that identified the same Ch1 and Ch8 loci. We then generated a new reference quality genome assembly of parental isolate MorSM40-3 with annotation supported by deep transcriptome sequencing of infection time points. The annotation identified candidate genes predicted to encode small, secreted proteins, one or more of which are likely responsible for overcoming the CIho5791 resistance.

The foliar disease net form net blotch (NFNB), caused by the necrotrophic fungal pathogen Pyrenophora teres f. teres (Ptt), causes significant yield and quality losses of barley worldwide. Dominant resistance conferred by the Resistance to Pyrenophora teres 5 (Rpt5) gene from barley line CI5791 is the broadest and most effective resistance reported in this pathosystem. The Rpt5 locus was identified in multiple independent genetic studies utilizing diverse host populations and Ptt isolates, and harbors both dominant Rpt5 resistance and isolate-specific susceptibility genes/alleles that are dominant in the absence of Rpt5, designated susceptibility to Pyrenophora teres 1 (Spt1). Ptt virulence and avirulence effectors from diverse pathogen isolates genetically interact with the Rpt5/Spt1 locus, suggesting a complex locus with a function targeted by the evolution of a diversity of pathogen effectors. High-resolution mapping utilizing 1,920 recombinant gametes from a CI5791 x Tifang biparental population, identified 12 candidate genes in an ~4.6 Mb delimited region in the cv Morex V3 genome assembly, but is 1.1 − 2.2 Mb in the pangenome assemblies, containing 5−12 genes. Analysis revealed a strong correlation between the CI5791 allele of a receptor-like protein (RLP), provisionally designated Rpt5 candidate gene 1, (Rcg1), and broad Rpt5−mediated resistance. Two independent transformants of the CI5791 Rcg1 allele in the susceptible cv Golden Promise background showed significantly increased resistance when challenged with Rpt5 avirulent Ptt isolates 6A, 15A, and 0−1 compared to the Golden Promise wildtype. Thus, Rpt5, encodes an RLP and is the first net blotch resistance gene cloned in barley.

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.

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. [ ] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Hybrid genotypes can provide significant yield gains over conventional inbred varieties due to heterosis or hybrid vigor. However, hybrids can also display unintended negative attributes or phenotypes such as extreme pathogen susceptibility. The necrotrophic pathogen Pyrenophora teres f. maculata (Ptm) causes spot form net blotch, which has caused significant yield losses to barley worldwide. Here, we report on a non-transgressive hybrid susceptibility locus in barley identified between the three parental lines CI5791, Tifang and Golden Promise that are resistant to Ptm isolate 13IM.3. However, F₂ progeny from CI5791 × Tifang and CI5791 × Golden Promise crosses exhibited extreme susceptibility. The susceptible phenotype segregated in a ratio of 1 resistant:1 susceptible representing a genetic segregation ratio of 1 parental (res):2 heterozygous (sus):1 parental (res) suggesting a single hybrid susceptibility locus. Genetic mapping using a total of 715 CI5791 × Tifang F₂ individuals (1430 recombinant gametes) and 149 targeted SNPs delimited the hybrid susceptibility locus designated Susceptibility to Pyrenophora teres 2 (Spt2) to an ~ 198 kb region on chromosome 5H of the Morex V3 reference assembly. This single locus was independently mapped with 83 CI5791 × Golden Promise F₂ individuals (166 recombinant gametes) and 180 genome wide SNPs that colocalized to the same Spt2 locus. The CI5791 genome was sequenced using PacBio Continuous Long Read technology and comparative analysis between CI5791 and the publicly available Golden Promise genome assembly determined that the delimited region contained a single high confidence Spt2 candidate gene predicted to encode a pentatricopeptide repeat-containing protein.