Variability in Chromosome 1 of Select Moroccan Pyrenophora teres f. teres Isolates Overcomes a Highly Effective Barley Chromosome 6H Source of Resistance.

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. [Formula: see text] The author(s) have dedicated the work to the public domain under the Creative Commons CC0 "No Rights Reserved" license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law, 2024.

A Moroccan Pyrenophora teres f. teres Population Defeats Rpt5, the Broadly Effective Resistance on Barley Chromosome 6H.

Net form net blotch (NFNB), caused by Pyrenophora teres f. teres, is an important barley disease. The centromeric region of barley chromosome 6H has often been associated with resistance or susceptibility to NFNB, including the broadly effective dominant resistance gene Rpt5 derived from barley line CIho 5791. We characterized a population of Moroccan P. teres f. teres isolates that had overcome Rpt5 resistance and identified quantitative trait loci (QTL) that were effective against these isolates. Eight Moroccan P. teres f. teres isolates were phenotyped on barley lines CIho 5791 and Tifang. Six isolates were virulent on CIho 5791, and two were avirulent. A CIho 5791 × Tifang recombinant inbred line (RIL) population was phenotyped with all eight isolates and confirmed the defeat of the 6H resistance locus formerly mapped as Rpt5 in barley line CI9819. A major QTL on chromosome 3H with the resistance allele derived from Tifang, as well as minor QTL, was identified and provided resistance against these isolates. F2 segregation ratios supported dominant inheritance for both the 3H and 6H resistance. Furthermore, inoculation of progeny isolates derived from a cross of P. teres f. teres isolates 0-1 (virulent on Tifang/avirulent on CIho 5791) and MorSM 40-3 (avirulent on Tifang/virulent on CIho 5791) onto the RIL and F2 populations determined that recombination between isolates can generate novel genotypes that overcome both resistance genes. Markers linked to the QTL identified in this study can be used to incorporate both resistance loci into elite barley cultivars for durable resistance.

Development of a Greenhouse Assay to Screen Soybean Varieties for Resistance to Aerial Blight Caused by Rhizoctonia solani Anastomosis Group 1-IA.

Aerial blight, caused by the fungus Rhizoctonia solani anastomosis group (AG) 1-IA, is an economically important soybean disease in the mid-Southern United States. Management has relied on fungicide applications during the season, but there is an increasing prevalence of resistance to commonly used strobilurin fungicides and an urgent need to identify soybean varieties resistant to aerial blight. Because the patchy distribution of the pathogen complicates field variety screening, the present study aimed to develop a greenhouse screening protocol to identify soybean varieties resistant to aerial blight. For this, 88 pathogen isolates were collected from commercial fields and research farms across five Louisiana parishes, and 77% were confirmed to be R. solani AG1-IA. Three polymorphic codominant microsatellite markers were used to explore the genetic diversity of 43 R. solani AG1-IA isolates, which showed high genetic diversity, with 35 haplotypes in total and only two haplotypes common to two other locations. Six genetically diverse isolates were chosen and characterized for their virulence and fungicide sensitivity. The isolate AC2 was identified as the most virulent and was resistant to both active ingredients, azoxystrobin and pyraclostrobin, tested. The six isolates were used in greenhouse variety screening trials using a millet inoculation protocol. Of the 31 varieties screened, only Armor 48-D25 was classified as moderately resistant, and plant height to the first node influenced final disease severity. The study provides short-term solutions for growers to choose less susceptible varieties for planting and lays the foundation to characterize host resistance against this important soybean pathogen.

Mutations in a barley cytochrome P450 gene enhances pathogen induced programmed cell death and cutin layer instability.

Disease lesion mimic mutants (DLMMs) are characterized by the spontaneous development of necrotic spots with various phenotypes designated as necrotic (nec) mutants in barley. The nec mutants were traditionally considered to have aberrant regulation of programmed cell death (PCD) pathways, which have roles in plant immunity and development. Most barley nec3 mutants express cream to orange necrotic lesions contrasting them from typical spontaneous DLMMs that develop dark pigmented lesions indicative of serotonin/phenolics deposition. Barley nec3 mutants grown under sterile conditions did not exhibit necrotic phenotypes until inoculated with adapted pathogens, suggesting that they are not typical DLMMs. The F2 progeny of a cross between nec3-γ1 and variety Quest segregated as a single recessive susceptibility gene post-inoculation with Bipolaris sorokiniana, the causal agent of the disease spot blotch. Nec3 was genetically delimited to 0.14 cM representing 16.5 megabases of physical sequence containing 149 annotated high confidence genes. RNAseq and comparative analysis of the wild type and five independent nec3 mutants identified a single candidate cytochrome P450 gene (HORVU.MOREX.r2.6HG0460850) that was validated as nec3 by independent mutations that result in predicted nonfunctional proteins. Histology studies determined that nec3 mutants had an unstable cutin layer that disrupted normal Bipolaris sorokiniana germ tube development.

A High-Quality Genome Assembly for Cercospora janseana, Causal Agent of Narrow Brown Leaf Spot of Rice.

Cercospora janseana causes narrow brown leaf spot of rice. A nearly complete telomere-to-telomere reference genome was assembled with a combination of Oxford Nanopore and Illumina sequences. The genome assembly has a total length of 39,075,509 bp and consists of 15 chromosomes, 14 of which have telomeric repeats at both ends. The assembly N50 is 2.97 Mb and the L50 is five contigs. RNA-seq-mediated gene annotation identified 10,850 genes, including 955 predicted secreted proteins and 361 predicted effector proteins. This highly contiguous and almost complete C. janseana reference genome will be a vital resource for further investigation of host-pathogen interactions and genome evolution within this pathosystem. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Association mapping reveals a reciprocal virulence/avirulence locus within diverse US Pyrenophora teres f. maculata isolates.

BACKGROUND: Spot form net blotch (SFNB) caused by the necrotrophic fungal pathogen Pyrenophora teres f. maculata (Ptm) is an economically important disease of barley that also infects wheat. Using genetic analysis to characterize loci in Ptm genomes associated with virulence or avirulence is an important step to identify pathogen effectors that determine compatible (virulent) or incompatible (avirulent) interactions with cereal hosts. Association mapping (AM) is a powerful tool for detecting virulence loci utilizing phenotyping and genotyping data generated for natural populations of plant pathogenic fungi. RESULTS: Restriction-site associated DNA genotyping-by-sequencing (RAD-GBS) was used to generate 4,836 single nucleotide polymorphism (SNP) markers for a natural population of 103 Ptm isolates collected from Idaho, Montana and North Dakota. Association mapping analyses were performed utilizing the genotyping and infection type data generated for each isolate when challenged on barley seedlings of thirty SFNB differential barley lines. A total of 39 marker trait associations (MTAs) were detected across the 20 barley lines corresponding to 30 quantitative trait loci (QTL); 26 novel QTL and four that were previously mapped in Ptm biparental populations. These results using diverse US isolates and barley lines showed numerous barley-Ptm genetic interactions with seven of the 30 Ptm virulence/avirulence loci falling on chromosome 3, suggesting that it is a reservoir of diverse virulence effectors. One of the loci exhibited reciprocal virulence/avirulence with one haplotype predominantly present in isolates collected from Idaho increasing virulence on barley line MXB468 and the alternative haplotype predominantly present in isolates collected from North Dakota and Montana increasing virulence on barley line CI9819. CONCLUSIONS: Association mapping provided novel insight into the host pathogen genetic interactions occurring in the barley-Ptm pathosystem. The analysis suggests that chromosome 3 of Ptm serves as an effector reservoir in concordance with previous reports for Pyrenophora teres f. teres, the causal agent of the closely related disease net form net blotch. Additionally, these analyses identified the first reported case of a reciprocal pathogen virulence locus. However, further investigation of the pathosystem is required to determine if multiple genes or alleles of the same gene are responsible for this genetic phenomenon.

A triple threat: the Parastagonospora nodorum SnTox267 effector exploits three distinct host genetic factors to cause disease in wheat.

Parastagonospora nodorum is a fungal pathogen of wheat. As a necrotrophic specialist, it deploys effector proteins that target dominant host susceptibility genes to elicit programmed cell death (PCD). Here we identify and functionally validate the effector targeting the host susceptibility genes Snn2, Snn6 and Snn7. We utilized whole-genome sequencing, association mapping, gene-disrupted mutants, gain-of-function transformants, virulence assays, bioinformatics and quantitative PCR to characterize these interactions. A single proteinaceous effector, SnTox267, targeted Snn2, Snn6 and Snn7 to trigger PCD. Snn2 and Snn6 functioned cooperatively to trigger PCD in a light-dependent pathway, whereas Snn7-mediated PCD functioned in a light-independent pathway. Isolates harboring 20 SnTox267 protein isoforms quantitatively varied in virulence. The diversity and distribution of isoforms varied between populations, indicating adaptation to local selection pressures. SnTox267 deletion resulted in the upregulation of effector genes SnToxA, SnTox1 and SnTox3. We validated a novel effector operating in an inverse-gene-for-gene manner to target three genetically distinct host susceptibility genes and elicit PCD. The discovery of the complementary gene action of Snn2 and Snn6 indicates their potential function in a guard or decoy model. Additionally, differences in light dependency in the elicited pathways and upregulation of unlinked effectors sheds new light onto a complex fungal necrotroph-host interaction.

The Parastagonospora nodorum necrotrophic effector SnTox5 targets the wheat gene Snn5 and facilitates entry into the leaf mesophyll.

Parastagonospora nodorum is an economically important necrotrophic fungal pathogen of wheat. Parastagonospora nodorum secretes necrotrophic effectors that target wheat susceptibility genes to induce programmed cell death (PCD). In this study, we cloned and functionally validated SnTox5 and characterized its role in pathogenesis. We used whole genome sequencing, genome-wide association study (GWAS) mapping, CRISPR-Cas9-based gene disruption, gain-of-function transformation, quantitative trait locus (QTL) analysis, haplotype and isoform analysis, protein modeling, quantitative PCR, and laser confocal microscopy to validate SnTox5 and functionally characterize SnTox5. SnTox5 is a mature 16.26 kDa protein with high structural similarity to SnTox3. Wild-type and mutant P. nodorum strains and wheat genotypes of SnTox5 and Snn5, respectively, were used to show that SnTox5 not only targets Snn5 to induce PCD but also facilitates the colonization of the mesophyll layer even in the absence of Snn5. Here we show that SnTox5 facilitates the efficient colonization of the mesophyll tissue and elicits PCD specific to host lines carrying Snn5. The homology to SnTox3 and the ability of SnTox5 to facilitate the colonizing of the mesophyll also suggest a role in the suppression of host defense before PCD induction.

Local adaptation drives the diversification of effectors in the fungal wheat pathogen Parastagonospora nodorum in the United States.

Filamentous fungi rapidly evolve in response to environmental selection pressures in part due to their genomic plasticity. Parastagonospora nodorum, a fungal pathogen of wheat and causal agent of septoria nodorum blotch, responds to selection pressure exerted by its host, influencing the gain, loss, or functional diversification of virulence determinants, known as effector genes. Whole genome resequencing of 197 P. nodorum isolates collected from spring, durum, and winter wheat production regions of the United States enabled the examination of effector diversity and genomic regions under selection specific to geographically discrete populations. 1,026,859 SNPs/InDels were used to identify novel loci, as well as SnToxA and SnTox3 as factors in disease. Genes displaying presence/absence variation, predicted effector genes, and genes localized on an accessory chromosome had significantly higher pN/pS ratios, indicating a higher rate of sequence evolution. Population structure analyses indicated two P. nodorum populations corresponding to the Upper Midwest (Population 1) and Southern/Eastern United States (Population 2). Prevalence of SnToxA varied greatly between the two populations which correlated with presence of the host sensitivity gene Tsn1 in the most prevalent cultivars in the corresponding regions. Additionally, 12 and 5 candidate effector genes were observed to be under diversifying selection among isolates from Population 1 and 2, respectively, but under purifying selection or neutrally evolving in the opposite population. Selective sweep analysis revealed 10 and 19 regions that had recently undergone positive selection in Population 1 and 2, respectively, involving 92 genes in total. When comparing genes with and without presence/absence variation, those genes exhibiting this variation were significantly closer to transposable elements. Taken together, these results indicate that P. nodorum is rapidly adapting to distinct selection pressures unique to spring and winter wheat production regions by rapid adaptive evolution and various routes of genomic diversification, potentially facilitated through transposable element activity.

Identification and mapping of a novel resistance gene to the rice pathogen, Cercospora janseana.

KEY MESSAGE: The genetic architecture of resistance to Cercospora janseana was examined, and a single resistance locus was identified. A SNP marker was identified and validated for utilization in U.S. breeding germplasm Cercospora janseana (Racib.) is a fungal pathogen that causes narrow brown leaf spot (NBLS) in rice. Although NBLS is a major disease in the southern United States and variation in resistance among U.S. rice germplasm exists, little is known about the genetic architecture underlying the trait. In this study, a recombinant inbred line population was evaluated for NBLS resistance under natural disease infestation in the field across three years. A single, large-effect QTL, CRSP-2.1, was identified that explained 81.4% of the phenotypic variation. The QTL was defined to a 532 kb physical interval and 13 single nucleotide polymorphisms (SNPs) were identified across the region to characterize the haplotype diversity present in U.S. rice germplasm. A panel of 387 U.S. rice germplasm was genotyped with the 13 haplotype SNPs and phenotyped over two years for NBLS resistance. Fourteen haplotypes were identified, with six haplotypes accounting for 94% of the panel. The susceptible haplotype from the RIL population was the only susceptible haplotype observed in the U.S. germplasm. A single SNP was identified that distinguished the susceptible haplotype from all resistant haplotypes, explaining 52.7% of the phenotypic variation for NBLS resistance. Pedigree analysis and haplotype characterization of historical germplasm demonstrated that the susceptible haplotype was introduced into Southern U.S. germplasm through the California line L-202 into the Louisiana variety Cypress. Cypress was extensively used as a parent over the last 25 years, resulting in the susceptible CRSP-2.1 allele increasing in frequency from zero to 44% in the modern U.S. germplasm panel.

Botrytis spp.: A Contemporary Perspective and Synthesis of Recent Scientific Developments of a Widespread Genus that Threatens Global Food Security.

This perspective presents a synopsis of the topics contained in the Phytopathology Pathogen Spotlight on Botrytis spp. causing gray mold, including pathogen biology and systematics, genomic characterization of new species, perspectives on genome editing, and fungicide resistance. A timely breakthrough to engineer host plant resistance against the gray mold fungus has been demonstrated in planta and may augment chemical controls in the near future. While B. cinerea has garnered much of the research attention, other economically important Botrytis spp. have been identified and characterized via morphological and genome-based approaches. Gray mold control is achieved primarily through fungicide applications but resistance to various chemical classes is a major concern that threatens global plant health and food security. In this issue, new information on molecular mechanism(s) of fungicide resistance and ways to manage control failures are presented. Finally, a significant leap in fundamental pathogen biology has been achieved via development of CRISPR/Cas9 to assess gene function in the fungus which likely will spawn new control mechanisms and facilitate gene discovery studies.

A Comparative Genomic Analysis of the Barley Pathogen Pyrenophora teres f. teres Identifies Subtelomeric Regions as Drivers of Virulence.

Pyrenophora teres f. teres causes net form net blotch of barley and is an economically important pathogen throughout the world. However, P. teres f. teres is lacking in the genomic resources necessary to characterize the mechanisms of virulence. Recently a high-quality reference genome was generated for P. teres f. teres isolate 0-1. Here, we present the reference quality sequence and annotation of four new isolates and we use the five available P. teres f. teres genomes for an in-depth comparison, resulting in the generation of hypotheses pertaining to the potential mechanisms and evolution of virulence. Comparative analyses were performed between all five P. teres f. teres genomes, examining genomic organization, structural variations, and core and accessory genomic content, specifically focusing on the genomic characterization of known virulence loci and the localization of genes predicted to encode secreted and effector proteins. We showed that 14 of 15 currently published virulence quantitative trait loci (QTL) span accessory genomic regions, consistent with these accessory regions being important drivers of host adaptation. Additionally, these accessory genomic regions were frequently found in subtelomeric regions of chromosomes, with 10 of the 14 accessory region QTL localizing to subtelomeric regions. Comparative analysis of the subtelomeric regions of P. teres f. teres chromosomes revealed translocation events in which homology was detected between nonhomologous chromosomes at a significantly higher rate than the rest of the genome. These results indicate that the subtelomeric accessory genomic compartments not only harbor most of the known virulence loci but, also, that these regions have the capacity to rapidly evolve.

Genetics of Variable Disease Expression Conferred by Inverse Gene-For-Gene Interactions in the Wheat-Parastagonospora nodorum Pathosystem.

The wheat-Parastagonospora nodorum pathosystem involves the recognition of pathogen-secreted necrotrophic effectors (NEs) by corresponding wheat NE sensitivity genes. This inverse gene-for-gene recognition leads to necrotrophic effector-triggered susceptibility and ultimately septoria nodorum blotch disease. Here, we used multiple pathogen isolates to individually evaluate the effects of the host gene-NE interactions Tan spot necrosis1-Stagonospora nodorum ToxinA (Tsn1-SnToxA), Stagonospora nodorum necrosis1-Stagonospora nodorum Toxin1 (Snn1-SnTox1), and Stagonospora nodorum necrosis3-B genome homeolog1-Stagonospora nodorum Toxin3 (Snn3-B1-SnTox3), alone and in various combinations, to determine the relative importance of these interactions in causing disease. Genetic analysis of a recombinant inbred wheat population inoculated separately with three P. nodorum isolates, all of which produce all three NEs, indicated that the Tsn1-SnToxA and Snn3-B1-SnTox3 interactions contributed to disease caused by all four isolates, but their effects varied and ranged from epistatic to additive. The Snn1-SnTox1 interaction was associated with increased disease for one isolate, but for other isolates, there was evidence that this interaction inhibited the expression of other host gene-NE interactions. RNA sequencing analysis in planta showed that SnTox1 was differentially expressed between these three isolates after infection. Further analysis of NE gene-knockout isolates showed that the effect of some interactions could be masked or inhibited by other compatible interactions, and the regulation of this occurs at the level of NE gene transcription. Collectively, these results show that the inverse gene-for-gene interactions leading to necrotrophic effector-triggered susceptibility in the wheat-P. nodorum pathosystem vary in their effects depending on the genetic backgrounds of the pathogen and host, and interplay among the interactions is complex and intricately regulated.

Genotyping-by-Sequencing for the Study of Genetic Diversity in Puccinia triticina.

Leaf rust, caused by Puccinia triticina Erikss., is globally the most widespread rust of wheat. Populations of P. triticina are highly diverse for virulence, with many different races found annually. The genetic diversity of P. triticina populations has been previously assessed using different types of DNA markers. Genotyping technologies that provide a higher density of markers distributed across the genome will be more powerful for analysis of genetic and phylogenetic relationships in P. triticina populations. In this study, we utilized restriction-associated DNA (RAD) genotyping-by-sequencing (GBS) adapted for the Ion Torrent sequencing platform for the study of population diversity in P. triticina. A collection of 102 isolates, collected mainly from tetraploid and hexaploid wheat, was used. The virulence phenotypes of the isolates were determined on 20 lines of Thatcher wheat near isogenic for leaf rust resistance genes. Seven races were found among 57 isolates collected from tetraploid wheat, and 21 races were observed among 40 hexaploid wheat type isolates. This is the first study to report durum wheat virulent races to Lr3bg in Tunisia, Lr14a in Morocco, and Lr3bg and Lr28 in Mexico. Ethiopian isolates with high virulence to durum wheat but avirulent on Thatcher (hexaploid wheat) were tested for virulence on a set of durum (tetraploid) differentials. A subset of 30 isolates representing most of the virulence phenotypes in the 102 isolates were genotyped using RAD-GBS. Phylogenetic analysis of 30 isolates using 2,125 single nucleotide polymorphism (SNP) markers showed nine distinct clusters. There was a general correlation between virulence phenotypes and SNP genotypes. The high bootstrap values between clusters of isolates in the phylogenetic tree indicated that RAD-GBS can be used as a new genotyping tool that is fast, simple, high throughput, cost effective, and provides a sufficient number of markers for the study of genetic diversity in P. triticina.[Formula: see text] Copyright © 2020 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Transcriptome-wide association study identifies putative elicitors/suppressor of Puccinia graminis f. sp. tritici that modulate barley rpg4-mediated stem rust resistance.

BACKGROUND: Stem rust is an economically important disease of wheat and barley. However, studies to gain insight into the molecular basis of these host-pathogen interactions have primarily focused on wheat because of its importance in human sustenance. This is the first extensive study utilizing a transcriptome-wide association mapping approach to identify candidate Puccinia graminis f. sp. tritici (Pgt) effectors/suppressors that elicit or suppress barley stem rust resistance genes. Here we focus on identifying Pgt elicitors that interact with the rpg4-mediated resistance locus (RMRL), the only effective source of Pgt race TTKSK resistance in barley. RESULTS: Thirty-seven Pgt isolates showing differential responses on RMRL were genotyped using Restriction Site Associated DNA-Genotyping by Sequencing (RAD-GBS), identifying 24 diverse isolates that were used for transcript analysis during the infection process. In planta RNAseq was conducted with the 24 diverse isolates on the susceptible barley variety Harrington, 5 days post inoculation. The transcripts were mapped to the Pgt race SCCL reference genome identifying 114 K variants in predicted genes that would result in nonsynonymous amino acid substitutions. Transcriptome wide association analysis identified 33 variants across 28 genes that were associated with dominant RMRL virulence, thus, representing candidate suppressors of resistance. Comparative transcriptomics between the 9 RMRL virulent -vs- the 15 RMRL avirulent Pgt isolates identified 44 differentially expressed genes encoding candidate secreted effector proteins (CSEPs), among which 38 were expressed at lower levels in virulent isolates suggesting that they may represent RMRL avirulence genes. Barley transcript analysis after colonization with 9 RMRL virulent and 15 RMRL avirulent isolates inoculated on the susceptible line Harrington showed significantly lower expression of host biotic stress responses specific to RMRL virulent isolates suggesting virulent isolates harbor effectors that suppress resistance responses. CONCLUSIONS: This transcriptomic study provided novel findings that help fill knowledge gaps in the understanding of stem rust virulence/avirulence and host resistance in barley. The pathogen transcriptome analysis suggested RMRL virulence might depend on the lack of avirulence genes, but evidence from pathogen association mapping analysis and host transcriptional analysis also suggested the alternate hypothesis that RMRL virulence may be due to the presence of suppressors of defense responses.

Characterization of genes required for both Rpg1 and rpg4-mediated wheat stem rust resistance in barley.

BACKGROUND: Puccinia graminis f. sp. tritici (Pgt) race TTKSK and its lineage pose a threat to barley production world-wide justifying the extensive efforts to identify, clone, and characterize the rpg4-mediated resistance locus (RMRL), the only effective resistance to virulent Pgt races in the TTKSK lineage. The RMRL contains two nucleotide-binding domain and leucine-rich repeat (NLR) resistance genes, Rpg5 and HvRga1, which are required for resistance. The two NLRs have head-to-head genome architecture with one NLR, Rpg5, containing an integrated C-terminal protein kinase domain, characteristic of an "integrated sensory domain" resistance mechanism. Fast neutron mutagenesis of line Q21861 was utilized in a forward genetics approach to identify genetic components that function in the RMRL or Rpg1 resistance mechanisms, as Q21861 contains both genes. A mutant was identified that compromises both RMRL and Rpg1-mediated resistances and had stunted seedling roots, designated required for P. graminis resistance 9 (rpr9). RESULTS: The rpr9 mutant generated in the Q21861 background was crossed with the Swiss landrace Hv584, which carries RMRL but contains polymorphism across the genome compared to Q21861. To map Rpr9, a Hv584 x rpr9 F6:7 recombinant inbred line (RIL) population was developed. The RIL population was phenotyped with Pgt race QCCJB. The Hv584 x rpr9 RIL population was genotyped with the 9 k Illumina Infinium iSelect marker panel, producing 2701 polymorphic markers. A robust genetic map consisting of 563 noncosegregating markers was generated and used to map Rpr9 to an ~ 3.4 cM region on barley chromosome 3H. The NimbleGen barley exome capture array was utilized to capture rpr9 and wild type Q21861 exons, followed by Illumina sequencing. Comparative analysis, resulting in the identification of a 1.05 Mbp deletion at the chromosome 3H rpr9 locus. The identified deletion contains ten high confidence annotated genes with the best rpr9 candidates encoding a SKP1-like 9 protein and a F-box family protein. CONCLUSION: Genetic mapping and exome capture rapidly identified candidate gene/s that function in RMRL and Rpg1 mediated resistance pathway/s. One or more of the identified candidate rpr9 genes are essential in the only two known effective stem rust resistance mechanisms, present in domesticated barley.

Mapping of barley susceptibility/resistance QTL against spot form net blotch caused by Pyrenophora teres f. maculata using RIL populations.

Spot form net blotch (SFNB) caused by the necrotrophic fungal pathogen Pyrenophora teres f. maculata (Ptm) is an important disease of barley worldwide including the major barley production regions of North America. To characterize SFNB resistance/susceptibility quantitative trait loci (QTL), three recombinant inbred line (RIL) populations were developed from crosses between the malting barley cultivars, Tradition (six row) and Pinnacle (two row), and the two world barley core collection lines, PI67381 and PI84314. Tradition and Pinnacle were susceptible to many North American Ptm isolates, while PI67381 and PI84314 carry resistances to diverse Ptm isolates from across the globe. The RIL populations, Tradition/PI67381, Pinnacle/PI67381, and Pinnacle/PI84314 were genotyped using polymerase chain reaction-mediated genotype-by-sequencing single nucleotide polymorphism marker panels and phenotyped at the seedling stage with six geographically distinct Ptm isolates: FGOB10Ptm-1 (North Dakota, USA), Pin-A14 (Montana, USA), Cel-A17 (Montana, USA), SG1 (Australia), NZKF2 (New Zealand) and DEN2.6 (Denmark). The goal was to determine if the susceptible elite lines contained common susceptibility genes/QTL or if the resistant lines had common resistant genes/QTL effective against diverse Ptm isolates. The QTL analyses identified a total of 12 resistance and/or susceptibility loci on chromosomes 2H, 3H, 4H, 6H, and 7H of which three had not been previously reported. Common major QTL were detected on chromosome 2H (R2 = 14-40%) and 7H (R2 = 24-80%) in all three RIL populations, suggesting underlying genes with broad resistance specificity. The major 7H QTL was shown to be a dominant susceptibility gene in both susceptible malting barley varieties.

Genetic analysis of virulence in the Pyrenophora teres f. teres population BB25×FGOH04Ptt-21.

Pyrenophora teres f. teres is the causal agent of net form net blotch (NFNB) of barley. In order to map the genetics of avirulence/virulence in P. teres f. teres, a fungal population was developed using P. teres f. teres isolates BB25 (Denmark) and FGOH04Ptt-21 (North Dakota, USA) due to these two isolates differing in virulence on several common barley lines. 109 progeny isolates were obtained from the BB25 by FGOH04Ptt-21 cross that were then used for NFNB disease evaluation across eight barley lines, four of which have been used commonly as NFNB differential lines as well as four cultivars commonly used in barley production in the Northern Great Plains. BB25 was virulent on one of the barley lines and avirulent on seven of the barley lines whereas, FGOH04Ptt-21 was virulent on all eight barley lines evaluated. Genetic maps were generated with single nucleotide polymorphism (SNP) markers obtained using a restriction associated DNA genotyping by sequencing (RAD-GBS) approach. Sixteen linkage groups were formed and were used to identify quantitative trait loci (QTL) associated with avirulence/virulence. Nine unique QTL were identified on eight linkage groups out of which three QTL had major effects (R2≥45%) while the remaining six QTL were relatively minor (R2<20%). One or two major effect loci were identified for the lines commonly used as differentials. Conversely, variation in virulence on the local barley cultivars was mostly associated with small effect loci that contributed quantitatively to disease.

Association mapping utilizing diverse barley lines reveals net form net blotch seedling resistance/susceptibility loci.

KEY MESSAGE: A diverse collection of barley lines was phenotyped with three North American Pyrenophora teres f. teres isolates and association analyses detected 78 significant marker-trait associations at 16 genomic loci. Pyrenophora teres f. teres is a necrotrophic fungal pathogen and the causal agent of the economically important foliar disease net form net blotch (NFNB) of barley. The deployment of effective and durable resistance against P. teres f. teres has been hindered by the complexity of quantitative resistance and susceptibility. Several bi-parental mapping populations have been used to identify QTL associated with NFNB disease on all seven barley chromosomes. Here, we report the first genome-wide association study (GWAS) to detect marker-trait associations for resistance or susceptibility to P. teres f. teres. Geographically diverse barley genotypes from a world barley core collection (957) were genotyped with the Illumina barley iSelect chip and phenotyped with three P. teres f. teres isolates collected in two geographical regions of the USA (15A, 6A and LDNH04Ptt19). The best of nine regression models tested were identified for each isolate and used for association analysis resulting in the identification of 78 significant marker-trait associations (MTA; -log10p value >3.0). The MTA identified corresponded to 16 unique genomic loci as determined by analysis of local linkage disequilibrium between markers that did not meet a correlation threshold of R 2 ≥ 0.1, indicating that the markers represented distinct loci. Five loci identified represent novel QTL and were designated QRptts-3HL, QRptts-4HS, QRptts-5HL.1, QRptts-5HL.2, and QRptts-7HL.1. In addition, 55 of the barley lines examined exhibited a high level of resistance to all three isolates and the SNP markers identified will provide useful genetic resources for barley breeding programs.