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Spring barley production and quality in the United States is severely impacted by Fusarium head blight (FHB), primarily caused by Fusarium graminearum. Management of FHB is best accomplished using an integrated approach including resistant varieties and fungicides. Field experiments were established from 2014 to 2019 to quantify the effects of host resistance, fungicide, and fungicide timing on management of FHB and deoxynivalenol (DON) and yield protection in spring barley. Three separate research objectives were investigated using multiyear and multilocation experiments. These included investigating the efficacy of prothioconazole + tebuconazole applied at either Feekes growth stage (FGS) 10.5 (full head) or 3 to 7 days after FGS 10.5; the efficacy of sequential fungicide applications occurring at both FGS 10.5 and 3 to 7 days after FGS 10.5 using prothioconazole + tebuconazole, metconazole, prothioconazole, or tebuconazole; and the efficacy and timing of pydiflumetofen + propiconazole. Pooled analysis results suggest that metconazole, prothioconazole + tebuconazole, and pydiflumetofen + propiconazole are all effective fungicides for reducing DON, reducing FHB, and protecting yield. The use of sequential fungicide applications with one occurring at FGS 10.5 and another at 3 to 7 days after FGS 10.5 provided greater suppression than a single application at FGS 10.5. The greatest DON reduction from pydiflumetofen + propiconazole occurred when applied at full head or 3 to 7 days after full head. Across all research objectives, the most effective single fungicide application timing to reduce FHB, reduce DON, and protect yield in barley is 3 to 7 days after FGS 10.5.

Plant breeding aims to develop elite crop varieties appropriate for various environments with higher quality and quantity of production. Researchers use quantitative trait loci (QTL) mapping and association studies to identify regions in the genome responsible for the variation of the quantitative traits of interest. However, mapped regions do not always translate to functional proteins, which makes it challenging to identify genes associated with traits of interest. The biological functions of proteins are strongly dependent on their 3D structure. Alternatively, if proteins can be directly linked with the phenotypes, the effect of mutations on phenotypic changes can be assessed. Innovation of deep learning models in biology opens new avenues of exploration. AlphaFold is an AI system that predicts the 3D structure of a protein from its amino acid sequence with near experimental accuracy and was used in this study. Point mutations with a significant influence on the 3D structure of a protein can capture the effect on phenotypes through association study, and this provides insights into the regions that are of functional importance. In the current study, 534 plants were selected based on plant vigor, and 154 missense variants that change amino acid sequences, including 5 significant hits from previous study, were included. The changes in protein 3D structure were assessed by association with the phenotype. The analysis identified five significant associations, four of which were also identified in previous study of SNPs GWAS, however, a new fifth association was also identified which was annotated as disease resistance gene in Medicago truncatula. This study helps to associate SNPs that could be missed by GWAS due to stringent Bonferroni corrected p-values by providing a more robust filter for SNPs using features from predicted protein 3D structures.

Alfalfa primarily produces seeds through cross-pollination among different individuals. Self-pollination results in severe inbreeding depression, such as weak seedlings and termination of growth. Identifying the genetic loci associated with vigorous plant growth could enable the elimination of deleterious alleles and eventually develop inbred alfalfa lines for hybrid production. In this study, 215 alfalfa accessions were self-pollinated for three generations. Within accessions, pairs of weak and strong plants were sampled and bulked for exome sequencing. We extracted individual DNA from 534 plants that included parental clones, strong and weak pairs of plants, and plants selected based on the number of seeds produced. Among them, we formed 42 pools, including 16 with strong plants and 17 with weak plants, 3 top-seeded plants, 3 low-seeded plants, and 3 no-seeded plants. Along with 79 individuals, these pools were sequenced in the target regions covering 112,626 contigs across the entire alfalfa genome. From the 121 samples, (42+79) genotyped, 13.2 million SNPs including indels were generated. After filtering for MAF (>5%), depth (

A diverse sexual population of wheat stem rust, Puccinia graminis f. sp. tritici, exists in the Pacific Northwest region of the United States because of the natural presence of Mahonia spp. that serves as alternate hosts to complete its sexual life cycle. The region appears to be a center of stem rust diversity in North America where novel virulence gene combinations can emerge that could overcome deployed barley and wheat stem rust resistances. A total of 100 single pustule isolates derived from stem rust samples collected from barley in Eastern Washington during the 2019 growing season were assayed for virulence on the two known effective barley stem rust resistance genes/loci, Rpg1 and the rpg4/5-mediated resistance locus (RMRL) at the seedling stage. Interestingly, 99% of the P. graminis f. sp. tritici isolates assayed were virulent on barley variety Morex carrying the Rpg1 gene, and 62% of the isolates were virulent on the variety Golden Promise transformant (H228.2c) that carries a single-copy insertion of the Rpg1 gene from Morex and is more resistant than Morex to many Rpg1 avirulent isolates. Also, 16% of the isolates were virulent on the near isogenic line HQ-1, which carries the RMRL introgression from the barley line Q21861 in the susceptible Harrington background. Alarmingly, 10% of the isolates were virulent on barley line Q21861, which contains both Rpg1 and RMRL. Thus, we report on the first P. graminis f. sp. tritici isolates worldwide with virulence on both Rpg1 and RMRL when stacked together, representing the most virulent P. graminis f. sp. tritici isolates reported on barley.

Stripe rust ​​(caused by Puccinia striiformis f. sp. tritici) is one of the most devastating diseases of wheat and causes large-scale epidemics and severe yield loss. Applying fungicides during early epidemic development is crucial to controlling the disease but is often challenged by resource-limited human visual scouting. Deep learning has the potential to process images and videos captured from affordable devices to empower high-throughput phenotyping for early detection of stripe rust for timely application of fungicides and improve control efficiency. Here, we developed RustNet, a neural network-based image classifier, for efficiently monitoring fields for stripe rust. RustNet was built on a ResNet-18 architecture pre-trained with ImageNet Large-Scale Visual Recognition Challenge (ILSVRC) dataset using transfer learning. RGB images and videos of multiple wheat fields with different wheat types (winter and spring wheat), conditions (irrigated and non-irrigated), and locations were acquired using smartphones or unmanned aerial vehicles near the canopy. A semi-automated image labeling approach was conducted to improve labeling efficiency by combining automated machine labeling and human correction. Cross-validations across multiple categories (sensor platforms, wheat types, and locations) achieved Area Under Curve from 0.72 to 0.87. Independent validation on a published dataset from Germany achieved accuracies ranging from 0.79 to 0.86. The visualization of the last convolutional layer of RustNet demonstrated the identification of pixels with stripe rust. RustNet is freely available at https://zzlab.net/RustNet.

BackgroundIn plants, the plasma membrane is enclosed by the cell wall and anchors RLK and RLP proteins, which play a fundamental role in perception of developmental and environmental cues and are crucial in plant development and immunity. These plasma membrane receptors belong to large gene/protein families that are not easily classified computationally. This detailed analysis of these plasma membrane proteins brings a new source of information to the legume genetic, physiology and breeding research communities.ResultsA computational approach to identify and classify RLK and RLP proteins is presented. The strategy was evaluated using experimentally-validated RLK and RLP proteins and was determined to have a sensitivity of over 0.85, a specificity of 1.00, and a Matthews correlation coefficient of 0.91. The computational approach can be used to develop a detailed catalog of plasma membrane receptors (by type and domains) in several legume/crop species. The exclusive domains identified in legumes for RLKs are WaaY, APH Pkinase_C, LRR_2, and EGF, and for RLP are L-lectin LPRY and PAN_4. The RLK-nonRD and RLCK subclasses are also discovered by the methodology. In both classes, less than 20% of the total RLK predicted for each species belong to this class. Among the 10-species evaluated ~ 40% of the proteins in the kinome are RLKs. The exclusive legume domain combinations identified are B-Lectin/PR5K domains inG. max,M. truncatula,V. angularis, andV. unguiculataand a three-domain combination B-lectin/S-locus/WAK inC. cajan,M. truncatula,P. vulgaris,V. angularis. andV. unguiculata.ConclusionsThe analysis suggests that about 2% of the proteins of each genome belong to the RLK family and less than 1% belong to RLP family. Domain diversity combinations are greater for RLKs compared with the RLP proteins and LRR domains, and the dual domain combination LRR/Malectin were the most frequent domain for both groups of plasma membrane receptors among legume and non-legume species. Legumes exclusively show Pkinase extracellular domains, and atypical domain combinations in RLK and RLP compared with the non-legumes evaluated. The computational logic approach is statistically well supported and can be used with the proteomes of other plant species.

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 .

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.

Summary Pyrenophora teres f. teres and P. teres f. maculata are significant pathogens that cause net blotch of barley. An increased number of loci involved in P. teres resistance or susceptibility responses of barley as well as interacting P. teres virulence effector loci have recently been identified through biparental and association mapping studies of both the pathogen and host. Characterization of the resistance/susceptibility loci in the host and the interacting effector loci in the pathogen will provide a path for targeted gene validation for better‐informed release of resistant barley cultivars. This review assembles concise consensus maps for all loci published for both the host and pathogen, providing a useful resource for the community to be used in pathogen characterization and barley breeding for resistance to both forms of P. teres., Pyrenophora teres causes net blotch of barley. Here we review the latest progress on host resistance and pathogen virulence, providing a valuable resource for the barley net blotch research community.

BackgroundStem 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 candidatePuccinia graminisf. sp.tritici(Pgt) effectors/suppressors that elicit or suppress barley stem rust resistance genes. Here we focus on identifyingPgtelicitors that interact with therpg4-mediated resistance locus (RMRL), the only effective source ofPgtrace TTKSK resistance in barley.ResultsThirty-sevenPgtisolates 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 plantaRNAseq was conducted with the 24 diverse isolates on the susceptible barley variety Harrington, 5 days post inoculation. The transcripts were mapped to thePgtrace 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 avirulentPgtisolates 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.ConclusionsThis 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.

Sphaerulina musiva, the causal agent of Septoria leaf spot and stem canker, is responsible for mortality and yield loss in Populus plantations. However, little is known about the mode of infection and the mechanisms of resistance in this pathosystem. To characterize these phenomena, microscopic, biochemical, and transcriptome comparisons were performed between leaves of moderately resistant and susceptible genotypes of Populus inoculated with S. musiva conidia. Using scanning electron, cryofracture, and laser-scanning confocal microscopy, the infection and colonization of Populus leaves by S. musiva were examined across five time points (48 h, 96 h, 1 week, 2 weeks, and 3 weeks). The infection process was similar regardless of the host genotype. Differences in host colonization between susceptible and moderately resistant genotypes were apparent by 1 week postinoculation. However, the germination of conidia was greater on the susceptible than on the moderately resistant genotype (P < 0.008). Diaminobenzidine staining, a measure of hydrogen peroxide accumulation, was different (P < 0.001) between the host genotypes by 2 weeks postinoculation. Transcriptome differences between genotypes indicated that the speed and amplitude of the defense response were faster and more extensive in the moderately resistant genotype. Changes in gene expression support the microscopic and biochemical observations.

Spot form of net blotch (SFNB), caused by the necrotrophic fungus Pyrenophora teres f. maculata (Ptm), is considered one of the emerging and devastating foliar diseases of barley. Deployment of host resistance against this pathogen remains one of the most effective strategies for its sustainable management. The SFNB resistance in a panel of 336 diverse barley genotypes (AM-2014) was evaluated against three isolates (FGO-Ptm [USA], SM4-2 [Morocco], and SM30-1 [Morocco]), at the seedling stage and under natural conditions in seven environments in Morocco at the adult-plant stage. We identified nine barley genotypes that confer high SFNB resistance in at least five distinct environments and against two isolates. The AM-2014 panel was genotyped with 9K single nucleotide polymorphism (SNP) using an Illumina Infinium select assay. Genome-wide association studies (GWAS) were carried out using mixed linear models accounting for population structure as covariates. Significant marker-trait associations were subject to false discovery rate (FDR) at qQRpts4, QRpt6, QRpt7, and SFNB-3H-58.64, while the remaining may represent novel loci. Together, the associated SNPs and resistance sources identified in this study will be useful resources for SFNB resistance breeding.

A diverse sexual population of wheat stem rust

Spot form net blotch (SFNB), caused by the necrotrophic fungal pathogen Pyrenophora teres f. maculata (Ptm), is a foliar disease of barley that results in significant yield losses in major growing regions worldwide. Understanding the host-parasite interactions between pathogen virulence/avirulence genes and the corresponding host susceptibility/resistance genes is important for the deployment of genetic resistance against SFNB. Two recombinant inbred mapping populations were developed to characterize genetic resistance/susceptibility to the Ptm isolate 13IM8.3, which was collected from Idaho (ID). An Illumina Infinium array was used to produce a genome-wide marker set. Quantitative trait loci (QTL) analysis identified ten significant resistance/susceptibility loci, with two of the QTL being common to both populations. One of the QTL on 5H appears to be novel, while the remaining loci have been reported previously. Single nucleotide polymorphisms (SNPs) closely linked to or delimiting the significant QTL have been converted to user-friendly markers. Loci and associated molecular markers identified in this study will be useful in genetic mapping and deployment of the genetic resistance to SFNB in barley.

Unimproved landraces and wild relatives of crops are sources of genetic diversity that were lost post domestication in modern breeding programs. To tap into this rich resource, genome-wide association studies in large plant genomes have enabled the rapid genetic characterization of desired traits from natural landrace and wild populations. Wild barley (Hordeum spontaneum), the progenitor of domesticated barley (Hordeum vulgare), is dispersed across Asia and North Africa, and has co-evolved with the ascomycetous fungal pathogens Pyrenophora teres f. teres and P. teres f. maculata, the causal agents of the diseases net form of net blotch and spot form of net blotch, respectively. Thus, these wild and local adapted barley landraces from the region of origin of both the host and pathogen represent a diverse gene pool to identify new sources of resistance, due to millions of years of co-evolution. The barley—P. teres pathosystem is governed by complex genetic interactions with dominant, recessive, and incomplete resistances and susceptibilities, with many isolate-specific interactions. Here, we provide the first genome-wide association study of wild and landrace barley from the Fertile Crescent for resistance to both forms of P. teres. A total of 14 loci, four against P. teres f. maculata and 10 against P. teres f. teres, were identified in both wild and landrace populations, showing that both are genetic reservoirs for novel sources of resistance. We also highlight the importance of using multiple algorithms to both identify and validate additional loci.

Unimproved landraces and wild relatives of crops are sources of genetic diversity that were lost post domestication in modern breeding programs. To tap into this rich resource, genome wide association studies in large plant genomes have enabled the rapid genetic characterization of desired traits from natural landrace and wild populations. Wild barley (Hordeum spontaneum), the progenitor of domesticated barley (H. vulgare), is dispersed across Asia and North Africa, and has co-evolved with the ascomycetous fungal pathogensPyrenophora teresf.teresandP. teresf.maculata, the casual agents of the diseases net form of net blotch and spot form of net blotch, respectively. Thus, these wild and local adapted barley landraces from the region of origin of both the host and pathogen represent a diverse gene pool to identify new sources of resistance, due to millions of years of co-evolution. The barley -P. terespathosystem is governed by complex genetic interactions with dominant, recessive, and incomplete resistances and susceptibilities, with many isolate-specific interactions. Here we provide the first genome wide association study of wild and landrace barley from the Fertile Crescent for resistance to both forms ofP. teres. A total of 14 loci, four againstP. teresf.maculataand ten againstPyrenophora teresf.teres,were identified in both wild and landrace populations, showing that both are genetic reservoirs for novel sources of resistance. We also highlight the importance of using multiple algorithms to both identify and validate additional loci.

Seed vigour is considered a critical stage for barley production, and cultivars with early seedling vigour (ESV) facilitate rapid canopy formation. In this study, QTLs for 12 ESV-related traits were mapped using 185 RILs derived from a Xena x H94061120 evaluated across six independent environments. DArT markers were used to develop a genetic map (1075.1 cM, centimorgans) with an average adjacent-marker distance of 3.28 cM. In total, 46 significant QTLs for ESV-related traits were detected. Fourteen QTLs for biomass yield were found on all chromosomes, two of them co-localized with QTLs on 1H for grain yield. The related traits: length of the first and second leaves and dry weight of the second leaf, biomass yield and grain yield, had high heritability (&gt, 30%). Meanwhile, a significant correlation was observed between grain yield and biomass yield, which provided a clear image of these traits in the selection process. Our results demonstrate that a pleiotropic QTL related to the specific leaf area of the second leaf, biomass yield, and grain yield was linked to the DArT markers bPb-9280 and bPb-9108 on 1H, which could be used to significantly improve seed vigour by marker-assisted selection and facilitate future map-based cloning efforts.

We report genomic regions that significantly control resistance to scald, net form (NFNB) and spot form net blotch (SFNB) in barley. Barley genotypes from Ethiopia, ICARDA, and the United States were evaluated in Ethiopia and North Dakota State University (NDSU). Genome-wide association studies (GWAS) were conducted using 23,549 single nucleotide polymorphism (SNP) markers for disease resistance in five environments in Ethiopia. For NFNB and SFNB, we assessed seedling resistance in a glasshouse at NDSU. A large proportion of the Ethiopian landraces and breeding genotypes were resistant to scald and NFNB. Most of genotypes resistant to SFNB were from NDSU. We identified 17, 26, 7, and 1 marker-trait associations (MTAs) for field-scored scald, field-scored net blotch, greenhouse-scored NFNB, and greenhouse-scored SFNB diseases, respectively. Using the genome sequence and the existing literature, we compared the MTAs with previously reported loci and genes for these diseases. For leaf scald, only a few of our MTAs overlap with previous reports. However, the MTAs found for field-scored net blotch as well as NFNB and SFNB mostly overlap with previous reports. We scanned the barley genome for identification of candidate genes within 250 kb of the MTAs, resulting in the identification of 307 barley genes for the 51 MTAs. Some of these genes are related to plant defense responses such as subtilisin-like protease, chalcone synthase, lipoxygenase, and defensin-like proteins.

Asexual urediniospore infection of primary cereal hosts by Puccinia graminis f. sp. tritici (Pgt), the wheat stem rust pathogen, was considered biphasic. The first phase, spore germination and appressoria formation, requires a dark period and moisture. The second phase, host entry by the penetration peg originating from the appressoria formed over the guard cells, was thought to require light to induce natural stomata opening. Previous studies concluded that inhibition of colonization by the dark was due to lack of penetration through closed stomata. A sensitive WGA-Alexa Fluor 488 fungal staining, surface creation and biovolume analysis method was developed enabling visualization and quantification of fungal growth in planta at early infection stages surpassing visualization barriers using previous methods. The improved method was used to investigate infection processes of Pgt during stomata penetration and colonization in barley and wheat showing that penetration is light independent. Based on the visual growth and fungal biovolume analysis it was concluded that the differences in pathogen growth dynamics in both resistant and susceptible genotypes was due to light induced pathogen growth after penetration into the substomatal space. Thus, light induced plant or pathogen cues triggers pathogen growth in-planta post penetration.

Five microsatellite (SSR) markers were used to evaluate the genetic diversity of six Aegilops species from Azerbaijan and Georgia. A total of 39 alleles were generated with an average of 7.8 alleles per primer. Twenty markers were species-specific and 6 were accession-specific. The transferability of SSR markers across six species was 100%, with exception of gwm210. The mean polymorphism information content (PIC) and expected heterozygosity (He) values for the entire collection were 0.688 and 0.725, respectively. The average PIC value was the highest in Ae. biuncialis accessions (0.55). The genetic distance (GD) indices, based on five SSR markers, ranged from 0 to 0.83, with a mean value of 0.47. The highest genetic similarity was noted between Ae. neglecta and Ae. triuncialis (GD = 0.26), and the lowest between Ae. neglecta and Ae. tauschii (GD = 0.66). The dendrogram created based on SSR data grouped 72 Aegilops accessions into six clusters according to their taxonomic classification. The accessions from the same province were often placed in the same subclusters, indicating that grouping based on genetic parameters was closely related to the geographic region within countries. The PCoA analysis could differentiate Aegilops accessions according to their species and confirmed subgrouping obtained by cluster analysis.

Fusarium head blight (FHB) and the occurrence of mycotoxins is the largest food safety threat to malting and brewing grains. Worldwide surveys of commercial beers have reported that the trichothecene mycotoxin deoxynivalenol (DON) is the most frequent contaminant in beer. Although the DON content of grain generally declines during steeping due to its solubilization, Fusarium spp. can continue to grow and produce DON from steeping through the early kilning stage of malting. DON present on malt is largely extracted into beer. The objective of the current study was to localize the growth of Fusarium spp. within FHB-infected kernels by developing an improved method and to associate fungal growth with the production of DON during malting. FHB-infected barley, wheat, rye, and triticale grains that exhibited large increases in the amount of Fusarium Tri5 DNA and trichothecene mycotoxins following malting were screened for hyphal localization. The growth of fungal hyphae associated with grain and malt was imaged by scanning electron microscopy and confocal laser-scanning microscopy assisted with WGA-Alexa Fluor 488 staining, respectively. In barley, hyphae were present on or within the husk, vascular bundle, and pericarp cavities. Following malting, vast hyphal growth was observed not only in these regions but also in the aleurone layer, endosperm, and embryo. Extensive fungal growth was also observed following malting of wheat, rye, and triticale. However, these grains already had an extensive internal presence of Fusarium hyphae in the unmalted grain, thus representing an enhanced chance of fungal expansion during the malting. [Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY 4.0 International license .

Disease lesion mimic mutants (DLMMs) are characterized by 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. Also, 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 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 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.AUTHOR SUMMARYAt the site of pathogen infection, plant defense mechanisms rely on controlled programmed cell death (PCD) to sequester biotrophic pathogens that require living cells to extract nutrient from the host. However, these defense mechanisms are hijacked by necrotrophic plant pathogens that purposefully induce PCD mechanism to feed from the dead cells facilitating further disease development. Thus, understanding PCD responses is important for resistance to both classes of pathogens. We characterized five independent disease lesion mimic mutants of barley designated necrotic 3 (nec3) that show aberrant regulation of PCD responses upon pathogen challenge. A cytochrome P450 gene was identified as Nec3 encoding a Tryptamine 5-Hydroxylase that functions as a terminal serotonin biosynthetic enzyme in the Tryptophan pathway of plants. The nec3 mutants have disrupted serotonin biosynthesis resulting in expansive PCD, necrotrophic pathogen susceptibility and cutin layer instability. The nec3 mutants lacking serotonin deposition in pathogen induced necrotic lesions show expansive PCD and disease susceptibility suggesting a role of serotonin to sequester PCD and suppress pathogen colonization. The identification of Nec3 will facilitate functional analysis to elucidate the role serotonin plays in the elicitation or suppression of PCD immunity responses to diverse pathogens and effects it has on cutin layer biosynthesis.

Septoria nodorum blotch (SNB), a disease caused by the necrotrophic fungal pathogen Parastagonospora nodorum, is a threat to wheat (Triticum aestivum) production worldwide. Multiple inverse gene-for-gene interactions involving the recognition of necrotrophic effectors (NEs) by wheat sensitivity genes play major roles in causing SNB. One interaction involves the wheat gene Snn3 and the P. nodorum NE SnTox3. Here, we used a map-based strategy to clone the Snn3-D1 gene from Aegilops tauschii, the D-genome progenitor of common wheat. Snn3-D1 contained protein kinase and major sperm protein domains, both of which were essential for function as confirmed by mutagenesis. As opposed to other characterized interactions in this pathosystem, a compatible Snn3-D1-SnTox3 interaction was light-independent, and Snn3-D1 transcriptional expression was downregulated by light and upregulated by darkness. Snn3-D1 likely emerged in Ae. tauschii due to an approximately 218-kb insertion that occurred along the west bank of the Caspian Sea. The identification of this new class of NE sensitivity genes combined with the previously cloned sensitivity genes demonstrates that P. nodorum can take advantage of diverse host targets to trigger SNB susceptibility in wheat.

Barley is an important cereal crop worldwide because of its use in the brewing and distilling industry. However, adequate supplies of quality malting barley are threatened by global climate change due to drought in some regions and excess precipitation in others, which facilitates epidemics caused by fungal pathogens. The disease net form net blotch caused by the necrotrophic fungal pathogen Pyrenophora teres f. teres (Ptt) has emerged as a global threat to barley production and diverse populations of Ptt have shown a capacity to overcome deployed genetic resistances. The barley line CI5791 exhibits remarkably effective resistance to diverse Ptt isolates from around the world that maps to two major QTL on chromosomes 3H and 6H. To identify genes involved in this effective resistance, CI5791 seed were γ-irradiated and two mutants, designated CI5791-γ3 and CI5791-γ8, with compromised Ptt resistance were identified from an M2 population. Phenotyping of CI5791-γ3 and -γ8 × Heartland F2 populations showed three resistant to one susceptible segregation ratios and CI5791-γ3 × -γ8 F1 individuals were susceptible, thus these independent mutants are in a single allelic gene. Thirty-four homozygous mutant (susceptible) CI5791-γ3 × Heartland F2 individuals, representing 68 recombinant gametes, were genotyped via PCR genotype by sequencing. The data were used for single marker regression mapping placing the mutation on chromosome 3H within an approximate 75 cM interval encompassing the 3H CI5791 resistance QTL. Sequencing of the mutants and wild-type (WT) CI5791 genomic DNA following exome capture identified independent mutations of the HvWRKY6 transcription factor located on chromosome 3H at ∼50.7 cM, within the genetically delimited region. Post transcriptional gene silencing of HvWRKY6 in barley line CI5791 resulted in Ptt susceptibility, confirming that it functions in NFNB resistance, validating it as the gene underlying the mutant phenotypes. Allele analysis and transcript regulation of HvWRKY6 from resistant and susceptible lines revealed sequence identity and upregulation upon pathogen challenge in all genotypes analyzed, suggesting a conserved transcription factor is involved in the defense against the necrotrophic pathogen. We hypothesize that HvWRKY6 functions as a conserved signaling component of defense mechanisms that restricts Ptt growth in barley.

Fusarium head blight (FHB) and the occurrence of mycotoxins is the largest food safety threat to malting and brewing grains. Objectives of the current study were to localize the growth of Fusarium within FHB infected kernels and to associate it with the production of DON that occurred during malting. FHB infected barley, wheat, rye, and triticale grains that exhibited large increases in Fusarium Tri5 DNA and trichothecene mycotoxins following malting, were screened for hyphal localization. The growth of hyphae, both on the surface of kernels and within tissues of grain and malt was, imagined by scanning electron microscopy and confocal laser scanning microscopy assisted with WGA-Alexa Fluor 488 pre-staining, respectively. In barley, hyphae were primarily present on or within husk, vascular bundle, and pericarp cavities. Following malting, large amounts of hyphal growth were observed in not only these regions, but also in the aleurone layer, endosperm, and embryo. Extensive fungal growth was also observed following malting of wheat, rye, and triticale. Interestingly, these grains already had an extensive internal presence of hyphae in unmalted grain, occurring in the pericarp, testa, vascular bundle, nucellar projection, aleurone layer, endosperm, pericarp and endosperm cavities, and embryo. Shotgun sequencing followed by metagenomics analysis verified that Fusarium spp. accounted for above 90% of the fungal hyphae growing in the interior of grains during malting, which coincided with the significant production of mycotoxins.

Background In situ analysis of biomarkers such as DNA, RNA and proteins are important for research and diagnostic purposes. At the RNA level, plant gene expression studies rely on qPCR, RNAseq and probe-based in situ hybridization (ISH). However, for ISH experiments poor stability of RNA and RNA based probes commonly results in poor detection or poor reproducibility. Recently, the development and availability of the RNAscope RNA-ISH method addressed these problems by novel signal amplification and background suppression. This method is capable of simultaneous detection of multiple target RNAs down to the single molecule level in individual cells, allowing researchers to study spatio-temporal patterning of gene expression. However, this method has not been optimized thus poorly utilized for plant specific gene expression studies which would allow for fluorescent multiplex detection. Here we provide a step-by-step method for sample collection and pretreatment optimization to perform the RNAscope assay in the leaf tissues of model monocot plant barley. We have shown the spatial distribution pattern of HvGAPDH and the low expressed disease resistance gene Rpg1 in leaf tissue sections of barley and discuss precautions that should be followed during image analysis. Results We have shown the ubiquitous HvGAPH and predominantly stomatal guard cell associated subsidiary cell expressed Rpg1 expression pattern in barley leaf sections and described the improve RNAscope methodology suitable for plant tissues using confocal laser microscope. By addressing the problems in the sample collection and incorporating additional sample backing steps we have significantly reduced the section detachment and experiment failure problems. Further, by reducing the time of protease treatment, we minimized the sample disintegration due to over digestion of barley tissues. Conclusions RNAscope multiplex fluorescent RNA-ISH detection is well described and adapted for animal tissue samples, however due to morphological and structural differences in the plant tissues the standard protocol is deficient and required optimization. Utilizing barley specific HvGAPDH and Rpg1 RNA probes we report an optimized method which can be used for RNAscope detection to determine the spatial expression and semi-quantification of target RNAs. This optimized method will be immensely useful in other plant species such as the widely utilized Arabidopsis.

Plant biotrophic pathogen disease resistances rely on immunity receptor-mediated programmed cell death (PCD) responses, but specialized necrotrophic/hemi-biotrophic pathogens hijack these mechanisms to colonize the resulting dead tissue in their necrotrophic phase. Thus, immunity receptors can become necrotrophic pathogen dominant susceptibility targets but resistance mechanisms that resist necrotroph manipulation are recessive resistance genes. The barley rcs5 QTL imparts recessive resistance against the disease spot blotch caused by the hemi-biotrophic fungal pathogen Bipolaris sorokiniana. The rcs5 genetic interval was delimited to ~0.23 cM, representing an ~234 kb genomic region containing four wall-associated kinase (WAK) genes, designated HvWak2, Sbs1, Sbs2 (susceptibility to Bipolaris sorokiniana1&2), and HvWak5. Post-transcriptional gene silencing of Sbs1&2 in susceptible barley cultivars resulted in resistance showing dominant susceptibility function. Allele analysis of Sbs1&2 from resistant and susceptible barley cultivars identified sequence polymorphisms associated with phenotypes in their primary coding sequence and promoter regions, suggesting differential transcriptional regulation may contribute to susceptibility. Transcript analysis of Sbs1&2 showed nearly undetectable expression in resistant and susceptible cultivars prior to pathogen challenge; however, upregulation of both genes occurred specifically in susceptible cultivars post-inoculation with a virulent isolate. Apoplastic wash fluids collected from barley infected with a virulent isolate induced Sbs1, suggesting regulation by an apoplastic-secreted effector. Thus, Sbs1&2 function as B. sorokiniana susceptibility targets and non-functional alleles or alleles that resist induction by the pathogen mediate rcs5-recessive resistance. The sbs1&2 alleles underlying the rcs5 QTL that the pathogen is unable to manipulate are the first resistance genes identified against spot blotch.SIGNIFICANCE STATEMENTThe rcs5 locus in barley confers a high level of seedling resistance and a moderate level of adult plant resistance to spot blotch. It is part of a complex that has provided durable spot blotch resistance in many North American barley cultivars (cv) for more than 50 years. Genetic characterization and positional cloning of rcs5 identified the dominant susceptibility genes, Sbs1 and Sbs2 (susceptibility to Bipolaris sorokiniana 1 and 2) as wall-associated kinases. These genes are hijacked by the hemibiotrophic pathogen in its necrotrophic phase to induce programmed cell death, facilitating disease development. We report the first spot blotch resistance/susceptibility genes cloned that function via alleles that cannot be specifically induced and hijacked by virulent isolates of the pathogen.

A barley core collection can be studied extensively and the derived information can be used to identify loci/genes for the genetic improvement of quantitative and qualitative traits. To assess genetic diversity, allelic variation and population structure of Egyptian barley, 134 barley genotypes collected from a different region along with 19 cultivated genotypes obtained from of the Egyptian Agricultural Research Center. All genotypes were analyzed with 261 polymorphic SSR and SNP alleles. The mean number of alleles per locus was 4 and PIC was 0.49, while the level of genetic diversity was 0.55 ranging from 0.03 to 0.82. The genotypes were assigned to three subpopulations that were consistent with their origins. The genetic variation within population was higher (51%) than among population at the molecular levels (FST = 0.491 when P

Genetic diversity of 139 accessions of diploid Triticum species including Triticum urartu, Triticum boeoticum and Triticum monococcum was studied using 11 SSR (simple sequence repeats) markers. A total of 111 alleles with an average of 10 alleles per locus were detected. The polymorphism information content (PIC) of each SSR marker ranged from 0.30 to 0.90 with an average value of 0.62. Among the three Triticum species T. urartu had the highest number of total alleles (Na = 81), private alleles (Npa = 15) and showed higher genetic diversity (Hex = 0.58; PIC = 0.54). The genotypes from Turkey exhibited the highest genetic diversity (PIC = 0.6), while the least diversity was observed among 4 Georgian accessions (PIC = 0.11). Cluster analysis was able to distinguish 139 wheat accessions at the species level. The highest genetic similarity (GS) was noted between T. boeticum and T. monococcum (GS = 0.84), and the lowest between T. urartu and T. monococcum (GS = 0.46). The grouping pattern of the PCoA analysis corresponded with cluster analysis. No significant differences were found in clustering of T. urartu and T. monococcum accessions with respect to their geographic regions, while within T. boeoticum species, accessions from Iran were somewhat associated with their geographical origin and clustered as a close and separate group. The results from our study demonstrated that SSR markers were good enough for further genetic diversity analysis in einkorn wheat species.

Pyrenophora teres f. teres, the causal agent of net form net blotch (NFNB) of barley, is a destructive pathogen in barley-growing regions throughout the world. Typical yield losses due to NFNB range from 10 to 40%; however, complete loss has been observed on highly susceptible barley lines where environmental conditions favor the pathogen. Currently, genomic resources for this economically important pathogen are limited to a fragmented draft genome assembly and annotation, with limited RNA support of the P. teres f. teres isolate 0-1. This research presents an updated 0-1 reference assembly facilitated by long-read sequencing and scaffolding with the assistance of genetic linkage maps. Additionally, genome annotation was mediated by RNAseq analysis using three infection time points and a pure culture sample, resulting in 11,541 high-confidence gene models. The 0-1 genome assembly and annotation presented here now contains the majority of the repetitive content of the genome. Analysis of the 0-1 genome revealed classic characteristics of a “two-speed” genome, being compartmentalized into GC-equilibrated and AT-rich compartments. The assembly of repetitive AT-rich regions will be important for future investigation of genes known as effectors, which often reside in close proximity to repetitive regions. These effectors are responsible for manipulation of the host defense during infection. This updated P. teres f. teres isolate 0-1 reference genome assembly and annotation provides a robust resource for the examination of the barley–P. teres f. teres host–pathogen coevolution.

Changes in the relative genetic performance of genotypes across environments are referred to as genotype × environment interactions (GEIs). GEIs can affect barley breeding improvement for salt tolerance because it often complicates the evaluation and selection of superior genotypes. The present study evaluated the GEIs over 60 barley genotypes for yield components and grain yield in six salinity environments in North Delta, Egypt. Data were analyzed using the additive main effects and multiplicative interaction (AMMI) and Tai’s stability parameters. GEIs effects on yield explained 20.3, 20.1, 14.6, and 33.0% of the total variation besides, the first two principal components account for 67.3, 56.3, 64.3, and 83.7% of the explained variance in the four sets, respectively. Six genotypes namely G-4, G-7, G-20, G-34, G-36, and G-39 were found to be most stable and high yielding across environments (GY >2.00 t ha-1), and located close to zero projection onto the AEC ordinate. Tai’s stability parameters demonstrated that these genotypes were more responsive to the environmental changes. The genotypes G-50 and G-53 showed perfect/static stability (α = -0.95, -0.91, respectively). In contrast, the genotype; G-36 had α = 0 and λ = 1.10, indicating parallel with the environmental effects followed by G-44. Overall, we found that GEIs for grain yield are highly significant in all sets, suggesting that responded differently across environments. This interaction may be a result of changes in genotypes’ relative performance across environments, due to their differential responses to various abiotic factors.

Pyrenophora teres f. maculata is the cause of the foliar disease spot form net blotch (SFNB) on barley. To evaluate pathogen genetics underlying the P. teres f. maculata–barley interaction, we developed a 105-progeny population by crossing two globally diverse isolates, one from North Dakota and the other from Western Australia. Progeny were phenotyped on a set of four barley genotypes showing a differential reaction to the parental isolates, then genotyped using a restriction site-associated-genotype-by-sequencing (RAD-GBS) approach. Genetic maps were developed for use in quantitative trait locus (QTL) analysis to identify virulence-associated QTL. Six QTL were identified on five different linkage groups and individually accounted for 20–37% of the disease variation, with the number of significant QTL ranging from two to four for the barley genotypes evaluated. The data presented demonstrate the complexity of virulence involved in the P. teres f. maculata–barley pathosystem and begins to lay the foundation for understanding this important interaction.

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 sub-telomeric regions of chromosomes with 10 of the 14 accessory region QTL localizing to sub-telomeric regions. Comparative analysis of the sub-telomeric regions of P. teres f. teres chromosomes revealed translocation events where homology was detected between non-homologous chromosomes at a significantly higher rate than the rest of the genome. These results indicate that the sub-telomeric accessory genomic compartments not only harbor most of the known virulence loci, but also that these regions have the capacity to rapidly evolve.

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. Electronic supplementary material The online version of this article (10.1186/s12864-019-5858-z) contains supplementary material, which is available to authorized users.

Stem rust, caused by Puccinia graminis f. sp. tritici (Pgt) is an economically important disease of wheat and barley. Rpg1 is the only resistance gene deployed in Midwestern US barley varieties and provides remarkable resistance to most North American races, except Pgt race QCCJB. Rpg1 is also ineffective against Pgt race TTKSK and its lineage that originated in Africa. The barley rpg4-mediated resistance locus (RMRL) conferring resistance to Pgt races QCCJB and TTKSK was isolated from line Q21861, which is resistant to all known Pgt races due to Rpg1 and RMRL. To develop elite barley varieties RMRL was pyramided into the varieties, Pinnacle and Conlon (both contain Rpg1), producing the near isogenic lines (NILs), Pinnacle RMRL-NIL (PRN) and Conlon RMRL-NIL (CRN). The CRN was resistant to Pgt races QCCJB (RMRL specific) and HKHJC (Rpg1 specific) at the seedling stage and Pgt race TTKSK (RMRL specific) at the adult stage. In contrast, PRN was susceptible to QCCJB and HKHJC at the seedling stage and TTKSK at the adult stage. Interestingly, PRN's susceptibility to QCCJB and HKHJC showed that RMRL was non-functional in the Pinnacle background but its presence also suppressed Rpg1-mediated resistance. Thus, in the absence of a gene/s found in the Q21861 background, Rpg1 becomes non-functional if RMRL is present, suggesting that another polymorphic gene, that we designated Rrr1 (required for rpg4-mediated resistance 1), is required for RMRL resistance and Rpg1-mediated resistance in the presence of RMRL. Utilizing a PRN/Q21861 derived recombinant inbred line (RIL) population, Rrr1 was delimited to a ∼0.5 MB physical region, slightly proximal (∼1.8 MB) of RMRL on barley chromosome 5H. A second gene, designated required for Rpg1-mediated resistance 2 (Rrr2), with duplicate gene action to Rrr1 in Rpg1-mediated resistance function, was genetically delimited to a physical region of ∼0.7 MB, slightly distal (∼3.1 MB) to Rpg1 on the short arm of barley chromosome 7H. Thus, Rrr1 is required for RMRL resistance and Rrr1 or Rrr2 is required for functional Rpg1-mediated resistance in the presence of the RMRL introgression. Candidate Rrr1 and Rrr2 genes were identified that need to be considered when pyramiding Rpg1 and RMRL in barley.

Parastagonospora nodorum is a necrotrophic fungal pathogen causing Septoria nodorum blotch on wheat. We have identified nine necrotrophic effector–host dominant sensitivity gene interactions, and we have cloned three of the necrotrophic effector genes, including SnToxA, SnTox1, and SnTox3. Because sexual populations of P. nodorum are difficult to develop under lab conditions, genome-wide association study (GWAS) is the best population genomic approach to identify genomic regions associated with traits using natural populations. In this article, we used a global collection of 191 P. nodorum isolates from which we identified 2,983 single-nucleotide polymorphism (SNP) markers and gene markers for SnToxA and SnTox3 to evaluate the power of GWAS on two popular wheat breeding lines that were sensitive to SnToxA and SnTox3. Strong marker trait associations (MTA) with P. nodorum virulence that mapped to SnTox3 and SnToxA were first identified using the marker set described above. A novel locus in the P. nodorum genome associated with virulence was also identified as a result of this analysis. To evaluate whether a sufficient level of marker saturation was available, we designed a set of primers every 1 kb in the genomic regions containing SnToxA and SnTox3. Polymerase chain reaction amplification was performed across the 191 isolates and the presence/absence polymorphism was scored and used as the genotype. The marker proximity necessary to identify MTA flanking SnToxA and SnTox3 ranged from 4 to 5 and 1 to 7 kb, respectively. Similar analysis was performed on the novel locus. Using a 45% missing data threshold, two more SNP were identified spanning a 4.6-kb genomic region at the novel locus. These results showed that the rate of linkage disequilibrium (LD) decay in P. nodorum and, likely, other fungi is high compared with plants and animals. The fast LD decay in P. nodorum is an advantage only if sufficient marker density is attained. Based on our results with the SnToxA and SnTox3 regions, markers are needed every 9 or 8 kb, respectively, or in every gene, to guarantee that genes associated with a quantitative trait such as virulence are not missed.