High resolution mapping of a novel non-transgressive hybrid susceptibility locus in barley exploited by P. teres f. maculata.

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

Variability in Chromosome 1 of select Moroccan P. teres f. teres isolates enables isolates to overcome 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.

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.

Wild barley (Hordeum spontaneum) and landraces (Hordeum vulgare) from Turkey contain an abundance of novel Rhynchosporium commune resistance loci.

KEY MESSAGE: Rhynchosporium commune is a globally devastating pathogen of barley. Wild and landrace barley are underutilized, however, contain an abundance of loci that can be used as potential sources of resistance. Rhynchosporium commune, the causal agent of the disease scald or leaf blotch of barley, is a hemibiotrophic fungal pathogen of global importance, responsible for yield losses ranging from 30 to 40% on susceptible varieties. To date, over 150 resistance loci have been characterized in barley. However, due to the suspected location of the R. commune host jump in Europe, European germplasm has been the primary source used to screen for R. commune resistance leaving wild (Hordeum spontaneum) and landrace (H. vulgare) barley populations from the center of origin largely underutilized. A diverse population consisting of 94 wild and 188 barley landraces from Turkey were genotyped using PCR-GBS amplicon sequencing and screened with six Turkish R. commune isolates. The isolates were collected from distinct geographic regions of Turkey with two from the Aegean region, two from central Turkey and two from the Fertile Crescent region. The data set was utilized for association mapping analysis with a total of 21 loci identified, of which 12 were novel, indicating that these diverse primary barley gene pools contain an abundance of novel R. commune resistances that could be utilized for resistance breeding.

Host and pathogen genetics reveal an inverse gene-for-gene association in the P. teres f. maculata-barley pathosystem.

KEY MESSAGE: Pathogen and host genetics were used to uncover an inverse gene-for-gene interaction where virulence genes from the pathogen Pyrenophora teres f. maculata target barley susceptibility genes, resulting in disease. Although models have been proposed to broadly explain how plants and pathogens interact and coevolve, each interaction evolves independently, resulting in various scenarios of host manipulation and plant defense. Spot form net blotch is a foliar disease of barley caused by Pyrenophora teres f. maculata. We developed a barley population (Hockett × PI 67381) segregating for resistance to a diverse set of P. teres f. maculata isolates. Quantitative trait locus analysis identified major loci on barley chromosomes (Chr) 2H and 7H associated with resistance/susceptibility. Subsequently, we used avirulent and virulent P. teres f. maculata isolates to develop a pathogen population, identifying two major virulence loci located on Chr1 and Chr2. To further characterize this host-pathogen interaction, progeny from the pathogen population harboring virulence alleles at either the Chr1 or Chr2 locus was phenotyped on the Hockett × PI 67381 population. Progeny harboring only the Chr1 virulence allele lost the barley Chr7H association but maintained the 2H association. Conversely, isolates harboring only the Chr2 virulence allele lost the barley Chr2H association but maintained the 7H association. Hockett × PI 67381 F2 individuals showed susceptible/resistant ratios not significantly different than 15:1 and results from F2 inoculations using the single virulence genotypes were not significantly different from a 3:1 (S:R) ratio, indicating two dominant susceptibility genes. Collectively, this work shows that P. teres f. maculata virulence alleles at the Chr1 and Chr2 loci are targeting the barley 2H and 7H susceptibility alleles in an inverse gene-for-gene manner to facilitate colonization.

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.

Genome-wide association mapping of Pyrenophora teres f. maculata and Pyrenophora teres f. teres resistance loci utilizing natural Turkish wild and landrace barley populations.

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.

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.

Genetic mapping of host resistance to the Pyrenophora teres f. maculata isolate 13IM8.3.

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.

Expansion of Internal Hyphal Growth in Fusarium Head Blight-Infected Grains Contributes to the Elevated Mycotoxin Production During the Malting Process.

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.

Visualization of spatial gene expression in plants by modified RNAscope fluorescent in situ hybridization.

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.

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.

The Barley HvWRKY6 Transcription Factor Is Required for Resistance Against Pyrenophora teres f. teres.

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.

Research advances in the Pyrenophora teres-barley interaction.

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.

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.

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.

Shedding Light on Penetration of Cereal Host Stomata by Wheat Stem Rust Using Improved Methodology.

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.

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.

Association mapping, transcriptomics, and transient expression identify candidate genes mediating plant-pathogen interactions in a tree.

Invasive microbes causing diseases such as sudden oak death negatively affect ecosystems and economies around the world. The deployment of resistant genotypes for combating introduced diseases typically relies on breeding programs that can take decades to complete. To demonstrate how this process can be accelerated, we employed a genome-wide association mapping of ca 1,000 resequenced Populus trichocarpa trees individually challenged with Sphaerulina musiva, an invasive fungal pathogen. Among significant associations, three loci associated with resistance were identified and predicted to encode one putative membrane-bound L-type receptor-like kinase and two receptor-like proteins. A susceptibility-associated locus was predicted to encode a putative G-type D-mannose-binding receptor-like kinase. Multiple lines of evidence, including allele analysis, transcriptomics, binding assays, and overexpression, support the hypothesized function of these candidate genes in the P. trichocarpa response to S. musiva.