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.

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.

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.

Identification of Fungal Species Associated with Crown and Root Rots of Wheat and Evaluation of Plant Reactions to the Pathogens in North Dakota.

Common root rot (CRR) and crown rot (CR), caused by Bipolaris sorokiniana and Fusarium species, respectively, can cause significant yield losses in cereal crops. To assess the prevalence, incidence, and severity of these diseases in North Dakota, wheat samples were collected from spring wheat fields across the state in 2012, 2013, and 2014. Based on subcrown internode symptoms, a greater incidence and severity of CRR was observed in 2012 (warm and dry year) than in 2013 and 2014. Also, the Northwestern Glaciated Plains and Northwestern Great Plains ecoregions showed greater CRR incidence and severity compared with the Northern Glaciated Plains and Lake Agassiz Plains ecoregions in the state. B. sorokiniana and Fusarium species, including Fusarium acuminatum, Fusarium avenaceum, Fusarium culmorum, Fusarium graminearum, Fusarium equiseti, Fusarium pseudograminearum, Fusarium oxysporum, Fusarium redolens, Fusarium sporotrichioides, and Fusarium solani were isolated and identified from the root and crown tissues of the wheat samples. B. sorokiniana was isolated more frequently than other fungal species in all sampled years and ecoregions of North Dakota. F. acuminatum, F. avenaceum, F. culmorum, F. equiseti, F. graminearum, F. pseudograminearum, and F. redolens were pathogenic causing infections on seedlings of the two wheat genotypes (ND652 and Alsen), whereas isolates of F. oxysporum and F. solani were nonpathogenic and considered as secondary invaders associated with the root and CR diseases. Evaluation of some spring wheat genotypes for reactions to one B. sorokiniana isolate at seedling and adult plant stages, and one F. culmorum isolate at the seedling stage, indicated that susceptibility to these pathogens varied among different wheat genotypes tested. This study provides useful information on fungal species associated with CRR and CR of wheat in North Dakota and on resistant/susceptible reactions of some spring wheat lines to the different fungal isolates evaluated.

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

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

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.

Microscopic, Biochemical, and Molecular Comparisons of Moderately Resistant and Susceptible Populus Genotypes Inoculated with Sphaerulina musiva.

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.

Characterization of recombinants of the Aegilops peregrina-derived Lr59 translocation of common wheat.

KEY MESSAGE: A compensating, recombined Lr59 translocation with greatly reduced alien chromatin was identified. Microsatellite locus Xdupw217 occurs within the remaining segment and can be used as a co-dominant marker for Lr59. In earlier studies, leaf rust (caused by Puccinia triticina Eriks.) resistance gene Lr59 was transferred from Aegilops peregrina (Hackel) Maire et Weiler to chromosome arm 1AL of common wheat (Triticum aestivum L.). The resistance gene was then genetically mapped on the translocated chromosome segment following homoeologous pairing induction. Eight recombinants that retained the least alien chromatin apparently resulted from crossover within a terminal region of the translocation that was structurally different from 1AL. These recombinants could not be differentiated by size, and it was not clear whether they were compensating in nature. The present study determined that the distal part of the original translocation has group 6 chromosome homoeology and a 6BS telomere (with the constitution of the full translocation chromosome being 1AS·1L(P)·6S(P) ·6BS). During the allosyndetic pairing induction experiment to map and shorten the full size translocation, a low frequency of quadrivalents involving 1A, the 1A translocation, and two 6B chromosomes was likely formed. Crossover within such quadrivalents apparently produced comparatively small compensating alien chromatin inserts within the 6BS satellite region on chromosome 6B of seven of the eight recombinants. It appears that the Gli-B2 storage protein locus on 6BS has not been affected by the recombination events, and the translocations are therefore not expected to affect baking quality. Simple sequence repeat marker results showed that Lr59-151 is the shortest recombinant, and it will therefore be used in breeding. Marker DUPW217 detects a homoeo-allele within the remaining alien chromatin that can be used for marker-assisted selection of Lr59.

The Tarnished Legacy of a Wonder Drug: Revisiting the Complicated History of Clozapine.

Anecdotal evidence of superior efficacy and lack of extrapyramidal symptoms in treating schizophrenia made clozapine a promising therapy in the United States during the early 1970s. In 1975, however, numerous fatal cases of clozapine-related agranulocytosis in Finland nearly ended the drug's development. Convinced of the significant benefits to patients, some clinicians in the United States advocated having clozapine available on a case-by-case humanitarian basis, which eventually helped resurrect the drug for Food and Drug Administration approval in 1989. This article builds on previous literature by utilizing oral histories from clinicians, researchers, and a patient's family member to understand how clozapine was saved. Exploring these stakeholders' perspectives has value to modern clinicians, who underprescribe the drug despite demonstrable benefits for treatment-resistant schizophrenia and suicide prevention.

Population and genome-wide association studies of Sclerotinia sclerotiorum isolates collected from diverse host plants throughout the United States.

Introduction: Sclerotinia sclerotiorum is a necrotrophic fungal pathogen causing disease and economic loss on numerous crop plants. This fungus has a broad host range and can infect over 400 plant species, including important oilseed crops such as soybean, canola, and sunflower. S. sclerotiorum isolates vary in aggressiveness of lesion formation on plant tissues. However, the genetic basis for this variation remains to be determined. The aims of this study were to evaluate a diverse collection of S. sclerotiorum isolates collected from numerous hosts and U.S. states for aggressiveness of stem lesion formation on sunflower, to evaluate the population characteristics, and to identify loci associated with isolate aggressiveness using genome-wide association mapping. Methods: A total of 219 S. sclerotiorum isolates were evaluated for stem lesion formation on two sunflower inbred lines and genotyped using genotyping-by-sequencing. DNA markers were used to assess population differentiation across hosts, regions, and climatic conditions and to perform a genome-wide association study of isolate aggressiveness. Results and discussion: We observed a broad range of aggressiveness for lesion formation on sunflower stems, and only a moderate correlation between aggressiveness on the two lines. Population genetic evaluations revealed differentiation between populations from warmer climate regions compared to cooler regions. Finally, a genome-wide association study of isolate aggressiveness identified three loci significantly associated with aggressiveness on sunflower. Functional characterization of candidate genes at these loci will likely improve our understanding of the virulence strategies used by this pathogen to cause disease on a wide array of agriculturally important host plants.

A Type II hybrid effectiveness-implementation study of an integrated CHW intervention to address maternal healthcare in rural Nepal.

Skilled care during pregnancy, childbirth, and postpartum is essential to prevent adverse maternal health outcomes, yet utilization of care remains low in many resource-limited countries, including Nepal. Community health workers (CHWs) can mitigate health system challenges and geographical barriers to achieving universal health coverage. Gaps remain, however, in understanding whether evidence-based interventions delivered by CHWs, closely aligned with WHO recommendations, are effective in Nepal's context. We conducted a type II hybrid effectiveness-implementation, mixed-methods study in two rural districts in Nepal to evaluate the effectiveness and the implementation of an evidence-based integrated maternal and child health intervention delivered by CHWs, using a mobile application. The intervention was implemented stepwise over four years (2014-2018), with 65 CHWs enrolling 30,785 families. We performed a mixed-effects Poisson regression to assess institutional birth rate (IBR) pre-and post-intervention. We used the Reach, Effectiveness, Adoption, Implementation, and Maintenance framework to evaluate the implementation during and after the study completion. There was an average 30% increase in IBR post-intervention, adjusting for confounding variables (p<0.0001). Study enrollment showed 35% of families identified as dalit, janjati, or other castes. About 78-89% of postpartum women received at least one CHW-counseled home visit within 60 days of childbirth. Ten (53% of planned) municipalities adopted the intervention during the study period. Implementation fidelity, measured by median counseled home visits, improved with intervention time. The intervention was institutionalized beyond the study period and expanded to four additional hubs, albeit with adjustments in management and supervision. Mechanisms of intervention impact include increased knowledge, timely referrals, and longitudinal CHW interaction. Full-time, supervised, and trained CHWs delivering evidence-based integrated care appears to be effective in improving maternal healthcare in rural Nepal. This study contributes to the growing body of evidence on the role of community health workers in achieving universal health coverage.

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.

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.

RNAseq Analysis of Rhizomania-Infected Sugar Beet Provides the First Genome Sequence of Beet Necrotic Yellow Vein Virus from the USA and Identifies a Novel Alphanecrovirus and Putative Satellite Viruses.

"Rhizomania" of sugar beet is a soilborne disease complex comprised of beet necrotic yellow vein virus (BNYVV) and its plasmodiophorid vector, Polymyxa betae. Although BNYVV is considered the causal agent of rhizomania, additional viruses frequently accompany BNYVV in diseased roots. In an effort to better understand the virus cohort present in sugar beet roots exhibiting rhizomania disease symptoms, five independent RNA samples prepared from diseased beet seedlings reared in a greenhouse or from field-grown adult sugar beet plants and enriched for virus particles were subjected to RNAseq. In all but a healthy control sample, the technique was successful at identifying BNYVV and provided sequence reads of sufficient quantity and overlap to assemble > 98% of the published genome of the virus. Utilizing the derived consensus sequence of BNYVV, infectious RNA was produced from cDNA clones of RNAs 1 and 2. The approach also enabled the detection of beet soilborne mosaic virus (BSBMV), beet soilborne virus (BSBV), beet black scorch virus (BBSV), and beet virus Q (BVQ), with near-complete genome assembly afforded to BSBMV and BBSV. In one field sample, a novel virus sequence of 3682 nt was assembled with significant sequence similarity and open reading frame (ORF) organization to members within the subgenus Alphanecrovirus (genus Necrovirus; family Tombusviridae). Construction of a DNA clone based on this sequence led to the production of the novel RNA genome in vitro that was capable of inducing local lesion formation on leaves of Chenopodium quinoa. Additionally, two previously unreported satellite viruses were revealed in the study; one possessing weak similarity to satellite maize white line mosaic virus and a second possessing moderate similarity to satellite tobacco necrosis virus C. Taken together, the approach provides an efficient pipeline to characterize variation in the BNYVV genome and to document the presence of other viruses potentially associated with disease severity or the ability to overcome resistance genes used for sugar beet rhizomania disease management.

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.

Pyramiding rpg4- and Rpg1-Mediated Stem Rust Resistance in Barley Requires the Rrr1 Gene for Both to Function.

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.

Identification and Validation of a New Source of Low Grain Cadmium Accumulation in Durum Wheat.

Cadmium (Cd) is a heavy metal that has no known biological function and is toxic for many living organisms. The maximum level of Cd concentration allowed in the international market for wheat grain is 0.2 mg kg-1 Because phenotyping for Cd uptake is expensive and time consuming, molecular markers associated with genes conferring low Cd uptake would expedite selection and lead to the development of durum cultivars with reduced Cd concentrations. Here, we identified single nucleotide polymorphisms (SNPs) associated with a novel low Cd uptake locus in the durum experimental line D041735, which has hexaploid common wheat in its pedigree. Genetic analysis revealed a single major QTL for Cd uptake on chromosome arm 5BL within a 0.3 cM interval flanked by SNP markers. Analysis of the intervening sequence revealed a gene with homology to an aluminum-induced protein as a candidate gene. Validation and allelism tests revealed that the low Cd uptake gene identified in this study is different from the closely linked Cdu1-B gene, which also resides on 5BL. This study therefore showed that the durum experimental line D041735 contains a novel low Cd uptake gene that was likely acquired from hexaploid wheat.