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A repeatable and reliable reverse transcription quantitative PCR (RT-qPCR) experiment depends upon proper reference genes (RGs) selection. This study aims to examine the expression stability of nine candidate RGs for the Avena sativa - Blumeria graminis experimental setup. B. graminis causes powdery mildew - the most devastating and economically important fungal disease of crops worldwide. RGs were evaluated in Pm3 and Pm4 oat differential lines and the susceptible cultivar Fuchs during compatible and incompatible interactions with different pathotypes of Blumeria graminis f. sp. avenae in six-time points post inoculation. The identification of genes exhibiting high expression stability was done by four algorithms (geNorm, NormFinder, BestKeeper and deltaCt). The results indicated that regardless of the analysed group, two most stable RGs are required for data normalization. The most sufficient RGs combination was HNR (heterogeneous nuclear ribonucleoprotein 27 C) + EIF4A (eukaryotic initiation factor 4 A‑3). ARF (ADP‑ribosylation factor) could also be pondered as demonstrating high expression stability. These genes can be considered universal candidates for RT-qPCR normalization to study interaction with B. graminis as well as Puccinia coronata and Puccinia graminis, as confirmed by our previous research. The worst candidate for data standardisation was TUA (α- tubulin). To our best knowledge, this is the first report regarding RGs' selection in this pathosystem. Identified RGs are proper normalisation candidates for gene expression studies in the A. sativa infected by B. graminis as well as other related pathogens., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

The population structure and evolution of basidiomycetes like rust fungi are influenced by complex reproductive cycles and dikaryotic life stages where two independent nuclear haplotypes are present in the cell. The ability to alternate between asexual (clonal) and sexual reproduction increases the evolutionary capacity in these species. Furthermore, exchange of intact nuclei (somatic hybridization) in rust fungi can allow for rapid generation of genetic variability outside of the sexual cycle. Puccinia coronata f. sp. avenae (Pca), the causal agent of oat crown rust, is a pathogen of global economic importance that is difficult to control due to rapid breakdown of host genetic resistance. The contribution of sexuality, clonality, and migration to virulence evolution varies across Pca populations. As such, the Pca pathosystem is ideal to address the role of mating type, recombination, mutation, and somatic hybridization in host adaptation. We expanded the existing resources for USA and South African populations by generating whole genome sequencing data of Taiwanese and Australian isolates. An atlas of 30 chromosome-level, fully-phased nuclear haplotypes from six USA isolates and nine Australian isolates was created to capture the genomic composition of key Pca lineages. At the haplotype level, we confirmed previous reports of genetic recombination in the USA population and additionally detected either sexual or cryptic recombination between Australian isolates, contrasting previous evaluations that suggested Pca populations in Australia to be purely clonal. We also identified somatic hybridization events in Pca that are not only associated with significant changes in fitness but also imply intercontinental migration of haplotypes, which provides further impetus for molecular monitoring of rust pathogen populations on a global scale., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: KL, HM, MS, and IL are employees of Phase Genomics, a company commercializing proximity ligation sequencing technology., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

The nutritional quality and yield of oats (Avena sativa) are often compromised by plant diseases such as red leaf, powdery mildew, and leaf spot. Sugars Will Eventually be Exported Transporters (SWEETs) are newly identified sugar transporters involved in regulating plant growth and stress responses. However, the roles of SWEET genes in biotic stress responses remain uncharacterized in oats. In this study, 13 AsSWEET genes were identified across nine chromosomes of the oat genome, all of which were predicted to contain seven transmembrane regions. Phylogenetic analysis revealed four clades of AsSWEET proteins, with high homology to SWEET proteins in the Poaceae family. Collinearity analysis demonstrated strong relationships between oat and Zea mays SWEETs. Using subcellular localization prediction tools, AsSWEET proteins were predicted to localize to the plasma membrane. Promoter analysis revealed cis-acting elements associated with light response, growth, and stress regulation. Six AsSWEET proteins were predicted to interact in a network centered on AsSWEET1a and AsSWEET11. Gene expression analysis of two oat varieties, 'ForagePlus' and 'Molasses', indicated significant expression differences in several AsSWEET genes following infection with powdery mildew or leaf spot, including AsSWEET1a, AsSWEET1b, AsSWEET2b, AsSWEET3a, AsSWEET11, and AsSWEET16. These SWEET genes are potential candidates for disease resistance in oats. This study provides a foundation for understanding the regulatory mechanisms of AsSWEET genes, particularly in response to powdery mildew and leaf spot, and offers insights for enhancing oat molecular breeding., (© 2024. The Author(s).)

Puccinia coronata f. sp. avenae is the causal agent of the disease known as crown rust, which represents a bottleneck in oat production worldwide. Characterization of pathogen populations often involves race (pathotype) assignments using differential sets, which are not uniform across countries. This study compared the virulence profiles of 25 P. coronata f. sp. avenae isolates from Australia using two host differential sets, one from Australia and one from the United States. These differential sets were also genotyped using diversity arrays technology sequencing technology. Phenotypic and genotypic discrepancies were detected on 8 out of 29 common lines between the two sets, indicating that pathogen race assignments based on those lines are not comparable. To further investigate molecular markers that could assist in the stacking of rust resistance genes important for Australia, four published Pc91 -linked markers were validated across the differential sets and then screened across a collection of 150 oat cultivars. Drover, Aladdin, and Volta were identified as putative carriers of the Pc91 locus. This is the first report to confirm that the cultivar Volta carries Pc91 and demonstrates the value of implementing molecular markers to characterize materials in breeding pools of oat. Overall, our findings highlight the necessity of examining seed stocks using pedigree and molecular markers to ensure seed uniformity and bring robustness to surveillance methodologies. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license., Competing Interests: The author(s) declare no conflict of interest.

Main Conclusion: A gene-to-metabolite approach afforded new insights regarding defence mechanisms in oat plants that can be incorporated into plant breeding programmes for the selection of markers and genes related to disease resistance. Monitoring metabolite levels and changes therein can complement and corroborate transcriptome (mRNA) data on plant-pathogen interactions, thus revealing mechanisms involved in pathogen attack and host defence. A multi-omics approach thus adds new layers of information such as identifying metabolites with antimicrobial properties, elucidating metabolomic profiles of infected and non-infected plants, and reveals pathogenic requirements for infection and colonisation. In this study, two oat cultivars (Dunnart and SWK001) were inoculated with Pseudomonas syringae pathovars, pathogenic and non-pathogenic on oat. Following inoculation, metabolites were extracted with methanol from leaf tissues at 2, 4 and 6 days post-infection and analysed by multiple reaction monitoring (MRM) on a triple quadrupole mass spectrometer system. Relatedly, mRNA was isolated at the same time points, and the cDNA analysed by quantitative PCR (RT-qPCR) for expression levels of selected gene transcripts associated with avenanthramide (Avn) biosynthesis. The targeted amino acids, hydroxycinnamic acids and Avns were successfully quantified. Distinct cultivar-specific differences in the metabolite responses were observed in response to pathogenic and non-pathogenic strains. Trends in aromatic amino acids and hydroxycinnamic acids seem to indicate stronger activation and flux through these pathways in Dunnart as compared to SWK001. A positive correlation between hydroxycinnamoyl-CoA:hydroxyanthranilate N-hydroxycinnamoyl transferase (HHT) gene expression and the abundance of Avn A in both cultivars was documented. However, transcript profiling of selected genes involved in Avn synthesis did not reveal a clear pattern to distinguish between the tolerant and susceptible cultivars., (© 2024. The Author(s).)

Food insecurity and malnutrition have reached critical levels with increased human population, climate fluctuations, water shortage; therefore, higher-yielding crops are in the spotlight of numerous studies. Abiotic factors affect the yield of staple food crops; among all, wheat stem sawfly ( Cephus cinctus Norton) and orange wheat blossom midge ( Sitodiplosis mosellana ) are two of the most economically and agronomically harmful insect pests which cause yield loss in cereals, especially in wheat in North America. There is no effective strategy for suppressing this pest damage yet, and only the plants with intrinsic tolerance mechanisms such as solid stem phenotypes for WSS and antixenosis and/or antibiosis mechanisms for OWBM can limit damage. A major QTL and a causal gene for WSS resistance were previously identified in wheat, and 3 major QTLs and a causal gene for OWBM resistance. Here, we present a comparative analysis of coding and non-coding features of these loci of wheat across important cereal crops, barley, rye, oat, and rice. This research paves the way for our cloning and editing of additional WSS and OWBM tolerance gene(s), proteins, and metabolites.

Key Message: Six new PCR-based markers for the Pc39 crown rust resistance gene in Avena sativa L. were developed. Pc39 was mapped to Mrg11 of the oat consensus map using BLASTn analysis. The aim of this study was the identification of molecular markers for the Pc39 gene in cultivated oat (Avena sativa L.). Pc39 is a major race-specific crown rust resistance gene originally found in an Israeli accession of the wild hexaploid Avena sterilis. The effectiveness of this gene in Europe has decreased in recent years, but is still relatively high and breeding programs would benefit from the availability of molecular markers to aid in its mapping and deployment. The complexity of the oat genome poses a significant obstacle to genetic research. No oat rust resistance genes have yet been cloned, and even the number of relevant molecular markers is very limited. Here, genotyping of a segregating population derived from a cross 'Celer' (Pc39)/STH9210 (susceptible) was conducted using RAPD- and SRAP-PCR-based methods, as well as microarray-based DArT™ and next-generation sequencing DArTseq™ techniques. Markers associated with Pc39 were placed on the hexaploid oat consensus linkage group Mrg11 at 3.7-6.7 cM. Six new PCR-based markers were developed to allow identification of the resistant Pc39 allele. These tightly linked markers will be useful in marker-assisted selection, with the closest, SCAR_3456624, being within 0.37 cM of Pc39. The newly developed markers could find applications in the fine mapping or positional cloning of this gene. Moreover, easy-to-use PCR-based markers linked to Pc39 could facilitate the utilization of this gene in oat breeding programs, especially as a component of crown rust resistance gene pyramids.

Key Message: SNP loci linked to the crown rust resistance gene Pc98 were identified by linkage analysis and KASP assays were developed for marker-assisted selection in breeding programs. Crown rust is among the most damaging diseases of oat and is caused by Puccinia coronata var. avenae f. sp. avenae (Urban and Marková) (Pca). Host resistance is the preferred method to prevent crown rust epidemics. Pc98 is a race-specific, seedling crown rust resistance gene obtained from the wild oat Avena sterilis accession CAV 1979 that is effective at all growth stages of oat. Virulence to Pc98 has been very low in the Pca populations that have been tested. The objectives of this study were to develop SNP markers linked to Pc98 for use in marker-assisted selection and to locate Pc98 on the oat consensus map. The Pc98 gene was mapped using F 2:3 populations developed from the crosses Pc98/Bingo and Pc98/Kasztan, where Pc98 is a single-gene line carrying Pc98. Both populations were evaluated in seedling inoculation experiments. Pc98 was mapped relative to Kompetitive Allele-Specific PCR SNP markers in both populations, placing Pc98 on the Mrg20 linkage group of the consensus map. Pc98 was bracketed by two SNP markers GMI_ES22_c3052_382_kom399 and GMI_ES14_lrc18344_662_kom398 in the Pc98/Bingo mapping population with genetic distances of 0.9 cM and 0.3 cM, respectively. Pc98 co-segregated with four SNP markers in the Pc98/Kasztan population, and the closest flanking markers were GMI_DS_LB_6017_kom367 and avgbs2_153634.1.59_kom410 with genetic distances of 0.7 cM and 0.3 cM, respectively. Two SNP loci defined a haplotype that accurately predicted Pc98 status in a diverse group of oat germplasm, which will be valuable for marker-assisted selection of Pc98 in breeding of new oat cultivars.

The appropriate selection of various traits in valuable plants is very important for modern plant breeding. Effective resistance to fungal diseases, such as powdery mildew, is an example of such a trait in oats. Marker-assisted selection is an important tool that reduces the time and cost of selection. The aims of the present study were the identification of dominant DArTseq markers associated with a new resistance gene, annotated as Pm11 and derived from Avena sterilis genotype CN113536, and the subsequent conversion of these markers into a PCR-based assay. Among the obtained 30,620 silicoDArT markers, 202 markers were highly associated with resistance in the analysed population. Of these, 71 were selected for potential conversion: 42 specific to resistant and 29 to susceptible individuals. Finally, 40 silicoDArT markers were suitable for primer design. From this pool, five markers, 3 for resistant and 2 for susceptible plants, were selected for product amplification in the expected groups. The developed method, based on 2 selection markers, provides certain identification of resistant and susceptible homozygotes. Also, the use of these markers allowed the determination of heterozygotes in the analysed population. Selected silicoDArT markers were also used for chromosomal localization of new resistance genes. Five out of 71 segregating silicoDArT markers for the Pm11 gene were found on the available consensus genetic map of oat. Five markers were placed on linkage groups corresponding to Mrg12 on the Avena sativa consensus map.

Key Message: The widely deployed, oat stem rust resistance gene Pg13 was mapped by linkage analysis and association mapping, and KASP markers were developed for marker-assisted selection in breeding programs. Pg13 is one of the most extensively deployed stem rust resistance genes in North American oat cultivars. Identification of markers tightly linked to this gene will be useful for routine marker-assisted selection, identification of gene pyramids, and retention of the gene in backcrosses and three-way crosses. To this end, high-density linkage maps were constructed in four bi-parental mapping populations using SNP markers identified from 6K oat Infinium iSelect and genotyping-by-sequencing platforms. Additionally, genome-wide associations were identified using two sets of association panels consisting of diverse elite oat lines in one set and landrace accessions in the other. The results showed that Pg13 was located at approximately 67.7 cM on linkage group Mrg18 of the consensus genetic map. The gene co-segregated with the 7C-17A translocation breakpoint and with crown rust resistance gene Pc91. Co-segregating markers with the best prediction accuracy were identified at 67.7-68.5 cM on Mrg18. KASP assays were developed for linked SNP loci for use in oat breeding.

Background: Protein kinases play a key role in plant cell homeostasis and the activation of defense mechanisms. Partial resistance to fungi in plants is interesting because of its durability. However, the variable number of minor loci associated with this type of resistance hampers the reliable identification of the full range of genes involved. The present work reports the technique of protein kinase (PK)-profiling for the identification of the PK genes induced in the partially resistant oats line MN841801-1 following exposure to the fungus Puccinia coronata. This is the first time this technique has been used with cDNA (complementary DNA) from a suppression subtractive hybridization library obtained after the hybridization of cDNAs from inoculated and mock-inoculated plants., Results: Six degenerate primers based on the conserved domains of protein kinases were used in a PK-profiling assay including cDNA from mock-inoculated leaves and subtracted cDNA. Of the 75.7% of sequences cloned and sequenced that showed significant similarity to resistance genes, 76% were found to code for PKs. Translation and ClustalW2 alignment of each sequence cloned with the complete sequences of the most similar B. distachyon PKs allowed those of the partially resistant oat line to be deduced and characterized. Further, a phylogenetic study carried out after alignment of these B. distachyon PK sequences with the most similar protein sequences of related species also allowed to deduce different functions for the PK cloned. RT-qPCR (Reverse Transcription-quantitative PCR) was analyzed on nine representative sequences to validate the reliability of the employed PK-profiling method as a tool for identifying the expression of resistance-associated genes., Conclusions: PK-profiling would appear to be a useful tool for the identification of the PKs expressed in oats after challenge by P. coronata, and perhaps other pathogens. Most of the PKs studied are related to receptor-like protein kinases expressed shortly after infection. This is in agreement with previous studies indicating a close relationship between partial resistance and the first layer of defense against pathogen used by plants.

Crown rust, caused by Puccinia coronata f. sp. avenae, is the most widespread and harmful fungal disease of oat. The best defense against the pathogen is use of cultivars with genetic resistance, which is effective, economic, and an environmentally friendly alternative to chemical control. However, the continuous evolution of the pathogen can rapidly overcome major gene resistance, creating an urgent need to identify new sources. Wild oat accessions have already proven to be valuable donors of many resistance genes, but the weed species Avena fatua remains underexploited. Its abundance across multiple environments and the frequent occurrence of herbicide-resistant populations demonstrate its ready ability to adapt to biotic and abiotic stresses; yet, surprisingly, there are no extensive studies which describe crown rust resistance occurrence in gene bank stocks of A. fatua. In this study, 204 accessions of A. fatua maintained in the collections of the United States Department of Agriculture (USDA) and Polish National Centre for Plant Genetic Resources were evaluated at the seedling stage for crown rust reaction using host-pathogen tests with five highly diverse and virulent races of P. coronata. Of tested genotypes, 85% showed a heterogeneous infection pattern, while 61% were susceptible or moderately susceptible to all races. Of the 79 resistant A. fatua accessions, seedling resistance to at least two P. coronata isolates was recognized within 19 accessions, with 13 displaying a homogeneously resistant phenotype to one or two races. Accessions showing multiple single seedling resistance to three or four isolates were observed. Based on the seedling reaction to isolates used in the study, 18 infection profiles (IP) were determined. Using UPGMA clustering, resistant accessions were divided into six main clusters encompassing samples with similar IPs. Twelve of 18 patterns allowed us to postulate the likely presence of novel crown rust resistance genes, whose origin was predominantly from Kenya or Egypt. Future work will clarify the genetic basis of the resistances observed here, as well as confirm their potential utility in breeding resistant oat cultivars.

Key Message: Oat crown rust is one of the most damaging diseases of oat. We identified a new source of resistance and developed KASP and TaqMan markers for selection in breeding programs. A new highly effective resistance to oat crown rust (Puccinia coronata f. sp. avenae) was identified in the diploid oat Avena strigosa PI 258731 and introgressed into hexaploid cultivated oat. Young plants with this resistance show moderate susceptibility, whereas older plant tissues and adult plants are resistant with no virulent isolates encountered in over 8 years of testing. Resistance was incorporated into hexaploid oat by embryo rescue, colchicine chromosome doubling followed by backcrosses with a hexaploid parent, and selection for stable transmission of resistance. To mitigate flag leaf and panicle chlorosis/necrosis associated with the resistance, crosses were made with derived resistant lines to breeding lines of divergent parentage followed by selection. Subsequently, two F 2 sister lines, termed MNBT1020-1 and MNBT1021-1, were identified in which the chlorosis/necrosis was reduced. These two lines performed well in replicated multi-location state trials in 2015 and 2016 out-yielding all cultivar entries. Segregating F 2:3 plants resulting from crosses of MNBT lines to susceptible parents were genotyped with the oat 6K SNP array, and SNP loci with close linkage to the resistance were identified. KASP assays generated from linked SNPs showed accurate discrimination of the resistance in derivatives of the resistant MNBT lines crossed to susceptible breeding lines. A TaqMan marker was developed and correctly identified homozygous resistance in over 95% of 379 F 4 plants when rust was scored in F 4:5 plants in the field. Thus, a novel highly effective resistance and associated molecular markers are available for use in breeding, genetic analysis, and functional studies.

Barley yellow dwarf viruses (BYDVs) are responsible for the disease barley yellow dwarf (BYD) and affect many cereals including oat (Avena sativa L.). Until recently, the molecular marker technology in oat has not allowed for many marker-trait association studies to determine the genetic mechanisms for tolerance. A genome-wide association study (GWAS) was performed on 428 spring oat lines using a recently developed high-density oat single nucleotide polymorphism (SNP) array as well as a SNP-based consensus map. Marker-trait associations were performed using a Q-K mixed model approach to control for population structure and relatedness. Six significant SNP-trait associations representing two QTL were found on chromosomes 3C (Mrg17) and 18D (Mrg04). This is the first report of BYDV tolerance QTL on chromosome 3C (Mrg17) and 18D (Mrg04). Haplotypes using the two QTL were evaluated and distinct classes for tolerance were identified based on the number of favorable alleles. A large number of lines carrying both favorable alleles were observed in the panel.

Crown rust, caused by Puccinia coronata Corda f. sp. avenae Eriks., is a serious menace in oats, for which resistance is an effective means of control. Wild diploid oat accessions are a source of novel resistances that first need to be characterised prior to introgression into locally adapted oat cultivars. A genetic analysis of resistance to crown rust was carried out in three diverse diploid oat accessions (CIav6956, CIav9020, PI292226) and two cultivars (Saia and Glabrota) of A. strigosa. A single major gene conditioning resistance to Australian crown rust pathotype (Pt) 0000-2 was identified in each of the three accessions. Allelism tests suggested that these genes are either the same, allelic, or tightly linked with less than 1 % recombination. Similarly, a single gene was identified in Glabrota, and possibly two genes in Saia; both cultivars previously reported to carry two and three crown rust resistance genes, respectively. The identified seedling resistance genes could be deployed in combination with other resistance gene(s) to enhance durability of resistance to crown rust in hexaploid oat. Current diploid and hexaploid linkage maps and molecular anchor markers (simple sequence repeat [SSR] and diversity array technology [DArT] markers) should facilitate their mapping and introgression into hexaploid oat.

Viral pathogenicity has often been correlated to the expression of the viral encoded-RNA silencing suppressor protein (SSP). The silencing suppressor activity of the P0 protein encoded by cereal yellow dwarf virus-RPV (CYDV-RPV) and -RPS (CYDV-RPS), two poleroviruses differing in their symptomatology was investigated. CYDV-RPV displays milder symptoms in oat and wheat whereas CYDV-RPS is responsible for more severe disease. We showed that both P0 proteins (P0(CY-RPV) and P0(CY-RPS)) were able to suppress local RNA silencing induced by either sense or inverted repeat transgenes in an Agrobacterium tumefaciens-mediated expression assay in Nicotiana benthamiana. P0(CY-RPS) displayed slightly higher activity. Systemic spread of the silencing signal was not impaired. Analysis of short-interfering RNA (siRNA) abundance revealed that accumulation of primary siRNA was not affected, but secondary siRNA levels were reduced by both CYDV P0 proteins, suggesting that they act downstream of siRNA production. Correlated with this finding we showed that both P0 proteins partially destabilized ARGONAUTE1. Finally both P0(CY-RPV) and P0(CY-RPS) interacted in yeast cells with ASK2, a component of an E3-ubiquitin ligase, with distinct affinities., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Developing oat cultivars with partial resistance to crown rust would be beneficial and cost-effective for disease management. Two recombinant inbred-line populations were generated by crossing the susceptible cultivar Provena with two partially resistant sources, CDC Boyer and breeding line 94197A1-9-2-2-2-5. A third mapping population was generated by crossing the partially resistant sources to validate the quantitative trait locus (QTL) results. The three populations were evaluated for crown rust severity in the field at Louisiana State University (LSU) in 2009 and 2010 and at the Cereal Disease Laboratory (CDL) in St. Paul, MN, in 2009, 2010, and 2011. An iSelect platform assay containing 5,744 oat single nucleotide polymorphisms was used to genotype the populations. From the 2009 CDL test, linkage analyses revealed two QTLs for partial resistance in the Provena/CDC Boyer population on chromosome 19A. One of the 19A QTLs was also detected in the 2009 LSU test. Another QTL was detected on chromosome 12D in the CDL 2009 test. In the Provena/94197A1-9-2-2-2-5 population, only one QTL was detected, on chromosome 13A, in the CDL 2011 test. The 13A QTL from the Provena/94197A1-9-2-2-2-5 population was validated in the CDC Boyer/94197A1-9-2-2-2-5 population in the CDL 2010 and 2011 tests. Comparative analysis of the significant marker sequences with the rice genome database revealed 15 candidate genes for disease resistance on chromosomes 4 and 6 of rice. These genes could be potential targets for cloning from the two resistant parents.

Key Message: Genetic analysis and genome mapping of a major seedling oat crown rust resistance gene, designated PcKM, are described. The chromosomal location of the PcKM gene was identified and linked markers were validated. Crown rust (Puccinia coronata Corda f. sp. avenae Eriks) is the most important foliar disease of oats and can cause considerable yield loss in the absence of appropriate management practices. Utilization of novel resistant genes is the most effective, economic and environmentally sound approach to control the disease. Crown rust resistance present in the cultivar 'Morton' was evaluated in a population developed from the cross OT3019 × 'Morton' to elucidate the genetic basis of resistance. Crown rust reaction evaluated in field nurseries and greenhouse tests demonstrated that resistance provided by 'Morton' was controlled by a single gene, temporarily designated as PcKM. The gene was initially linked to a random amplified polymorphic DNA band and subsequently converted into a sequence characterized amplified region (SCAR) marker. Genotyping with the PcKM SCAR on the 'Kanota' × 'Ogle' population, used to create the first oat chromosome-anchored linkage map, placed the PcKM gene on chromosome 12D. Consensus map markers present in the same region as the PcKM SCAR were tested on the OT3019 × 'Morton' population and two additional phenotyped populations segregating for PcKM to identify other markers useful for marker-assisted selection. Three markers were perfectly linked to the PcKM phenotype from which TaqMan and KBioscience competitive allele-specific PCR assays were developed and validated on a set of 25 oat lines. The assays correctly identified PcKM carriers. The markers developed in this study will facilitate fine mapping of the PcKM gene and simplify selection for this crown rust resistance.

A trade-off between a pathogen's ability to infect many hosts and its reproductive capacity on each host genotype is predicted to limit the evolution of an expanded host range, yet few empirical results provide evidence for the magnitude of such trade-offs. Here, we test the hypothesis for a trade-off between the number of host genotypes that a fungal pathogen can infect (host genotype range) and its reproductive capacity on susceptible plant hosts. We used strains of the oat crown rust fungus that carried widely varying numbers of virulence (avr) alleles known to determine host genotype range. We quantified total spore production and the expression of four pathogen life-history stages: infection efficiency, time until reproduction, pustule size, and spore production per pustule. In support of the trade-off hypothesis, we found that virulence level, the number of avr alleles per pathogen strain, was correlated with significant delays in the onset of reproduction and with smaller pustule sizes. Modeling from our results, we conclude that trade-offs have the capacity to constrain the evolution of host genotype range in local populations. In contrast, long-term trends in virulence level suggest that the continued deployment of resistant host lines over wide regions of the United States has generated selection for increased host genotype range., (© 2014 The Author(s). Evolution © 2014 The Society for the Study of Evolution.)

The genetics of resistance to powdery mildew caused by Blumeria graminis f. sp. avenae of four cultivated oats was studied using monosomic analysis. Cultivar 'Bruno' carries a gene (Pm6) that shows a recessive mode of inheritance and is located on chromosome 10D. Cultivar 'Jumbo' possesses a dominant resistance gene (Pm1) on chromosome 1C. In cultivar 'Rollo', in addition to the gene Pm3 on chromosome 17A, a second dominant resistance gene (Pm8) was identified and assigned to chromosome 4C. In breeding line APR 122, resistance was conditioned by a dominant resistance gene (Pm7) that was allocated to chromosome 13A. Genetic maps established for resistance genes Pm1, Pm6 and Pm7 employing amplified fragment length polymorphism (AFLP) markers indicated that these genes are independent of each other, supporting the results from monosomic analysis.

Mycotoxins caused by Fusarium spp. is a major concern on food and feed safety in oats, although Fusarium head blight (FHB) is often less apparent than in other small grain cereals. Breeding resistant cultivars is an economic and environment-friendly way to reduce toxin content, either by the identification of resistance QTL or phenotypic evaluation. Both are little explored in oats. A recombinant-inbred line population, Hurdal × Z595-7 (HZ595, with 184 lines), was used for QTL mapping and was phenotyped for 3 years. Spawn inoculation was applied and deoxynivalenol (DON) content, FHB severity, days to heading and maturity (DH and DM), and plant height (PH) were measured. The population was genotyped with DArTs, AFLPs, SSRs and selected SNPs, and a linkage map of 1,132 cM was constructed, covering all 21 oat chromosomes. A QTL for DON on chromosome 17A/7C, tentatively designated as Qdon.umb-17A/7C, was detected in all experiments using composite interval mapping, with phenotypic effects of 12.2–26.6 %. In addition, QTL for DON were also found on chromosomes 5C, 9D, 13A, 14D and unknown_3, while a QTL for FHB was found on 11A. Several of the DON/FHB QTL coincided with those for DH, DM and/or PH. A half-sib population of HZ595, Hurdal × Z615-4 (HZ615, with 91 lines), was phenotyped in 2011 for validation of QTL found in HZ595, and Qdon.umb-17A/7C was again localized with a phenotypic effect of 12.4 %. Three SNPs closely linked to Qdon.umb-17A/7C were identified in both populations, and one each for QTL on 5C, 11A and 13A were identified in HZ595. These SNPs, together with those yet to be identified, could be useful in marker-assisted selection to pyramiding resistance QTL.

To elucidate genetic mechanisms of host species specificity between graminicolous anthracnose fungi and gramineous plants, infection assays were performed with a Sorghum isolate (Colletotrichum sublineolum), a Digitaria isolate (C. hanaui), a Polypogon isolate (C. cereale), and an Avena isolate (C. cereale). They were specifically virulent on the plants from which they were isolated. When 72 wheat lines were inoculated with an unadapted isolate from Asia Minor bluegrass (Cgp29), however, some exceptional cultivars were recognized. Although most cultivars were resistant to Cgp29, 'Hope' was susceptible. In F2 populations derived from crosses between three resistant cultivars-'Norin 4' (N4), 'Chinese Spring' (CS), and 'Shin-chunaga' (Sch)-and the susceptible Hope, resistant and susceptible seedlings segregated in a 3:1 ratio, suggesting that a major gene is involved in the resistance of each cultivar to Cgp29. In F2 populations derived from crosses between the three resistant cultivars, all seedlings were resistant, suggesting that these three cultivars carry the same gene. This resistance gene was designated as "resistance to Colletotrichum cereale 1" (Rcc1). Analysis with the CS-Hope chromosome substitution lines and molecular mapping revealed that Rcc1 was located on the long arm of chromosome 5A. Cytologically, Rcc1 was mainly associated with hypersensitive reaction. These results suggest that major genes similar to those controlling cultivar specificity are involved in the resistance of wheat against the unadapted isolate of C. cereale.

Two of the domains most widely shared among R genes are the nucleotide binding site (NBS) and protein kinase (PK) domains. The present study describes and maps a number of new oat resistance gene analogues (RGAs) with two purposes in mind: (1) to identify genetic regions that contain R genes and (2) to determine whether RGAs can be used as molecular markers for qualitative loci and for QTLs affording resistance to Puccinia coronata. Such genes have been mapped in the diploid A. strigosa × A. wiestii (Asw map) and the hexaploid MN841801-1 × Noble-2 (MN map). Genomic and cDNA NBS-RGA probes from oat, barley and wheat were used to produce RFLPs and to obtain markers by motif-directed profiling based on the NBS (NBS profiling) and PK (PK profiling) domains. The efficiency of primers used in NBS/PK profiling to amplify RGA fragments was assessed by sequencing individual marker bands derived from genomic and cDNA fragments. The positions of 184 markers were identified in the Asw map, while those for 99 were identified in the MN map. Large numbers of NBS and PK profiling markers were found in clusters across different linkage groups, with the PK profiling markers more evenly distributed. The location of markers throughout the genetic maps and the composition of marker clusters indicate that NBS- and PK-based markers cover partly complementary regions of oat genomes. Markers of the different classes obtained were found associated with the two resistance loci, PcA and R-284B-2, mapped on Asw, and with five out of eight QTLs for partial resistance in the MN map. 53 RGA-RFLPs and 187 NBS/PK profiling markers were also mapped on the hexaploid map A. byzantina cv. Kanota × A. sativa cv. Ogle. Significant co-localization was seen between the RGA markers in the KO map and other markers closely linked to resistance loci, such as those for P. coronata and barley yellow dwarf virus (Bydv) that were previously mapped in other segregating populations.

Powdery mildew is a prevalent fungal disease affecting oat (Avena sativa L.) production in Europe. Common oat cultivar Rollo was previously shown to carry the powdery mildew resistance gene Eg-3 in common with cultivar Mostyn. The resistance gene was mapped with restriction fragment length polymorphism (RFLP) markers from Triticeae group-1 chromosomes using a population of F(3) lines from a cross between A. byzantina cv. Kanota and A. sativa cv. Rollo. This comparative mapping approach positioned Eg-3 between cDNA-RFLP marker loci cmwg706 and cmwg733. Since both marker loci were derived from the long arm of barley chromosome 1H, the subchromosomal location of Eg-3 was assumed to be on the long arm of oat chromosome 17. Amplified fragment length polymorphism (AFLP) marker technology featured as an efficient means for obtaining markers closely linked to Eg-3.

Segregating F(3) families, derived from a cross between oat cultivar Swan and the putative single gene line PC68, were used to determine the association of seed storage protein loci and resistance gene analogues (RGAs) with the crown rust resistance gene Pc68. SDS-PAGE analysis detected three avenin loci, AveX, AveY, and AveZ, closely linked to Pc68. Their diagnostic alleles are linked in coupling to Pc68 and were also detected in three additional lines carrying Pc68. Another protein locus was linked in repulsion to Pc68. In complementary studies, three wheat RGA clones (W2, W4, and W10) detected restriction fragment length polymorphisms (RFLPs) between homozygous resistant and homozygous susceptible F(3) DNA bulks. Four oat homologues of W2 were cloned and sequenced. RFLPs detected with two of them were mapped using F(3) and F(4) populations. Clone 18 detected a locus, Orga2, linked in repulsion to Pc68. Clone 22 detected several RFLPs including Orga1 (the closest locus to Pc68) and three RGA loci (Orga22-2, Orga22-3, and Orga22-4) loosely linked to Pc68. The diagnostic RFLPs linked in coupling to Pc68 were detected by clone 22 in three additional oat lines carrying Pc68 and have potential utility in investigating and improving crown rust resistance of oat.

Crown rust is an important disease of oat caused by Puccinia coronata Corda f. sp. avenae Eriks. Crown rust is efficiently and effectively managed through the development of resistant oat varieties. Pc91 is a seedling crown rust resistance gene that is highly effective against the current P. coronata population in North America. The primary objective of this study was to develop DNA markers linked to Pc91 for purposes of marker-assisted selection in oat breeding programs. The Pc91 locus was mapped using a population of F7-derived recombinant inbred lines developed from the cross 'CDC Sol-Fi'/'HiFi' made at the Crop Development Centre, University of Saskatchewan. The population was evaluated for reaction to P. coronata in field nurseries in 2008 and 2009. Pc91 mapped to a linkage group consisting of 44 Diversity Array Technology (DArT) markers. DArTs were successfully converted to sequence characterized amplified region (SCAR) markers. Five robust SCARs were developed from three non-redundant DArTs that co-segregated with Pc91. SCAR markers were developed for different assay systems, such that SCARs are available for agarose gel electrophoresis, capillary electrophoresis, and Taqman single nucleotide polymorphism detection. The SCAR markers accurately postulated the Pc91 status of 23 North American oat breeding lines.

Management of oat crown rust disease with host resistance is challenging because major gene resistance is generally short lived. Partially resistant oat cultivars could benefit oat growers by providing more durable resistance. The objective of this study was to validate and discover quantitative trait loci (QTL) affecting crown rust resistance in the partially resistant oat line MN841801-1 using conventional and molecular assessments of disease produced in single-race greenhouse inoculations, single-race polycyclic field tests, and under natural infection in disease-conducive environments. Crown rust was assessed on 150 F(6:9) MN841801-1/'Noble-2' recombinant inbred lines. In total, eight QTL associated with MN841801-1 alleles were detected. Of these, seven matched QTL previously identified while a new QTL (Prq8) was detected on linkage group MN13. Four QTL (Prq1a, Prq2, Prq7, and Prq8) were consistently detected and predicative genetic assays for these QTL should be developed for future validation in additional genetic backgrounds.

Two previously isolated resistance gene analogs (RGAs) of oat have been located as RFLPs in the reference map of Avena byzantina 'Kanota' x Avena sativa 'Ogle' in regions either homologous or homoeologous to loci for resistance to Puccinia coronata, the causal agent of crown rust. In this study, the RGAs were mapped in two recombinant inbred line (RIL) populations that segregate for crown rust resistance: the diploid Avena strigosa x Avena wiestii RIL population (Asw), which has been used for mapping the complex locus PcA, and the hexaploid MN841801-1 x Noble-2 RIL population (MN), in which QTLs have been located. To obtain single-locus markers, RGAs were converted to sequence tagged site (STS) markers using a procedure involving extension of the original RGA sequence lengths by PCR genome walking, amplification and cloning of the parental fragments, and identification of single nucleotide polymorphisms. The procedure successfully obtained STSs from different members of the L7M2 family of sequences, the initial NBS of which have nucleotide similarities of >83%. However, for RGA III2.18, the parental lines were not polymorphic for the STSs assayed. A sequence characterized amplified region (SCAR) marker with features of an RGA had been previously identified for gene Pc94. This marker was also mapped in the above RIL populations. Markers based on RGA L7M2 co-localized with markers defining the QTL Prq1a in linkage group MN3, and were located 15.2 cM from PcA in linkage group AswAC. The SCAR marker for Pc94 was also located in the QTL Prq1a but at 39.5 cM from PcA in AswAC, indicating that the NBS-LRR sequence represented by this marker is not related to PcA. L7M2 was also excluded as a member of the PcA cluster, although it could be an appropriate marker for the Prq1a cluster if chromosome rearrangements are postulated.

Mapping disease resistance loci relies on the type and precision of phenotypic measurements. For crown rust of oat, disease severity is commonly assessed based on visual ratings of infection types (IT) and/or diseased leaf area (DLA) of infected plants in the greenhouse or field. These data can be affected by several variables including; (i) non-uniform disease development in the field; (ii) atypical symptom development in the greenhouse; (iii) the presence of multiple pathogenic races or pathotypes in the field, and (iv) rating bias. To overcome these limitations, we mapped crown rust resistance to single isolates in the Ogle/TAM O-301 (OT) recombinant inbred line (RIL) population using detailed measurements of IT, uredinia length (UL) and relative fungal DNA (FDNA) estimates determined by q-PCR. Measurements were taken on OT parents and recombinant inbred lines (RIL) inoculated with Puccinia coronata pathotypes NQMG and LGCG in separate greenhouse and field tests. Qualitative mapping identified an allele conferred by TAM O-301 on linkage group (LG) OT-11, which produced a bleached fleck phenotype to both NQMG and LGCG. Quantitative mapping identified two major quantitative trait loci (QTL) originating from TAM O-301 on LGs OT-11 and OT-32 which reduced UL and FDNA of both isolates in all experiments. Additionally, minor QTLs that reduced UL and FDNA were detected on LGs OT-15 and OT-8, originating from TAM O-301, and on LG OT-27, originating from Ogle. Detailed assessments of the OT population using two pathotypes in both the greenhouse and field provided comprehensive information to effectively map the genes responsible for crown rust resistance in Ogle and TAM O-301 to NQMG and LGCG.

CYP51 sterol demethylases are the only cytochrome P450 enzymes with a conserved function across the animal, fungal, and plant kingdoms (in the synthesis of essential sterols). These highly conserved enzymes, which are important targets for cholesterol-lowering drugs, antifungal agents, and herbicides, are regarded as the most ancient member cytochrome P450 family. Here we present a report of a CYP51 enzyme that has acquired a different function. We show that the plant enzyme AsCYP51H10 is dispensable for synthesis of essential sterols and has been recruited for the production of antimicrobial compounds (avenacins) that confer disease resistance in oats. The AsCyp51H10 gene is synonymous with Sad2, a gene that we previously had defined by mutation as being required for avenacin synthesis. In earlier work, we showed that Sad1, the gene encoding the first committed enzyme in the avenacin pathway (beta-amyrin synthase), had arisen by duplication and divergence of a cycloartenol synthase-like gene. Together these data indicate an intimate evolutionary connection between the sterol and avenacin pathways. Sad1 and Sad2 lie within 70 kb of each other and are expressed specifically in the epidermal cells of the root tip, the site of accumulation of avenacins. These findings raise intriguing questions about the recruitment, coevolution, and regulation of the components of this specialized defense-related metabolic pathway.

To facilitate the detection of quantitative trait loci (QTLs) for partial resistance to oat crown rust, Puccinia coronata f. sp. avenae Eriks., a genetic map was generated in a population of 158 F(6)-derived oat recombinant inbred lines from a cross of a partial resistance line MN841801-1 by a susceptible cultivar selection 'Noble-2'. The map, developed using 230 marker loci, mostly restriction fragment length polymorphism and amplified fragment length polymorphism markers, spanned 1,509 cM (Haldane) arranged into 30 linkage groups of 2-18 markers each. Four consistently detected major QTLs for partial rust resistance, Prq1a, Prq1b, Prq2, and Prq7, and three minor QTLs, Prq3, Prq5, and Prq6, were found in tests involving three field and two greenhouse environments. In addition, two major QTLs for flowering time, Ftq1 and Ftq7, and five weaker QTLs, Ftq2, Ftq3, Ftq4, Ftq5, and Ftq6, were revealed. Overlapping of the map segments of Ftq1 and Prq1 and of Ftq7 and Prq7 suggested either linkage between the flowering time QTLs and resistance QTLs or a pleiotropic effect of the Ftq QTLs on rust resistance. Relatively low heritability estimates (0.30) obtained for partial resistance to crown rust in the field indicate a potential value for marker-assisted selection.

Bacterial attack is a serious agricultural problem for growth of rice seedlings in the nursery and field. The thionins purified from seed and etiolated seedlings of barley are known to have antimicrobial activity against necrotrophic pathogens; however, we found that no endogenous rice thionin genes alone are enough for resistance to two major seed-transmitted phytopathogenic bacteria, Burkholderia plantarii and B. glumae, although rice thionin genes constitutively expressed in coleoptile, the target organ of the bacteria. Thus, we isolated thionin genes from oat, one of which was overexpressed in rice. When wild-type rice seed were germinated with these bacteria, all seedlings were wilted with severe blight. In the seedling infected with B. plantarii, bacterial staining was intensively marked around stomata and intercellular spaces. However, transgenic rice seedlings accumulating a high level of oat thionin in cell walls grew almost normally with bacterial staining only on the surface of stomata. These results indicate that the oat thionin effectively works in rice plants against bacterial attack.