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Leaf rust caused by Puccinia hordei is a major disease of barley and is best managed by deploying effective resistance genes. The effect of post-inoculation temperature [18 ± 2°C (low), 22 ± 2°C (m...

Barley is an intermediate or near nonhost to many cereal rust pathogens that infect grasses, making it a highly suitable model to understand the evolution and genetic basis of nonhost resistance (NHR) in plants. To characterize the genetic architecture of NHR in barley, we used the Oregon Wolfe Barley doubled haploid and Morex × SusPtrit recombinant inbred line mapping populations. To elicit a wide array of NHR responses, we tested 492 barley accessions and both mapping populations with pathogenically diverse cereal rust isolates representing distinct formae speciales adapted to Avena, Hordeum, Triticum, and Lolium spp.: P. coronata f. sp. avenae (oat crown rust pathogen) and P. coronata f. sp. lolii (ryegrass crown rust pathogen), P. graminis f. sp. avenae (oat stem rust pathogen) and P. graminis f. sp. lolii (the ryegrass stem rust pathogen), and P. striiformis f. sp. tritici (wheat stripe rust pathogen) and P. striiformis f. sp. pseudo-hordei (barley grass stripe rust pathogen). With the exception of P. coronata f. sp. lolii and P. coronata f. sp. avenae, susceptibility and segregation for NHR was observed in the barley accessions and both mapping populations. Quantitative trait loci (QTLs) for NHR were mapped on all seven chromosomes. NHR in barley to the heterologous rusts tested was attributable to a combination of QTLs with either or both overlapping and distinct specificities. Across both mapping populations, broadly effective NHR loci were also identified that likely play a role in host specialization.

For effective varietal improvement of horticultural crops peach (Prunus persica) and nectarine (Prunus persica var. nucipersica), information about their population structure and genetic relatedness plays an important role. In this study we used retrotransposon-based markers (iPBS) to estimate the genetic diversity and population structure of 48 peach and nectarine genotypes from various distinct geographical regions of Punjab and Khyber Pakhtunkhwa, Pakistan. A total of 461 alleles were identified from PCR amplicons derived from nine iPBS primer pairs with an average of 8.5 alleles/locus. Among all four groups the genotypes collected from Swat and Hunza had the highest and the lowest expected heterozygosity, unbiased expected heterozygosity and Shannon’s information index, respectively. We constructed a Neighbour-Joining dendrogram and performed principal coordinate analysis based on the distance matrices, and both forms of analysis grouped the 48 genotypes into two distinct clusters. The STRUCTURE software distributed the forty-eight genotypes into two main populations (k = 2) indicating a low diversity between genotypes collected from Chakwal, Swat, Mansehra and Hunza.

Summary Leaf rust, caused by Puccinia hordei, is a devastating fungal disease affecting barley (Hordeum vulgare subsp. vulgare) production globally. Despite the effectiveness of genetic resistance, the deployment of single genes often compromises durability due to the emergence of virulent P. hordei races, prompting the search for new sources of resistance. Here we report on the cloning of Rph15, a resistance gene derived from barley’s wild progenitor H. vulgare subsp. spontaneum. We demonstrate using introgression mapping, mutation and complementation that the Rph15 gene from the near‐isogenic line (NIL) Bowman + Rph15 (referred to as BW719) encodes a coiled‐coil nucleotide‐binding leucine‐rich repeat (NLR) protein with an integrated Zinc finger BED (ZF‐BED) domain. A predicted KASP marker was developed and validated across a collection of Australian cultivars and a series of introgression lines in the Bowman background known to carry the Rph15 resistance. Rph16 from HS‐680, another wild barley derived leaf rust resistance gene, was previously mapped to the same genomic region on chromosome 2H and was assumed to be allelic with Rph15 based on genetic studies. Both sequence analysis, race specificity and the identification of a knockout mutant in the HS‐680 background suggest that Rph15‐ and Rph16‐mediated resistances are in fact the same and not allelic as previously thought. The cloning of Rph15 now permits efficient gene deployment and the production of resistance gene cassettes for sustained leaf rust control.

Enhancing the photosynthetic induction response to fluctuating light has been suggested as a key target for improvement in crop breeding programmes, with the potential to substantially increase whole-canopy carbon assimilation and contribute to crop yield potential. Rubisco activation may be the main physiological process that will allow us to achieve such a goal. In this study, we assessed the phenotype of Rubisco activation rate in a doubled haploid (DH) barley mapping population [131 lines from a Yerong/Franklin (Y/F) cross] after a switch from moderate to saturating light. Rates of Rubisco activation were found to be highly variable across the mapping population, with a median activation rate of 0.1 min−1 in the slowest genotype and 0.74 min−1 in the fastest genotype. A unique quantitative trait locus (QTL) for Rubisco activation rate was identified on chromosome 7H. This is the first report on the identification of a QTL for Rubisco activation rate in planta and the discovery opens the door to marker-assisted breeding to improve whole-canopy photosynthesis of barley. This also suggests that genetic factors other than the previously characterized Rubisco activase (RCA) isoforms on chromosome 4H control Rubisco activity. Further strength is given to this finding as this QTL co-localized with QTLs identified for steady-state photosynthesis and stomatal conductance. Several other distinct QTLs were identified for these steady-state traits, with a common overlapping QTL on chromosome 2H, and distinct QTLs for photosynthesis and stomatal conductance identified on chromosomes 4H and 5H, respectively. Future work should aim to validate these QTLs under field conditions so that they can be used to aid plant breeding efforts., Speeding up the photosynthetic induction response to fluctuating light has been highlighted as a key target for crop improvement programmes. We identified significant variation in the photosynthetic induction response after a switch from moderate to saturating light across a barley doubled haploid population. A quantitative trait locus (QTL) for Rubisco activation rate was identified on chromosome 7H, as well as overlapping QTLs for steady-state photosynthesis and stomatal conductance. This is the first report on the identification of a QTL for Rubisco activation rate in planta and the discovery opens the door to marker-assisted breeding to improve whole-canopy photosynthesis of barley.

A new gene Rph28 conferring resistance to barley leaf rust was discovered and fine-mapped on chromosome 5H from wild barley. Leaf rust is a highly destructive disease of barley caused by the fungal pathogen Puccinia hordei. Genetic resistance is considered to be the most effective, economical and eco-friendly approach to minimize losses caused by this disease. A study was undertaken to characterize and fine map a seedling resistance gene identified in a Hordeum vulgare ssp. spontaneum-derived barley line, HEB-04-101, that is broadly effective against a diverse set of Australian P. hordei pathotypes. Genetic analysis of an F3 population derived from a cross between HEB-04-101 and the H. vulgare cultivar Flagship (seedling susceptible) confirmed the presence of a single dominant gene for resistance in HEB-04-101. Selective genotyping was performed on representative plants from non-segregating homozygous resistant and homozygous susceptible F3 families using the targeted genotyping-by-sequencing (tGBS) assay. Putatively linked SNP markers with complete fixation were identified on the long arm of chromosome 5H spanning a physical interval between 622 and 669 Mb based on the 2017 Morex barley reference genome assembly. Several CAPS (cleaved amplified polymorphic sequences) markers were designed from the pseudomolecule sequence of the Morex assembly (v1.0 and v2.0), and 16 polymorphic markers were able to delineate the RphHEB locus to a 0.05 cM genetic interval spanning 98.6 kb. Based on its effectiveness and wild origin, RphHEB is distinct from all other designated Rph genes located on chromosome 5H and therefore the new locus symbol Rph28 is recommended for RphHEB in accordance with the rules and cataloguing system of barley gene nomenclature.

Enhancing the photosynthetic induction response to fluctuating light has been suggested as a key target for improvement in crop breeding programs, with the potential to substantially increase whole canopy carbon assimilation and contribute to crop yield potential. Rubisco activation may be the main physiological process that will allow us to achieve such a goal. In this study, we phenotypically assessed the rubisco activation rate in a doubled haploid (DH) barley mapping population [131 lines from a Yerong/Franklin (Y/F) cross] after a switch from moderate to saturating light. Rates of rubisco activation were found to be highly variable across the mapping population, with a median activation rate of 0.1 min−1 in the slowest genotype and 0.74 min−1 in the fastest genotype. A QTL for rubisco activation rate was identified on chromosome 7H. This is the first report on the identification of a QTL for rubisco activation rate in planta and the discovery opens the door to marker assisted breeding to improve whole canopy photosynthesis of barley. Further strength is given to this finding as this QTL colocalised with QTLs identified for steady state photosynthesis and stomatal conductance. Several other distinct QTLs were identified for these steady state traits, with a common overlapping QTL on chromosome 2H, and distinct QTLs for photosynthesis and stomatal conductance identified on chromosomes 4H and 5H respectively. Future work should aim to validate these QTLs under field conditions so that they can be used to aid plant breeding efforts.HighlightSignificant variation exists in the photosynthetic induction response after a switch from moderate to saturating light across a barley doubled haploid population. A QTL for rubisco activation rate was identified on chromosome 7H, as well as overlapping QTLs for steady state photosynthesis and stomatal conductance.

Improving resistance to barley leaf rust (caused by Puccinia hordei) is an important breeding objective in most barley growing regions worldwide. The development and subsequent utilisation of high-throughput PCR-based co-dominant molecular markers remains an effective approach to select genotypes with multiple effective resistance genes, permitting efficient gene deployment and stewardship. The genes Rph20 and Rph24 confer widely effective adult plant resistance (APR) to leaf rust, are common in European and Australian barley germplasm (often in combination), and act interactively to confer high levels of resistance (Dracatos et al. 2015; Zeims et al. 2017; Singh et al. 2018). Here we report on the development and validation of co-dominant insertion-deletion (indel) based PCR markers that are highly predictive for the Rph20 and Rph24 resistances.

The barley cultivar Quinn was previously reported to carry two genes for resistance to

Unraveling and exploiting mechanisms of disease resistance in cereal crops is currently limited by their large repeat-rich genomes and the lack of genetic recombination or cultivar (cv)-specific sequence information. We cloned the first leaf rust resistance gene Rph1 (Rph1.a) from cultivated barley (Hordeum vulgare) using “MutChromSeq,” a recently developed molecular genomics tool for the rapid cloning of genes in plants. Marker-trait association in the CI 9214/Stirling doubled haploid population mapped Rph1 to the short arm of chromosome 2H in a physical region of 1.3 megabases relative to the barley cv Morex reference assembly. A sodium azide mutant population in cv Sudan was generated and 10 mutants were confirmed by progeny-testing. Flow-sorted 2H chromosomes from Sudan (wild type) and six of the mutants were sequenced and compared to identify candidate genes for the Rph1 locus. MutChromSeq identified a single gene candidate encoding a coiled-coil nucleotide binding site Leucine-rich repeat (NLR) receptor protein that was altered in three different mutants. Further Sanger sequencing confirmed all three mutations and identified an additional two independent mutations within the same candidate gene. Phylogenetic analysis determined that Rph1 clustered separately from all previously cloned NLRs from the Triticeae and displayed highest sequence similarity (89%) with a homolog of the Arabidopsis (Arabidopsis thaliana) disease resistance protein 1 protein in Triticum urartu. In this study we determined the molecular basis for Rph1-mediated resistance in cultivated barley enabling varietal improvement through diagnostic marker design, gene editing, and gene stacking technologies.

Barley is a host to Puccinia striiformis f. sp. hordei, and is an intermediate or near nonhost to the formae speciales adapted to wheat (P. striiformis f. sp. tritici) and to barley grass (P. striiformis f. sp. pseudo-hordei). The genetic basis of resistance to these forms of P. striiformis is not well understood. Accordingly, a recombinant inbred line (RIL) population was developed using a P. striiformis-susceptible accession (Biosaline-19) and the immune cultivar Pompadour. We investigated the genetic basis of resistance to four diverse P. striiformis isolates (P. striiformis f. sp. pseudo-hordei, and P. striiformis f. sp. tritici pathotypes 104 E137 A−, 134 E16 A+, and 64 E0 A−). and determined that the immunity in Pompadour at the seedling stage to the different P. striiformis isolates was due to quantitative trait loci (QTL) on chromosomes 1H, 3H, 5H, and 7H with both overlapping and distinct specificities. Further histological analysis confirmed the presence of isolate specificity. The RILs were also assessed in the field for resistance to P. striiformis f. sp. pseudo-hordei, P. striiformis f. sp. hordei, and the leaf rust pathogen (P. hordei) to identify pleiotropic QTL loci effective at the adult plant stage and determine whether the leaf rust resistance in Pompadour (Rph20) was also effective to P. striiformis. RILs that were seedling susceptible to P. striiformis f. sp. pseudo-hordei were resistant in the field, implicating the involvement of adult plant resistance (APR). Additional QTLs were identified on chromosome 7H at the same genetic position as Rph23 (APR to leaf rust), suggesting either pleiotropic resistance or the presence of a stripe rust resistance gene closely linked to or allelic with Rph23. Unlike many pleiotropic APR genes identified and isolated in wheat, our data suggest that the Rph20 locus does not confer resistance to the P. striiformis isolates used in this study (P. striiformis f. sp. hordei [χ2 (independence) = 2.47 P > 0.12] and P. striiformis f. sp. pseudo-hordei [χ2 (independence) = 0.42 P > 0.60]).

Individual plants encounter a vast number of microbes including bacteria, viruses, fungi and oomycetes through their growth cycle, yet few of these pathogens are able to infect them. Plant species have diverged over millions of years, co-evolving with few specific pathogens. The host boundaries of most pathogen species can be clearly defined. In general, the greater the genetic divergence from the preferred host, the less likely that pathogen would be able to infect that plant species. Co-evolution and divergence also occur within pathogen species, leading to highly specialized subspecies with narrow host ranges. For example, cereal rust and mildew pathogens (Puccinia and Blumeria spp.) display high host specificity as a result of ongoing co-evolution with a narrow range of grass species. In rare cases, however, some plant species are in a transition from host to nonhost or are intermediate hosts (near nonhost). Barley was reported as a useful model for genetic and molecular studies of nonhost resistance due to rare susceptibility to numerous heterologous rust and mildew fungi. This review evaluates host specificity in numerous Puccinia/Blumeria-cereal pathosystems and discusses various approaches for transferring nonhost resistance (NHR) genes between crop species to reduce the impact of important diseases in food production.

Oat stem rust, caused by Puccinia graminis f. sp. avenae (Pga), is one of the most severe diseases of oats worldwide. Population studies are scarce for this pathogen, mainly due to the lack of polymorphic molecular markers suitable for genetic analysis. We sequenced an Australian Pga isolate, determined the abundance of simple sequence repeats (SSRs), and developed PCR-based polymorphic markers suitable for genetic diversity analysis. The amplification of 194 primer pairs was initially assessed across a validation panel including 12 isolates of different cereal rust species and their formae speciales. A high cross-species amplification frequency was observed for most markers, however 19% SSRs were diagnostic for only P. graminis. A subset of 19 genomic-derived SSRs were deemed useful for genetic diversity analysis of Pga and were assessed on 66 Pga isolates from Australia, Brazil and Sweden. Brazilian and Australian isolates were characterized by one and two predominant clonal lineages, respectively. In contrast, the Swedish isolates previously shown to undergo sexual recombination were highly diverse (nine distinct genotypes out of 10 isolates) and divided into two sub-populations. The genomic-derived SSR markers developed in this study were well suited to the population studies undertaken, and have diagnostic capabilities that should aid in the identification of unknown rust pathogen species. This article is protected by copyright. All rights reserved.

We identified Rph24 as a locus in barley (Hordeum vulgare L.) controlling adult plant resistance (APR) to leaf rust, caused by Puccinia hordei. The locus was previously reported as a quantitative trait locus in barley line ND24260-1 and named qRphND. We crossed ND24260-1 to the leaf-rust-susceptible standard Gus and determined inheritance patterns in the progeny. For the comparative marker frequency analysis (MFA), resistant and susceptible tails of the F2 were genotyped with Diversity Arrays Technology genotyping-by-sequencing (DArT-Seq) markers. The Rph24 locus was positioned at 55.5 centimorgans on chromosome 6H on the DArT-Seq consensus map. Evaluation of F2:3 families confirmed that a single locus from ND24260-1 conferred partial resistance. The haploblock strongly associated with the Rph24 locus was used to estimate the allele frequency in a collection of 282 international barley cultivars. Rph24 was frequently paired with APR locus Rph20 in cultivars displaying high levels of APR to leaf rust. The markers identified in this study for Rph24 should be useful for marker-assisted selection.

Leaf rust, caused byPuccinia hordeiis a devastating fungal disease affecting barley (Hordeum vulgaresubsp.vulgare) production globally. Race-specific resistance (R) genes have been deployed widely; however, their durability is often compromised due to the rapid emergence of virulentP. hordeiraces, prompting the search for new sources of broad-spectrum resistance. Here we report on the cloning ofRph15, a broadly effective resistance gene derived from the wild progenitorHordeum vulgaresubsp.spontaneum. We demonstrate using introgression mapping, mutation and complementation thatRph15encodes a coiled-coil nucleotide-binding leucine-rich repeat (NLR) protein with an integrated Zinc-finger BED (ZF-BED) domain. The allelic variation at theRph15locus was assessed using barley exome capture data that traced its origin to the western region of the Fertile Crescent bordering Jordan and Israel. To unravel the genetic relationship of two other leaf rust resistance genes (Rph14andRph16) mapped at similar locus on chromosome 2H, we re-sequenced theRph15gene from the near-isogenic line forRph15(Bowman+Rph15) and the two donor accessions ofRph14(PI 584760) andRph16, (PI 405292, Hs 680). Both whole genome and Sanger sequencing confirmed that Hs 680 carriedRph15, whileRph14in PI 584760 was an independent locus. A perfect diagnostic KASP marker was developed and validated to permit efficient introduction ofRph15into cultivated barley.

Fine mapping of the barley leaf rust resistance locus Rph13 on chromosome 3HL facilitates its use in breeding programs through marker-assisted selection. Barley leaf rust (BLR—caused by Puccinia hordei) is a widespread fungal disease that can be effectively controlled by genetic resistance. There is an ongoing need to both diversify and genetically characterise resistance loci to provide effective and durable control given the ongoing threat of rapidly evolving P. hordei populations. Here, we report on the molecular genetic characterisation of the Rph13 locus, originally derived from wild barley and transferred to barley accession Berac (then referred to as PI 531849). The 2017 reference genome of cv. Morex was used as a road map to rapidly narrow both a genetic and physical intervals around the Rph13 resistance locus. Using recombination-based mapping, we narrowed the physical interval to 116.6 kb on chromosome 3H in a segregating population of a cross of the Rph13 carrying resistant line PI 531849 with the leaf rust-susceptible cultivar Gus. We identified two nucleotide-binding leucine-rich repeat genes as likely candidates for the Rph13 resistance. Sequences from the candidate genes enabled the development of a KASP marker that distinguished resistant and susceptible progeny and was found to be predictive and useful for MAS.

This is the first genetic study reporting on the interaction and molecular mapping of resistance to the barley grass stripe rust pathogen (Puccinia striiformis f. sp. pseudo-hordei, Psph) in common wheat. Seedlings of 638 wheat accessions were tested and it was determined that wheat is a near-nonhost to Psph based on rare susceptibility observed in

Breeding for adult plant resistance (APR) is currently impeded by the low frequency of annual field-based testing and variable environmental conditions. We developed and implemented a greenhouse-based methodology for the rapid phenotyping of APR to leaf rust in barley to improve the efficacy of gene discovery and cloning. We assessed the effects of temperature (18 and 23°C) and growth stage (1–5 weeks) on the expression of APR in the greenhouse using 28 barley genotypes with both known and uncharacterized APR. All lines were susceptible in week 1, while lines carrying Rph20 and several with uncharacterized resistance expressed resistance as early as week 2. In contrast, lines lacking Rph20 and carrying either Rph23 and/or Rph24 expressed resistance from week 4. Resistant phenotypes were clearest at 18°C. A subset of 16 of the 28 lines were assessed for leaf rust across multiple national and international field sites. The greenhouse screening data reported in this study were highly correlated to most of the field sites, indicating that they provide comparable data on APR phenotypes for screening purposes.

Eighty-eight Australian and 10 international barley cultivars were assessed for resistance to the barley stripe (yellow) rust pathogen, Puccinia striiformis f. sp. hordei (Psh). All cultivars were tested for seedling resistance to two UK-derived isolates of Psh (11.01 and 83.39) that were shown to differ in virulence based on responses on 16 differential barley genotypes. The 98 barley cultivars differed substantially in stripe rust response; 45% were susceptible to Psh 11.01, 53% to Psh 83.39 and 44% to both isolates. The observed diverse infection types (ITs) suggest the presence of both known and uncharacterized resistance. However, further multipathotype tests are required for accurate gene postulation. The Yerong × Franklin (Y×F) doubled haploid (DH) population was phenotypically assessed as seedlings using both Psh isolates. Yerong and Franklin were immune and highly resistant, respectively, to both isolates used in this study. Marker-trait and QTL mapping identified a major effect on the long arm of chromosome 7H contributed by Franklin in response to all isolates. The resistance of Yerong was mapped to 113·96 and 169·38 cM on chromosome 5HL in response to Psh 11.01 and 83.39, respectively. The Psh resistance sources identified in this study can be used for further genetic analysis and introgression for varietal improvement.

Leaf rust of barley is caused by the macrocyclic, heteroecious rust pathogen Puccinia hordei, with aecia reported from selected species of the genera Ornithogalum, Leopoldia, and Dipcadi, and uredinia and telia occurring on Hordeum vulgare, H. vulgare ssp. spontaneum, Hordeum bulbosum, and Hordeum murinum, on which distinct parasitic specialization occurs. Although Puccinia hordei is sporadic in its occurrence, it is probably the most common and widely distributed rust disease of barley. Leaf rust has increased in importance in recent decades in temperate barley-growing regions, presumably because of more intensive agricultural practices. Although total crop loss does not occur, under epidemic conditions yield reductions of up to 62% have been reported in susceptible varieties. Leaf rust is primarily controlled by the use of resistant cultivars, and, to date, 21 seedling resistance genes and two adult plant resistance (APR) genes have been identified. Virulence has been detected for most seedling resistance genes but is unknown for the APR genes Rph20 and Rph23. Other potentially new sources of APR have been reported, and additivity has been described for some of these resistances. Approaches to achieving durable resistance to leaf rust in barley are discussed.

We report on a new adult plant resistance (APR) gene Rph23 conferring resistance to leaf rust in barley. The gene was identified and characterized from a doubled haploid population derived from an intercross between the Australian barley varieties Yerong (Y) and Franklin (F). Genetic analysis of adult plant field leaf rust scores of the Y/F population collected over three successive years indicated involvement of two highly additive genes controlling APR, one of which was named Rph23. The gene was mapped to chromosome 7HS positioned at a genetic distance 36.6 cM. Rph23 is closely linked to marker Ebmac0603, which is flanked by markers bPb-8660 and bPb-9601 with linkage distances of 0.8 and 5.1 cM, respectively. A PCR-based marker was optimized for marker-assisted selection of Rph23, and on the basis of this marker, the gene was postulated as being common in Australian and global barley germplasm. Pedigree and molecular marker analyses indicated that the six-rowed black Russian landrace ‘LV-Taganrog’ is the likely origin of Rph23.

Barley leaf rust, caused by Puccinia hordei, is one of the most destructive foliar pathogens of cultivated barley, causing significant yield losses in many regions throughout the world. In this study, 521 seedling-susceptible barley germplasm obtained from diverse breeding material sourced from the Australian Winter Cereals Collection (AWCC), the University of Western Australia (UWA), and international collections from Australia, China, Germany, Spain, and Uruguay were assessed for adult plant resistance (APR) in field nurseries over multiple growing seasons. Lines (213) that consistently showed APR over multiple seasons were screened with PCR-based markers closely linked to Rph20 (bPb0837) and Rph23 (EBmac0603). About 93 % of the lines that were resistant in the field carried one or more uncharacterised APR genes with or without Rph20 and Rph23. There was high variability for APR within specific international germplasm collections. The presence of bPb0837 was strongly correlated with high APR (TR-MR) responses in the field, while lines that were positive for the EBmac0603 allele had intermediate resistance (MRMS). Both EBmac0603 and bPb0837 were present in three German lines (Lenka, Line 17 and Volla) and in the Australian variety Macquarie, all were thus were postulated to carry both Rph20 and Rph23 and had TR-20MR responses in the field. Molecular markers closely linked to Rph20 and Rph23 provide a valuable resource that can be used to assist the incorporation of these genes into new cultivars and identify uncharacterised APR.

The success of breeding for barley leaf rust (BLR) resistance relies on regular discovery, characterization and mapping of new resistance sources. Greenhouse and field studies revealed that the barley cultivars Baronesse, Patty and RAH1995 carry good levels of adult plant resistance (APR) to BLR. Doubled haploid populations [(Baronesse/Stirling (B/S), Patty/Tallon (P/T) and RAH1995/Baudin (R/B)] were investigated in this study to understand inheritance and map resistance to BLR. The seedlings of two populations (B/S and R/B) segregated for leaf rust response that conformed to a single gene ratio ( $${\text{X}}_{1:1}^{2}$$ = 0.12, P > 0.7 for B/S and $${\text{X}}_{1:1}^{2}$$ = 0.34, P > 0.5 for R/B) whereas seedlings of third population (P/T) segregated for two-gene ratio ( $${\text{X}}_{1:1}^{2}$$ = 0.17, P > 0.6) when tested in greenhouse. It was concluded that the single gene in Baudin and one of the two genes in Tallon is likely Rph12, whereas gene responsible for seedling resistance in Stirling is Rph9.am (allele of Rph12). The second seedling gene in Tallon is uncharacterized. In the field, APR was noted in lines that were susceptible as seedlings. A range of disease responses (CI 5–90) was observed in all three populations. Marker trait association analysis detected three QTLs each in populations B/S (QRph.sun-2H.1, QRph.sun-5H.1 and QRph.sun-6H.1) and R/B (QRph.sun-1H, QRph.sun-2H.2, QRph.sun-3H and QRph.sun-6H.2), and four QTLs in population P/T (QRph.sun-6H.2, QRph.sun-1H.2, QRph.sun-5H.2 and QRph.sun-7H) that significantly contributed to low leaf rust disease coefficients. High frequency of QRph. sun-5H.1, QRph. sun-6H.1, QRph. sun-1H.1, QRph. sun-2H.2, QRph. sun-6H.2, QRph. sun-7H (based on presence of the marker, closely associated to the respective QTLs) was observed in international commercial barley germplasm and hence providing an opportunity for rapid integration into breeding programmes. The identified candidate markers closely linked to these QTLs will assist in selecting and assembling new APR gene combinations; expectantly this will help in achieving good levels of durable resistance for controlling BLR.

Nicotiana alata defensins 1 and 2 (NaD1 and NaD2) are plant defensins from the ornamental tobacco that have antifungal activity against a variety of fungal pathogens. Some plant defensins interact with fungal cell wall O-glycosylated proteins. Therefore, we investigated if this was the case for NaD1 and NaD2, by assessing the sensitivity of the three Aspergillus nidulans (An) O-mannosyltransferase (pmt) knockout (KO) mutants (AnDpmtA, AnDpmtB, and AnDpmtC). AnDpmtA was resistant to both defensins, while AnDpmtC was resistant to NaD2 only, suggesting NaD1 and NaD2 are unlikely to have a general interaction with O-linked side chains. Further evidence of this difference in the antifungal mechanism was provided by the dissimilarity of the NaD1 and NaD2 sensitivities of the Fusarium oxysporum f. sp. lycopersici (Fol) signalling knockout mutants from the cell wall integrity (CWI) and high osmolarity glycerol (HOG) mitogen-activated protein kinase (MAPK) pathways. HOG pathway mutants were sensitive to both NaD1 and NaD2, while CWI pathway mutants only displayed sensitivity to NaD2

Barley is a near-nonhost to numerous heterologous (nonadapted) rust pathogens because a small proportion of genotypes are somewhat susceptible. We assessed 66 barley accessions and three mapping populations (Vada × SusPtrit, Cebada Capa × SusPtrit, and SusPtrit × Golden Promise) for response to three Swedish oat stem rust (Puccinia graminis f. sp. avenae) fungal isolates and determined that barley is a near-nonhost to P. graminis f. sp. avenae and that resistance was polygenically inherited. The parental genotypes Vada and Golden Promise were immune to all three isolates, whereas Cebada Capa was immune to two isolates and moderately resistant to the third. Phenotypic data from the Vada × SusPtrit mapping population and the barley accessions tested also demonstrated isolate-specific resistance. In particular, the SusPtrit parent and several other accessions allowed sporulation by isolate Ingeberga but were resistant to isolate Evertsholm. Nine chromosomal regions carried quantitative trait loci (QTL) (Rpgaq1 to Rpgaq9) of varying effect, most of which colocated to previously identified QTL for resistance to other heterologous rust pathogens. Rpgaq1 on chromosome 1H (Vada and Golden Promise) was effective toward all isolates tested. Microscopic examination indicated that resistance was prehaustorial in Vada whereas, in SusPtrit, both pre- and posthaustorial mechanisms play a role.

The most serious foliar disease of perennial ryegrass (Lolium perenne L.) is crown rust (caused by Puccinia coronata Corda f.sp. lolii Brown). Progress in resistance breeding using recurrent selection has been slow, due to lack of genetic knowledge. Puccinia coronata is a basidiomycete fungus with a complex life-cycle involving both asexual and sexual reproductive modes. Pathotype variation may exist, but confi rmation is complicated by the outbreeding nature of the host grass and consequent intrapopulation diversity. Qualitative and quantitative resistance mechanisms may hence be due to either major resistance genes responding to mixed pathogen populations, or minor (quantitative) resistance genes. Advances in molecular biology have permitted analysis of both host and pathogen genetics. Development of pathogen-derived simple sequence repeat (SSR) genetic markers allowed detailed analysis of genetic variation, as well as generation of homogenized inoculum for detailed trait-dissection studies. Multiple genomic regions were identifi ed as containing quantitative trait loci (QTLs) for pathogen resistance, and specifi c single nucleotide polymorphism (SNP) markers for defense response (DR) and disease resistance (R) genes have been evaluated for QTL co-location. The current knowledge status of the crown rust pathogen and genetics of host resistance is reviewed, as well as future prospects for facilitated rust resistance breeding.

Complementary genes for resistance to wheat stripe rust in an Avocet selection mapped to chromosome arms 3DL and 5BL. Susceptible Avocet selections lacked the 5BL gene due to a chromosomal deletion. This study reports the inheritance and genetic mapping of the YrA (temporary name of convenience to describe the specificity) seedling resistance to wheat stripe rust (caused by Puccinia striiformis f. sp. tritici; Pst) in a resistant selection of the Australian cv. Avocet [Avocet R (AvR)-AUS 90660]. Genetic analysis was performed on F2 populations and F3 generation families from crosses between wheats that carried and lacked the YrA resistance. Greenhouse seedling tests with two avirulent Pst pathotypes (104 E137 A- and 108 E141 A-) confirmed that the YrA resistance was inherited as two complementary dominant genes. Ninety-two doubled haploid (DH) lines from a cross between the Australian cv. Teal (Pst susceptible) and AvR were used for DArT-Seq genotypic analysis to map the seedling resistance. Marker-trait association analysis using 9035 DArT-Seq loci mapped the genes to the long arms of chromosomes 3D (3DL) and 5B (5BL), respectively. F2 populations from crosses between susceptible DH lines that carried either the 3DL or 5BL marker genotypes confirmed the complementary gene model. Fluorescence in situ hybridization (FISH) analysis determined that Teal carries a reciprocal T5B-7B translocation. FISH analysis also identified a 5BL chromosomal deletion in Avocet S relative to AvR that further validated the complementary gene model and possibly explained the heterogeneity of closely related wheats carrying the YrA resistance. The individual loci of the complementary YrA resistance were designated Yr73 (3DL) and Yr74 (5BL). Candidate single gene reference stocks will be permanently accessioned following cytological analysis to avoid the T5B-7B translocation.

In barley, gene Rpg5 was first identified for providing resistance to the rye stem rust pathogen (Puccinia graminis f. sp. secalis). A subsequent study determined that Rpg5 is required for rpg4-mediated resistance to the wheat stem rust pathogen (P. graminis f. sp. tritici) including pathotype TTKSK (“Ug99”), which poses a major threat to global wheat and barley production. Based on the effectiveness of Rpg5 against P. graminis f. sp. tritici and P. graminis f. sp. secalis, we assessed whether it also conferred resistance to the oat stem rust pathogen (P. graminis f. sp. avenae). A barley F8 recombinant inbred line (RIL) population was produced by crossing ‘Q21861’ (Rpg1 and Rpg5) with ‘73-G1’ (Rpg1), which is susceptible to P. graminis f. sp. avenae, P. graminis f. sp. secalis, and some pathotypes of P. graminis f. sp. tritici. Seedling tests were performed on the F8 RIL population using Australian pathotypes of P. graminis f. sp. tritici, P. graminis f. sp. secalis, P. graminis f. sp. avenae, and a putative somatic hybrid between P. graminis f. sp. tritici and P. graminis f. sp. secalis known as the ‘Scabrum’ rust. Segregation in the responses to all rust isolates for the RILs was identical (50 resistant: 52 susceptible), and fitted a 1:1 ratio (X2 = 0.039, P = 0.843), indicating that resistance to all isolates was monogenetically inherited. Screening of the RILs and the parental lines with perfect markers for the functional Rpg1 and Rpg5 resistance alleles indicated that Rpg1 was fixed, while Rpg5 was positive in all resistant lines and negative in all susceptible lines. This suggests that different formae speciales of P. graminis may share common effectors, and that the Rpg5 locus confers resistance to both P. graminis f. sp. tritici and P. graminis f. sp. secalis and the heterologous formae speciales of P. graminis, P. graminis f. sp. avenae.

Summary Defensins are a large family of small, cysteine-rich, basic proteins, produced by most plants and plant tissues. They have a primary function in defence against fungal disease, although other functions have been described. This study reports the isolation and characterization of a class I secreted defensin (NaD2) from the flowers of Nicotiana alata, and compares its antifungal activity with the class II defensin (NaD1) from N. alata flowers, which is stored in the vacuole. NaD2, like all other class I defensins, lacks the C-terminal pro-peptide (CTPP) characteristic of class II defensins. NaD2 is most closely related to Nt-thionin from N. tabacum (96% identical) and shares 81% identity with MtDef4 from alfalfa. The concentration required to inhibit in vitro fungal growth by 50% (IC50) was assessed for both NaD1 and NaD2 for the biotrophic basidiomycete fungi Puccinia coronata f. sp. avenae (Pca) and P. sorghi (Ps), the necrotrophic pathogenic ascomycetes Fusarium oxysporum f. sp. vasinfectum (Fov), F. graminearum (Fgr), Verticillium dahliae (Vd) and Thielaviopsis basicola (Tb), and the saprobe Aspergillus nidulans. NaD1 was a more potent antifungal molecule than NaD2 against both the biotrophic and necrotrophic fungal pathogens tested. NaD2 was 5–10 times less effective at killing necrotrophs, but only two-fold less effective on Puccinia species. A new procedure for testing antifungal proteins is described in this study which is applicable to pathogens with spores that are not amenable to liquid culture, such as rust pathogens. Rusts are the most damaging fungal pathogens of many agronomically important crop species (wheat, barley, oats and soybean). NaD1 and NaD2 inhibited urediniospore germination, germ tube growth and germ tube differentiation (appressoria induction) of both Puccinia species tested. NaD1 and NaD2 were fungicidal on Puccinia species and produced stunted germ tubes with a granular cytoplasm. When NaD1 and NaD2 were sprayed onto susceptible oat plants prior to the plants being inoculated with crown rust, they reduced the number of pustules per leaf area, as well as the amount of chlorosis induced by infection. Similar to observations in vitro, NaD1 was more effective as an antifungal control agent than NaD2. Further investigation revealed that both NaD1 and NaD2 permeabilized the plasma membranes of Puccinia spp. This study provides evidence that both secreted (NaD2) and nonsecreted (NaD1) defensins may be useful for broad-spectrum resistance to pathogens.

Crown rust fungus, Puccinia coronata f.sp. lolii is an obligate biotrophic pathogen of ryegrasses which causes significant reductions of herbage yield, palatability and digestibility. Genetic diversity in virulence has been reported in all the major temperate regions of the world and is therefore a major problem for pasture and turf breeders developing varieties with durable resistance for crown rust. Knowledge of the genetic variation present both within and between Australasian crown rust populations is essential for the efficient production of resistant varieties. A total of 11 efficient simple sequence repeat (SSR) markers developed from a urediniospore-derived expressed sequence tag (EST) resource have been used for Australasian intraspecific genetic diversity analysis. Seventy-two single pustule samples comprising three main populations from both North and South Islands of New Zealand and from south-eastern Australia were genotyped. The analysis identified 59 distinct genotypes, high levels of genetic diversity being detected both within and between populations. All methods of analysis detected no significant difference between isolates from the North and South Islands of New Zealand (p > 0.05), and high intrapopulation diversity between Victorian isolates. However high population differentiation (p < 0.001) was detected between Victorian isolates and those from the South Island (PhiPT [estimate of genetic variability = 0.101) and especially the North Islands (PhiPT = 0.162) of New Zealand. Genetic dissection of crown rust population structure within Australasia will inform the magnitude of gene pyramiding required for the development of varieties with durable resistance.

Susceptibility to foliar pathogens commonly causes significant reductions in productivity of the important temperate forage perennial ryegrass. Breeding for durable disease resistance involves not only the deployment of major genes but also the additive effects of minor genes. An approach based on in vitro single nucleotide polymorphism (SNP) discovery in candidate defence response (DR) genes has been used to develop potential diagnostic genetic markers. SNPs were predicted, validated and mapped for representatives of the pathogenesis-related (PR) protein-encoding and reactive oxygen species (ROS)-generating gene classes. The F(1)(NA(6) x AU(6)) two-way pseudo-test cross population was used for SNP genetic mapping and detection of quantitative trait loci (QTLs) in response to a crown rust field infection. Novel resistance QTLs were coincident with mapped DR gene SNPs. QTLs on LG3 and LG7 also coincided with both herbage quality QTLs and candidate genes for lignin biosynthesis. Multiple DR gene SNP loci additionally co-located with QTLs for grey leaf spot, bacterial wilt and crown rust resistance from other published studies. Further functional validation of DR gene SNP loci using methods such as fine-mapping and association genetics will improve the efficiency of parental selection based on superior allele content.

The causative organism of crown rust in ryegrasses (Puccinia coronata f.sp. lolii) is an obligate biotroph that causes significant economic losses within the temperate grazing industries of dairy, meat, and wool production. This study reports on the development, transferability, and utility of gene-associated simple sequence repeat (SSR) molecular markers for crown rust. Analysis of 1100 expressed sequence tag (EST) sequences from a urediniospore-derived cDNA library detected 55 SSR loci. The majority of EST-SSR arrays contained perfect trinucleotide repeats with consistently low repeat numbers, and the motifs (ACC)n and (CAT)n were most commonly represented. DNA extraction from single pustules, in conjunction with multiple displacement amplification, provided the basis for PCR-based screening to evalu- ate genetic marker performance. An example of the identification of intraspecific genetic diversity was obtained from the analysis of 16 P. coronata isolates originating from the United Kingdom, Australia, New Zealand, and Japan. A subset of 12 robust EST-SSR markers was informative for determination of pathogen diversity within and between these localities. It was also demonstrated that crown rust EST-SSR markers were capable of cross-amplification in closely related fungal taxa (Puccinia spp.) and filamentous fungi within the Ascomycota. Resume : L'agent causal de la rouille couronnee chez les ray-grass (P. coronata f. sp. lolii) est un biotrophe obliga- toire qui occasionne des pertes economiques importantes dans les zones temperees de production fourragere pour les fins de production de lait, de viande et de laine. Cette etude rapporte le developpement, la transportabilite et l'utilite de marqueurs microsatellites (SSR) associes a des genes chez la rouille couronnee. L'analyse de 1 100 etiquettes de sequences exprimees (EST) issus d'une banque d'ADNc provenant d'uredospores a decele 55 SSR. La plupart de ces EST-SSR contenaient des repetitions trinucleotidiques parfaites dont le nombre de repetitions etait constamment faible et chez lesquelles les motifs (ACC)n et (CAT)n etaient les plus communs. L'extraction d'ADN de pustules individuels, en conjonction avec une amplification a deplacement multiple (MDA), a rendu possible un crible PCR pour evaluer la performance des marqueurs genetiques. Un exemple de detection de diversite genetique intraspecifique a ete obtenu en analysant 16 isolats du P. coronata provenant du Royaume-Uni, de l'Australie, de la Nouvelle-Zelande et du Japon. Un sous-ensemble de 12 marqueurs EST-SSR robustes s'est avere utilisable chez des taxons fongiques proches (Puccinia spp.) ainsi que chez des champignons filamenteux appartenant aux ascomycetes. Mots cles : Puccinia coronata, microsatellite, etiquette de sequence exprimee, uredospore, diversite genetique, pathogene.

Puccinia coronata (crown rust) is a damaging pathogen of ryegrasses used for both forage and turf applications. Alternative sources of crown rust pathogen resistance with enhanced durability are required for perennial ryegrass breeding programs. This approach may require pyramiding of major and minor resistance gene combinations into varieties, opposing the capacity of evolving pathogen populations to overcome complex resistance mechanisms. Crown rust pathogen populations have been homogenised into distinct lineages on detached leaves through the use of molecular genotyping technology. Subsequent propagation and inoculation of such spore lineages on defined host populations and individuals requires high inoculation efficiency, given the delivery of initially small spore quantities. In this study, an inoculation protocol was developed for effective uniform infection on susceptible host plant genotypes at both seedling and adult developmental stages. A randomised complete block design was used to test the protocol on glasshouse-grown plants from 10 elite perennial ryegrass cultivars. Significant quantitative differences in pustule number and latency period were detected between cultivars, suggesting that the protocol was highly effective at providing a uniform distribution of pustules in low and high urediniospore suspension volumes. The inoculation and quantification methods provide a framework for standardised assessment of crown rust infection under controlled environment conditions, and may in principle be applied to any Puccinia—host interaction.

Background Qualitative pathogen resistance in both dicotyledenous and monocotyledonous plants has been attributed to the action of resistance (R) genes, including those encoding nucleotide binding site – leucine rich repeat (NBS-LRR) proteins and receptor-like kinase enzymes. This study describes the large-scale isolation and characterisation of candidate R genes from perennial ryegrass. The analysis was based on the availability of an expressed sequence tag (EST) resource and a functionally-integrated bioinformatics database. Results Amplification of R gene sequences was performed using template EST data and information from orthologous candidate using a degenerate consensus PCR approach. A total of 102 unique partial R genes were cloned, sequenced and functionally annotated. Analysis of motif structure and R gene phylogeny demonstrated that Lolium R genes cluster with putative ortholoci, and evolved from common ancestral origins. Single nucleotide polymorphisms (SNPs) predicted through resequencing of amplicons from the parental genotypes of a genetic mapping family were validated, and 26 distinct R gene loci were assigned to multiple genetic maps. Clusters of largely non-related NBS-LRR genes were located at multiple distinct genomic locations and were commonly found in close proximity to previously mapped defence response (DR) genes. A comparative genomics analysis revealed the co-location of several candidate R genes with disease resistance quantitative trait loci (QTLs). Conclusion This study is the most comprehensive analysis to date of qualitative disease resistance candidate genes in perennial ryegrass. SNPs identified within candidate genes provide a valuable resource for mapping in various ryegrass pair cross-derived populations and further germplasm analysis using association genetics. In parallel with the use of specific pathogen virulence races, such resources provide the means to identify gene-for-gene mechanisms for multiple host pathogen-interactions and ultimately to obtain durable field-based resistance.

Genetic analysis of host resistance to the crown rust pathogen of ryegrasses has traditionally involved the use of composite urediniospore collections, which may contain multiple genotypes and virulence races. Consequently, discrimination between major gene responses to alternative races and quantitative resistance has proved challenging for disease trait-dissection studies.Amethod of propagation on detached leaves has been used to derive genetically homogenised crown rust pathogen lines from bulked spore collections obtained from five distinct Australian locations. Spores from individual pustules were repeatedly reinoculated onto fresh leaves. Amplicon complexity was assessed at the conclusion of each cycle using crown rust pathogen expressed sequence tag (EST)-derived simple sequence repeat (SSR) primer pairs. The mean number of amplification products per isolate declined over successive rounds of reinfection. The number of rounds of sequential infection required for decay to stable levels was dependent on the diversity present within the bulked spore collection. Large-scale propagation of the putative homogenised populations developed in the present study may provide inoculum for infection studies to allow quantitative trait loci mapping of specific gene-for-gene responses. The methodology has significant applications for the pyramiding of resistance genes specific to diverse crown rust pathogen genotypes during cultivar development and for pathotyping of existing elite germplasm. This approach is also applicable to other Puccinia species.

Background Barley is an important cereal crop cultivated for malt and ruminant feed and in certain regions it is used for human consumption. It is vulnerable to numerous foliar diseases including barley leaf rust caused by the pathogen Puccinia hordei. Results A temporarily designated resistance locus RphCantala (RphC) identified in the Australian Hordeum vulgare L. cultivar ‘Cantala’ displayed an intermediate to low infection type (“;12 = N”) against the P. hordei pathotype 253P- (virulent on Rph1, Rph2, Rph4, Rph6, Rph8 and RphQ). Phenotypic assessment of a ‘CI 9214’ (susceptible) x ‘Stirling’ (RphC) (CI 9214/Stirling) doubled haploid (DH) population at the seedling stage using P. hordei pathotype 253P-, confirmed that RphC was monogenically inherited. Marker-trait association analysis of RphC in the CI 9214/Stirling DH population using 4,500 DArT-seq markers identified a highly significant (−log10Pvalue > 17) single peak on the long arm of chromosome 5H (5HL). Further tests of allelism determined that RphC was genetically independent of Rph3, Rph7, Rph11, Rph13 and Rph14, and was an allele of Rph12 (Rph9.z), which also maps to 5HL. Conclusion Multipathotype tests and subsequent pedigree analysis determined that 14 related Australian barley varieties (including ‘Stirling’ and ‘Cantala’) carry RphC and that the likely source of this resistance is via a Czechoslovakian landrace LV-Kvasice-NA-Morave transferred through common ancestral cultivars ‘Hanna’ and ‘Abed Binder’. RphC is an allele of Rph12 (Rph9.z) and is therefore designated Rph9.am. Bioinformatic analysis using sequence arrays from DArT-seq markers in linkage disequilibrium with Rph9.am identified possible candidates for further gene cloning efforts and marker development at the Rph9/Rph12/Rph9.am locus. Electronic supplementary material The online version of this article (doi:10.1186/s12870-014-0382-4) contains supplementary material, which is available to authorized users.