Expression patterns of candidate genes for the Lr46/Yr29 "slow rust" locus in common wheat (Triticum aestivum L.) and associated miRNAs inform of the gene conferring the Puccinia triticina resistance trait.

Leaf rust caused by Puccinia triticina (Pt) is one of the most impactful diseases causing substantial losses in common wheat (Triticum aestivum L.) crops. In adult plants resistant to Pt, a horizontal adult plant resistance (APR) is observed: APR protects the plant against multiple pathogen races and is distinguished by durable persistence under production conditions. The Lr46/Yr29 locus was mapped to chromosome 1B of common wheat genome, but the identity of the underlying gene has not been demonstrated although several candidate genes have been proposed. This study aimed to analyze the expression of nine candidate genes located at the Lr46/Yr29 locus and their four complementary miRNAs (tae-miR5384-3p, tae-miR9780, tae-miR9775, and tae-miR164), in response to Pt infection. The plant materials tested included five reference cultivars in which the molecular marker csLV46G22 associated with the Lr46/Yr29-based Pt resistance was identified, as well as one susceptible control cultivar. Biotic stress was induced in adult plants by inoculation with fungal spores under controlled conditions. Plant material was sampled before and at 6, 12, 24, 48 hours post inoculation (hpi). Differences in expression of candidate genes at the Lr46/Yr29 locus were analyzed by qRT-PCR and showed that the expression of the genes varied at the analyzed time points. The highest expression of Lr46/Yr29 candidate genes (Lr46-Glu1, Lr46-Glu2, Lr46-Glu3, Lr46-RLK1, Lr46-RLK2, Lr46-RLK3, Lr46-RLK4, Lr46-Snex, and Lr46-WRKY) occurred at 12 and 24 hpi and such expression profiles were obtained only for one candidate gene among the nine genes analyzed (Lr46-Glu2), indicating that it may be a contributing factor in the resistance response to Pt infection.

Mapping rust resistance in European winter wheat: many QTLs for yellow rust resistance, but only a few well characterized genes for stem rust resistance.

KEY MESSAGE: Stem rust resistance was mainly based on a few, already known resistance genes; for yellow rust resistance there was a combination of designated genes and minor QTLs. Yellow rust (YR) caused by Puccinia striiformis f. sp. tritici (Pst) and stem rust (SR) caused by Puccinia graminis f. sp. tritici (Pgt) are among the most damaging wheat diseases. Although, yellow rust has occurred regularly in Europe since the advent of the Warrior race in 2011, damaging stem rust epidemics are still unusual. We analyzed the resistance of seven segregating populations at the adult growth stage with the parents being selected for YR and SR resistances across three to six environments (location-year combinations) following inoculation with defined Pst and Pgt races. In total, 600 progenies were phenotyped and 563 were genotyped with a 25k SNP array. For SR resistance, three major resistance genes (Sr24, Sr31, Sr38/Yr17) were detected in different combinations. Additional QTLs provided much smaller effects except for a gene on chromosome 4B that explained much of the genetic variance. For YR resistance, ten loci with highly varying percentages of explained genetic variance (pG, 6-99%) were mapped. Our results imply that introgression of new SR resistances will be necessary for breeding future rust resistant cultivars, whereas YR resistance can be achieved by genomic selection of many of the detected QTLs.

A Putative Effector Pst-18220, from Puccinia striiformis f. sp. tritici, Participates in Rust Pathogenicity and Plant Defense Suppression.

Stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), stands out as one of the most devastating epidemics impacting wheat production worldwide. Resistant wheat varieties had swiftly been overcome due to the emergence of new virulent Pst strains. Effectors secreted by Pst interfere with plant immunity, and verification of their biological function is extremely important for controlling wheat stripe rust. In this study, we identified an effector, Pst-18220, from Puccinia striiformis f. sp. tritici (Pst), which was induced during the early infection stage of Pst. Silencing the expression of Pst-18220 through virus-mediated host-induced gene silencing (HIGS) resulted in a decreased number of rust pustules. In Nicotiana benthamiana, it significantly suppressed cell death induced by Pseudomonas syringae pv. tomato (Pto) DC3000. In Arabidopsis, plants with stable overexpression of Pst-18220 showed increased susceptibility to Pto DC3000, accompanied by a decrease in the expression level of pattern-triggered immunity (PTI)/effector-triggered immunity (ETI)-related genes, namely, AtPCRK1, AtPCRK2, and AtBIK1. These results emphasize the significant role of the Pst candidate effector, Pst-18220, in rust pathogenicity and the suppression of plant defense mechanisms. This broadens our understanding of effectors without any known motif.

Comprehensive analysis of hairpin small RNAs involved in resistance and pathogenesis during wheat-Puccinia triticina interactions.

Wheat leaf rust, caused by the fungus Puccinia triticina (Pt), severely affects the grain quality and quantity of bread wheat (Triticum aestivum L.). Hairpin small(s)RNAs, like micro(mi)RNAs and their variants [including isomiRNAs (isomiRs) and microRNA-like RNAs (milRNAs)], along with their corresponding target genes, bestow leaf rust disease resistance, development and progression from both interacting species. However, the regulatory networks remain inadequately understood. Thirteen differentially expressed novel miRNAs, including two isomiRs and three milRNAs were discerned from induced reads of wheat sRNA libraries, and a further 5,393 and 1,275 candidate target genes were predicted in wheat and Pt, respectively. Functional annotation divulged that wheat-originated miRNAs/isomiRs were involved in resistance, while Pt-derived milRNAs imparted pathogenesis. The identified milRNAs- Tae-Pt-milR5, Tae-Pt-milR12, and Tae-Pt-milR14b and their cleavage sites on Pt target gene MEP5 were confirmed through degradome library screening, suggesting cross-kingdom translocation of Pt virulent genes in wheat host. Co-expression analysis of miRNAs/isomiRs-target genes provided insights into combating leaf rust disease, while co-expression analysis of milRNAs-target gene pairs reflected the extent of pathogenicity exerted by Pt with varied expression levels at the analyzed time points. The analysis pinpointed leaf rust-responsive candidate hairpin sRNAs- Tae-miR8, Tae-Pt-miR12, Tae-Pt-miR14a, and Tae-Pt-miR14b in wheat and Tae-Pt-milR12 in Pt. This study provides new insights into the hairpin sRNAs involved in the resistance and pathogenesis of wheat and Pt, respectively. Furthermore, crucial hairpin sRNAs and their promising targets for future biotechnological interventions to augment stress resilience have been identified.

Identification and functional characterization of the npc-2-like domain containing rust effector protein that suppresses cell death in plants.

The MD-2-related lipid-recognition (ML/Md-2) domain is a lipid/sterol-binding domain that are involved in sterol transfer and innate immunity in eukaryotes. Here we report a genome-wide survey of this family, identifying 84 genes in 30 fungi including plant pathogens. All the studied species were found to have varied ML numbers, and expansion of the family was observed in Rhizophagus irregularis (RI) with 33 genes. The molecular docking studies of these proteins with cholesterol derivatives indicate lipid-binding functional conservation across the animal and fungi kingdom. The phylogenetic studies among eukaryotic ML proteins showed that Puccinia ML members are more closely associated with animal (insect) npc2 proteins than other fungal ML members. One of the candidates from leaf rust fungus Puccinia triticina, Pt5643 was PCR amplified and further characterized using various studies such as qRT-PCR, subcellular localization studies, yeast functional complementation, signal peptide validation, and expression studies. The Pt5643 exhibits the highest expression on the 5th day post-infection (dpi). The confocal microscopy of Pt5643 in onion epidermal cells and N. benthamiana shows its location in the cytoplasm and nucleus. The functional complementation studies of Pt5643 in npc2 mutant yeast showed its functional similarity to the eukaryotic/yeast npc2 gene. Furthermore, the overexpression of Pt5643 also suppressed the BAX, NEP1, and H2O2-induced program cell death in Nicotiana species and yeast. Altogether the present study reports the novel function of ML domain proteins in plant fungal pathogens and their possible role as effector molecules in host defense manipulation.

Puccinia triticina avirulence protein AvrLr21 directly interacts with wheat resistance protein Lr21 to activate wheat immune response.

Wheat leaf rust, caused by Puccinia triticina (Pt), remains a constant threat to wheat production worldwide. Deployment of race-specific leaf rust (Lr) resistance genes in wheat provides effective protection against leaf rust, but often leads to selective pressures that drive the rapid emergence of new virulent Pt isolates in nature. However, the molecular mechanisms underlying the evasion of Lr-delivered resistance by leaf rust remain largely unknown. Here, we identify an avirulence gene AvrLr21 in Pt that triggers Lr21-dependent immune responses. BSMV (Barley stripe mosaic virus)-mediated host-induced gene silencing assay shows that silencing AvrLr21 compromises Lr21-mediated immunity. AvrLr21 interacts directly with Lr21 protein to induce a hypersensitive response in tobacco leaves. The evolved Lr21-breaking Pt isolates can suppress Lr21-mediated immunity. Our data provide a basis for studying the molecular determinants in Pt-wheat incompatible interaction and monitoring natural Pt populations to prioritize the deployment of Lr resistance genes in the field.

Translationally controlled tumor protein interacts with TaCIPK23 to positively regulate wheat resistance to Puccinia triticina.

Hypersensitive response-programmed cell death (HR-PCD) regulated by Ca2+ signal is considered the major regulator of resistance against Puccinia triticina (Pt.) infection in wheat. In this study, the bread wheat variety Thatcher and its near-isogenic line with the leaf rust resistance locus Lr26 were infected with the Pt. race 260 to obtain the compatible and incompatible combinations, respectively. The expression of translationally controlled tumor protein (TaTCTP) was upregulated upon infection with Pt., through a Ca2+-dependent mechanism in the incompatible combination. The knockdown of TaTCTP markedly increased the area of dying cell and the number of Pt. haustorial mother cells (HMCs) at the infection sites, whereas plants overexpressing the gene exhibited enhanced resistance. The interaction between TaTCTP and calcineurin B-like protein-interacting protein kinase 23 (TaCIPK23) was also investigated, and the interaction was found occurred in the endoplasmic reticulum. TaCIPK23 phosphorylated TaTCTP in vitro. The expression of a phospho-mimic TaTCTP mutant in Nicotiana benthamiana promoted HR-like cell death. Silencing TaCIPK23 or TaCIPK23/TaTCTP co-silencing resulted in the same results as silencing TaTCTP. This suggested that TaTCTP is a novel phosphorylation target of TaCIPK23, and both participate in the resistance of wheat to Pt. in the same pathway.

Virulent Effector Hasp155 of Puccinia striiformis f. sp. tritici Suppresses Plant Immunity and Promotes Fungus Infection.

As a kind of obligate biotrophic fungus, Puccinia striiformis f. sp. tritici (Pst) secretes vast effectors via haustoria to host cells during the infection to inhibit host defense responses and promote fungal invasion. In this study, based on the completion of genome sequencing and haustorial transcriptome sequencing of Pst, we identified a Pst effector (Hasp155) that is significantly induced in the early stage of Pst infection to wheat. The 18 N-terminal amino acids of Hasp155 encoded a signal peptide with a secretory function. Transient expression of Hasp155 in Nicotiana benthamiana inhibited Bax-induced cell death as well as chitin-triggered callose deposition and defense-related gene expression. Moreover, delivery of the Hasp155 protein into wheat cells via type three secretion systems (TTSS) led to reduced plant immunity to nonpathogenic bacteria and to the avirulent Pst race with decreased H2O2 accumulation and promoted Pst development. Furthermore, transgenic overexpression of Hasp155 significantly renders wheat resistance susceptible, resulting in a decreased defense response and increased Pst pathogenicity. Overall, these results indicate that Hasp155 is an important effector of Pst pathogenicity by suppressing plant immunity.

Identification of stripe rust resistance gene YrBDT in Chinese landrace wheat Baidatou using BSE-seq and BSR-seq.

KEY MESSAGE: A new stripe rust resistance gene YrBDT in Chinese landrace wheat Baidatou was mapped to a 943.6-kb interval on chromosome arm 6DS and co-segregated with a marker CAPS3 developed from candidate gene TraesCS6D03G0027300. Stripe rust caused by Puccinia striiformis f. sp. tritici (Pst) is a devastating foliar disease of wheat. Chinese landrace wheat Baidatou has shown high resistance to a broad spectrum of Pst races at both the seedling and adult-plant stages for decades in the Longnan region of Gansu province, a hot spot for stripe rust epidemics. Here, we report fine mapping and candidate gene analysis of stripe rust resistance gene YrBDT in Baidatou. Analysis of F1, F2 plants and F2:3 lines indicated that resistance in Baidatou to Pst race CYR31 was conferred by a single dominant gene, temporarily designated YrBDT. Bulked segregant exome capture sequencing (BSE-seq) analysis revealed 61 high-confidence polymorphic SNPs concentrated in a 5.4-Mb interval at the distal of chromosome arm 6DS. Several SNPs and InDels were also identified by genome mining of DNA sampled from the parents and contrasting bulks. The YrBDT locus was mapped to a 943.6-kb (4,658,322-5,601,880 bp) genomic region spanned by markers STS2 and STS3 based on IWGSC RefSeq v2.1, including five putative disease resistance genes. There was high collinearity of the target interval among Chinese Spring RefSeq v2.1, Ae. tauschii AL8/78 and Fielder genomes. The expression level of TraesCS6D03G0027300 showed significant association with Pst infection, and a gene-specific marker CAPS3 developed from TraesCS6D03G0027300 co-segregated with YrBDT suggesting this gene as a candidate of YrBDT. The resistance gene and flanking markers can be used in marker-assisted selection for improvement of stripe rust resistance.

Fine mapping of stem rust resistance derived from soft red winter wheat cultivar AGS2000 to an NLR gene cluster on chromosome 6D.

The Puccinia graminis f. sp. tritici (Pgt) Ug99-emerging virulent races present a major challenge to global wheat production. To meet present and future needs, new sources of resistance must be found. Identification of markers that allow tracking of resistance genes is needed for deployment strategies to combat highly virulent pathogen races. Field evaluation of a DH population located a QTL for stem rust (Sr) resistance, QSr.nc-6D from the breeding line MD01W28-08-11 to the distal region of chromosome arm 6DS where Sr resistance genes Sr42, SrCad, and SrTmp have been identified. A locus for seedling resistance to Pgt race TTKSK was identified in a DH population and an RIL population derived from the cross AGS2000 × LA95135. The resistant cultivar AGS2000 is in the pedigree of MD01W28-08-11 and our results suggest that it is the source of Sr resistance in this breeding line. We exploited published markers and exome capture data to enrich marker density in a 10 Mb region flanking QSr.nc-6D. Our fine mapping in heterozygous inbred families identified three markers co-segregating with resistance and delimited QSr.nc-6D to a 1.3 Mb region. We further exploited information from other genome assemblies and identified collinear regions of 6DS harboring clusters of NLR genes. Evaluation of KASP assays corresponding to our co-segregating SNP suggests that they can be used to track this Sr resistance in breeding programs. However, our results also underscore the challenges posed in identifying genes underlying resistance in such complex regions in the absence of genome sequence from the resistant genotypes.

Association mapping with a diverse population of Puccinia graminis f. sp. tritici identified avirulence loci interacting with the barley Rpg1 stem rust resistance gene.

BACKGROUND: Wheat stem rust, caused by Puccinia graminis f. sp. tritici (Pgt), is an important disease of barley and wheat. A diverse sexual Pgt population from the Pacific Northwest (PNW) region of the US contains a high proportion of individuals with virulence on the barley stem rust resistance (R) gene, Rpg1. However, the evolutionary mechanisms of this virulence on Rpg1 are mysterious considering that Rpg1 had not been deployed in the region and the gene had remained remarkably durable in the Midwestern US and prairie provinces of Canada. METHODS AND RESULTS: To identify AvrRpg1 effectors, genome wide association studies (GWAS) were performed using 113 Pgt isolates collected from the PNW (n = 89 isolates) and Midwest (n = 24 isolates) regions of the US. Disease phenotype data were generated on two barley lines Morex and the Golden Promise transgenic (H228.2c) that carry the Rpg1 gene. Genotype data was generated by whole genome sequencing (WGS) of 96 isolates (PNW = 89 isolates and Midwest = 7 isolates) and RNA sequencing (RNAseq) data from 17 Midwestern isolates. Utilizing ~1.2 million SNPs generated from WGS and phenotype data (n = 96 isolates) on the transgenic line H228.2c, 53 marker trait associations (MTAs) were identified. Utilizing ~140 K common SNPs generated from combined analysis of WGS and RNAseq data, two significant MTAs were identified using the cv Morex phenotyping data. The 55 MTAs defined two distinct avirulence loci, on supercontig 2.30 and supercontig 2.11 of the Pgt reference genome of Pgt isolate CRL 75-36-700-3. The major avirulence locus designated AvrRpg1A was identified with the GWAS using both barley lines and was delimited to a 35 kb interval on supercontig 2.30 containing four candidate genes (PGTG_10878, PGTG_10884, PGTG_10885, and PGTG_10886). The minor avirulence locus designated AvrRpg1B identified with cv Morex contained a single candidate gene (PGTG_05433). AvrRpg1A haplotype analysis provided strong evidence that a dominant avirulence gene underlies the locus. CONCLUSIONS: The association analysis identified strong candidate AvrRpg1 genes. Further analysis to validate the AvrRpg1 genes will fill knowledge gaps in our understanding of rust effector biology and the evolution and mechanism/s of Pgt virulence on Rpg1.

Evaluation of effectiveness resistance genes in wheat genotypes using marker-assisted selection for stripe rust resistance breeding".

Stripe rust, induced by Puccinia striiformis f. sp. tritici, is the most harmful and prevalent disease in temperate regions worldwide, affecting wheat production areas globally. An effective strategy for controlling the disease involves enhancing genetic resistance against stripe rust, achieved through Egyptian breeding efforts not previously conducted on wheat genotypes. The resistance level to stripe rust in thirty-eight wheat genotypes was assessed using marker-assisted selection methods. The investigation suggests that wheat breeding programs can utilize slow-rusting Yr genes, which are effective resistance genes, to develop novel genotypes with stripe rust resistance through marker-assisted breeding. Based on the four disease responses of the wheat genotypes under investigation, the results categorized the genotypes into three groups. The first group included resistant genotypes, the second group exhibited a slow-rusting character with the lowest disease symptom rates, and the last group displayed the highest disease characteristics rates throughout the three seasons, comprising fast-rusting genotypes. The rust-resistant genes identified were Yr5, Yr9, Yr10, Yr15, Yr17, Yr18, Yr26, Yr29, Yr30, and Yr36. Genes Yr26, Yr30, and Yr36 were present in all genotypes. Genotypes Misr3, Misr4, Giza168, Giza167, Giza170, Giza171, Gemmeiza9, and Gemmeiza10 carried the Yr9 gene. Only one genotype, Sids13, was found to have the Yr17 gene. Genes Yr18 and Yr29 were identified in Sids14, Giza168, Giza170, Gemmeiza9, and Gemmeiza10. However, none of the wheat genotypes showed the presence of Yr5, Yr10, or Yr15. Several backcrossing generations were conducted to introduce the Yr5 and Yr10 genes into susceptible genotypes (Misr1, Misr2, and Gemmeiza11). These genotypes are cultivated globally and are known for producing high-quality flour, making them of great importance to farmers. The study demonstrates significant potential for enhancing wheat genotypes for stripe rust resistance and increased production.

Isocitrate lyase promotes Puccinia striiformis f. sp. tritici susceptibility in wheat (Triticum aestivum) by suppressing accumulation of glyoxylate cycle intermediates.

Plant fungal parasites manipulate host metabolism to support their own survival. Among the many central metabolic pathways altered during infection, the glyoxylate cycle is frequently upregulated in both fungi and their host plants. Here, we examined the response of the glyoxylate cycle in bread wheat (Triticum aestivum) to infection by the obligate biotrophic fungal pathogen Puccinia striiformis f. sp. tritici (Pst). Gene expression analysis revealed that wheat genes encoding the two unique enzymes of the glyoxylate cycle, isocitrate lyase (TaICL) and malate synthase, diverged in their expression between susceptible and resistant Pst interactions. Focusing on TaICL, we determined that the TaICL B homoeolog is specifically upregulated during early stages of a successful Pst infection. Furthermore, disruption of the B homoeolog alone was sufficient to significantly perturb Pst disease progression. Indeed, Pst infection of the TaICL-B disruption mutant (TaICL-BY400*) was inhibited early during initial penetration, with the TaICL-BY400* line also accumulating high levels of malic acid, citric acid, and aconitic acid. Exogenous application of malic acid or aconitic acid also suppressed Pst infection, with trans-aconitic acid treatment having the most pronounced effect by decreasing fungal biomass 15-fold. Thus, enhanced TaICL-B expression during Pst infection may lower accumulation of malic acid and aconitic acid to promote Pst proliferation. As exogenous application of aconitic acid and malic acid has previously been shown to inhibit other critical pests and pathogens, we propose TaICL as a potential target for disruption in resistance breeding that could have wide-reaching protective benefits for wheat and beyond.

Evaluation of genetic variation and host resistance to wheat stem rust pathogen (Puccinia graminis f. sp. tritici) in bread wheat (Triticum aestivum L.) varieties grown in Türkiye.

Wheat stem rust, which is caused by Puccinia graminis f. sp. tritici (Pgt), is a highly destructive disease that affects wheat crops on a global scale. In this study, the reactions of 150 bread wheat varieties were evaluated for natural Pgt infection at the adult-plant stage in the 2019-2020 and 2020-2021 growing seasons, and they were analyzed using specific molecular markers to detect stem rust resistance genes (Sr22, Sr24, Sr25, Sr26, Sr31, Sr38, Sr50, and Sr57). Based on phenotypic data, the majority of the varieties (62%) were resistant or moderately resistant to natural Pgt infection. According to molecular results, it was identified that Sr57 was present in 103 varieties, Sr50 in nine varieties, Sr25 in six varieties, and Sr22, Sr31, and Sr38 in one variety each. Additionally, their combinations Sr25 + Sr50, Sr31 + Sr57, Sr38 + Sr50, and Sr38 + Sr57 were detected in these varieties. On the other hand, Sr24 and Sr26 were not identified. In addition, many varieties had low stem rust scores, including a large minority that lacked Sr57. These varieties must have useful resistance to stem rust and could be the basis for selecting greater, possibly durable resistance.

Evasion of wheat resistance gene Lr15 recognition by the leaf rust fungus is attributed to the coincidence of natural mutations and deletion in AvrLr15 gene.

Employing race-specific resistance genes remains an effective strategy to protect wheat from leaf rust caused by Puccinia triticina (Pt) worldwide, while the newly emerged Pt races, owing to rapid genetic evolution, frequently overcome the immune response delivered by race-specific resistance genes. The molecular mechanisms underlying the newly evolved virulence Pt pathogen remain unknown. Here, we identified an avirulence protein AvrLr15 from Pt that induced Lr15-dependent immune responses. Heterologously produced AvrLr15 triggered pronounced cell death in Lr15-isogenic wheat leaves. AvrLr15 contains a functional signal peptide, localized to the plant nucleus and cytosol and can suppress BAX-induced cell death. Evasion of Lr15-mediated resistance in wheat was associated with a deletion and point mutations of amino acids in AvrLr15 rather than AvrLr15 gene loss in the Lr15-breaking Pt races, implying that AvrLr15 is required for the virulence function of Pt. Our findings identified the first molecular determinant of wheat race-specific immunity and facilitated the identification of the first AVR/R gene pair in the Pt-wheat pathosystem, which will provide a molecular marker to monitor natural Pt populations and guide the deployment of Lr15-resistant wheat cultivars in the field.

Genome wide association study of seedling and adult plant leaf rust resistance in two subsets of barley genetic resources.

Leaf rust (LR) caused by Puccinia hordei is a serious disease of barley worldwide, causing significant yield losses and reduced grain quality. Discovery and incorporation of new sources of resistance from gene bank accessions into barley breeding programs is essential for the development of leaf rust resistant varieties. To identify Quantitative Trait Loci (QTL) conferring LR resistance in the two barley subsets, the Generation Challenge Program (GCP) reference set of 142 accessions and the leaf rust subset constructed using the Focused Identification of Germplasm Strategy (FIGS) of 76 barley accessions, were genotyped to conduct a genome-wide association study (GWAS). The results revealed a total of 59 QTL in the 218 accessions phenotyped against barley leaf rust at the seedling stage using two P. hordei isolates (ISO-SAT and ISO-MRC), and at the adult plant stage in four environments in Morocco. Out of these 59 QTL, 10 QTL were associated with the seedling resistance (SR) and 49 QTL were associated with the adult plant resistance (APR). Four QTL showed stable effects in at least two environments for APR, whereas two common QTL associated with SR and APR were detected on chromosomes 2H and 7H. Furthermore, 39 QTL identified in this study were potentially novel. Interestingly, the sequences of 27 SNP markers encoded the candidate genes (CGs) with predicted protein functions in plant disease resistance. These results will provide new perspectives on the diversity of leaf rust resistance loci for fine mapping, isolation of resistance genes, and for marker-assisted selection for the LR resistance in barley breeding programs worldwide.

Combined Genome-Wide Association Study and Transcriptome Analysis Reveal Candidate Genes for Resistance to Rust (Puccinia graminis) in Dactylis glomerata.

Rust disease is a common plant disease that can cause wilting, slow growth of plant leaves, and even affect the growth and development of plants. Orchardgrass (Dactylis glomerata L.) is native to temperate regions of Europe, which has been introduced as a superior forage grass in temperate regions worldwide. Orchardgrass has rich genetic diversity and is widely distributed in the world, which may contain rust resistance genes not found in other crops. Therefore, we collected a total of 333 orchardgrass accessions from different regions around the world. Through a genome-wide association study (GWAS) analysis conducted in four different environments, 91 genes that overlap or are adjacent to significant single nucleotide polymorphisms (SNPs) were identified as potential rust disease resistance genes. Combining transcriptome data from susceptible (PI292589) and resistant (PI251814) accessions, the GWAS candidate gene DG5C04160.1 encoding glutathione S-transferase (GST) was found to be important for orchardgrass rust (Puccinia graminis) resistance. Interestingly, by comparing the number of GST gene family members in seven species, it was found that orchardgrass has the most GST gene family members, containing 119 GST genes. Among them, 23 GST genes showed significant differential expression after inoculation with the rust pathogen in resistant and susceptible accessions; 82% of the genes still showed significantly increased expression 14 days after inoculation in resistant accessions, while the expression level significantly decreased in susceptible accessions. These results indicate that GST genes play an important role in orchardgrass resistance to rust (P. graminis) stress by encoding GST to reduce its oxidative stress response.

Mapping of Aegilops speltoides derived leaf rust and stripe rust resistance genes using 35K SNP array.

Wheat is an essential food commodity cultivated throughout the world. However, this crop faces continuous threats from fungal pathogens, leaf rust (LR) and stripe rust (YR). To continue feeding the growing population, these major destructors of wheat must be effectively countered by enhancing the genetic diversity of cultivated germplasm. In this study, an introgression line with hexaploid background (ILsp3603) carrying resistance against Pt pathotypes 77-5 (121R63-1), 77-9 (121R60-1) and Pst pathotypes 46S119 (46E159), 110S119 (110E159), 238S119 (238E159) was developed from donor wheat wild progenitor, Aegilops speltoides acc pau 3603. To understand the genetic basis of resistance and map these genes (named Lrsp3603 and Yrsp3603), inheritance studies were carried out in F6 and F7 mapping population, developed by crossing ILsp3603 with LR and YR susceptible cultivar WL711, which revealed a monogenic (single gene) inheritance pattern for each of these traits. Bulk segregant analysis combined with 35 K Axiom SNP array genotyping mapped both genes as separate entities on the short arm of chromosome 6B. A genetic linkage map, comprising five markers, 1 SNP, 1 PLUG and three gene based SSRs, covered a genetic distance of 12.65 cM. Lrsp3603 was flanked by markers Tag-SSR14 (located proximally at 2.42 cM) and SNP AX-94542331 (at 3.28 cM) while Yrsp3603 was mapped at one end closest to AX-94542331 at 6.62 cM distance. Functional annotation of Lrsp3603 target region (∼ 1 Mbp) revealed 10 gene IDs associated with disease resistance mechanisms including three encoding typical R gene domains.

Development and identification of a novel wheat-Thinopyrum ponticum disomic substitution line DS5Ag(5D) with new genes conferring resistance to powdery mildew and leaf rust.

BACKGROUND: Powdery mildew (caused by Blumeria graminis f. sp. tritici (Bgt)) and leaf rust (caused by Puccinia triticina (Pt)) are prevalent diseases in wheat (Triticum aestivum L.) production. Thinopyrum ponticum (2n = 10x = 70, EeEeEbEbExExStStStSt) contains genes that confer high levels of resistance to these diseases. RESULTS: An elite wheat-Th. ponticum disomic substitution line, DS5Ag(5D), was developed in the Bainong Aikang 58 (AK58) background. The line was assessed using genomic in situ hybridization (GISH), oligo-nucleotide probe multiplex (ONPM) fluorescence in situ hybridization (FISH), and molecular markers. Twenty eight chromosome-specific molecular markers were identified for the alien chromosome, and 22 of them were co-dominant. Additionally, SNP markers from the wheat 660 K SNP chip were utilized to confirm chromosome identification and they provide molecular tools for tagging the chromosome in concern. The substitution line demonstrated high levels of resistance to powdery mildew throughout its growth period and to leaf rust at the adult stage. Based on the resistance evaluation of five F5 populations between the substitution lines and wheat genotypes with different levels of sensitivity to the two diseases. Results showed that the resistance genes located on 5Ag confered stable resistance against both diseases across different backgrounds. Resistance spectrum analysis combined with diagnostic marker detection of known resistance genes of Th. ponticum revealed that 5Ag contained two novel genes, Pm5Ag and Lr5Ag, which conferred resistance to powdery mildew and leaf rust, respectively. CONCLUSIONS: In this study, a novel wheat-Th. ponticum disomic substitution line DS5Ag(5D) was successfully developed. The Th. ponticum chromosome 5Ag contain new resistance genes for powdery mildew and leaf rust. Chromosomic-specific molecular markers were generated and they can be used to track the 5Ag chromosome fragments. Consequently, this study provides new elite germplasm resources and molecular markers to facilitate the breeding of wheat varieties that is resistant to powdery mildew and leaf rust.

Characterization of Quantitative Trait Loci for Leaf Rust Resistance from CI 13227 in Three Winter Wheat Populations.

Leaf rust is a widespread foliar wheat disease causing substantial yield losses worldwide. Slow rusting is "adult plant" resistance that significantly slows epidemic development and thereby reduces yield loss. Wheat accession CI 13227 was previously characterized as having slow-rusting resistance. To validate the quantitative trait loci (QTLs) and develop diagnostic markers for slow rusting resistance in CI 13227, a new population of recombinant inbred lines of CI 13227 × Everest was evaluated for latent period, final severity, area under the disease progress curve, and infection type in greenhouses and genotyped using genotyping-by-sequencing. Four QTLs were identified on chromosome arms 2BL, 2DS, 3BS, and 7BL, explaining 6.82 to 28.45% of the phenotypic variance for these traits. Seven kompetitive allele-specific polymorphism markers previously reported to be linked to the QTLs in two other CI 13227 populations were validated. In addition, the previously reported QLr.hwwg-7AL was remapped to 2BL (renamed QLr.hwwg-2BL) after adding new markers in this study. Phenotypic data showed that the recombinant inbred lines harboring two or three of the QTLs had a significantly longer latent period. QLr.hwwg-2DS on 2DS showed a major effect on all rust resistance traits and was finely mapped to a 2.7-Mb interval by two newly developed flanking markers from exome capture. Three disease-resistance genes and two transporter genes were identified as the putative candidates for QLr.hwwg-2DS. The validated QTLs can be used as slow-rusting resistance resources, and the markers developed in this study will be useful for marker-assisted selection.