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.

CONCLUSIONS: 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.

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.
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.

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.

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.

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.

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.

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.

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.

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.