Screening global wheat germplasm with a diverse collection of pathogen races expands the catalog of novel Yr loci and identifies new sources of broad-spectrum resistance against evolving Pst populations. Newly emerging highly virulent races of Puccinia striiformis f. sp. tritici (Pst) often defeat deployed resistance genes (Yr), highlighting the need for novel sources of durable resistance. A global diversity panel of 377 spring wheat (Triticum aestivum L.) lines was screened for all-stage resistance (ASR) against a panel of diverse 20 Pst isolates at the seedling stage and for adult-plant-stage resistance (APR) against natural mix of field races. Genome-wide association mapping identified 77 unique Yr loci. Of these, 34 overlapped with the previously mapped 1150 Yr loci, confirming the robustness of our GWAS results, while 43 were likely novel. Comparison of the nine adult-plant-stage Yr loci mapped in our study with known APR genes identified only one overlap, with Yr29. Except for Yr29, APR genes Yr18 and Yr36 were detected at low frequencies, indicating that resistance in our panel may arise from less characterized or novel sources. Two wheat lines, lacking widely effective Yr5 and Yr15 alleles, exhibited resistance to all 20 Pst races at the seedling stage and natural field races at the adult stage, suggesting that they may carry novel, broad-spectrum ASR alleles. Wheat improvement had no effect on the frequency of ASR alleles but resulted in a threefold increase in the frequency of APR alleles, suggesting that the latter were subjected to more consistent breeding selection over time. Our findings underscore the value of combined screening of diverse germplasm with diverse pathogen races to identify novel sources of broad-spectrum resistance for breeding stripe rust resistant cultivars.

Stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is a devastating wheat disease worldwide, and novel stripe rust resistance genes that can be readily used in wheat breeding are needed for sustainable wheat production. Winter wheat cultivar Samara exhibits high resistance to predominant Pst races in the United States. An F 2:3 population and an F 7 RIL population derived from the cross Samara × Jagalene were evaluated for responses to Pst race PSTv-37 at the seedling stage, leading to the conclusion that a single dominant gene conditions stripe rust resistance in Samara. A subset of F 2 plants was genotyped using genotyping-by-sequencing (GBS), and allele distribution analyses at each GBS-SNP locus revealed a genomic region harboring a stripe rust resistance gene, temporarily designated YrSA, in the terminal region of chromosome arm 2AL in Samara. Some GBS-SNPs in the target region were converted to KASP markers and linkage analysis delimited YrSA to an interval of 1.65 Mb between 784.71 Mb and 786.36 Mb in the Chinese Spring IWGSC RefSeq v2.1 reference sequence. The mapping results were further confirmed in the F 7 RIL population. YrSA is a new stripe rust resistance gene different from other genes previously reported on chromosome arm 2AL based on their locations, origins, and responses to Pst races. The physical order of YrSA with other permanently named Yr genes on 2AL is Yr1-Yr86-Yr32-YrSA. YrSA can be widely used to enhance wheat stripe rust resistance in the USA, and the newly developed KASP markers can be used in marker-assisted selection.

Abstract Stripe rust and leaf rust, caused by Puccinia striiformis f. sp. tritici and P. triticina , respectively, are the most destructive wheat diseases in the southern Great Plains. ‘Green Hammer’ is a hard red winter wheat (HRWW) cultivar released by Oklahoma State University in 2018 and has demonstrated a stable adult plant resistance to stripe rust and race-specific seedling resistance to leaf rust. To identify and map rust resistance loci, 109 doubled haploid (DH) lines derived from the cross between Green Hammer and another HRWW cultivar, ‘Lonerider’, were developed. Lonerider showed adult plant resistance to stripe rust but was susceptible to multiple P. triticina races. The DH lines were evaluated for stripe rust at the adult plant stage in greenhouse and field environments across Oklahoma, Kansas, and Washington, and for leaf rust at the seedling stage against seven U.S. P. triticina races and at the adult plant stage in Oklahoma and Texas. Genotyping-by-sequencing generated 6,078 polymorphic single-nucleotide polymorphisms used for genetic mapping. Quantitative trait loci (QTL) analysis identified 14 stripe rust and 8 leaf rust resistance QTL. For stripe rust, a major QTL in Green Hammer, QYr.osughln-2AS , was identified in the proximity of the 2N v S translocation. Three other major stripe rust resistance QTL were identified in Lonerider on chromosomes 2AL (two QTL) and 2BS (one QTL). For leaf rust, QLr.osughln-1DS and QLr.osughln-2DS.1 were the two major QTL identified in Green Hammer and most likely correspond to the all-stage resistance genes Lr21 and Lr39 , respectively. In this study, we identified previously characterized genes as well as unknown genes that can be utilized in wheat breeding programs to enhance resistance to leaf rust and stripe rust.

Puccinia striiformis f. sp. tritici, causing stripe rust, is one of the most prominent pathogens of wheat worldwide. The biotrophic and obligate fungus is capable of rapidly developing new virulent races that can overcome race-specific resistance in host plants. The traditional virulence characterization of the pathogen requires strict conditions for testing isolates on wheat differentials with specific resistance genes, which is time-consuming. Developing molecular markers for avirulence genes could provide an efficient method for monitoring virulence changes in the pathogen population. In this study, secreted protein gene-based single-nucleotide polymorphism markers previously identified to be associated with avirulence genes of the pathogen were converted to kompetitive allele-specific PCR (KASP) markers. The KASP markers were screened with a diverse panel of 192 isolates selected from various countries based on their virulent races and molecular genotypes. The markers significantly correlated with the avirulence/virulence phenotypic data of the 192 isolates were further validated with 845 isolates collected from the United States in 2019 to 2021. Based on the results of both the screening and validation data, 21 KASP markers significantly associated with different avirulence genes were developed. Seventeen of the 21 markers were significantly associated with two or more avirulence genes, and except AvrYr10, and the remaining 15 avirulence genes had two or more markers. Different combinations of up to three markers could be used for specific detection of 16 avirulence genes in monitoring the pathogen population.

Stripe rust, caused by Puccinia striiformis f. sp. tritici, is among the most destructive wheat (Triticum aestivum L.) diseases. Identifying resistance genes is crucial for the development of resistant cultivars. Baker's Ann, a hard winter wheat cultivar developed by Oklahoma State University, has shown stable adult plant resistance to stripe rust. To dissect the genetic basis underlying stripe rust resistance in Baker's Ann, 125 doubled haploid lines, derived from the cross OK12D22004-016 × Baker's Ann, were evaluated at the adult plant stage in the greenhouse and in field environments in Oklahoma, Kansas, and Washington. This population was genotyped using genotyping‑by‑sequencing, which produced 7268 single‑nucleotide polymorphisms for genetic mapping. Quantitative trait loci (QTLs) analysis identified six loci, four from Baker's Ann on chromosomes 2DL, 4BS, 4BL, and 7BL, and two from OK12D22004-016 on chromosomes 2AS and 2AL. Although OK12D22004-016 is susceptible in the US Great Plains, it was found to carry QYr.osu-2AS, which was linked to Yr17 on the 2NvS translocation and explained up to 30% of the phenotypic variation but was effective in a single location in Washington. Two major QTLs were identified in Baker's Ann: QYr.osu-2DL on chromosome 2DL that explained up to 57% of the phenotypic variation, and QYr.osu-4BL on chromosome 4BL that explained up to 15% of the phenotypic variation. Resistance in Baker's Ann resulted from additive effects of the four QTLs. Kompetitive allele-specific PCR markers were developed for QYr.osu-2DL to facilitate marker-assisted selection for stripe rust resistance.

Wheat stripe rust, caused by the biotrophic fungal pathogen Puccinia striiformis f. sp. tritici (Pst), is among the top crop diseases incurring huge economic losses worldwide. Identification of new stripe rust-resistant sources that can be easily used in wheat cultivar development is essential for food security. PI 622129, an Iranian wheat landrace, exhibits high resistance to the predominant U.S. Pst races. A recombinant inbred line (RIL) population from the cross PI 622129 × Stardust was genotyped using single-nucleotide polymorphisms generated by genotyping-by-sequencing. The RIL population was evaluated for responses to the Pst race PSTv-37 at the seedling stage in three environments, and quantitative trait loci (QTLs) analysis revealed four QTLs for stripe rust resistance on chromosome arms 2DS, 5BS, 2AL, and 7BL. Of these, QYr.stars-2DS and QYr.stars-5BS are major QTLs that explained 21 to 38% and 11.6 to 27.2% of the total phenotypic variance, respectively, in three experiments. QYr.stars-2DS is a new stripe rust resistance locus that was identified in the interval of 2.58 to 5.54 Mb on chromosome arm 2DS based on the Chinese Spring IWGSC RefSeq v.2.1 reference genome. Another QTL, QYr.stars-5BS, is close to Yr47 and was delimited to the interval 8.1 to 9.0 Mb in the reference genome. QYr.stars-2AL and QYr.stars-7BL were mapped to the terminal and QTL-rich regions on chromosome arms 2AL (750.8 to 752.5 Mb) and 7BL (718.1 to 721.2 Mb), respectively. KASP markers were developed to facilitate rapid introgression of these QTLs into locally adapted lines via marker-assisted selection.

Puccinia striiformis f. sp. hordei (Psh), causing stripe rust epidemics on barley, has been continually changing in virulence since its introduction into the United States in the early 1990s. To monitor virulent changes, 344 Psh isolates recovered from stripe rust samples collected from barley, grasses, and wheat from 2010 to 2020 were tested on the set of 12 barley differentials. Among the 12 virulence factors, the frequency of virulence to Topper was the highest (99.4%), followed by those to Abed Binder 12 (77.9%), Trumpf (35.2%), and Bancroft (27.6%). The virulence frequencies of all remaining differentials were below 20.0%, with 16.6% on Hiproly, 15.7% on Bigo, 14.5% on Varunda, 10.8% on Emir, 9.9% on Astrix and Heils Franken, 8.1% on Muzurka, and 2.6% on I 5. Except for the consistently high frequencies of the virulence to Topper from different years, epidemiological regions, and hosts, virulence frequencies of other differentials varied from year to year, region to region, and host to host. A total of 41 Psh races were identified from the isolates, with the numbers of virulence factors ranging from 0 to 12. Among these races, 12 were first identified in the United States in the present study, whereas the other 29 races were first detected before 2010. Race PSH-33 (virulent on differentials Topper and Abed Binder 12) had the highest frequency (36.0%), followed by PSH-48 (virulent only on differential Topper) with a frequency of 20.6%. Cluster analysis and discriminant analysis of principal components revealed differentiations among years, with major changes occurring between 2016 and 2017, and to a lesser extent to regions and hosts. The results should be useful for developing resistant barley cultivars for control of stripe rust.

Puccinia striiformis f. sp. tritici (Pst) and P. striiformis f. sp. hordei (Psh) cause stripe rust epidemics on wheat and barley, respectively. Demethylation inhibitor (DMI) fungicides have been used for decades in managing stripe rust in the United States, but the pathogen tolerance was not clear. To determine the dynamics of DMI fungicide targeting gene Cyp51 mutants, Pst isolates collected from 1968 to 2021 and Psh isolates from 1993 to 2021 were tested using a Kompetitive allele-specific PCR (KASP) marker for the Y134F point mutation in the Cyp51 gene. The mutant allele was found in the Pst and Psh populations as early as in 1968 and 1993, respectively, and the mutant frequencies fluctuated from year to year. The KASP test of the Pst-infected leaf samples from fungicide-testing plots in 2024 revealed that the application of the DMI fungicide Tilt increases the mutant frequency. The urediniospore germination tests with 22 selected isolates at different concentrations of Tilt showed that the mean half-maximal effective concentration (EC50) value of the homozygous mutant isolates was 2.7 times higher than that of the wild-type isolates, but those of the heterozygous and wild-type isolates were not significantly different from each other. The results indicate that the KASP marker is useful in monitoring the DMI fungicide targeting gene mutants, and DMI fungicides are likely less effective in controlling the homozygous mutant population than the wild-type and heterozygous populations. The information is useful for managing stripe rust using diverse fungicides and growing resistant cultivars.

Plant pathogens rapidly evolve in response to changes in their hosts and environments, resulting in destructive epidemics. The fungus Puccinia striiformis f. sp. tritici ( Pst ), responsible for wheat stripe rust disease, exemplifies such evolutionary dynamics. Host population changes impose selective pressures on pathogens; however, the selection footprints and demographic patterns of Pst are not well understood. In this study, we performed whole‐genome sequencing of 69 Pst isolates collected from diverse regions across China to investigate genome‐wide patterns of gene flow and divergence. Our findings reveal significant gene flow within China but limited exchange between Chinese and international Pst populations, characterized by a slower decay of linkage disequilibrium compared to fungi with known sexual reproduction. We identified extensive hard and soft sweeps associated with Pst adaptation. The genes involved in these sweeps are enriched for secreted proteins and effectors, with functions related to pathogenicity, temperature tolerance, and fungicide resistance, indicating positive selection driven by both host and abiotic pressures. Demographic reconstruction shows strong bottlenecks in Pst populations during the domestication of wheat approximately 10,000 years ago and the advent of modern agriculture 100 years ago, suggesting that crop domestication and breeding programs have significantly influenced pathogen population dynamics. Our study provides valuable insights into the genomic evolution of Pst in China and highlights the impact of modern agricultural practices on pathogen demography.

Introducing and characterizing variation through mutagenesis plus functional genomics can accelerate resistance breeding as well as our understanding of crop plant immunity. To reveal new germplasm resources for fungal disease resistance breeding in elite durum wheat, we challenged the diverse alleles in a sequenced and cataloged ethyl methanesulfonate mutagenized population of elite tetraploid wheat Triticum turgidum subsp. durum cv ‘Kronos’ with stripe rust. We screened 2,000 mutant lines and identified sixteen enhanced disease resistance (EDR) lines with persistent resistance to stripe rust over four years of field testing. To find broad-spectrum resistance, we challenged these lines with other major biotrophic and necrotrophic pathogens, including those causing Septoria tritici blotch, tan spot, Fusarium head blight and leaf rust. Enhanced resistance to multiple fungi was found in 13 of 16 EDR lines. Five EDR lines showed spontaneous lesion formation in the absence of pathogens, providing new mutant resources to study plant stress response in the absence of the confounding effects of pathogen infection. We mapped exome capture sequencing data of the EDR lines to a recently released long-read Kronos genome to aid in the identification of causal mutations. We located an EDR resistance locus to an 175 Mb interval on chromosome 1B. Importantly, these phenotypically characterized EDR lines are newly described durum germplasm coupled with improved functional genomics resources that are readily available for both wheat fungal resistance breeding and basic plant immunity research.

Newly emerging highly virulent races of Puccinia striiformis f. sp. tritici (Pst) often defeat deployed resistance genes ( Yr ), highlighting the need for novel sources of durable resistance. A global diversity panel of 377 spring wheat ( Triticum aestivum L.) lines was screened for all-stage resistance (ASR) against 20 diverse Pst isolates at the seedling stage and adult plant resistance (APR) against natural mix of field races. Genome-wide association mapping identified 77 unique Yr loci. Of these, 33 overlapped with the previously reported ~1,100 loci, while 44 were likely novel. Nine mapped Yr loci were effective at the adult plant stage and showed no overlap with the known APR genes. Two wheat lines, though lacked the widely effective Yr5 and Yr15 genes, exhibited resistance to all 20 Pst races at the seedling stage and natural field races at the adult stage, suggesting that they may carry novel, broad-spectrum resistance alleles. APR genes Yr18 , Yr29 , and Yr36 were detected at low frequencies, indicating that much of the observed resistance may arise from less characterized or novel sources. Wheat improvement had no effect on the frequency of ASR alleles but resulted in a three-fold increase in the frequency of APR alleles, suggesting that the latter were subjected to more consistent breeding selection over time. Our findings underscore the value of combined screening of diverse germplasm with the diverse panels of pathogen races to identify novel sources of broad-spectrum resistance for breeding stripe rust resistant cultivars. ### Competing Interest Statement The authors have declared no competing interest. Gates Foundation, https://ror.org/0456r8d26, INV-004430 USDA National Institute of Food and Agriculture, 2022–68013-36439 (WheatCAP)

Stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is a destructive disease of wheat worldwide. William Som (WS), an Argentinian spring wheat landrace, has consistently exhibited high-level resistance to stripe rust for over 20 years in our field evaluations in Washington state, USA. A previous study showed high-temperature adult-plant (HTAP) resistance in WS. To map the HTAP resistance quantitative trait loci (QTL) in WS, 114 F5-8 recombinant inbred lines (RILs) from the cross AvS/WS were evaluated for their stripe rust response in seven field environments in Washington. The RILs and parents were genotyped with the Infinium 90K SNP chip. Four stable QTL, QYrWS.wgp-1BL on chromosome 1B (669–682 Mb), QyrWS.wgp-2AL on 2A (611–684 Mb), QyrWS.wgp-3AS on 3A (9–13 Mb), and QyrWS.wgp-3BL on 3B (476–535 Mb), were identified, and they explained 10.0–19.0%, 10.2–16.7%, 7.0–15.9%, and 12.0–27.8% of the phenotypic variation, respectively. The resistance in WS was found to be due to additive interactions of the four QTL. For each QTL, two Kompetitive allele-specific PCR (KASP) markers were developed, and these markers should facilitate the introgression of the HTAP resistance QTL into new wheat cultivars.

Wheat stripe (yellow) rust, caused by the fungus Puccinia striiformis f. sp. tritici (Pst), is one of the most threatening wheat diseases worldwide. Monitoring the virulence of Pst population is essential for managing wheat stripe rust. In this study, 18 wheat Yr single-gene lines were used to identify the virulence patterns of 67 isolates collected from 13 provinces in China in 2020, from which 33 Pst races were identified. The frequency of virulence to different Yr genes varied from 1.49% to 97.01%, with 4.48% to Yr1, 26.87% to Yr6, 11.94% to Yr7, 95.52% to Yr8, 19.40% to Yr9, 11.94% to Yr17, 2.99% to Yr24, 35.82% to Yr27, 38.81% to Yr43, 97.01% to Yr44, 8.96% to YrSP, 1.49% to Yr85, 95.52% to YrExp2, and 7.46% to Yr76. None of the isolates were virulent to Yr5, Yr10, Yr15, and Yr32. Among the 33 races, PstCN-062 (with virulence to Yr8, Yr44, and YrExp2) and PstCN-001 (with virulence to Yr8, Yr43, Yr44, and YrExp2) were the prevalent races, with frequencies of 28.36% and 11.94%, respectively. These results provide valuable information for breeding resistant wheat cultivars for controlling stripe rust.

The genome-wide association study utilizing a diverse global collection of 318 barley accessions identified 44 loci for stripe rust resistance, including 14 potentially novel loci. Stripe rust is an important disease of barley in temperate regions worldwide. Identification and genetic characterization of stripe rust resistance are essential for development and deployment of durable resistance in barley cultivars. A total of 318 spring barley accessions from a global barley collection were evaluated for resistance to the stripe rust pathogen Puccinia striiformis f. sp. hordei (Psh) at the seedling stage in the greenhouse (4–20 °C) using five Psh races (PSH-33, PSH-48, PSH-72, PSH-117 and PSH-118) and at the adult-plant stage in the greenhouse under a high-temperature profile (15–25 °C) and in four field environments in Washington, USA. The frequencies of resistant accessions ranged from 15 to 54

The GWAS and testing with Yr gene linked markers identified 109 loci including 40 novel loci for all-stage and adult plant stage resistance to stripe rust in 459 US contemporary hard winter wheat genotypes. Stripe rust is a destructive wheat disease, caused by Puccinia striiformis f. sp. tritici (Pst). To identify sources of stripe rust resistance in US contemporary hard winter wheat, a panel of 459 Great Plains wheat genotypes was evaluated at the seedling stage against five US Pst races and at the adult plant stage in field environments in Oklahoma, Kansas, and Washington. The results showed that 7–14

Stripe rust of wheat is a serious disease caused by Puccinia striiformis f. sp. tritici (Pst). Growing resistant cultivars is the most preferred approach to control the disease. To identify wheat genotypes with quantitative trait loci (QTL) for durable resistance to stripe rust, 465 winter wheat entries that were presumed to have high-temperature adult-plant (HTAP) resistance were used in this study. In the greenhouse seedling tests with seven Pst races, 16 entries were resistant to all the tested races. The 465 entries were also phenotyped for stripe rust responses at the adult-plant stage under natural infection of Pst in multiple field locations from 2018 to 2021 in the Washington state, and 345 entries were found to have stable resistance. The contrast of the susceptibility in the greenhouse seedling tests and the resistance in the field adult-plant stage for most of the entries indicated predominantly HTAP resistance in this panel. The durability of the resistance was demonstrated by a subset of 175 entries that were tested in multiple locations from 2007 to 2021. The 465 entries were genotyped through genotyping by multiplexed sequencing of single-nucleotide polymorphism (SNP) markers. Combining the stripe rust response and SNP marker data, a genome-wide association study (GWAS) was conducted, resulting in 143 marker-trait associations, from which 28 QTL that were detected at least with two races or in two field environments were identified, including seven for all-stage resistance and 21 for HTAP resistance. These QTL each explained 6.0% to 40.0% of the phenotypic variation. Compared with previously reported Yr genes and QTL based on their genomic positions, five QTL including two for HTAP resistance were identified as new. A total of 10 user-friendly Kompetitive allele specific PCR (KASP) markers were developed for eight of the HTAP resistance loci. In addition, molecular markers were used to detect 13 previously reported HTAP resistance genes/QTL, including two also identified in the GWAS analyses, and their frequencies ranged from 0.86% to 88.17% in the panel. The durable resistant genotypes, the genes/QTL identified, and the KASP markers developed in this study should be useful to develop wheat cultivars with long-lasting resistance to stripe rust.

Stripe rust (also called yellow rust, Yr) of wheat is caused by Puccinia striiformis f. sp. tritici (Pst), and stripe rust of barley is caused by Puccinia striiformis f. sp. hordei (Psh). These fungal pathogens continually produce new virulent races that may circumvent race-specific resistance genes in commercially grown cultivars of the crops. To effectively develop new resistant cultivars and manage diseases, it is essential to monitor virulence changes and characterize the races of the pathogen. This chapter describes the protocols used to identify races of the pathogens in the United States. Currently, a set of 18 Yr single-gene lines is used to differentiate races of Pst, and a set of 12 barley genotypes is used to differentiate races of Psh. As both Pst and Psh can infect other cereal crops and grasses, some Pst races can infect few barley varieties, and some Psh races may infect few wheat varieties, a scheme we have been using to address the across virulence issue is presented and discussed.

The adult plant resistance gene Yr78 provides durable resistance to stripe rust ( Puccinia striiformis f. sp. tritici ) in common wheat but has not been detected in durum wheat or other tetraploid wheat accessions. To introduce Yr78 into durum wheat, we introgressed it into the cultivar Kronos by marker‐assisted backcrossing. Field evaluation revealed that the introgression of Yr78 reduced the mean infection type from 6.27 to 3.79 and the mean disease severity from 63.91% to 38.71%. Using germplasm excluding other resistance quantitative trait locus found in Kronos, we quantified the effect of Yr78 and observed that Yr78 alone significantly reduced stripe rust infection type and severity by 14.72% and 25.76%, respectively ( p < 0.05), but the infection type (IT) score remained high (mean IT = 6.75), indicating Yr78 should be combined with additional resistance genes for enhanced effectiveness. To achieve this, we combined the linked Yr36 and Yr78 in coupling in common wheat to enhance the Pst resistance and facilitate their simultaneous deployment in breeding programs. The combined Yr36–Yr78 genes significantly reduced the mean IT score by 42.86% compared to lines without these two genes. To enhance the value of this combination, we also combined the linked Yr36–Yr78 with seedling resistance genes Yr5 and Yr15 , so all four genes can be transferred in a single cross. Molecular markers for these genes are provided to facilitate their deployment. The germplasm developed in this study has been deposited to GRIN‐Global to accelerate its use in durum and common wheat breeding programs.

Rusts of the genus Puccinia are wheat pathogens. Stem (black; Sr), leaf (brown; Lr), and stripe (yellow; Yr) rust, caused by Puccinia graminis f. sp. tritici (Pgt), Puccinia triticina (Pt), and Puccinia striiformis f. sp. tritici (Pst), can occur singularly or in mixed infections and pose a threat to wheat production globally in terms of the wide dispersal of their urediniospores. The development of durable resistant cultivars is the most sustainable method for controlling them. Many resistance genes have been identified, characterized, genetically mapped, and cloned; several quantitative trait loci (QTLs) for resistance have also been described. However, few studies have considered resistance to all three rust pathogens in a given germplasm. A genome-wide association study (GWAS) was carried out to identify loci associated with resistance to the three rusts in a collection of 230 inbred lines of tetraploid wheat (128 of which were Triticum turgidum ssp. durum) genotyped with SNPs. The wheat panel was phenotyped in the field and subjected to growth chamber experiments across different countries (USA, Mexico, Morocco, Italy, and Spain); then, a mixed linear model (MLM) GWAS was performed. In total, 9, 34, and 5 QTLs were identified in the A and B genomes for resistance to Pgt, Pt, and Pst, respectively, at both the seedling and adult plant stages. Only one QTL on chromosome 4A was found to be effective against all three rusts at the seedling stage. Six QTLs conferring resistance to two rust species at the adult plant stage were mapped: three on chromosome 1B and one each on 5B, 7A, and 7B. Fifteen QTLs conferring seedling resistance to two rusts were mapped: five on chromosome 2B, three on 7B, two each on 5B and 6A, and one each on 1B, 2A, and 7A. Most of the QTLs identified were specific for a single rust species or race of a species. Candidate genes were identified within the confidence intervals of a QTL conferring resistance against at least two rust species by using the annotations of the durum (cv. ‘Svevo’) and wild emmer wheat (‘Zavitan’) reference genomes. The 22 identified loci conferring resistance to two or three rust species may be useful for breeding new and potentially durable resistant wheat cultivars.

Aegilops umbellulata serve as an important reservoir for novel biotic and abiotic stress tolerance for wheat improvement. However, chromosomal rearrangements and evolutionary trajectory of this species remain to be elucidated. Here, we present a comprehensive investigation into Ae. umbellulata genome by generating a high-quality near telomere-to-telomere genome assembly of PI 554389 and resequencing 20 additional Ae. umbellulata genomes representing diverse geographical and phenotypic variations. Our analysis unveils complex chromosomal rearrangements, most prominently in 4U and 6U chromosomes, delineating a distinct evolutionary trajectory of Ae. umbellulata from wheat and its relatives. Furthermore, our data rectified the erroneous naming of chromosomes 4U and 6U in the past and highlighted multiple major evolutionary events that led to the present-day U-genome. Resequencing of diverse Ae. umbellulata accessions revealed high genetic diversity within the species, partitioning into three distinct evolutionary sub-populations and supported by extensive phenotypic variability in resistance against several races/pathotypes of five major wheat diseases. Disease evaluations indicated the presence of several novel resistance genes in the resequenced lines for future studies. Resequencing also resulted in the identification of six new haplotypes for Lr9, the first resistance gene cloned from Ae. umbellulata. The extensive genomic and phenotypic resources presented in this study will expedite the future genetic exploration of Ae. umbellulata, facilitating efforts aimed at enhancing resiliency and productivity in wheat.