Yuxiang Li

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.

Aegilops species are wheat relatives that harbor valuable disease resistance genes for wheat breeding. The wheat Yr8 near-isogenic line AvSYr8NIL has long been believed to carry only Yr8 for race-specific all-stage resistance to stripe rust, caused by Puccinia striiformis f. sp. tritici, derived from Aegilops comosa. However, AvSYr8NIL has been found to have high-temperature adult-plant (HTAP) resistance in our field and greenhouse tests. To confirm both HTAP and Yr8 resistance, seeds from AvSYr8NIL were treated with ethyl methanesulfonate to generate mutant lines. The mutant lines with only Yr8 (M641) and only HTAP resistance (M488) were crossed with the susceptible recurrent parent Avocet S (AvS). The F₁ and F₄ lines of AvS/M641 were phenotyped with Yr8-avirulent races in the seedling stage at the low-temperature (4 to 20°C) profile, while the F₁, F₂, F₄, and F₅ lines of AvS/M488 were phenotyped with Yr8-virulent races in the adult-plant stage at the high-temperature (10 to 30°C) profile. Both Yr8 and the HTAP resistance gene (YrM488) were recessive. The F₄ populations of AvS/M641 and AvS/M488 were genotyped using polymorphic Kompetitive allele-specific PCR markers converted from single-nucleotide polymorphisms. Yr8 was mapped to a 0.66-cM fragment, and YrM488 was mapped to a 1.22-cM interval on chromosome 2D. The physical distance between the two resistance genes was estimated to be more than 500 Mb, indicating their distinct loci. The mutant lines with separated resistance genes would be useful in enhancing our understanding of different types of resistance and in further studying the interactions between wheat and the stripe rust pathogen.

Wheat near-isogenic line AvSYr17NIL carrying Yr17, originally from Aegilops ventricosa for all-stage resistance to Puccinia striiformis f. sp. tritici, also shows nonrace-specific, high-temperature adult-plant (HTAP) resistance to the stripe rust pathogen. To separate and identify the HTAP resistance gene, seeds of AvSYr17NIL were treated with ethyl methanesulfonate. Mutant lines with only HTAP resistance were obtained, and one of the lines, M1225, was crossed with the susceptible recurrent parent Avocet S (AvS). Field responses of the F₂ plants and F₃ lines, together with the parents, were recorded at the adult-plant stage in Pullman and Mount Vernon, WA under natural P. striiformis f. sp. tritici infection. The parents and the F₄ population were phenotyped with a Yr17-virulent P. striiformis f. sp. tritici race in the adult-plant stage under the high-temperature profile in the greenhouse. The phenotypic results were confirmed by testing the F₅ population in the field under natural P. striiformis f. sp. tritici infection. The F₂ data indicated a single recessive gene, temporarily named YrM1225, for HTAP resistance. The F₄ lines were genotyped with Kompetitive allele-specific PCR markers converted from single-nucleotide polymorphism markers polymorphic between M1225 and AvS. The HTAP resistance gene was mapped on the short arm of chromosome 2A in an interval of 7.5 centimorgans using both linkage and quantitative trait locus mapping approaches. The separation of the HTAP resistance gene from Yr17 should improve the understanding and utilization of the different types of resistance.

Stripe rust, caused by Puccinia striiformis f. sp. tritici, is a devastating disease of wheat (Triticum aestivum) in the United States. The fungal pathogen can rapidly evolve, producing new virulent races infecting previously resistant cultivars and genotypes adapting to different environments. The objective of this study was to investigate the long-term population dynamics of P. striiformis f. sp. tritici in the United States. Through genotyping 1,083 isolates taken from 1968 to 2009, using 14 simple sequence repeat (SSR) markers and 92 secreted protein single nucleotide polymorphism (SP-SNP) markers, 614 and 945 genotypes were detected, respectively. In general, the two types of markers produced consistent genetic relationships among the P. striiformis f. sp. tritici populations over the 40-year period. The prior-to-2000 and the 2000-to-2009 populations were significantly different, with the latter showing higher genotypic diversity and higher heterozygosity than the earlier populations. Clustering analyses using genotypes of either SSR or SP-SNP markers revealed three molecular groups (MGs), MG1, MG2, and MG3. The prior-to-2000 and the 2000-to-2009 groups both had evidence of MG1 and MG2; however, MG3 was only found in the 2000-to-2009 population. Some of the isolates in the period of 2000 to 2009 formed individual clusters, suggesting exotic incursions. Other isolates of the same period were clustered with prior-to-2000 isolates, indicating that they were developed from the previously established populations. The data suggest the coexistence of newly introduced populations alongside established populations in the United States. Twenty SP-SNP markers were significantly associated to individual avirulence genes. These results are useful for developing more accurate monitoring systems and provide guidance for disease management.

Background: The stripe rust pathogen,Puccinia striiformisf. sp.tritici(Pst), threats world wheat production. Resistance toPstis often overcome by pathogen virulence changes, but the mechanisms of variation are not clearly understood. To determine the role of mutation inPstvirulence changes, in previous studies 30 mutant isolates were developed from a least virulent isolate using ethyl methanesulfonate (EMS) mutagenesis and phenotyped for virulence changes. The progenitor isolate was sequenced, assembled and annotated for establishing a high-quality reference genome. In the present study, the 30 mutant isolates were sequenced and compared to the wide-type isolate to determine the genomic variation and identify candidates for avirulence (Avr) genes.Results: The sequence reads of the 30 mutant isolates were mapped to the wild-type reference genome to identify genomic changes. After selecting EMS preferred mutations, 264,630 and 118,913 single nucleotide polymorphism (SNP) sites and 89,078 and 72,513 Indels (Insertion/deletion) were detected among the 30 mutant isolates compared to the primary scaffolds and haplotigs of the wild-type isolate, respectively. Deleterious variants including SNPs and Indels occurred in 1866 genes. Genome wide association analysis identified 754 genes associated with avirulence phenotypes. A total of 62 genes were found significantly associated to 16 avirulence genes after selection through six criteria for putative effectors and degree of association, including 48 genes encoding secreted proteins (SPs) and 14 non-SP genes but with high levels of association (P ≤ 0.001) to avirulence phenotypes. Eight of the SP genes were identified as avirulence-associated effectors with high-confidence as they met five or six criteria used to determine effectors.Conclusions: Genome sequence comparison of the mutant isolates with the progenitor isolate unraveled a large number of mutation sites along the genome and identified high-confidence effector genes as candidates for avirulence genes inPst.Since the avirulence gene candidates were identified from associated SNPs and Indels caused by artificial mutagenesis, these avirulence gene candidates are valuable resources for elucidating the mechanisms of the pathogen pathogenicity, and will be studied to determine their functions in the interactions between the wheat host and thePstpathogen.

Stripe rust caused by Puccinia striiformis is a disastrous disease of cereal crops and various grasses. To date, 14 stripe rust genomes are publicly available, including 13 P. striiformis f. sp. tritici and 1 P. striiformis f. sp. hordei. In this study, one isolate (11-281) of P. striiformis collected from wheatgrass (Agropyron cristatum), which is avirulent to most of standard differential genotypes of wheat and barley, was sequenced, assembled, and annotated. The sequences were assembled to a draft genome of 84.75 Mb, which is comparable with previously sequenced P. striiformis f. sp. tritici and P. striiformis f. sp. hordei isolates. The draft genome comprised 381 scaffolds and contained 1,829 predicted secreted proteins. The high-quality draft genome of the isolate is a valuable resource in shedding light on the evolution and pathogenicity of P. striiformis.