Molecular Mechanisms of the Stripe Rust Interaction with Resistant and Susceptible Wheat Genotypes.

Rust fungi cause significant damage to wheat production worldwide. In order to mitigate disease impact and improve food security via durable resistance, it is important to understand the molecular basis of host-pathogen interactions. Despite a long history of research and high agricultural importance, still little is known about the interactions between the stripe rust fungus and wheat host on the gene expression level. Here, we present analysis of the molecular interactions between a major wheat pathogen-Puccinia striiformis f. sp. tritici (Pst)-in resistant and susceptible host backgrounds. Using plants with durable nonrace-specific resistance along with fully susceptible ones allowed us to show how gene expression patterns shift in compatible versus incompatible interactions. The pathogen showed significantly greater number and fold changes of overexpressed genes on the resistant host than the susceptible host. Stress-related pathways including MAPK, oxidation-reduction, osmotic stress, and stress granule formation were, almost exclusively, upregulated in the resistant host background, suggesting the requirement of the resistance-countermeasure mechanism facilitated by Pst. In contrast, the susceptible host background allowed for broad overrepresentation of the nutrient uptake pathways. This is the first study focused on the stripe rust pathogen-wheat interactions, on the whole transcriptome level, from the pathogen side. It lays a foundation for the better understanding of the resistant/susceptible hosts versus pathogenic fungus interaction in a broader sense.

Molecular Mapping of Yr85 and Comparison with Other Genes for Resistance to Stripe Rust on Wheat Chromosome 1B.

Wheat stripe rust, caused by Puccinia striiformis f. sp. tritici, is one of the most serious plant diseases worldwide. Resistant cultivars are the most effective way to control the disease. YrTr1 is an important stripe rust resistance gene that has been used in wheat breeding programs and is represented in the host differential set to identify P. striiformis f. sp. tritici races in the United States. To map YrTr1, AvSYrTr1NIL was backcrossed to its recurrent parent Avocet S (AvS). Seedlings of BC7F2, BC7F3, and BC8F1 populations were tested with YrTr1-avirulent races under controlled conditions, and BC7F2 plants were genotyped using simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) markers. YrTr1 was mapped to the short arm of chromosome 1B using four SSR and seven SNP markers. The genetic distances of YrTr1 from the nearest flanking markers IWA2583 and IWA7480 were 1.8 and 1.3 centimorgans (cM), respectively. DNA amplification of a set of 21 Chinese Spring (CS) nulli-tetrasomic lines and seven CS 1B deletion lines with three SSR markers confirmed the chromosome arm location and further placed the gene in chromosomal bin region 1BS18 (0.5). The gene was determined to be about 7.4 cM proximal to Yr10. Based on multirace response array and chromosomal location, YrTr1 was determined to be different from other permanently named stripe rust resistance genes in chromosome arm 1BS and was named Yr85.

Genotyping by Multiplexed Sequencing (GMS) Using SNP Markers.

SNP-based genotyping has become the most effective approach to generate target-specific data for use in genetic studies. In this chapter, we will describe a high-throughput genotyping method that multiplexes hundreds to thousands of SNP markers in a two-step PCR protocol that can be customized to fit the specific needs of a study.

Characterization and Molecular Mapping of a Gene Conferring High-Temperature Adult-Plant Resistance to Stripe Rust Originally from Aegilops ventricosa.

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.

Changes of Barley Stripe Rust Populations in the United States from 1993 to 2017.

Barley stripe rust is a relatively new disease in the United States. The pathogen, Puccinia striiformis f. sp. hordei (Psh), was first observed in Texas in 1991 and has spread north and westwards and mainly caused epidemics in the western United States. A total of 447 isolates collected from 1993 to 2017 were identified as 382 multilocus genotypes (MLGs) using 14 simple sequence repeat markers. The MLGs were clustered into six molecular groups (MGs) using the discriminant analysis of principal components and the hierarchical cluster analysis, and the MGs had significant differences in frequency in different years. MG1 was present in the population prior to the year 2000. MG2, MG3, and MG4 became predominate after 2000. MG5 was detected in all 24 years but more frequent from 2010 to 2017. MG6 was the most recent group detected mainly from 2011 to 2017 and had the highest correlation coefficient with the virulence phenotypes among the MGs. The heterozygosity and genotypic diversity of the Psh populations increased from 2000 to 2017, even more from 2010 to 2017. The results indicate rapid genetic changes from year to year, with major molecular group changes around 2000 and 2010. The possible mechanisms underlying the population changes are discussed.

Identification of Secreted Protein Gene-Based SNP Markers Associated with Virulence Phenotypes of Puccinia striiformis f. sp. tritici, the Wheat Stripe Rust Pathogen.

Stripe rust caused by Puccinia striiformis f. sp. tritici (Pst) is a destructive disease that occurs throughout the major wheat-growing regions of the world. This pathogen is highly variable due to the capacity of virulent races to undergo rapid changes in order to circumvent resistance in wheat cultivars and genotypes and to adapt to different environments. Intensive efforts have been made to study the genetics of wheat resistance to this disease; however, no known avirulence genes have been molecularly identified in Pst so far. To identify molecular markers for avirulence genes, a Pst panel of 157 selected isolates representing 126 races with diverse virulence spectra was genotyped using 209 secreted protein gene-based single nucleotide polymorphism (SP-SNP) markers via association analysis. Nineteen SP-SNP markers were identified for significant associations with 12 avirulence genes: AvYr1, AvYr6, AvYr7, AvYr9, AvYr10, AvYr24, AvYr27, AvYr32, AvYr43, AvYr44, AvYrSP, and AvYr76. Some SP-SNPs were associated with two or more avirulence genes. These results further confirmed that association analysis in combination with SP-SNP markers is a powerful tool for identifying markers for avirulence genes. This study provides genomic resources for further studies on the cloning of avirulence genes, understanding the mechanisms of host-pathogen interactions, and developing functional markers for tagging specific virulence genes and race groups.

Population structure and genetic diversity of U.S. wheat varieties.

The United States is a major wheat producer with more than a century of wheat (Triticum aestivum L.) research and breeding. Using a panel of 753 historical and modern wheat varieties grown in the United States from the early 1800s to present day, we examined population structure and changes in genetic diversity. We used previously mapped high-quality single-nucleotide polymorphism (SNP) markers from the wheat 90K SNP array for genotyping. The wheat varieties had a slight hierarchical population structure based on growth habit and then by kernel color within spring varieties and by kernel hardness within winter varieties, which corresponds with geographical distribution of the varieties. Classifying varieties by market class, which is a combination of habit, hardness, and color, accounted for the greatest amount of variation (13.3%). We did not find evidence of decreased genetic diversity of either spring or winter varieties after the release of the first semidwarf wheat variety in 1961. On the contrary, northern and Pacific spring varieties, hard red spring (HRS), hard white spring (HWS), and soft white winter (SWW) had increases in both SNP and haplotype genetic diversity after 1961. The soft white spring (SWS) and soft red winter (SRW) market classes already had high genetic diversity in varieties before 1961 and showed some evidence of decreased diversity after 1961. Examination of temporal trends in genetic diversity also did not indicate long-term decline in diversity despite occasional fluctuations.

Identification and Mapping of Quantitative Trait Loci Associated with Stripe Rust Resistance in Spring Club Wheat Cultivar JD.

Puccinia striiformis Westend. f. sp. tritici, commonly known as stripe rust, is an economically important pathogen of wheat (Triticum aestivum L.). The hexaploid club spring wheat cultivar JD contains both all-stage and adult plant resistance (APR) genes and exhibited consistent high resistance to stripe rust in the field. In this study, we aimed to identify the quantitative trait loci (QTL) for stripe rust resistance using a BC1F7 back-cross inbred-line population derived from the cross of JD and the recurrent parental line 'Avocet'. The population was phenotyped in field plots in Washington State at the Spillman Agronomy Farm in Pullman and Mount Vernon Northwest Washington Research and Extension Center in between 2014 and 2016. A major QTL tentatively designated as QYrJD.wsu-1B, conferring all-stage resistance in JD background, was identified and mapped at the telomere region on the short arm of chromosome 1B using the genotyping-by-sequencing method. This QTL was further characterized with simple sequence repeat (SSR) markers and found to have the greatest logarithm-of-the-odds score and phenotypic effect, using SSR marker wmc798 on chromosome 1BS. Seven additional QTLs associated with APR were identified in the JD background on chromosomes 2D, 3A, 3B, 4A, 6B, and 7A with partial phenotypic effects.

Late-maturity α-amylase (LMA): exploring the underlying mechanisms and end-use quality effects in wheat.

MAIN CONCLUSION: A comprehensive understanding of LMA from the underlying molecular aspects to the end-use quality effects will greatly benefit the global wheat industry and those whose livelihoods depend upon it. Late-maturity α-amylase (LMA) leads to the expression and protein accumulation of high pI α-amylases during late grain development. This α-amylase is maintained through harvest and leads to an unacceptable low falling number (FN), the wheat industry's standard measure for predicting end-use quality. Unfortunately, low FN leads to significant financial losses for growers. As a result, wheat researchers are working to understand and eliminate LMA from wheat breeding programs, with research aims that include unraveling the genetic, biochemical, and physiological mechanisms that lead to LMA expression. In addition, cereal chemists and quality scientists are working to determine if and how LMA-affected grain impacts end-use quality. This review is a comprehensive overview of studies focused on LMA and includes open questions and future directions.

Molecular Characterization of Wheat Stripe Rust Pathogen (Puccinia striiformis f. sp. tritici) Collections from Nine Countries.

Stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is one of the most important diseases of wheat worldwide. To understand the worldwide distribution of its molecular groups, as well as the diversity, differentiation, and migration of the Pst populations, 567 isolates collected from nine countries (China, Pakistan, Italy, Egypt, Ethiopia, Canada, Mexico, Ecuador, and the U.S.) in 2010-2018 were genotyped using 14 codominant simple sequence repeat markers. A total of 433, including 333 new multi-locus genotypes (MLGs), were identified, which were clustered into ten molecular groups (MGs). The MGs and country-wise populations differed in genetic diversity, heterozygosity, and correlation coefficient between the marker and virulence data. Many isolates from different countries, especially the isolates from Mexico, Ecuador, and the U.S., were found to be identical or closely related MLGs, and some of the MGs were present in all countries, indicating Pst migrations among different countries. The analysis of molecular variance revealed 78% variation among isolates, 12% variation among countries, and 10% variation within countries. Only low levels of differentiation were found by the pairwise comparisons of country populations. Of the 10 MGs, 5 were found to be involved in sexual and/or somatic recombination. Identical and closely related MLGs identified from different countries indicated international migrations. The study provides information on the distributions of various Pst genetic groups in different countries and evidence for the global migrations, which should be useful in understanding the pathogen evolution and in stressing the need for continual monitoring of the disease and pathogen populations at the global scale.

Population Diversity, Dynamics, and Differentiation of Wheat Stripe Rust Pathogen Puccinia striiformis f. sp. tritici From 2010 to 2017 and Comparison With 1968 to 2009 in the United States.

Stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is a serious disease on wheat in the United States, especially after 2000. In the present study, 2,247 Pst isolates collected over all stripe rust epidemiological regions in the United States from 2010 to 2017 were genotyped at 14 simple sequence repeat (SSR) loci to investigate the population diversity, dynamics, and differentiation. A total of 1,454 multilocus genotypes (MLGs) were detected. In general, the populations in the west (regions 1-6) had more MLGs and higher diversities than the populations in the east (regions 7-12). The populations of 2010 and 2011 were more different from the other years. Genetic variation was higher among years than among regions, indicating the fast changes of the population. The divergence (Gst) was bigger between the west population and east population than among regions within either the west or east population. Gene flow was stronger among the regional populations in the east than in the west. Clustering analyses revealed 3 major molecular groups (MGs) and 10 sub-MGs by combining the genotypic data of 2010-2017 isolates with those of 1968-2009. MG1 contained both 1968-2009 isolates (23.1%) and 2010-2017 isolates (76.9%). MG2 had 99.4% of isolates from 1968-2009. MG3, which was the most recent and distinct group, had 99.1% of isolates from 2010-2017. Of the 10 sub-MGs, 5 (MG1-3, MG1-5, MG3-2, MG3-3, and MG3-4) were detected only from 2011 to 2017. The SSR genotypes had a moderate, but significant correlation (r = 0.325; p < 0.0001) with the virulence phenotype data. The standard index values of association (rbarD = 0.11) based on either regional or yearly populations suggest clonal reproduction. This study indicated high diversity, fast dynamics, and various levels of differentiation of the Pst population over the years and among epidemiological regions, and the results should be useful for managing wheat stripe rust.

QTL Analysis of Adult Plant Resistance to Stripe Rust in a Winter Wheat Recombinant Inbred Population.

Stripe rust, caused by the fungus Puccinia striiformis f. sp. tritici, is a worldwide disease of wheat that causes devastating crop losses. Resistant cultivars have been developed over the last 40 years that have significantly reduced the economic impact of the disease on growers, but in heavy infection years it is mostly controlled through the intensive application of fungicides. The Pacific Northwest of the United States has an ideal climate for stripe rust and has one of the most diverse race compositions in the country. This has resulted in many waves of epidemics that have overcome most of the resistance genes traditionally used in elite germplasm. The best way to prevent high yield losses, reduce production costs to growers, and reduce the heavy application of fungicides is to pyramid multiple stripe rust resistance genes into new cultivars. Using genotyping-by-sequencing, we identified 4662 high quality variant positions in a recombinant inbred line population of 196 individuals derived from a cross between Skiles, a highly resistant winter wheat cultivar, and Goetze, a moderately to highly susceptible winter wheat cultivar, both developed at Oregon State University. A subsequent genome wide association study identified two quantitative trait loci (QTL) on chromosomes 3B and 3D within the predicted locations of stripe rust resistance genes. Resistance QTL, when combined together, conferred high levels of stripe rust resistance above the level of Skiles in some locations, indicating that these QTL would be important additions to future breeding efforts of Pacific Northwest winter wheat cultivars.

Genotyping Puccinia striiformis f. sp. tritici Isolates with SSR and SP-SNP Markers Reveals Dynamics of the Wheat Stripe Rust Pathogen in the United States from 1968 to 2009 and Identifies Avirulence-Associated Markers.

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.

Mapping Quantitative Trait Loci for High-Temperature Adult-Plant Resistance to Stripe Rust in Spring Wheat PI 197734 Using a Doubled Haploid Population and Genotyping by Multiplexed Sequencing.

Stripe rust caused by Puccinia striiformis f. sp. tritici (Pst) is a global concern for wheat production. Spring wheat cultivar PI 197734, of Sweden origin, has shown high-temperature adult-plant resistance (APR) to stripe rust for many years. To map resistance quantitative trait loci (QTL), 178 doubled haploid lines were developed from a cross of PI 197734 with susceptible AvS. The DH lines and parents were tested in fields in 2017 and 2018 under natural infection of Pst and genotyped with genotyping by multiplexed sequencing (GMS). Kompetitive allele specific PCR (KASP) and simple sequence repeat (SSR) markers from specific chromosomal regions were also used to genotype the population to validate and saturate resistance QTL regions. Two major QTL on chromosomes 1AL and 3BL and one minor QTL on 2AL were identified. The two major QTL, QYrPI197734.wgp-1A and QYrPI197734.wgp-3B, were detected in all tested environments explaining up to 20.7 and 46.8% phenotypic variation, respectively. An awnletted gene mapped to the expected distal end of chromosome 5AL indicated the accuracy of linkage mapping. The KASP markers converted from the GMS-SNPs in the 1A and 3B QTL regions were used to genotype 95 US spring wheat cultivars and breeding lines, and they individually showed different percentages of polymorphisms. The haplotypes of the three markers for the 1A QTL and four markers for the 3B QTL identified 37.9 and 21.1% of the wheat cultivar/breeding lines possibly carrying these two QTL, indicating their usefulness in marker-assisted selection (MAS) for incorporating the two major QTL into new wheat cultivars.

Genome-Wide Association Study and Gene Specific Markers Identified 51 Genes or QTL for Resistance to Stripe Rust in U.S. Winter Wheat Cultivars and Breeding Lines.

Stripe (yellow) rust, caused by fungal pathogen Puccinia striiformis f. sp. tritici (Pst), is a serious disease of wheat in the United States and many other countries. Growing resistant cultivars has been approved to be the best approach for control of stripe rust. To determine stripe rust resistance genes in U.S. winter wheat cultivars and breeding lines, we analyzed a winter wheat panel of 857 cultivars and breeding lines in a genome-wide association study (GWAS) using genotyping by multiplexed sequencing (GMS) and by genotyping with molecular markers of 18 important stripe rust resistance genes or quantitative trait loci (QTL). The accessions were phenotyped for stripe rust response at adult-plant stage under natural infection in Pullman and Mount Vernon, Washington in 2018 and 2019, and in the seedling stage with six predominant or most virulent races of Pst. A total of 51 loci were identified to be related to stripe rust resistance, and at least 10 of them (QYrww.wgp.1D-3, QYrww.wgp.2B-2, QYrww.wgp.2B-3, QYrww.wgp.2B-4, QYrww.wgp.3A, QYrww.wgp.5A, QYrww.wgp.5B, QYrww.wgp.5D, QYrww.wgp.6A-2 and QYrww.wgp.7B-3) were previously reported. These genes or QTL were found to be present at different frequencies in breeding lines and cultivars developed by breeding programs in various winter wheat growing regions. Both Yr5 and Yr15, which are highly resistant to all races identified thus far in the U.S., as well as Yr46 providing resistance to many races, were found absent in the breeding lines and commercially grown cultivars. The identified genes or QTL and their markers are useful in breeding programs to improve the level and durability of resistance to stripe rust.

Identification of Stripe Rust Resistance Loci in U.S. Spring Wheat Cultivars and Breeding Lines Using Genome-Wide Association Mapping and Yr Gene Markers.

Stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), poses a major threat to wheat production worldwide, especially in the United States. To identify loci for effective stripe rust resistance in U.S. wheat, a genome-wide association study (GWAS) was conducted using a panel of 616 spring wheat cultivars and breeding lines. The accessions in this panel were phenotyped for stripe rust response in the greenhouse at seedling stage with five predominant and highly virulent races of Pst and in different field environments at adult-plant stage in 2017 and 2018. In total, 2,029 single-nucleotide polymorphism markers that cover the whole genome were generated with genotyping by multiplexed sequencing and used in GWAS. In addition, 23 markers of previously reported resistance genes or quantitative trait loci (QTLs) were used to genotype the population. This spring panel was grouped into three subpopulations based on principal component analysis. A total of 37 genes or QTLs including 10 potentially new QTLs for resistance to stripe rust were detected by GWAS and linked marker tests. The frequencies of the resistance genes or QTLs in various nurseries were determined, indicating different intensities of these genes or QTLs used in breeding programs of different regions. These resistance loci and the information on their markers, effectiveness, and distributions should be useful for improving stripe rust resistance in wheat cultivars.

Genotyping by multiplexed sequencing (GMS): A customizable platform for genomic selection.

As genotyping technologies continue to evolve, so have their throughput and multiplexing capabilities. In this study, we demonstrate a new PCR-based genotyping technology that multiplexes thousands of single nucleotide polymorphism (SNP) markers with high-throughput capabilities in a simple protocol using a two-step PCR approach. The bioinformatic pipeline is user friendly and yields results that are intuitive to interpret. This method was tested on two recombinant inbred line (RIL) populations that had previous genotyping data from the Illumina Infinium assay for Triticum aestivum L. and the two data sets were found to be 100% in agreement. The genotyping by multiplexed sequencing (GMS) protocol multiplexes 1,656 wheat SNP markers, 207 syntenic barley SNP markers, and 49 known informative markers, which generate a possible 2,433 data points (including homoeoalleles and paralogs). This genotyping approach has the flexibility of being sequenced on either the Ion Torrent or Illumina next generation sequencing (NGS) platforms. Products are the result of direct sequencing and are therefore more reliable than scatter plot analysis which is the output of other genotyping methods such as the Illumina Infinium assay, komeptitive allele specific PCR and other like technologies.

Whole-Genome Mapping of Stripe Rust Resistance Quantitative Trait Loci and Race Specificity Related to Resistance Reduction in Winter Wheat Cultivar Eltan.

Winter wheat cultivar Eltan has been one of the most widely grown cultivars in the U.S. Pacific Northwest. It has shown variable levels of resistance to stripe rust in different years since it was released in 1990. To map all currently effective and defeated resistance genes in Eltan and understand the factors causing the resistance changes, 112 F2:5 recombinant inbred lines (RILs) were developed from a cross of Eltan with cultivar Avocet S. The RILs were evaluated in fields of Pullman, Washington in 2015, 2016, 2017, and 2018 and Mount Vernon, Washington in 2016 and 2017 under natural infections; they were also evaluated in the greenhouse with races PSTv-4 and PSTv-40 of Puccinia striiformis f. sp. tritici. The RILs were genotyped with the 90K Illumina iSelect wheat single-nucleotide polymorphism chip. A total of five quantitative trait loci (QTLs) were identified in Eltan. Two major QTLs on chromosome arms 2BS and 4AL were detected in the greenhouse tests, explaining up to 28.0 and 42.0% of phenotypic variation, respectively. The two race-specific QTLs were also detected in some field experiments but with reduced effects. A minor QTL on 5BS was detected in the greenhouse and field tests, explaining 10.0 to 14.8% of the phenotypic variation. The other two minor QTLs were mapped on 6AS and 7BL and detected only in field experiments, explaining up to 20.5 and 13.5% of phenotypic variation, respectively. All stripe rust samples collected in the experimental fields in 2015 and 2016 were identified as P. striiformis f. sp. tritici races virulent on seedlings of Eltan. The resistance reduction of Eltan was caused by changes of the P. striiformis f. sp. tritici population from avirulent to virulent, overcoming the race-specific all-stage resistance in Eltan.

Ethyl-methanesulfonate mutagenesis generated diverse isolates of Puccinia striiformis f. sp. tritici, the wheat stripe rust pathogen.

Puccinia striiformis f. sp. tritici (Pst) is an obligate biotrophic fungal pathogen causing stripe rust, one of the most important diseases of wheat worldwide. Mutation is considered as one of the major mechanisms causing virulence changes in the pathogen population, but experimental evidence is limited. To study the effect of mutation on pathogen variation, we developed 33 mutant isolates by treating urediniospores of Pst race PSTv-18, avirulent to all of the 18 Yr single-gene lines used to differentiate Pst races, with ethyl methanesulfonate (EMS). These isolates were characterized as 24 races, including 19 new races, through virulence testing on the set of 18 wheat Yr single-gene differential lines; and as 21 multi-locus genotypes with 19 simple sequence repeat and 48 single-nucleotide polymorphism markers. Most of the mutant isolates had more than one avirulence gene and more than one marker locus changed compared to the wild type isolate, indicating that EMS is able to cause mutations at multiple genome sites. The results showed that mutation can cause substantial changes in both avirulence and other genomic regions. The different frequencies of virulence among the mutant isolates suggested homozygous or heterozygous avirulence loci in the parental isolate, or relative ease of mutation at some avirulence loci. The results are useful for understanding evolutionary mechanisms of the important fungal pathogen.

Development, Validation, and Re-selection of Wheat Lines with Pyramided Genes Yr64 and Yr15 Linked on the Short Arm of Chromosome 1B for Resistance to Stripe Rust.

Stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is one of the most destructive diseases of wheat worldwide. The disease is most preferably managed by developing and growing cultivars with high-level, durable resistance. To achieve high-level and long-lasting resistance, we developed a wheat line, RIL-Yr64/Yr15, by pyramiding Yr64 and Yr15, both on the chromosome 1BS and providing high resistance to all tested Pst races. To validate RIL-Yr64/Yr15 possessing both genes, we crossed it to Avocet S (AvS). The F4 RILs from this cross were phenotyped with Pst races under controlled greenhouse conditions and also under natural Pst infection in the field. The population was genotyped with SSR markers previously reported to be linked to the resistance gene loci and with additional SSR and SNP-KASP markers along chromosome 1B. Both phenotype and genotype data confirmed the copresence of Yr64 and Yr15 in RIL-Yr64/Yr15, and the high-resolution linkage map dissected the chromosomal regions and traced their origins. New lines possessing these genes were selected from the F5 population of cross AvS × RIL-Yr64/Yr15 by marker-assisted selection. These lines with the two highly effective genes should be more useful than individual gene lines for developing high-level, durable resistant wheat cultivars.