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.

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 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.

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.

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.

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.

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.

Exome sequencing of bulked segregants identified a novel TaMKK3-A allele linked to the wheat ERA8 ABA-hypersensitive germination phenotype.

KEY MESSAGE: Using bulked segregant analysis of exome sequence, we fine-mapped the ABA-hypersensitive mutant ERA8 in a wheat backcross population to the TaMKK3-A locus of chromosome 4A. Preharvest sprouting (PHS) is the germination of mature grain on the mother plant when it rains before harvest. The ENHANCED RESPONSE TO ABA8 (ERA8) mutant increases seed dormancy and, consequently, PHS tolerance in soft white wheat 'Zak.' ERA8 was mapped to chromosome 4A in a Zak/'ZakERA8' backcross population using bulked segregant analysis of exome sequenced DNA (BSA-exome-seq). ERA8 was fine-mapped relative to mutagen-induced SNPs to a 4.6 Mb region containing 70 genes. In the backcross population, the ERA8 ABA-hypersensitive phenotype was strongly linked to a missense mutation in TaMKK3-A-G1093A (LOD 16.5), a gene associated with natural PHS tolerance in barley and wheat. The map position of ERA8 was confirmed in an 'Otis'/ZakERA8 but not in a 'Louise'/ZakERA8 mapping population. This is likely because Otis carries the same natural PHS susceptible MKK3-A-A660S allele as Zak, whereas Louise carries the PHS-tolerant MKK3-A-C660R allele. Thus, the variation for grain dormancy and PHS tolerance in the Louise/ZakERA8 population likely resulted from segregation of other loci rather than segregation for PHS tolerance at the MKK3 locus. This inadvertent complementation test suggests that the MKK3-A-G1093A mutation causes the ERA8 phenotype. Moreover, MKK3 was a known ABA signaling gene in the 70-gene 4.6 Mb ERA8 interval. None of these 70 genes showed the differential regulation in wild-type Zak versus ERA8 expected of a promoter mutation. Thus, the working model is that the ERA8 phenotype results from the MKK3-A-G1093A mutation.

Molecular Characterization of International Collections of the Wheat Stripe Rust Pathogen Puccinia striiformis f. sp. tritici Reveals High Diversity and Intercontinental Migration.

Puccinia striiformis f. sp. tritici causes stripe rust (yellow rust), one of the most important wheat diseases worldwide. To understand the genetic variation of the pathogen in a global scale, 283 P. striiformis f. sp. tritici isolates collected from 16 countries in eight geographic regions were genotyped using 24 codominant simple sequence repeat markers. The overall collection had a high level of genetic diversity, and the diversity levels in the Asian populations were generally higher than those of the other regions. Heterozygosity of isolates ranged from 0 to 75%, with an average of 46%. Mean heterozygosity in individual countries ranged from 34 to 59%. A total of 265 multilocus genotypes (MLGs) were detected, which were classified into eight molecular groups. Some of the molecular groups were present in all geographic regions. Moreover, many isolates from different regions were found to be identical or very closely related MLGs. Analysis of molecular variance revealed high variation within countries and intermediate variation between countries, but it revealed low and insignificant variation among geographic regions. Pairwise comparisons of regional populations detected considerable effective migrants and only low to moderate levels of differentiation. The molecular genotypes had a moderate level of correlation with the virulence phenotypes, and some of the molecular/virulence groups contained isolates from different continents. The results indicate tremendous migrations of P. striiformis f. sp. tritici and warrant the development of management strategies considering the global pathogen population.

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.

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.

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.

Introgression of a Novel Ug99-Effective Stem Rust Resistance Gene into Wheat and Development of Dasypyrum villosum Chromosome-Specific Markers via Genotyping-by-Sequencing (GBS).

Dasypyrum villosum is a wild relative of common wheat (Triticum aestivum L.) with resistance to Puccinia graminis f. tritici, the causal agent of stem rust, including the highly virulent race TTKSK (Ug99). In order to transfer resistance, T. durum-D. villosum amphiploids were initially developed and used as a bridge to create wheat-D. villosum introgression lines. Conserved ortholog set (COS) markers were used to identify D. villosum chromosome introgression lines, which were then subjected to seedling P. graminis f. tritici resistance screening with race TTKSK. A COS marker-verified line carrying chromosome 2V with TTKSK resistance was further characterized by combined genomic in situ and fluorescent in situ analyses to confirm a monosomic substitution line MS2V(2D) (20″ + 1' 2V[2D]). This is the first report of stem rust resistance on 2V, which was temporarily designated as SrTA10276-2V. To facilitate the use of this gene in wheat improvement, a complete set of previously developed wheat-D. villosum disomic addition lines was subjected to genotyping-by-sequencing analysis to develop D. villosum chromosome-specific markers. On average, 350 markers per chromosome were identified. These markers can be used to develop diagnostic markers for D. villosum-derived genes of interest in wheat improvement.

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.

Characterization of Novel Gene Yr79 and Four Additional Quantitative Trait Loci for All-Stage and High-Temperature Adult-Plant Resistance to Stripe Rust in Spring Wheat PI 182103.

Stripe rust, caused by Puccinia striiformis f. sp. tritici, is an important disease of wheat worldwide. Exploring new resistance genes is essential for breeding resistant wheat cultivars. PI 182103, a spring wheat landrace originally from Pakistan, has shown a high level of resistance to stripe rust in fields for many years, but genes for resistance to stripe rust in the variety have not been studied. To map the resistance gene(s) in PI 182103, 185 recombinant inbred lines (RILs) were developed from a cross with Avocet Susceptible (AvS). The RIL population was genotyped with simple sequence repeat (SSR) and single nucleotide polymorphism markers and tested with races PST-100 and PST-114 at the seedling stage under controlled greenhouse conditions and at the adult-plant stage in fields at Pullman and Mt. Vernon, Washington under natural infection by the stripe rust pathogen in 2011, 2012, and 2013. A total of five quantitative trait loci (QTL) were detected. QyrPI182103.wgp-2AS and QyrPI182103.wgp-3AL were detected at the seedling stage, QyrPI182103.wgp-4DL was detected only in Mt. Vernon field tests, and QyrPI182103.wgp-5BS was detected in both seedling and field tests. QyrPI182103.wgp-7BL was identified as a high-temperature adult-plant resistance gene and detected in all field tests. Interactions among the QTL were mostly additive, but some negative interactions were detected. The 7BL QTL was mapped in chromosomal bin 7BL 0.40 to 0.45 and identified as a new gene, permanently designated as Yr79. SSR markers Xbarc72 and Xwmc335 flanking the Yr79 locus were highly polymorphic in various wheat genotypes, indicating that the molecular markers are useful for incorporating the new gene for potentially durable stripe rust resistance into new wheat cultivars.

Inheritance of Virulence, Construction of a Linkage Map, and Mapping Dominant Virulence Genes in Puccinia striiformis f. sp. tritici Through Characterization of a Sexual Population with Genotyping-by-Sequencing.

Puccinia striiformis f. sp. tritici, the wheat stripe rust pathogen, is a dikaryotic, biotrophic, and macrocyclic fungus. Genetic study of P. striiformis f. sp. tritici virulence was not possible until the recent discovery of Berberis spp. and Mahonia spp. as alternate hosts. To determine inheritance of virulence and map virulence genes, a segregating population of 119 isolates was developed by self-fertilizing P. striiformis f. sp. tritici isolate 08-220 (race PSTv-11) on barberry leaves under controlled greenhouse conditions. The progeny isolates were phenotyped on a set of 29 wheat lines with single genes for race-specific resistance and genotyped with simple sequence repeat (SSR) markers, single nucleotide polymorphism (SNP) markers derived from secreted protein genes, and SNP markers from genotyping-by-sequencing (GBS). Using the GBS technique, 10,163 polymorphic GBS-SNP markers were identified. Clustering and principal component analysis grouped these markers into six genetic groups, and a genetic map, consisting of six linkage groups, was constructed with 805 markers. The six clusters or linkage groups resulting from these analyses indicated a haploid chromosome number of six in P. striiformis f. sp. tritici. Through virulence testing of the progeny isolates, the parental isolate was found to be homozygous for the avirulence loci corresponding to resistance genes Yr5, Yr10, Yr15, Yr24, Yr32, YrSP, YrTr1, Yr45, and Yr53 and homozygous for the virulence locus corresponding to resistance gene Yr41. Segregation was observed for virulence phenotypes in response to the remaining 19 single-gene lines. A single dominant gene or two dominant genes with different nonallelic gene interactions were identified for each of the segregating virulence phenotypes. Of 27 dominant virulence genes identified, 17 were mapped to two chromosomes. Markers tightly linked to some of the virulence loci may facilitate further studies to clone these genes. The virulence genes and their inheritance information are useful for understanding the host-pathogen interactions and for selecting effective resistance genes or gene combinations for developing stripe rust resistant wheat cultivars.

Virulence and Molecular Characterization of Experimental Isolates of the Stripe Rust Pathogen (Puccinia striiformis) Indicate Somatic Recombination.

Puccinia striiformis causes stripe rust on wheat, barley, and grasses. Natural population studies have indicated that somatic recombination plays a possible role in P. striiformis variation. To determine whether somatic recombination can occur, susceptible wheat or barley plants were inoculated with mixed urediniospores of paired isolates of P. striiformis. Progeny isolates were selected by passing through a series of inoculations of wheat or barley genotypes. Potential recombinant isolates were compared with the parental isolates on the set of 18 wheat or 12 barley genotypes that are used to differentiate races of P. striiformis f. sp. tritici (the wheat stripe rust pathogen) and P. striiformis f. sp. hordei (the barley stripe rust pathogen), respectively, for virulence changes. They were also tested with 51 simple-sequence repeat and 90 single-nucleotide polymorphism markers for genotype changes. From 68 possible recombinant isolates obtained from nine combinations of isolates based on virulence tests, 66 were proven to be recombinant isolates by molecular markers. Various types of recombinants were determined, including lost virulence from both virulent parental isolates, gained virulence from both avirulent isolates, combined virulences from both parents, and inherited virulence from one parent and avirulence from another. Marker data indicate that most of the recombinants were produced through chromosome reassortment and crossover after the hybridization of two parental isolates. The results demonstrate that somatic recombination is a mechanism by which new variants can be generated in P. striiformis.

Genome-Wide Association Mapping of Fusarium Head Blight Resistance in Spring Wheat Lines Developed in the Pacific Northwest and CIMMYT.

Fusarium head blight (FHB) is a destructive disease of wheat in humid and semihumid areas of the world. It has emerged in the Pacific Northwest (PNW) in recent years because of changing climate and crop rotation practices. Our objectives in the present study were to identify and characterize quantitative trait loci (QTL) associated with FHB resistance in spring wheat lines developed in the PNW and the International Maize and Wheat Improvement Center. In total, 170 spring wheat lines were evaluated in field and greenhouse trials in 2015 and 2016. Fourteen lines showing consistent resistance in multiple environments were identified. These lines are valuable resources in wheat variety improvement of FHB resistance because they have no Sumai 3 or Sumai 3-related background. The 170 lines were genotyped using a high-density Illumina 90K single-nucleotide polymorphisms (SNP) assay and 10 other non-SNP markers. A genome-wide association analysis was conducted with a mixed model (Q+K). Consistent, significant SNP associations with multiple traits were found on chromosomes 1B, 2B, 4B, 5A, 5B, and 6A. The locus on chromosome 5B for reduced deoxynivalenol content may be novel. The identified QTL are being validated in additional mapping studies and the identified resistant lines are being used in variety development for FHB resistance and facilitated by marker-assisted selection.