Enhancing thalassemia gene carrier identification in non-anemic populations using artificial intelligence erythrocyte morphology analysis and machine learning.

BACKGROUND: Non-anemic thalassemia trait (TT) accounted for a high proportion of TT cases in South China. OBJECTIVE: To use artificial intelligence (AI) analysis of erythrocyte morphology and machine learning (ML) to identify TT gene carriers in a non-anemic population. METHODS: Digital morphological data from 76 TT gene carriers and 97 controls were collected. The AI technology-based Mindray MC-100i was used to quantitatively analyze the percentage of abnormal erythrocytes. Further, ML was used to construct a prediction model. RESULTS: Non-anemic TT carriers accounted for over 60% of the TT cases. Random Forest was selected as the prediction model and named TT@Normal. The TT@Normal algorithm showed outstanding performance in the training, validation, and external validation sets and could efficiently identify TT carriers in the non-anemic population. The top three weights in the TT@Normal model were the target cells, microcytes, and teardrop cells. Elevated percentages of abnormal erythrocytes should raise a strong suspicion of being a TT gene carrier. TT@Normal could be promoted and used as a visualization and sharing tool. It is accessible through a URL link and can be used by medical staff online to predict the possibility of TT gene carriage in a non-anemic population. CONCLUSIONS: The ML-based model TT@Normal could efficiently identify TT carriers in non-anemic people. Elevated percentages of target cells, microcytes, and teardrop cells should raise a strong suspicion of being a TT gene carrier.

Identification of a Locus for High-Temperature Adult-Plant Resistance to Stripe Rust in the Wheat Yr8 Near-Isogenic Line through Mutagenesis and Molecular Mapping.

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 (4-20oC) 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 (10-30oC) 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 SNPs. Yr8 was mapped to a 0.66 cM fragment and YrM488 to a 1.22 cM interval on chromosome 2D. The physical distance between the two resistance genes was estimated to be over 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.

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.

Mining the Australian Grains Gene Bank for Rust Resistance in Barley.

Global barley production is threatened by plant pathogens, especially the rusts. In this study we used a targeted genotype-by-sequencing (GBS) assisted GWAS approach to identify rust resistance alleles in a collection of 287 genetically distinct diverse barley landraces and historical cultivars available in the Australian Grains Genebank (AGG) and originally sourced from Eastern Europe. The accessions were challenged with seven US-derived cereal rust pathogen races including Puccinia hordei (Ph-leaf rust) race 17VA12C, P. coronata var. hordei (Pch-crown rust) race 91NE9305 and five pathogenically diverse races of P. striiformis f. sp. hordei (Psh-stripe rust) (PSH-33, PSH-48, PSH-54, PSH-72 and PSH-100) and phenotyped quantitatively at the seedling stage. Novel resistance factors were identified on chromosomes 1H, 2H, 4H and 5H in response to Pch, whereas a race-specific QTL on 7HS was identified that was effective only to Psh isolates PSH-72 and PSH-100. A major effect QTL on chromosome 5HL conferred resistance to all Psh races including PSH-72, which is virulent on all 12 stripe rust differential tester lines. The same major effect QTL was also identified in response to leaf rust (17VA12C) suggesting this locus contains several pathogen specific rust resistance genes or the same gene is responsible for both leaf rust and stripe rust resistance. Twelve accessions were highly resistant to both leaf and stripe rust diseases and also carried the 5HL QTL. We subsequently surveyed the physical region at the 5HL locus for across the barley pan genome variation in the presence of known resistance gene candidates and identified a rich source of high confidence protein kinase and antifungal genes in the QTL region.

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.

Race Characterization of Puccinia striiformis f. sp. tritici in the United States from 2013 to 2017.

Stripe rust, caused by Puccinia striiformis f. sp. tritici, is an important disease of wheat. In this study, 1,567 isolates collected from the United States from 2013 to 2017 were tested for virulence on 18 wheat Yr single-gene lines to differentiate races. In total, 72 races, including 20 new, were identified, and their frequencies in different years and different epidemiological regions were determined and compared. The 20 new races had low frequencies, and 7 of them each were detected from only one sample and 10 only in a single year. Frequencies of virulence to Yr10, Yr24, and Yr32 were low (<10%); to Yr1, Yr76, YrTr1, and YrSP were moderate (10 to 40%); and to Yr6, Yr7, Yr8, Yr9, Yr17, Yr27, Yr43, Yr44, and Exp2 were high (>70%), although they varied from year to year and from region to region. No virulence was detected to either Yr5 or Yr15, indicating that these genes were still effective against the pathogen in the United States. Based on the virulence data, the diversity of the U.S. P. striiformis f. sp. tritici population was the highest in 2016 and lowest in 2015, and the diversity of the regional population was the highest in region 1 and lowest in region 11. The yearly populations between consecutive years were closer than nonconsecutive years, and the eastern populations were closer to each other than those among the western populations. The findings are useful for understanding the pathogen evolution and for developing resistant cultivars for control of the disease.

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.

Current Status and Future Perspectives of Genomics Research in the Rust Fungi.

Rust fungi in Pucciniales have caused destructive plant epidemics, have become more aggressive with new virulence, rapidly adapt to new environments, and continually threaten global agriculture. With the rapid advancement of genome sequencing technologies and data analysis tools, genomics research on many of the devastating rust fungi has generated unprecedented insights into various aspects of rust biology. In this review, we first present a summary of the main findings in the genomics of rust fungi related to variations in genome size and gene composition between and within species. Then we show how the genomics of rust fungi has promoted our understanding of the pathogen virulence and population dynamics. Even with great progress, many questions still need to be answered. Therefore, we introduce important perspectives with emphasis on the genome evolution and host adaptation of rust fungi. We believe that the comparative genomics and population genomics of rust fungi will provide a further understanding of the rapid evolution of virulence and will contribute to monitoring the population dynamics for disease management.

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.

Whole-genome sequencing of Puccinia striiformis f. sp. tritici mutant isolates identifies avirulence gene candidates.

BACKGROUND: The stripe rust pathogen, Puccinia striiformis f. sp. tritici (Pst), threats world wheat production. Resistance to Pst is often overcome by pathogen virulence changes, but the mechanisms of variation are not clearly understood. To determine the role of mutation in Pst virulence 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 in Pst. 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 the Pst pathogen.

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.

MARPLE, a point-of-care, strain-level disease diagnostics and surveillance tool for complex fungal pathogens.

BACKGROUND: Effective disease management depends on timely and accurate diagnosis to guide control measures. The capacity to distinguish between individuals in a pathogen population with specific properties such as fungicide resistance, toxin production and virulence profiles is often essential to inform disease management approaches. The genomics revolution has led to technologies that can rapidly produce high-resolution genotypic information to define individual variants of a pathogen species. However, their application to complex fungal pathogens has remained limited due to the frequent inability to culture these pathogens in the absence of their host and their large genome sizes. RESULTS: Here, we describe the development of Mobile And Real-time PLant disEase (MARPLE) diagnostics, a portable, genomics-based, point-of-care approach specifically tailored to identify individual strains of complex fungal plant pathogens. We used targeted sequencing to overcome limitations associated with the size of fungal genomes and their often obligately biotrophic nature. Focusing on the wheat yellow rust pathogen, Puccinia striiformis f.sp. tritici (Pst), we demonstrate that our approach can be used to rapidly define individual strains, assign strains to distinct genetic lineages that have been shown to correlate tightly with their virulence profiles and monitor genes of importance. CONCLUSIONS: MARPLE diagnostics enables rapid identification of individual pathogen strains and has the potential to monitor those with specific properties such as fungicide resistance directly from field-collected infected plant tissue in situ. Generating results within 48 h of field sampling, this new strategy has far-reaching implications for tracking plant health threats.

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.

Genome-Wide Mapping of Quantitative Trait Loci Conferring All-Stage and High-Temperature Adult-Plant Resistance to Stripe Rust in Spring Wheat Landrace PI 181410.

Stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is one of the most destructive diseases of wheat in the world. Genetic resistance is the best strategy for control of the disease. Spring wheat landrace PI 181410 has shown high level resistance to stripe rust. The present study characterized the landrace to have both race-specific all-stage resistance and nonrace-specific high-temperature adult-plant (HTAP) resistance. To map quantitative trait loci (QTL) for the resistance in PI 181410, it was crossed with Avocet S (AvS), from which a recombinant inbred line population was developed. The F5-F8 populations were consecutively phenotyped for stripe rust response in multiple field environments under natural Pst infection, and the F7 population was phenotyped in seedlings at low temperature and in adult-plant stage with selected Pst races in the greenhouse. The F7 population was genotyped using the 90K wheat SNP chip. Three QTL, QYrPI181410.wgp-4AS, QYrPI181410.wgp-4BL, and QYrPI181410.wgp-5BL.1, from PI 181410 for all-stage resistance, were mapped on chromosome arms 4AS, 4BL, and 5BL, respectively. Four QTL, QYrPI181410.wgp-1BL, QYrPI181410.wgp-4BL, QYrPI181410.wgp-5AS, and QYrPI181410.wgp-5BL.2, were identified from PI 181410 for HTAP resistance and mapped to 1BL, 4BL, 5AS, and 5BL, respectively. Two QTL with minor effects on stripe rust response were identified from AvS and mapped to 2BS and 2BL. Four of the QTL from PI 181410 and one from AvS were potentially new. As the 4BL QTL was most effective and likely a new gene for stripe rust resistance, three kompetitive allele specific PCR (KASP) markers were developed for incorporating this gene into new 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.

Genome Sequence Resource of a Puccinia striiformis Isolate Infecting Wheatgrass.

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.

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.

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.