Development of a Diagnostic Assay for Differentiation Between Genetic Groups in Clades I, II, III, and IV of Puccinia graminis f. sp. tritici.

Wheat stem rust has reemerged as a serious disease caused by new variants of Puccinia graminis f. sp. tritici. Variants with significant virulence and broad geographic distribution (Africa, Central Asia, and Europe) include the Ug99 race group, race TTRTF, and TKTTF race group. Genetic analysis has placed isolates representing these critical new virulent races into 12 genetic groups that make up clades I to IV. Development of molecular diagnostic assays for these 12 genetic groups will be an important component of global surveillance efforts. A single-nucleotide polymorphism database was mined for candidate markers that would differentiate between these 12 genetic groups. Thirty-five candidate markers were screened, and a core set of 17 markers was tested against a set of 94 isolates representing a broad range of genotypes and race phenotypes. These core markers were 100% accurate in identifying the 12 genetic groups for 52 isolates in clades I to IV, and no false positives were observed with nontarget isolates. The assay has built-in redundancy so that minor genetic changes or errors in genotyping calling will not affect the accuracy of the results. This assay is also effective in identifying the genetic groups in clade V from Germany and Georgia, the three main subgroups in North American clade VI, and clade VII consisting of race TTTTF found in North and South America. This assay provides a rapid diagnostic tool for both living and nonliving samples to detect these critical new races or race groups of P. graminis f. sp. tritici.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Distribution of Puccinia striiformis f. sp. tritici Races and Virulence in Wheat Growing Regions of Kenya from 1970 to 2014.

Stripe rust, caused by the fungal pathogen Puccinia striiformis f. sp. tritici, is a major threat to wheat (Triticum spp.) production worldwide. The objective of this study was to determine the virulence of P. striiformis f. sp. tritici races prevalent in the main wheat growing regions of Kenya, which includes Mt. Kenya, Eastern Kenya, and the Rift Valley (Central, Southern, and Northern Rift). Fifty P. striiformis f. sp. tritici isolates collected from 1970 to 1992 and from 2009 to 2014 were virulence phenotyped with stripe rust differential sets, and 45 isolates were genotyped with sequence characterized amplified region (SCAR) markers to differentiate the isolates and identify aggressive strains PstS1 and PstS2. Virulence corresponding to stripe rust resistance genes Yr1, Yr2, Yr3, Yr6, Yr7, Yr8, Yr9, Yr17, Yr25, and Yr27 and the seedling resistance in genotype Avocet S were detected. Ten races were detected in the P. striiformis f. sp. tritici samples obtained from 1970 to 1992, and three additional races were detected from 2009 to 2014, with a single race being detected in both periods. The SCAR markers detected both Pst1 and Pst2 strains in the collection. Increasing P. striiformis f. sp. tritici virulence was found in the Kenyan P. striiformis f. sp. tritici population, and different P. striiformis f. sp. tritici race groups were found to dominate different wheat growing regions. Moreover, recent P. striiformis f. sp. tritici races in East Africa indicated possible migration of some race groups into Kenya from other regions. This study is important in elucidating P. striiformis f. sp. tritici evolution and virulence diversity and useful in breeding wheat cultivars with effective resistance to stripe rust.

Identification of Winter Habit Bread Wheat Landraces in the National Small Grains Collection with Resistance to Emerging Stem Rust Pathogen Variants.

Wheat stem rust caused by Puccinia graminis f. sp. tritici is a widespread and recurring threat to wheat production. Emerging P. graminis f. sp. tritici variants are rapidly overcoming major gene resistance deployed in wheat cultivars and new sources of race-nonspecific resistance are urgently needed. The National Small Grains Collection (NSGC) contains thousands of wheat landrace accessions that may harbor unique and broadly effective sources of resistance to emerging P. graminis f. sp. tritici variants. All NSGC available facultative and winter-habit bread wheat landraces were tested in a field nursery in St. Paul, Minnesota, against a bulk collection of six common U.S. P. graminis f. sp. tritici races. Infection response and severity data were collected on 9,192 landrace accessions at the soft-dough stage and resistant accessions were derived from single spikes. Derived accessions were tested in St. Paul a second time to confirm resistance and in a field nursery in Njoro, Kenya against emerging races of P. graminis f. sp. tritici with virulence to many known resistance genes including Sr24, Sr31, Sr38, and SrTmp. Accessions resistant in the St. Paul field were also tested at the seedling stage with up to 13 P. graminis f. sp. tritici races, including TTKSK and TKTTF, and with 19 molecular markers linked with known stem rust resistance genes or genes associated with modern breeding practices. Forty-five accessions were resistant in both U.S. and Kenya field nurseries and lacked alleles linked with known stem rust resistance genes. Accessions with either moderate or strong resistance in the U.S. and Kenya field nurseries and with novel seedling resistance will be prioritized for further study.

Rust expression browser: an open source database for simultaneous analysis of host and pathogen gene expression profiles with expVIP.

BACKGROUND: Transcriptomics is being increasingly applied to generate new insight into the interactions between plants and their pathogens. For the wheat yellow (stripe) rust pathogen (Puccinia striiformis f. sp. tritici, Pst) RNA-based sequencing (RNA-Seq) has proved particularly valuable, overcoming the barriers associated with its obligate biotrophic nature. This includes the application of RNA-Seq approaches to study Pst and wheat gene expression dynamics over time and the Pst population composition through the use of a novel RNA-Seq based surveillance approach called "field pathogenomics". As a dual RNA-Seq approach, the field pathogenomics technique also provides gene expression data from the host, giving new insight into host responses. However, this has created a wealth of data for interrogation. RESULTS: Here, we used the field pathogenomics approach to generate 538 new RNA-Seq datasets from Pst-infected field wheat samples, doubling the amount of transcriptomics data available for this important pathosystem. We then analysed these datasets alongside 66 RNA-Seq datasets from four Pst infection time-courses and 420 Pst-infected plant field and laboratory samples that were publicly available. A database of gene expression values for Pst and wheat was generated for each of these 1024 RNA-Seq datasets and incorporated into the development of the rust expression browser ( http://www.rust-expression.com ). This enables for the first time simultaneous 'point-and-click' access to gene expression profiles for Pst and its wheat host and represents the largest database of processed RNA-Seq datasets available for any of the three Puccinia wheat rust pathogens. We also demonstrated the utility of the browser through investigation of expression of putative Pst virulence genes over time and examined the host plants response to Pst infection. CONCLUSIONS: The rust expression browser offers immense value to the wider community, facilitating data sharing and transparency and the underlying database can be continually expanded as more datasets become publicly available.

Genome-Wide Association Study for Multiple Biotic Stress Resistance in Synthetic Hexaploid Wheat.

Genetic resistance against biotic stress is a major goal in many wheat breeding programs. However, modern wheat cultivars have a limited genetic variation for disease and pest resistance and there is always a possibility of the evolution of new diseases and pests to overcome previously identified resistance genes. A total of 125 synthetic hexaploid wheats (SHWs; 2n = 6x = 42, AABBDD, Triticum aestivum L.) were characterized for resistance to fungal pathogens that cause wheat rusts (leaf; Puccinia triticina, stem; P. graminis f.sp. tritici, and stripe; P. striiformis f.sp. tritici) and crown rot (Fusarium spp.); cereal cyst nematode (Heterodera spp.); and Hessian fly (Mayetiola destructor). A wide range of genetic variation was observed among SHWs for multiple (two to five) biotic stresses and 17 SHWs that were resistant to more than two stresses. The genomic regions and potential candidate genes conferring resistance to these biotic stresses were identified from a genome-wide association study (GWAS). This GWAS study identified 124 significant marker-trait associations (MTAs) for multiple biotic stresses and 33 of these were found within genes. Furthermore, 16 of the 33 MTAs present within genes had annotations suggesting their potential role in disease resistance. These results will be valuable for pyramiding novel genes/genomic regions conferring resistance to multiple biotic stresses from SHWs into elite bread wheat cultivars and providing further insights on a wide range of stress resistance in wheat.

Kenyan Isolates of Puccinia graminis f. sp. tritici from 2008 to 2014: Virulence to SrTmp in the Ug99 Race Group and Implications for Breeding Programs.

Frequent emergence of new variants in the Puccinia graminis f. sp. tritici Ug99 race group in Kenya has made pathogen survey a priority. We analyzed 140 isolates from 78 P. graminis f. sp. tritici samples collected in Kenya between 2008 and 2014 and identified six races, including three not detected prior to 2013. Genotypic analysis of 20 isolates from 2013 and 2014 collections showed that the new races TTHST, TTKTK, and TTKTT belong to the Ug99 race group. International advanced breeding lines were evaluated against an isolate of TTKTT (Sr31, Sr24, and SrTmp virulence) at the seedling stage. From 169 advanced lines from Kenya, 23% of lines with resistance to races TTKSK and TTKST were susceptible to TTKTT and, from two North American regional nurseries, 44 and 91% of resistant lines were susceptible. Three lines with combined resistance genes were developed to facilitate pathogen monitoring and race identification. These results indicate the increasing virulence and variability in the Kenyan P. graminis f. sp. tritici population and reveal vulnerabilities of elite germplasm to new races.

Sources of Stem Rust Resistance in Wheat-Alien Introgression Lines.

Stem rust is one of the most devastating diseases of wheat. Widely virulent races of the pathogen in the Ug99 lineage (e.g., TTKSK) are threatening wheat production worldwide; therefore, there is an urgent need to enhance the diversity of resistance genes in the crop. The objectives of this study were to identify new sources of resistance in wheat-alien introgression derivatives from Secale cereale, Leymus mollis, L. racemosus, and Thinopyrum junceiforme, postulate genes conferring the resistance, and verify the postulated genes by use of molecular markers. From seedling tests conducted in the greenhouse, the presence of seven known stem rust resistance genes (Sr7b, Sr8a, Sr9d, Sr10, Sr31, Sr36, and SrSatu) was postulated in the wheat-alien introgression lines. More lines possessed a high level of resistance in the field compared with the number of lines that were resistant at the seedling stage. Three 2R (2D) wheat-rye substitution lines (SLU210, SLU238, and SLU239) seemed likely to possess new genes for resistance to stem rust based on their resistance pattern to 13 different stem rust races but the genes responsible could not be identified. Wheat-rye, wheat-L. racemosus, and wheat-L. mollis substitutions or translocations with single and multiple interchanges of chromosomes, in particular of the B and D chromosomes of wheat, were verified by a combination of genomic in situ hybridization and molecular markers. Thus, the present study identified novel resistance genes originating from different alien introgressions into the wheat genome of the evaluated lines. Such genes may prove useful in enhancing the diversity of stem rust resistance in wheat against widely virulent pathogen races such as those in the Ug99 lineage.

Association mapping of stem rust race TTKSK resistance in US barley breeding germplasm.

KEY MESSAGE: Loci conferring resistance to the highly virulent African stem rust race TTKSK were identified in advanced barley breeding germplasm and positioned to chromosomes 5H and 7H using an association mapping approach. African races of the stem rust pathogen (Puccinia graminis f. sp. tritici) are a serious threat to barley production worldwide because of their wide virulence. To discover and characterize resistance to African stem rust race TTKSK in US barley breeding germplasm, over 3,000 lines/cultivars were assessed for resistance at the seedling stage in the greenhouse and also the adult plant stage in the field in Kenya. Only 12 (0.3 %) and 64 (2.1 %) lines exhibited a resistance level comparable to the resistant control at the seedling and adult plant stage, respectively. To map quantitative trait loci (QTL) for resistance to race TTKSK, an association mapping approach was conducted, utilizing 3,072 single nucleotide polymorphism (SNP) markers. At the seedling stage, two neighboring SNP markers (0.8 cM apart) on chromosome 7H (11_21491 and 12_30528) were found significantly associated with resistance. The most significant one found was 12_30528; thus, the resistance QTL was named Rpg-qtl-7H-12_30528. At the adult plant stage, two SNP markers on chromosome 5H (11_11355 and 12_31427) were found significantly associated with resistance. This resistance QTL was named Rpg-qtl-5H-11_11355 for the most significant marker identified. Adult plant resistance is of paramount importance for stem rust. The marker associated with Rpg-qtl-5H-11_11355 for adult plant resistance explained only a small portion of the phenotypic variation (0.02); however, this QTL reduced disease severity up to 55.0 % under low disease pressure and up to 21.1 % under heavy disease pressure. SNP marker 11_11355 will be valuable for marker-assisted selection of adult plant stem rust resistance in barley breeding.

Identification of Ug99 stem rust resistance loci in winter wheat germplasm using genome-wide association analysis.

The evolution of a new race of stem rust, generally referred to as Ug99, threatens global wheat production because it can overcome widely deployed resistance genes that had been effective for many years. To identify loci conferring resistance to Ug99 in wheat, a genome-wide association study was conducted using 232 winter wheat breeding lines from the International Winter Wheat Improvement Program. Breeding lines were genotyped with diversity array technology, simple sequence repeat and sequence-tagged site markers, and phenotyped at the adult plant stage for resistance to stem rust in the stem rust resistance screening nursery at Njoro, Kenya during 2009-2011. A mixed linear model was used for detecting marker-trait associations. Twelve loci associated with Ug99 resistance were identified including markers linked to known genes Sr2 and Lr34. Other markers were located in the chromosome regions where no Sr genes have been previously reported, including one each on chromosomes 1A, 2B, 4A and 7B, two on chromosome 5B and four on chromosome 6B. The same data were used for investigating epistatic interactions between markers with or without main effects. The marker csSr2 linked to Sr2 interacted with wPt4930 on 6BS and wPt729773 in an unknown location. Another marker, csLV34 linked to Lr34, also interacted with wPt4930 on 6BS and wPt4916 on 2BS. The frequent involvement of wPt4916 on 2BS and wPt4930 on 6BS in interactions with other significant loci on the same or different chromosomes suggested complex genetic control for adult plant resistance to Ug99 in winter wheat germplasm.