Matthew N. Rouse

Detection of virulence to resistance gene Sr36 within the TTKS race lineage of Puccinia graminis f. sp. tritici

The stem rust resistance gene Sr36 confers a near-immune resistance reaction to many races of Puccinia graminis f. sp. tritici and is highly effective against race TTKSK (syn. Ug99), which possesses unusually broad virulence combinations. Because this gene is widely used in United States soft winter wheat germplasm and cultivars, it has been considered to be an important source of resistance to TTKSK. In 2007, moderately susceptible infection responses were observed on wheat lines and cultivars carrying Sr36 in a field screening nursery for stem rust at Njoro, Kenya. We derived 18 single-pustule isolates from stem rust samples collected from the 2007 Njoro nursery. The isolates were evaluated for virulence on 20 North American stem rust differential lines and on wheat lines and cultivars carrying Sr36, Sr31+Sr36, and Sr24+Sr31. Of the 18 isolates, 10 produced infection types 3+ to 4 on line W2691SrTt-1 (monogenic for Sr36) and other lines that carry Sr36 and belonged to a new virulence phenotype that was not detected in previous years. These isolates were identified as race TTTSK. The remaining eight isolates were identified as races TTKSK (five isolates) and TTKST (three isolates), with avirulence and virulence, respectively, to Sr24. Thirteen simple sequence repeat (SSR) markers were used to examine the genetic relationships among the three races in the TTKS lineage. All isolates in the lineage shared an identical SSR genotype and were clearly different from North American races. In all, 16 wheat cultivars and 60 elite breeding lines, postulated to possess Sr36, were susceptible to race TTTSK. The occurrence of race TTTSK with combined virulence on Sr31 and Sr36 has further broadened the virulence spectrum of the TTKS lineage and rendered an important source of resistance ineffective.

Identification of wheat gene Sr35 that confers resistance to Ug99 stem rust race group

Resistance May Not Be Futile Recently, Ug99, a particularly devastating strain of wheat stem rust fungus, has emerged, which could potentially threaten food security. Now, two genes have been cloned that offer resistance to Ug99. Saintenac et al. (p. 783, published online 27 June) cloned Sr35 from Triticum monococcum, a diploid wheat species not often cultivated. Periyannan et al. (p. 786, published online 27 June) cloned Sr33 from Aegilops tauschii, a diploid wild grass that contributed to the hexaploid genome of cultivated wheat. The genes both encode proteins that show features typical of other disease resistance proteins and offer opportunities to slow the pace of Ug99 progression. Two resistance genes are identified that could protect wheat from a virulent fungus that can severely reduce crop yields. Wheat stem rust, caused by Puccinia graminis f. sp. tritici (Pgt), is a devastating disease that can cause severe yield losses. A previously uncharacterized Pgt race, designated Ug99, has overcome most of the widely used resistance genes and is threatening major wheat production areas. Here, we demonstrate that the Sr35 gene from Triticum monococcum is a coiled-coil, nucleotide-binding, leucine-rich repeat gene that confers near immunity to Ug99 and related races. This gene is absent in the A-genome diploid donor and in polyploid wheat but is effective when transferred from T. monococcum to polyploid wheat. The cloning of Sr35 opens the door to the use of biotechnological approaches to control this devastating disease and to analyses of the molecular interactions that define the wheat-rust pathosystem.

Rapid cloning of disease-resistance genes in plants using mutagenesis and sequence capture

Wild relatives of domesticated crop species harbor multiple, diverse, disease resistance (R) genes that could be used to engineer sustainable disease control. However, breeding R genes into crop lines often requires long breeding timelines of 5–15 years to break linkage between R genes and deleterious alleles (linkage drag). Further, when R genes are bred one at a time into crop lines, the protection that they confer is often overcome within a few seasons by pathogen evolution. If several cloned R genes were available, it would be possible to pyramid R genes in a crop, which might provide more durable resistance. We describe a three-step method (MutRenSeq)-that combines chemical mutagenesis with exome capture and sequencing for rapid R gene cloning. We applied MutRenSeq to clone stem rust resistance genes Sr22 and Sr45 from hexaploid bread wheat. MutRenSeq can be applied to other commercially relevant crops and their relatives, including, for example, pea, bean, barley, oat, rye, rice and maize.

Discovery and molecular mapping of a new gene conferring resistance to stem rust, Sr53, derived from Aegilops geniculata and characterization of spontaneous translocation stocks with reduced alien chromatin.

This study reports the discovery and molecular mapping of a resistance gene effective against stem rust races RKQQC and TTKSK (Ug99) derived from Aegilops geniculata (2n = 4x = 28, UgUgMgMg). Two populations from the crosses TA5599 (T5DL-5MgL·5MgS)/TA3809 (ph1b mutant in Chinese Spring background) and TA5599/Lakin were developed and used for genetic mapping to identify markers linked to the resistance gene. Further molecular and cytogenetic characterization resulted in the identification of nine spontaneous recombinants with shortened Ae. geniculata segments. Three of the wheat–Ae. geniculata recombinants (U6154-124, U6154-128, and U6200-113) are interstitial translocations (T5DS·5DL-5MgL-5DL), with 20–30% proximal segments of 5MgL translocated to 5DL; the other six are recombinants (T5DL-5MgL·5MgS) have shortened segments of 5MgL with fraction lengths (FL) of 0.32–0.45 compared with FL 0.55 for the 5MgL segment in the original translocation donor, TA5599. Recombinants U6200-64, U6200-117, and U6154-124 carry the stem rust resistance gene Sr53 with the same infection type as TA5599, the resistance gene donor. All recombinants were confirmed to be genetically compensating on the basis of genomic in situ hybridization and molecular marker analysis with chromosome 5D- and 5Mg-specific SSR/STS-PCR markers. These recombinants between wheat and Ae. geniculata will provide another source for wheat stem rust resistance breeding and for physical mapping of the resistance locus and crossover hot spots between wheat chromosome 5D and chromosome 5MgL of Ae. geniculata.

A consensus map for Ug99 stem rust resistance loci in wheat

Key messageThis consensus map of stem rust genes, QTLs, and molecular markers will facilitate the identification of new resistance genes and provide a resource of information for development of new markers for breeding wheat varieties resistant to Ug99.AbstractThe global effort to identify new sources of resistance to wheat stem rust, caused by Pucciniagraminis f. sp. tritici race group Ug99 has resulted in numerous studies reporting both qualitative genes and quantitative trait loci. The purpose of our study was to assemble all available information on loci associated with stem rust resistance from 21 recent studies on Triticum aestivum L. (bread wheat) and Triticum turgidum subsp. durum desf. (durum wheat). The software LPmerge was used to construct a stem rust resistance loci consensus wheat map with 1,433 markers incorporating Single Nucleotide Polymorphism, Diversity Arrays Technology, Genotyping-by-Sequencing as well as Simple Sequence Repeat marker information. Most of the markers associated with stem rust resistance have been identified in more than one population. Several loci identified in these populations map to the same regions with known Sr genes including Sr2, SrND643, Sr25 and Sr57 (Lr34/Yr18/Pm38), while other significant markers were located in chromosome regions where no Sr genes have been previously reported. This consensus map provides a comprehensive source of information on 141 stem rust resistance loci conferring resistance to stem rust Ug99 as well as linked markers for use in marker-assisted selection.

Sources of Resistance to Stem Rust Race Ug99 in Spring Wheat Germplasm

Wheat stem rust (Puccinia graminis f. sp. tritici) race TTKSK (Ug99), with virulence to the majority of the worlds wheat (Triticum aestivum) cultivars, has spread from Uganda throughout eastern Africa, Yemen, and Iran. The identification and spread of variants of race TTKSK with virulence to additional stem rust resistance genes has reminded breeders and pathologists of the danger of deploying major resistance genes alone. In order to protect wheat from this rapidly spreading and adapting pathogen, multiple resistance genes are needed, preferably from improved germplasm. Preliminary screening of over 700 spring wheat breeding lines and cultivars developed at least 20 years ago identified 88 accessions with field resistance to Ug99. We included these resistant accessions in the stem rust screening nursery in Njoro, Kenya for two additional seasons. The accessions were also screened with a bulk of North American isolates of P. graminis f. sp. tritici in the field in St. Paul, MN. In order to further characterize the resistance in these accessions, we obtained seedling phenotypes for 10 races of P. graminis f. sp. tritici, including two races from the race TTKSK complex. This phenotyping led to the identification of accessions with either adult-plant or all-stage resistance to race TTKSK, and often North American races of P. graminis f. sp. tritici as well. These Ug99 resistant accessions can be obtained by breeders and introgressed into current breeding germplasm.

Races of Puccinia graminis f. sp. tritici with Combined Virulence to Sr13 and Sr9e in a Field Stem Rust Screening Nursery in Ethiopia

North American durum lines, selected for resistance to TTKSK (Ug99) and related races of Puccinia graminis f. sp. tritici in Kenya, became susceptible in Debre Zeit, Ethiopia, suggesting the presence of stem rust races that were virulent to the TTKSK-effective genes in durum. The objective of this study was to characterize races of P. graminis f. sp. tritici present in the Debre Zeit, Ethiopia stem rust nursery. Three races of P. graminis f. sp. tritici were identified from 34 isolates: JRCQC, TRTTF, and TTKSK. Both races JRCQC and TRTTF possess virulence on stem rust resistance genes Sr13 and Sr9e, which may explain why many TTKSK-resistant durum lines tested in Kenya became susceptible in Debre Zeit. The Sr9e-Sr13 virulence combination is of particular concern because these two genes constitute major components of stem rust resistance in North American durum cultivars. In addition to Sr9e and Sr13 virulence, race TRTTF is virulent to at least three stem rust resistance genes that are effective to race TTKSK, including Sr36, SrTmp, and resistance conferred by the 1AL.1RS rye translocation. Race TRTTF is the first known race with virulence to the stem rust resistance carried by the 1AL.1RS translocation, which represents one of the few effective genes against TTKSK in winter wheat cultivars in the United States. Durum entries exhibiting resistant to moderately susceptible infection response at the Debre Zeit nursery in 2009 were evaluated for reaction to races JRCQC, TRTTF, and TTKSK at the seedling stage. In all, 47 entries were resistant to the three races evaluated at the seedling stage, whereas 26 entries exhibited a susceptible reaction. These results suggest the presence of both major and adult plant resistance genes, which would be useful in durum-wheat-breeding programs. A thorough survey of virulence in the population of P. graminis f. sp. tritici in Ethiopia will allow characterization of the geographic distribution of the races identified in the Debre Zeit field nursery.

Identification of markers linked to the race Ug99 effective stem rust resistance gene Sr28 in wheat (Triticum aestivum L.).

Wheat stem rust caused by Puccinia graminis f. sp. tritici can cause devastating yield losses in wheat. Over the past several decades, stem rust has been controlled worldwide through the use of genetic resistance. Stem rust race TTKSK (Ug99), first detected in Uganda in 1998, threatens global wheat production because of its unique virulence combination. As the majority of the currently grown cultivars and advanced breeding lines are susceptible to race TTKSK, sources of resistance need to be identified and characterized to facilitate their use in agriculture. South Dakota breeding line SD 1691 displayed resistance to race TTKSK in the international wheat stem rust nursery in Njoro, Kenya. Seedling screening of progeny derived from SD 1691 crossed to susceptible LMPG-6 indicated that a single resistance gene was present. Allelism and race-specificity tests indicated the stem rust resistance gene in SD 1691 was Sr28. The chromosome arm location of Sr28 was previously demonstrated to be 2BL. We identified molecular markers linked to Sr28 and validated this linkage in two additional populations. Common spring wheat cultivars in the central United States displayed allelic diversity for markers flanking Sr28. These markers could be used to select for Sr28 in breeding populations and for combining Sr28 with other stem rust resistance genes.

Genomic selection for quantitative adult plant stem rust resistance in wheat

Quantitative adult plant resistance (APR) to stem rust (Puccinia graminis f. sp. tritici) is an important breeding target in wheat (Triticum aestivum L.) and a potential target for genomic selection (GS). To evaluate the relative importance of known APR loci in applying GS, we characterized a set of CIMMYT germplasm at important APR loci and on a genome‐wide profile using genotyping‐by‐sequencing (GBS). Using this germplasm, we describe the genetic architecture and evaluate prediction models for APR using data from the international Ug99 stem rust screening nurseries. Prediction models incorporating markers linked to important APR loci and seedling phenotype scores as fixed effects were evaluated along with the classic prediction models: Multiple linear regression (MLR), Genomic best linear unbiased prediction (G‐BLUP), Bayesian Lasso (BL), and Bayes Cπ (BCπ). We found the Sr2 region to play an important role in APR in this germplasm. A model using Sr2 linked markers as fixed effects in G‐BLUP was more accurate than MLR with Sr2 linked markers (p‐value = 0.12), and ordinary G‐BLUP (p‐value = 0.15). Incorporating seedling phenotype information as fixed effects in G‐BLUP did not consistently increase accuracy. Overall, levels of prediction accuracy found in this study indicate that GS can be effectively applied to improve stem rust APR in this germplasm, and if genotypes at Sr2 linked markers are available, modeling these genotypes as fixed effects could lead to better predictions.

Development and characterization of wheat-Ae. searsii Robertsonian translocations and a recombinant chromosome conferring resistance to stem rust

The emergence of a new highly virulent race of stem rust (Puccinia graminis tritici), Ug99, rapid evolution of new Ug99 derivative races overcoming resistance of widely deployed genes, and spread towards important wheat growing areas now potentially threaten world food security. Exploiting novel genes effective against Ug99 from wild relatives of wheat is one of the most promising strategies for the protection of the wheat crop. A new source of resistance to Ug99 was identified in the short arm of the Aegilopssearsii chromosome 3Ss by screening wheat- Ae.searsii introgression libraries available as individual chromosome and chromosome arm additions to the wheat genome. For transferring this resistance gene into common wheat, we produced three double-monosomic chromosome populations (3A/3Ss, 3B/3Ss and 3D/3Ss) and then applied integrated stem rust screening, molecular maker analysis, and cytogenetic analysis to identify resistant wheat-Ae. searsii Robertsonian translocation. Three Robertsonian translocations (T3AL·3SsS, T3BL·3SsS and T3DL·3SsS) and one recombinant (T3DS-3SsS·3SsL) with stem rust resistance were identified and confirmed to be genetically compensating on the basis of genomic in situ hybridization, analysis of 3A, 3B, 3D and 3SsS-specific SSR/STS-PCR markers, and C-banding. In addition, nine SSR/STS-PCR markers of 3SsS-specific were developed for marker-assisted selection of the resistant gene. Efforts to reduce potential linkage drag associated with 3SsS of Ae. searsii are currently under way.