Ug99 Resistance Research Articles

Chromosomal composition analysis and molecular marker development for the novel Ug99-resistant wheat-Thinopyrum ponticum translocation line WTT34.

A novel Ug99-resistant wheat-Thinopyrum ponticum translocation line was produced, its chromosomal composition was analyzed and specific markers were developed. Stem rust caused by Puccinia graminis f. sp. tritici Eriks. & E. Henn (Pgt) has seriously threatened global wheat production since Ug99 race TTKSK was first detected in Uganda in 1998. Thinopyrum ponticum is near immune to Ug99 races and may be useful for enhancing wheat disease resistance. Therefore, developing new wheat-Th. ponticum translocation lines that are resistant to Ug99 is crucial. In this study, a novel wheat-Th. ponticum translocation line, WTT34, was produced. Seedling and field evaluation revealed that WTT34 is resistant to Ug99 race PTKST. The resistance was derived from the alien parent Th. ponticum. Screening WTT34 with markers linked to Sr24, Sr25, Sr26, Sr43, and SrB resulted in the amplification of different DNA fragments from Th. ponticum, implying WTT34 carries at least one novel stem rust resistance gene. Genomic in situ hybridization (GISH), multicolor fluorescence in situ hybridization (mc-FISH), and multi-color GISH (mc-GISH) analyses indicated that WTT34 carries a T5DS·5DL-Th translocation, which was consistent with wheat660K single-nucleotide polymorphism (SNP) array results. The SNP array also uncovered a deletion event in the terminal region of chromosome 1D. Additionally, the homeology between alien segments and the wheat chromosomes 2A and 5D was confirmed. Furthermore, 51 PCR-based markers derived from the alien segments of WTT34 were developed based on specific-locus amplified fragment sequencing (SLAF-seq). These markers may enable wheat breeders to rapidly trace Th. ponticum chromosomal segments carrying Ug99 resistance gene(s).

Molecular Marker Based Design for Breeding Wheat Lines with Multiple Resistance and Superior Quality.

There has not been a major wheat stem rust epidemic worldwide since the 1970s, but the emergence of race TTKSK of Puccinia graminis f. sp. tritici in 1998 presented a great threat to the world wheat production. Single disease-resistance genes are usually effective for only several years before the pathogen changes genetically to overcome the resistance. Stripe rust caused by Puccinia striiformis f. sp. tritici (Pst) is one of the most common and persistent wheat diseases worldwide. The development of varieties with multiple resistance is the most economical and effective strategy for preventing stripe rust and stem rust, the two main rust diseases constraining wheat production. Plateau 448 has been widely used in the spring wheat growing region in northwest China, but it has become susceptible to stripe rust and is susceptible to TTKSK. To produce more durable resistance to race TTKSK as well as to stripe rust, four stem rust resistance genes (Sr33, Sr36, Sr-Cad, and Sr43) and three stripe rust resistance genes (Yr5, Yr18, and Yr26) were simultaneously introgressed into Plateau 448 to improve its stem rust (Ug99) and stripe rust resistance using a marker-assisted backcrossing strategy combined with phenotypic selection. We obtained 131 BC1F5 lines that pyramided two to four Ug99 resistance genes and one to two Pst resistance genes simultaneously. Thirteen of these lines were selected for their TTKSK resistance, and all of them exhibited near immunity or high resistance to TTKSK. Among the 131 pyramided lines, 95 showed high resistance to mixed Pst races. Nine lines exhibited not only high resistance to TTKSK and Pst but also better agronomic traits and high-molecular-weight glutenin subunit compositions than Plateau 448.

Identification and characterization of wheat stem rust resistance gene Sr21 effective against the Ug99 race group at high temperature.

Wheat stem rust, caused by Puccinia graminis f. sp. tritici (Pgt), is a devastating foliar disease. The Ug99 race group has combined virulence to most stem rust (Sr) resistance genes deployed in wheat and is a threat to global wheat production. Here we identified a coiled-coil, nucleotide-binding leucine-rich repeat protein (NLR) completely linked to the Ug99 resistance gene Sr21 from Triticum monococcum. Loss-of-function mutations and transgenic complementation confirmed that this gene is Sr21. Sr21 transcripts were significantly higher at high temperatures, and this was associated with significant upregulation of pathogenesis related (PR) genes and increased levels of resistance at those temperatures. Introgression of Sr21 into hexaploid wheat resulted in lower levels of resistance than in diploid wheat, but transgenic hexaploid wheat lines with high levels of Sr21 expression showed high levels of resistance. Sr21 can be a valuable component of transgenic cassettes or gene pyramids combining multiple resistance genes against Ug99.

Dissection of the multigenic wheat stem rust resistance present in the Montenegrin spring wheat accession PI 362698

BackgroundResearch to identify and characterize stem rust resistance genes in common wheat, Triticum aestivum, has been stimulated by the emergence of Ug99-lineage races of the wheat stem rust pathogen, Puccinia graminis f. sp. tritici (Pgt), in Eastern Africa. The Montenegrin spring wheat landrace PI 362698 was identified as a source of Pgt resistance. This accession exhibits resistance to multiple Ug99-lineage and North American Pgt races at seedling and adult-plant stages. A recombinant inbred population was developed by crossing the susceptible line LMPG-6 with a single plant selection of PI 362698. A genetic map was constructed using the Illumina iSelect 90 K wheat assay and the markers csLv34, NB-LRR3, and wMAS000003 and quantitative trait locus (QTL) analysis was performed.ResultsQTL analysis identified five significant QTLs (α = 0.05) on chromosomes 2B, 3B, 6A, 6D, and 7A associated with wheat stem rust resistance. The QTL on chromosome 3B was identified using both field data from Kenya (Pgt Ug99-lineage races) and seedling data from Pgt race MCCF. This QTL potentially corresponds to Sr12 or a new allele of Sr12. The multi-pathogen resistance gene Sr57 located on chromosome 7D is present in PI 362698 according to the diagnostic markers csLv34 and wMAS000003, however a significant QTL was not detected at this locus. The QTLs on chromosomes 2B, 6A, and 6D were identified during seedling trials and are thought to correspond to Sr16, Sr8a, and Sr5, respectively. The QTL identified on chromosome 7A was detected using MCCF seedling data and may be Sr15 or a potentially novel allele of recently detected Ug99 resistance QTLs.ConclusionsThe combination of resistance QTLs found in PI 362698 is like the resistance gene combination present in the broadly resistant cultivar Thatcher. As such, PI 362698 may not be a landrace as previously thought. PI 362698 has been crossed with North Dakota wheat germplasm for future breeding efforts. Additional work is needed to fully understand why the combination of genes present in PI 362698 and ‘Thatcher’ provide such durable resistance.

Identification and characterization of Sr13, a tetraploid wheat gene that confers resistance to the Ug99 stem rust race group

The Puccinia graminis f. sp. tritici (Pgt) Ug99 race group is virulent to most stem rust resistance genes currently deployed in wheat and poses a threat to global wheat production. The durum wheat (Triticum turgidum ssp. durum) gene Sr13 confers resistance to Ug99 and other virulent races, and is more effective at high temperatures. Using map-based cloning, we delimited a candidate region including two linked genes encoding coiled-coil nucleotide-binding leucine-rich repeat proteins designated CNL3 and CNL13. Three independent truncation mutations identified in each of these genes demonstrated that only CNL13 was required for Ug99 resistance. Transformation of an 8-kb genomic sequence including CNL13 into the susceptible wheat variety Fielder was sufficient to confer resistance to Ug99, confirming that CNL13 is Sr13CNL13 transcripts were slightly down-regulated 2-6 days after Pgt inoculation and were not affected by temperature. By contrast, six pathogenesis-related (PR) genes were up-regulated at high temperatures only when both Sr13 and Pgt were present, suggesting that they may contribute to the high temperature resistance mechanism. We identified three Sr13-resistant haplotypes, which were present in one-third of cultivated emmer and durum wheats but absent in most tested common wheats (Triticum aestivum). These results suggest that Sr13 can be used to improve Ug99 resistance in a large proportion of modern wheat cultivars. To accelerate its deployment, we developed a diagnostic marker for Sr13 The identification of Sr13 expands the number of Pgt-resistance genes that can be incorporated into multigene transgenic cassettes to control this devastating disease.

Identification and validation of single nucleotide polymorphic markers linked to Ug99 stem rust resistance in spring wheat.

Wheat stem rust (Puccinia graminis f. sp. tritici Eriks. and E. Henn.) is one of the most destructive diseases world-wide. Races belonging to Ug99 (or TTKSK) continue to cause crop losses in East Africa and threaten global wheat production. Developing and deploying wheat varieties with multiple race-specific genes or complex adult plant resistance is necessary to achieve durability. In the present study, we applied genome-wide association studies (GWAS) for identifying loci associated with the Ug99 stem rust resistance (SR) in a panel of wheat lines developed at the International Maize and Wheat Improvement Center (CIMMYT). Genotyping was carried out using the wheat 9K iSelect single nucleotide polymorphism (SNP) chip. Phenotyping was done in the field in Kenya by infection of Puccinia graminis f. sp. tritici race TTKST, the Sr24-virulent variant of Ug99. Marker-trait association identified 12 SNP markers significantly associated with resistance. Among them, 7 were mapped on five chromosomes. Markers located on chromosomes 4A and 4B overlapped with the location of the Ug99 resistance genes SrND643 and Sr37, respectively. Markers identified on 7DL were collocated with Sr25. Additional significant markers were located in the regions where no Sr gene has been reported. The chromosome location for five of the SNP markers was unknown. A BLASTN search of the NCBI database using the flanking sequences of the SNPs associated with Ug99 resistance revealed that several markers were linked to plant disease resistance analogues, while others were linked to regulatory factors or metabolic enzymes. A KASP (Kompetitive Allele Specific PCR) assay was used for validating six marker loci linked to genes with resistance to Ug99. Of those, four co-segregated with the Sr25-pathotypes while the rest identified unknown resistance genes. With further investigation, these markers can be used for marker-assisted selection in breeding for Ug99 stem rust resistance in wheat.

Genetic mapping of resistance to the Ug99 race group of Puccinia graminis f. sp. tritici in a spring wheat landrace CItr 4311.

A gene for Ug99 resistance from wheat landrace CItr 4311 was detected on the long arm of chromosome 2B. Wheat landrace CItr 4311 has seedling resistance to stem rust caused by Puccinia graminis f. sp. tritici race TTKSK and field resistance to the Ug99 race group. Parents, F1 seedlings, 121 doubled haploid (DH) lines, and 124 recombinant inbred lines (RILs) developed from a cross between CItr 4311 and the susceptible line LMPG-6 were evaluated for seedling resistance to race TTKSK. Goodness-of-fit tests indicated that a single dominant gene in CItr 4311 conditioned the TTKSK resistance. The 90K wheat iSelect SNP platform was used to genotype parents and the DH population. The seedling resistance locus was mapped to the chromosome arm 2BL. Parents and the DH population were evaluated for field resistance in Kenya. One major QTL for the field resistance was consistently detected in the same region on 2BL as the seedling resistance. Using KASP assays, five linked SNP markers were used to verify the result in the 124 RIL, 35 wheat accessions, 46 DH lines from the LMPG-6/PI 165194 cross and F1 seedlings, and susceptible bulks derived from crosses between six resistant landraces with LMPG-6. Race specificity, mapping results, and haplotype similarity with lines with Sr9h (Gabo 56, Timstein, and PI 670015), support the hypothesis that the Sr gene in CItr 4311 and the landraces is Sr9h. The KASP assays developed in this study will be useful for pyramiding the TTKSK resistance from CItr 4311 with other Sr genes effective against Ug99.

Mapping of SrTm4, a Recessive Stem Rust Resistance Gene from Diploid Wheat Effective to Ug99.

Race TTKSK (or Ug99) of Puccinia graminis f. sp. tritici, the causal agent of wheat stem rust, is a serious threat to wheat production worldwide. Diploid wheat, Triticum monococcum (genome Am), has been utilized previously for the introgression of stem rust resistance genes Sr21, Sr22, and Sr35. Multipathotype seedling tests of biparental populations demonstrated that T. monococcum accession PI 306540 collected in Romania contains a recessive resistance gene effective to all P. graminis f. sp. tritici races screened, including race TTKSK. We will refer to this gene as SrTm4, which is the fourth stem rust resistance gene characterized from T. monococcum. Using two mapping populations derived from crosses of PI 272557×PI 306540 and G3116×PI 306540, we mapped SrTm4 on chromosome arm 2AmL within a 2.1 cM interval flanked by sequence-tagged markers BQ461276 and DR732348, which corresponds to a 240-kb region in Brachypodium chromosome 5. The eight microsatellite and nine sequence-tagged markers linked to SrTm4 will facilitate the introgression and accelerate the deployment of SrTm4-mediated Ug99 resistance in wheat breeding programs.

Mapping resistance to the Ug99 race group of the stem rust pathogen in a spring wheat landrace.

A new gene for Ug99 resistance from wheat landrace PI 374670 was detected on the long arm of chromosome 7A. Wheat landrace PI 374670 has seedling and field resistance to stem rust caused by Puccinia graminis f. sp tritici Eriks. & E. Henn (Pgt) race TTKSK. To elucidate the inheritance of resistance, 216 BC1F2 families, 192 double haploid (DH) lines, and 185 recombinant inbred lines (RILs) were developed by crossing PI 374670 and the susceptible line LMPG-6. The parents and progeny were evaluated for seedling resistance to Pgt races TTKSK, MCCFC, and TPMKC. The DH lines were tested in field stem rust nurseries in Kenya and Ethiopia. The DH lines were genotyped with the 90K wheat iSelect SNP genotyping platform. Goodness-of-fit tests indicated that a single dominant gene in PI 374670 conditioned seedling resistance to the three Pgt races. The seedling resistance locus mapped to the long arm of chromosome 7A and this result was verified in the RIL population screened with the flanking SNP markers using KASP assays. In the same region, a major QTL for field resistance was detected in a 7.7 cM interval and explained 34-54 and 29-36% of the variation in Kenya and Ethiopia, respectively. Results from tests with specific Pgt races and the csIH81 marker showed that the resistance was not due to Sr22. Thus, a new stem rust resistance gene or allele, either closely linked or allelic to Sr15, is responsible for the seedling and field resistance of PI 374670 to Ug99.

Characterization of Sr9h, a wheat stem rust resistance allele effective to Ug99.

Wheat stem rust resistance gene SrWeb is an allele at the Sr9 locus that confers resistance to Ug99. Race TTKSK (Ug99) of Puccinia graminis f. sp. tritici, the causal fungus of stem rust, threatens global wheat production because of its broad virulence to current wheat cultivars. A recently identified Ug99 resistance gene from cultivar Webster, temporarily designated as SrWeb, mapped near the stem rust resistance gene locus Sr9. We determined that SrWeb is also present in Ug99 resistant cultivar Gabo 56 by comparative mapping and an allelism test. Analysis of resistance in a population segregating for both Sr9e and SrWeb demonstrated that SrWeb is an allele at the Sr9 locus, which subsequently was designated as Sr9h. Webster and Gabo 56 were susceptible to the Ug99-related race TTKSF+ from South Africa. Race TTKSF+ possesses unique virulence to uncharacterized Ug99 resistance in cultivar Matlabas. This result validated that resistance to Ug99 in Webster and Gabo 56 is conferred by the same gene: Sr9h. The emergence of pathogen virulence to several resistance genes that are effective to the original Ug99 race TTKSK, including Sr9h, suggests that resistance genes should be used in combinations in order to increase resistance durability.

Resistance to stem rust Ug99 in six bread wheat cultivars maps to chromosome 6DS

Identified SSR markers ( Xcfd49 and Xbarc183 ) linked with stem rust resistance for efficient use in marker-assisted selection and stacking of resistance genes in wheat breeding programs. More than 80 % of the worldwide wheat (Triticum aestivum L.) area is currently sown with varieties susceptible to the Ug99 race group of stem rust fungus. However, wheat lines Niini, Tinkio, Coni, Pfunye, Blouk, and Ripper have demonstrated Ug99 resistance at the seedling and adult plant stages. We mapped stem rust resistance in populations derived from crosses of a susceptible parent with each of the resistant lines. The segregation of resistance in each population indicated the presence of a single gene. The resistance gene in Niini mapped to short arm of chromosome 6D and was flanked by SSR markers Xcfd49 at distances of 3.9 cM proximal and Xbarc183 8.4 cM distal, respectively. The chromosome location of this resistance was validated in three other populations: PBW343/Coni, PBW343/Tinkio, and Cacuke/Pfunye. Resistance initially postulated to be conferred by the SrTmp gene in Blouk and Ripper was also linked to Xcfd49 and Xbarc183 on 6DS, but it was mapped proximal to Xbarc183 at a similar position to previously mapped genes Sr42 and SrCad. Based on the variation in diagnostic marker alleles, it is possible that Niini and Pfunye may carry different resistance genes/alleles. Further studies are needed to determine the allelic relationships between various genes located on chromosome arm 6DS. Our results provide valuable molecular marker and genetic information for developing Ug99 resistant wheat varieties in diverse germplasm and using these markers to tag the resistance genes in wheat 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.

Association mapping and gene–gene interaction for stem rust resistance in CIMMYT spring wheat germplasm

The recent emergence of wheat stem rust Ug99 and evolution of new races within the lineage threatens global wheat production because they overcome widely deployed stem rust resistance (Sr) genes that had been effective for many years. To identify loci conferring adult plant resistance to races of Ug99 in wheat, we employed an association mapping approach for 276 current spring wheat breeding lines from the International Maize and Wheat Improvement Center (CIMMYT). Breeding lines were genotyped with Diversity Array Technology (DArT) and microsatellite markers. Phenotypic data was collected on these lines for stem rust race Ug99 resistance at the adult plant stage in the stem rust resistance screening nursery in Njoro, Kenya in seasons 2008, 2009 and 2010. Fifteen marker loci were found to be significantly associated with stem rust resistance. Several markers appeared to be linked to known Sr genes, while other significant markers were located in chromosome regions where no Sr genes have been previously reported. Most of these new loci colocalized with QTLs identified recently in different biparental populations. Using the same data and Q+K covariate matrices, we investigated the interactions among marker loci using linear regression models to calculate P values for pairwise marker interactions. Resistance marker loci including the Sr2 locus on 3BS and the wPt1859 locus on 7DL had significant interaction effects with other loci in the same chromosome arm and with markers on chromosome 6B. Other resistance marker loci had significant pairwise interactions with markers on different chromosomes. Based on these results, we propose that a complex network of gene-gene interactions is, in part, responsible for resistance to Ug99. Further investigation may provide insight for understanding mechanisms that contribute to this resistance gene network.

Conflicting mapping results for stem rust resistance gene Sr13

Theor Appl Genet (2011) 122:659 DOI 10.1007/s00122-010-1495-2 COMMENT Conflicting mapping results for stem rust resistance gene Sr13 J. Dubcovsky • F. Ordon • D. Perovic B. Admassu • W. Friedt • Z. Abate • W. Zhang • S. Chao Published online: 14 December 2010 O The Author(s) 2010. This article is published with open access at Springerlink.com Two manuscripts with conflicting results on the mapping location of stem rust resistance gene Sr13 in wheat are published in this issue of TAG (Admassu et al. and Simons et al.). Both resistance genes were mapped on the long arm of chromosome 6AL, but the gene mapped by Admassu et al. between markers barc37 and wmc256 is more than 50 cM proximal to the one mapped by Simons et al. between markers CD926040 and BE471213. After the publication of the two manuscripts in TAG online, the authors of both manuscripts became aware of the conflicting results and they exchanged the critical hexaploid wheat cytogenetic stocks Khapstein/9*LMPG (developed by Dr. D. Knott) and the recurrent parent LMPG to deter- mine the source of the differences. In addition, they requested seeds for these two stocks from the U.S. Cereal Disease Laboratory in Saint Paul Minnesota (Dr. Yue Jin) and from Canada Agri-Food, Winnipeg (Dr. Tom Fetch). The online version of the original articles can be found under doi:10.1007/s00122-010-1433-3 and 10.1007/s00122-010-1444-0. J. Dubcovsky (&) Z. Abate W. Zhang Department of Plant Sciences, University of California, Davis, CA 95616, USA e-mail: jdubcovsky@ucdavis.edu F. Ordon D. Perovic B. Admassu Julius Kuehn-Institute, Federal Research Institute for Cultivated Plants (JKI), Institute for Resistance Research and Stress Tolerance, Erwin-Baur-Str. 27, 06484 Quedlinburg, Germany W. Friedt Institute of Crop Science and Plant Breeding I, Justus-Liebig-University Giessen, Heinrich-Buff-Ring 26-32, 35392 Giessen, Germany S. Chao USDA-ARS, Biosciences Research Laboratory, 1605 Albrecht Blvd N, Fargo, ND 58102-2765, USA Analysis of these stocks with molecular markers for the long arm of chromosome 6AL revealed that the Khapstein/ 9*LMPG seed stock used in the Admassu et al. paper was different from the other three Khapstein/9*LMPG stocks. Molecular markers barc37, barc107, barc118, barc1165, gwm570, wmc179, wmc256, wmc580, and cfd2 all showed different bands in the seed stock used in the Admassu paper relative to the seed stocks from the University of Califor- nia, the Cereal Disease Laboratory and Agri-Food Canada. These results indicate that the Khapstein/9*LMPG seed stock used in the Admassu paper is different from the other three. Since the Khapstein/9*LMPG hexaploid genetic stock defines the gene name Sr13, the gene mapped by Admassu et al. is most likely not Sr13 and should be renamed. The Khapstein/9*LMPG developed by Dr. D. Knott is polymorphic with the LMPG recurrent parent only for markers closely linked to the stem rust resistance gene mapped in the four tetraploid populations in the distal region of chromosome arm 6AL (barc104b and dupw167) by Simons et al. This observation, together with the fact that Sr13 was originated in tetraploid wheat (Khapli), provides indirect evidence that the gene mapped by Simons et al. is Sr13. Mapping the stem rust resistance gene present in the correct Khapstein/9*LMPG stock in an hexaploid segregating population will provide the final answer. Interestingly, both genes confer resistance to the new stem rust race Ug99 (TTKSK) suggesting that it would be possible to combine the two sources of resistance to Ug99 in the same 6AL arm. We have initiated crosses between the correct Khapstein/9*LMPG genetic stock and the source of the Admassu et al. Ug99 resistance to recombine both genes. Open Access This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which per- mits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.