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.
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Three members of the Puccinia genus, P. triticina (Pt), P. striiformis f.sp. tritici (Pst), and P. graminis f.sp. tritici (Pgt), cause the most common and often most significant foliar diseases of wheat. While similar in biology and life cycle, each species is uniquely adapted and specialized. The genomes of Pt and Pst were sequenced and compared to that of Pgt to identify common and distinguishing gene content, to determine gene variation among wheat rust pathogens, other rust fungi and basidiomycetes, and to identify genes of significance for infection. Pt had the largest genome of the three, estimated at 135 Mb with expansion due to mobile elements and repeats encompassing 50.9% of contig bases; by comparison repeats occupy 31.5% for Pst and 36.5% for Pgt. We find all three genomes are highly heterozygous, with Pst (5.97 SNPs/kb) nearly twice the level detected in Pt (2.57 SNPs/kb) and that previously reported for Pgt. Of 1,358 predicted effectors in Pt, 784 were found expressed across diverse life cycle stages including the sexual stage. Comparison to related fungi highlighted the expansion of gene families involved in transcriptional regulation and nucleotide binding, protein modification, and carbohydrate degradation enzymes. Two allelic homeodomain pairs, HD1 and HD2, were identified in each dikaryotic Puccinia species along with three pheromone receptor (STE3) mating-type genes, two of which are likely representing allelic specificities. The HD proteins were active in a heterologous Ustilago maydis mating assay and host induced gene silencing of the HD and STE3 alleles reduced wheat host infection.
The spring wheat belt of Western Siberia and Northern Kazakhstan covers more than 15 million ha. While moisture stress is the main factor limiting production, rusts also represent a major challenge, especially in years with higher rainfall. Stem rust was not considered economically important until 2015 when a local epidemic occurred in the Omsk region of Russia and neighboring areas of Kazakhstan and affected more than 1 million ha. It occurred again in 2016 though the spread, severity and losses were less. This study used 16 pathotypes and 17 molecular markers to characterize a set of 146 spring wheat varieties and breeding lines identified as stem rust resistant in Kenya and the Kazakhstan–Siberia region for the presence of major genes. The genetic basis of resistance in the material was limited to Sr25, Sr31, Sr36, Sr6Ai, Sr6Ai#2, and some unknown major genes. Genes Sr25 and Sr6Ai#2 also provided high levels of resistance to leaf rust through linkages with Lr19 and Lr6Ai#2. Adult plant resistance to stem rust was observed in 26 genotypes (16.5 %), including eight possessing Sr57 gene. The high risk of stem rust—as indicated by the 2015 Siberian epidemic—means that there is an urgent need to diversify the genetic bases of resistance and to promote resistant varieties with farmers.
Stem rust (Puccinia graminis f. sp. tritici, Pgt) races belonging to the Ug99 (TTKSK) race group pose a serious threat to global wheat (Triticum aestivum L.) production. To improve Pgt host resistance, the Ug99-effective resistance gene SrTA10187 previously identified in Aegilops tauschii Coss. was introgressed into wheat, and mapped to the short arm of wheat chromosome 6D. In this study, high-resolution mapping of SrTA10187 was done using a population of 1,060 plants. Pgt resistance was screened using race QFCSC. PCR-based SNP and STS markers were developed from genotyping-by-sequencing tags and SNP sequences available in online databases. SrTA10187 segregated as expected in a 3:1 ratio of resistant to susceptible individuals in three out of six BC3F2 families, and was fine-mapped to a 1.1 cM region on wheat chromosome 6DS. Marker context sequence was aligned to the reference Ae. tauschii genome to identify the physical region encompassing SrTA10187. Due to the size of the corresponding region, candidate disease resistance genes could not be identified with confidence. Comparisons with the Ae. tauschii genetic map developed by Luo et al. (PNAS 110(19):7940–7945, 2013) enabled identification of a discrete genetic locus and a BAC minimum tiling path of the region spanning SrTA10187. Annotation of pooled BAC library sequences led to the identification of candidate genes in the region of interest—including a single NB-ARC-LRR gene. The shorter genetic interval and flanking KASP™ and STS markers developed in this study will facilitate marker-assisted selection, gene pyramiding, and positional cloning of SrTA10187.
Wheat landrace PI 177906 has seedling resistance to stem rust caused by Puccinia graminis f. sp. tritici races TTKSK, TTKST, and BCCBC and field resistance to the Ug99 race group. Parents, 140 recombinant inbred lines, and 138 double haploid (DH) lines were evaluated for seedling resistance to races TTKSK and BCCBC. Parents and the DH population were evaluated for field resistance to Ug99 in Kenya. The 90K wheat single nucleotide polymorphism (SNP) genotyping platform was used to genotype the parents and populations. Goodness-of-fit tests indicated that two dominant genes in PI 177906 conditioned seedling resistance to TTKSK. Two major loci for seedling resistance were consistently mapped to the chromosome arms 2BL and 6DS. The BCCBC resistance was mapped to the same location on 2BL as the TTKSK resistance. Using field data from the three seasons, two major QTL were consistently detected at the same regions on 2BL and 6DS. Based on the mapping result, race specificity, and the infection type observed in PI 177906, the TTKSK resistance on 2BL is likely due to Sr28. One SNP marker (KASP_IWB1208) was found to be predictive for the presence of the TTKSK resistance locus on 2BL and Sr28.
Two new races of the wheat (Triticum aestivum L.) stem rust pathogen, representing the fifth and sixth variants described within the Ug99 lineage, were detected in South Africa. Races TTKSP and PTKST (North American notation) were detected in 2007 and 2009, respectively. Except for Sr24 virulence, race TTKSP is phenotypically identical to TTKSF, a commonly detected race of Puccinia graminis f. sp. tritici (Pgt) in South Africa. PTKST is similar to TTKSP except that it produces a lower infection type on the Sr21 differential and has virulence for Sr31. Simple sequence repeat (SSR) analysis confirmed the genetic relationship amongst TTKSF, TTKSP, PTKST and TTKSK (Ug99). TTKSK, PTKST and TTKSF grouped together with 99% similarity, while sharing 88% genetic resemblance with TTKSP. These four races in turn shared only 31% similarity with other South African races. It is proposed that both TTKSP and PTKST represent exotic introductions of Pgt to South Africa.
Isolates of Puccinia graminis f. sp. tritici belonging to the Ug99 race group are virulent to a broad spectrum of resistance genes, rendering most of the world's wheat germplasm susceptible to stem rust (3). Following the initial detection of Ug99 (TTKSK, North American [NA] race notation) in Uganda, virulence to the widely used Sr31 resistance gene has been reported from Kenya, Ethiopia, Sudan, and Iran (2,3). In November 2009, a wheat genotype suspected to carry Sr31 showed a susceptible response to stem rust in a disease nursery (29°08′05.02′′S, 30°38′29.18′′E), inoculated with race TTKSP, near Greytown in KwaZulu-Natal, South Africa. Inoculation of urediniospores of the field collection (isolate UVPgt60) onto seedlings of line Federation4*/Kavkaz confirmed virulence for Sr31. In three independent, replicated, and comparative seedling tests, eight single-pustule isolates of UVPgt60 all typed to race PTKST following the NA race nomenclature. These isolates produced compatible infection types (ITs) (3+ to 4) on the Sr31 testers Gamtoos, Sr31/6*LMPG, Federation4*/Kavkaz, Kavkaz, and Clement, whereas isolate UVPgt59 (TTKSP) was avirulent (ITs ;1 to 1) on these genotypes. In addition to Sr31 virulence, the new race differed from TTKSP by producing a lower IT (2 to 2++) on Cns_T.mono_ deriv., the accepted entry for Sr21 in the NA differential set. The UVPgt60 isolates were clearly avirulent on Einkorn (Sr21) (IT ;1=), a response that also differed from those produced by BPGSC, TTKSF, and TTKSP (IT 2). With the exception of Sr21, UVPgt60 isolates had a virulence pattern similar to race TTKST (1), notably the virulence combination for Sr24 and Sr31. Isolate UVPgt60.6 was randomly selected for testing on additional Sr genes and South African wheat cultivars and breeding lines. Similar to the race identification experiments seedling tests were duplicated and compared with reactions produced by TTKSP and other races. Greenhouse temperatures for all seedling tests ranged between 18 and 25°C. On the basis of primary leaf responses, PTKST is avirulent (ITs 0; to 2++) for Sr13, 14, 21, 22, 25, 26, 27, 29, 32, 33, 35, 36, 37, 39, 42, 43, 44, Em, Tmp, and Satu and virulent (ITs 3 to 4) for Sr5, 6, 7b, 8a, 8b, 9a, 9b, 9d, 9e, 9g, 10, 11, 16, 17, 24, 30, 31, 34, 38, 41, and McN. From 103 South African wheat cultivars and lines tested as seedlings, 59 and 47 were susceptible (IT ≥ 3) to races PTKST and TTKSP, respectively. Simple-sequence repeat analysis (4) with selected primer pairs showed that PTKST clusters with isolates belonging to the Ug99 lineage. Subsequent to the collection made at Greytown, stem rust sampled in December 2009 from naturally infected breeders' lines at Cedara (29°32′19.59′′S, 30°16′03.50′′E), KwaZulu-Natal, revealed five isolates with a virulence profile similar to PTKST. On the basis of current evidence it appears that PTKST may be an introduction to South Africa rather than a single-step mutation from local stem rust races.
During the 2000 to 2001 season, 27 stem rust samples were collected from wheat (Triticum aestivum), barley (Hordeum vulgare), and triticale (× Triticosecale) cultivars and lines in the Western Cape, South Africa. Following inoculation and multiplication on McNair 701 seedlings, 40 single pustule isolates of P. graminis f. sp. tritici were established. Twenty-six isolates obtained from wheat, barley, or triticale that produced a similar reaction pattern on a set of differentiating host lines, were designated as pathotype Pgt-2SA55. Pgt-2SA55 is avirulent to Sr5, -6, -7b, -8b, -9b, -9e, -9g, -23, -24, -27, -30, -38, and -Gt, and virulent to Sr11, and -Agi. The remaining 14 isolates, all from wheat and designated as pathotype Pgt-2SA88, were avirulent to Sr24, -27, and -Agi, and virulent to Sr5, -6, -7b, -8b, -9b, -9e, -9g, -11, -23, -30, -38, and -Gt. On an expanded differential set, representative isolates of each pathotype were all avirulent to Sr13, -15, -21, -22, -25, -26, -29, -31, -32, -33, -35, -39, -43, and -Em, and virulent to Sr7a, -8a, -9a, -9d, -9f, -10, -12, -14, -16, -19, -20, -34, and Lc. Pgt-2SA55 was avirulent on cv. Renown (Sr2, -7b, -9d, and -17), whereas Pgt-2SA88 was virulent on this cultivar and Line R Sel carrying only Sr17. Both pathotypes differ from those identified previously in South Africa (1) and to our knowledge, Pgt-2SA88 is the first local isolate to have virulence towards Sr8b and the T. ventricosum-derived gene Sr38. Virulence to Sr38 has been reported in a P. graminis f. sp. tritici isolate collected in Uganda (2). Pathotype Pgt-2SA88 is virulent to seedlings of the previously resistant South African cvs. SST 57 (heterogeneous), Tugela, Tugela DN, and PAN 3377. Furthermore, 20% of the elite breeding lines in the spring and winter wheat breeding program of the Small Grain Institute expressed susceptible seedling reactions to Pgt-2SA88. Triticale cvs. Rex and Kiewiet were heterogeneous in their seedling reaction towards Pgt-2SA55. Seedling and field reactions recorded for the barley cvs. Sterling and SSG 532 and the experimental varieties Puma and Jaguar, showed an increase in stem rust susceptibility to Pgt-2SA55 when compared with existing South African pathotypes. The higher incidence of stem rust in commercial fields and experimental plots of wheat and barley in the Western Cape may be attributed to a recent increase in the cultivation of stem rust-susceptible cultivars in the region. The detection of two new pathotypes of P. graminis f. sp. tritici is of concern to the local small grain industry and requires continued resistance breeding.
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.
The Ug99 race (TTKSK) of wheat stem rust was first detected in Uganda in 1998 (Pretorius et al. 2000) and since then, seven additional variants have been reported: TTKSF, TTKST, TTTSK, TTKSP, PTKSK, PTKST, and TTKSF+ (Pretorius et al. 2012). In this study, 84 stem rust samples from the 2014 surveys of wheat fields in Africa (Kenya, 9; Uganda, 28; Rwanda, 41; and Egypt, 6) were sent to the Global Rust Reference Center (GRRC, Denmark) for race analysis. Puccinia graminis f. sp. tritici (Pgt) samples were recovered on cv. Morocco, and resulting urediniospores of 53 single-pustule isolates were inoculated onto 20 North American stem rust differential lines using standard procedures (Jin et al. 2008). The pathotyping was repeated in two or three independent experiments. Twelve of the derived isolates were also typed at the USDA-ARS Cereal Disease Laboratory (USA) for an independent confirmation. Among the Kenyan samples, four collected from Njoro (Central Rift, cvs. Robin and Kwale) and two from Ntulumeti and Olgilai (South Rift, cv. Robin), were typed as TTKTK. Race TTKTK was similar to TTKSK except for additional virulence to SrTmp (Infection Type 4). An additional single-pustule isolate derived from one sample from Njoro showed a high infection type on LcSr24Ag and CnsSrTmp, testers for Sr24 and SrTmp, respectively, and was typed as TTKTT. These isolates were also tested on Siouxland (PI 483469, Sr24+Sr31), Sisson (PI 617053, Sr31+Sr36), and Triumph 64 (CI 13679, donor of SrTmp) to confirm their virulence/avirulence combinations to Sr24, Sr31, Sr36, and SrTmp. Race TTKTK was also detected at two locations in Uganda (Rubaya and Muko in Kabale region) and at five locations in Rwanda (Kinigi, Rwerere, Rufungo, Gatebe, and Kamenyo). Three isolates derived from stem rust samples collected on cv. PBW343 (carrying Sr31) in Sakha in the Nile Delta region in Egypt were also typed as TTKTK. In addition, DNA from isolates of race TTKTK were analyzed using a diagnostic qPCR assay (Ug99 RG stage-1, Szabo, unpublished data), which confirmed that these samples belong to the Ug99 lineage. The identification of SrTmp virulence in the Ug99 race group in several countries in one year emphasizes the relevance of coordinated international surveillance efforts and utilization of diverse sources of resistance to control stem rust in wheat. Further studies are in progress to determine the detailed relationship of the newly emerged races and other Pgt isolates identified in the Ug99 group.