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.     

Inheritance of resistance to Ug99 stem rust in wheat cultivar Norin 40 and genetic mapping of Sr42

Stem rust, caused by Puccinia graminis f. sp. tritici, is a devastating disease of wheat. The emergence of race TTKSK (Ug99) and new variants in Africa threatens wheat production worldwide. The best method of controlling stem rust is to deploy effective resistance genes in wheat cultivars. Few stem rust resistance (Sr) genes derived from the primary gene pool of wheat confer resistance to TTKSK. Norin 40, which carries Sr42, is resistant to TTKSK and variants TTKST and TTTSK. The goal of this study was to elucidate the inheritance of resistance to Ug99 in Norin 40 and map the Sr gene(s). A doubled haploid (DH) population of LMPG-6/Norin 40 was evaluated for resistance to the race TTKST. Segregation of 248 DH lines fitted a 1:1 ratio (χ (2) 1:1= 0.58, p = 0.45), indicating a single gene in Norin 40 conditioned resistance to Ug99. This was confirmed by an independent F(2:3) population also derived from the cross LMPG-6/Norin 40 where a 1:2:1 ratio (χ (2)1:2:1 = 0.69, p = 0.71) was observed following the inoculation with race TTKSK. Mapping with DNA markers located this gene to chromosome 6DS, the known location of Sr42. PCR marker FSD_RSA co-segregated with Sr42, and simple sequence repeat (SSR) marker BARC183 was closely linked (0.5 cM) to Sr42. A previous study found close linkage between FSD_RSA and SrCad, a temporarily designated gene that also confers resistance to Ug99, thus Sr42 may be the same gene or allelic. Marker FSD_RSA is suitable for marker-assisted selection (MAS) in wheat breeding programs to improve stem rust resistance, including Ug99.     

A consensus map for Ug99 stem rust resistance loci in wheat

Key message

This consensus map of stem rust genes, QTLs, and molecular markers will facilitate the identification of new resistance genes and provide a resource of in formation for development of new markers for breeding wheat varieties resistant to Ug99.

Abstract

The global effort to identify new sources of resistance to wheat stem rust, caused by Puccinia graminis 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.     

Genetics and mapping of stem rust resistance to Ug99 in the wheat cultivar Webster

New races of wheat stem rust, namely TTKSK (Ug99) and its variants, pose a threat to wheat production in the regions where they are found. The accession of the wheat cultivar Webster (RL6201) maintained at the Cereal Research Centre in Winnipeg, Canada, shows resistance to TTKSK and other races of stem rust. The purpose of this study was to study the inheritance of seedling resistance to stem rust in RL6201 and genetically map the resistance genes using microsatellite (SSR) markers. A population was produced by crossing the stem rust susceptible line RL6071 with Webster. The F₂ and F₃ were tested with TPMK, a stem rust race native to North America. The F₃ was also tested with TTKSK. Two independently assorting genes were identified in RL6201. Resistance to TPMK was conferred by Sr30, which was mapped with microsatellites on chromosome 5DL. The second gene, temporarily designated SrWeb, conferred resistance to TTKSK. SrWeb was mapped to chromosome 2BL using SSR markers. Comparison with previous genetic maps showed that SrWeb occupies a locus near Sr9. Further analysis will be required to determine if SrWeb is a new gene or an allele of a previously identified gene.     

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.     

Development and characterization of a compensating wheat-Thinopyrum intermedium Robertsonian translocation with Sr44 resistance to stem rust (Ug99)

The emergence of the highly virulent Ug99 race complex of the stem rust fungus (Puccinia graminis Pers. f. sp. tritici Eriks. and Henn.) threatens wheat (Triticum aestivum L.) production worldwide. One of the effective genes against the Ug99 race complex is Sr44, which was derived from Thinopyrum intermedium (Host) Barkworth and D.R. Dewey and mapped to the short arm of 7J (designated 7J#1S) present in the noncompensating T7DS-7J#1L?7J#1S translocation. Noncompensating wheat-alien translocations are known to cause genomic duplications and deficiencies leading to poor agronomic performance, precluding their direct use in wheat improvement. The present study was initiated to produce compensating wheat-Th. intermedium Robertsonian translocations with Sr44 resistance. One compensating RobT was identified consisting of the wheat 7DL arm translocated to the Th. intermedium 7J#1S arm resulting in T7DL?7J#1S. The T7DL?7J#1S stock was designated as TA5657. The 7DL?7J#1S stock carries Sr44 and has resistance to the Ug99 race complex. This compensating RobT with Sr44 resistance may be useful in wheat improvement. In addition, we identified an unnamed stem rust resistance gene located on the 7J#1L arm that confers resistance not only to Ug99, but also to race TRTTF, which is virulent to Sr44. However, the action of the second gene can be modified by the presence of suppressors in the recipient wheat cultivars.     

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

Key message

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.

Abstract

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.     

Searching for novel sources of field resistance to Ug99 and Ethiopian stem rust races in durum wheat via association mapping

Puccinia graminis f. sp. tritici, the causative agent of stem rust in wheat, is a devastating disease of durum wheat. While more than 50 stem rust resistance (Sr) loci have been identified in wheat, only a few of them have remained effective against Ug99 (TTKSK race) and other durum-specific Ethiopian races. An association mapping (AM) approach based on 183 diverse durum wheat accessions was utilized to identify resistance loci for stem rust response in Ethiopia over four field-evaluation seasons and artificial inoculation with Ug99 and a mixture of durum-specific races. The panel was profiled with simple sequence repeat, Diversity Arrays Technology and sequence-tagged site markers (1,253 in total). The resistance turned out to be oligogenic, with twelve QTL-tagging markers that were significant (P < 0.05) across three or four seasons. R ² values ranged from 1.1 to 11.3 %.Twenty-four additional single-marker/QTL regions were found to be significant over two seasons. The AM results confirmed the role of Sr13, previously described in bi-parental mapping studies, and the role of chromosome regions putatively harbouring Sr9, Sr14, Sr17 and Sr28. Three minor QTLs were coincident with those reported in hexaploid wheat and five overlapped with those recently reported in the Sebatel × Kristal durum mapping population. Thirteen single-marker/QTL regions were located in chromosome regions where no Sr genes/QTLs have been previously reported. The allelic variation identified in this study is readily available and can be exploited for marker-assisted selection, thus providing additional opportunities for a more durable stem rust resistance under field conditions.     

Development of co-dominant KASP markers co-segregating with Ug99 effective stem rust resistance gene <Emphasis Type="Italic">Sr26</Emphasis> in wheat

Stem rust of wheat, caused by Puccinia graminis f. sp. tritici (Pgt), is a threat to global food security due to its ability to cause total crop failures. The Pgt race TTKSK (Ug99) and its derivatives detected in East Africa carry virulence for many resistance genes present in modern cultivars. However, stem rust resistance gene Sr26 remains effective to all races of Pgt worldwide. Sr26 is carried on the Agropyron elongatum (syn. Thinopyrum ponticum) segment 6Ae#1L translocated to chromosome 6AL of wheat. In this study, a recombinant inbred line (RIL) population derived from a cross between the landrace Aus27969 and Avocet S, which carries Sr26, was used to develop co-dominant kompetitive allele-specific polymerase chain reaction (KASP) markers that co-segregate with Sr26. Four KASP markers (sunKASP_216, sunKASP_218, sunKASP_224 and sunKASP_225) were also shown to co-segregate with Sr26 in four additional RIL populations. When tested on Australian cultivars and breeding lines, these markers amplified alleles alternate to that linked with Sr26 in all cultivars known to lack this gene and Sr26-linked alleles in cultivars and genotypes known to carry Sr26. Genotypes WA-1 and WA-1/3*Yitpi carrying the shortest Sr26 translocation segment were positive only for markers sunKASP_224 and sunKASP_225. Our results suggest the four KASP markers are located on the original translocation and sunKASP_224 and sunKASP_225 are located on the shortened version. Therefore, sunKASP_224 and sunKASP_225 can be used for marker-assisted pyramiding of Sr26 with other stem rust resistance genes to achieve durable resistance in wheat.     

Simultaneous transfer, introgression, and genomic localization of genes for resistance to stem rust race TTKSK (Ug99) from Aegilops tauschii to wheat

Wheat production is currently threatened by widely virulent races of the wheat stem rust fungus, Puccinia graminis f. sp. tritici, that are part of the TTKSK (also known as ‘Ug99’) race group. The diploid D genome donor species Aegilops tauschii (2n = 2x = 14, DD) is a readily accessible source of resistance to TTKSK and its derivatives that can be transferred to hexaploid wheat, Triticum aestivum (2n = 6x = 42, AABBDD). To expedite transfer of TTKSK resistance from Ae. tauschii, a direct hybridization approach was undertaken that integrates gene transfer, mapping, and introgression into one process. Direct crossing of Ae. tauschii accessions with an elite wheat breeding line combines the steps of gene transfer and introgression while development of mapping populations during gene transfer enables the identification of closely linked markers. Direct crosses were made using TTKSK-resistant Ae. tauschii accessions TA1662 and PI 603225 as males and a stem rust-susceptible T. aestivum breeding line, KS05HW14, as a female. Embryo rescue enabled recovery of F₁ (ABDD) plants that were backcrossed as females to the hexaploid recurrent parent. Stem rust-resistant BC₁F₁ plants from each Ae. tauschii donor source were used as males to generate BC₂F₁ mapping populations. Bulked segregant analysis of BC₂F₁ genotypes was performed using 70 SSR loci distributed across the D genome. Using this approach, stem rust resistance genes from both accessions were located on chromosome arm 1DS and mapped using SSR and EST-STS markers. An allelism test indicated the stem rust resistance gene transferred from PI 603225 is Sr33. Race specificity suggests the stem rust resistance gene transferred from TA1662 is unique and this gene has been temporarily designated SrTA1662. Stem rust resistance genes derived from TA1662 and PI 603225 have been made available with selectable molecular markers in genetic backgrounds suitable for stem rust resistance breeding.     

Durable resistance to wheat stem rust needed

The recent outbreak of a new wheat stem rust race capable of parasitizing many commercial wheat cultivars highlights the need for durable disease resistance in crop plants. More advanced breeding approaches using quantitative disease resistance genes and resistance gene pyramids are being used to combat wheat stem rust and other diseases, though widespread adoption of these breeding methodologies is needed to maintain resistance efficacy. Advances in understanding the molecular basis of plant disease resistance at both host and nonhost levels offers further possibilities for stem rust resistance using biotechnological approaches. However, truly durable resistance to wheat stem rust and other phytopathogens seems an unlikely prospect in the face of continually evolving pathogen populations.     

QTL mapping of slow-rusting, adult plant resistance to race Ug99 of stem rust fungus in PBW343/Muu RIL population

Races of stem rust fungus pose a major threat to wheat production worldwide. We mapped adult plant resistance (APR) to Ug99 in 141 lines of a PBW343/Muu recombinant inbred lines (RILs) population by phenotyping them for three seasons at Njoro, Kenya in field trials and genotyping them with Diversity Arrays Technology (DArT) markers. Moderately susceptible parent PBW343 and APR parent Muu displayed mean stem rust severities of 66.6 and 5 %, respectively. The mean disease severity of RILs ranged from 1 to 100 %, with an average of 23.3 %. Variance components for stem rust severity were highly significant (p < 0.001) for RILs and seasons and the heritability (h ²) for the disease ranged between 0.78 and 0.89. Quantitative trait loci (QTL) analysis identified four consistent genomic regions on chromosomes 2BS, 3BS, 5BL, and 7AS; three contributed by Muu (QSr.cim-2BS, QSr.cim-3BS and QSr.cim-7AS) and one (QSr.cim-5BL) derived from PBW343. RILs with flanking markers for these QTLs had significantly lower severities than those lacking the markers, and combinations of QTLs had an additive effect, significantly enhancing APR. The QTL identified on chromosome 3BS mapped to the matching region as the known APR gene Sr2. Four additional QTLs on chromosomes 1D, 3A, 4B, and 6A reduced disease severity significantly at least once in three seasons. Our results show a complex nature of APR to stem rust where Sr2 and other minor slow rusting resistance genes can confer a higher level of resistance when present together.     

Diagnostic and co-dominant PCR markers for wheat stem rust resistance genes Sr25 and Sr26

Wheat stem rust, caused by Puccinia graminis f. sp. tritici, is one of the most destructive diseases of wheat. A new race of the pathogen named TTKSK (syn. Ug99) and its derivatives detected in East Africa are virulent to many designated and undesignated stem rust resistance genes. The emergence and spread of those races pose an imminent threat to wheat production worldwide. Genes Sr25 and Sr26 transferred into wheat from Thinopyrum ponticum are effective against these new races. DNA markers for Sr25 and Sr26 are needed to pyramid both genes into adapted germplasm. The previously published dominant markers Gb for Sr25 and Sr26#43 for Sr26 were validated with eight wheat lines with or without Sr25 or Sr26. We tested six published STS (sequence tagged site) markers amplifying diagnostic bands of Th. ponticum. Marker BF145935 consistently amplified well and can be used as a co-dominant marker for Sr25. Among 16 STS markers developed from wheat ESTs mapped to deletion bin 6AL8-0.90-1.00, none was co-dominant for tagging Sr26. However, five 6A-specific markers were identified. Multiplex PCR with marker Sr26#43 and 6A-specific marker BE518379 can be used as a co-dominant marker for Sr26. The co-dominant markers for Sr25 and Sr26 were validated with 37 lines with known stem rust resistance genes. A diverse set of germplasm consisting 170 lines from CIMMYT, China, USA and other counties were screened with the co-dominant markers for Sr25 and Sr26. Five lines with the diagnostic fragment for Sr25 were identified, and they all have ‘Wheatear’ in their pedigrees, which is known to carry Sr25. None of the 170 lines tested had Sr26, as expected.     

Anomalous segregations at the Sr6 locus for stem rust resistance in wheat

The action of the gene Sr6 for stem rust resistance in wheat is affected by temperature, light, and the particular susceptible parent with which a line carrying Sr6 has been crossed. Two experiments were carried out to determine whether the effect of the susceptible parents was due to modifier genes, the general genetic background, or interallelic interactions. The data indicated that the susceptible parents carried different sr6 alleles that interacted with Sr6, possibly in a paramutation-like process. In the course of the study, a number of anomalous results were obtained that may be due to the action of transposable elements. Received: 18 February 2000 / Accepted: 31 October 2000