Four Consistent Loci Confer Adult Plant Resistance to Leaf Rust in the Durum Wheat Lines Heller#1 and Dunkler.

The durum wheat lines Heller#1 and Dunkler from the International Maize and Wheat Improvement Center Global Wheat Program showed moderate and stable adult plant resistance to leaf rust under high disease pressure over field environments in northwestern Mexico. Leaf rust phenotyping was performed on two recombinant inbred line (RIL) populations derived from crosses of Heller#1 and Dunkler with the susceptible parent Atred#2, conducted under artificially induced Puccinia triticina epidemics in 2013, 2014, 2015, and 2016. The Atred#2 × Heller#1 and Atred#2 × Dunkler populations were genotyped by single nucleotide polymorphism (SNP) platforms and diversity arrays technology markers, respectively. Four leaf rust resistance quantitative trait loci were detected simultaneously in the two RIL populations: Lr46, QLr.cim-2BC, QLr.cim-5BL, and QLr.cim-6BL based on phenotypic data across all four crop seasons. They explained 11.7 to 46.8%, 7.2 to 26.1%, 8.4 to 24.1%, and 12.4 to 28.5%, respectively, of the phenotypic variation for leaf rust resistance in Atred#2 × Heller#1 and 16.3 to 56.6%, 6.7 to 15.7%, 4.1 to 10.1%, and 5.1 to 20.2% of the variation in the Atred#2 × Dunkler population. Only the resistance allele of QLr.cim-2BC was from the susceptible parent Atred#2, and resistance alleles at other loci came from the resistant parents Heller#1 and Dunkler. The SNP markers closely linked to Lr46 and QLr.cim-2BC were converted to kompetitive allele specific PCR markers for use in marker-assisted selection to improve leaf rust resistance through crosses with Heller#1 and Dunkler sources.

Characterization and Mapping of Leaf Rust and Stripe Rust Resistance Loci in Hexaploid Wheat Lines UC1110 and PI610750 under Mexican Environments.

Growing resistant wheat varieties is a key method of minimizing the extent of yield losses caused by the globally important wheat leaf rust (LR) and stripe rust (YR) diseases. In this study, a population of 186 F8 recombinant inbred lines (RILs) derived from a cross between a synthetic wheat derivative (PI610750) and an adapted common wheat line (cv. "UC1110") were phenotyped for LR and YR response at both seedling and adult plant stages over multiple seasons. Using a genetic linkage map consisting of single sequence repeats and diversity arrays technology markers, in combination with inclusive composite interval mapping analysis, we detected a new LR adult plant resistance (APR) locus, QLr.cim-2DS, contributed by UC1110. One co-located resistance locus to both rusts, QLr.cim-3DC/QYr.cim-3DC, and the known seedling resistance gene Lr26 were also mapped. QLr.cim-2DS and QLr.cim-3DC showed a marginally significant interaction for LR resistance in the adult plant stage. In addition, two previously reported YR APR loci, QYr.ucw-3BS and Yr48, were found to exhibit stable performances in rust environments in both Mexico and the United States and showed a highly significant interaction in the field. Yr48 was also observed to confer intermediate seedling resistance against Mexican YR races, thus suggesting it should be re-classified as an all-stage resistance gene. We also identified 5 and 2 RILs that possessed all detected YR and LR resistance loci, respectively. With the closely linked molecular markers reported here, these RILs could be used as donors for multiple resistance loci to both rusts in wheat breeding programs.

Genetic analysis and mapping of adult plant resistance loci to leaf rust in durum wheat cultivar Bairds.

KEY MESSAGE: New leaf rust adult plant resistance (APR) QTL QLr.cim - 6BL was mapped and confirmed the known pleotropic APR gene Lr46 effect on leaf rust in durum wheat line Bairds. CIMMYT-derived durum wheat line Bairds displays an adequate level of adult plant resistance (APR) to leaf rust in Mexican field environments. A recombinant inbred line (RIL) population developed from a cross of Bairds with susceptible parent Atred#1 was phenotyped for leaf rust response at Ciudad Obregon, Mexico, during 2013, 2014, 2015 and 2016 under artificially created epidemics of Puccinia triticina (Pt) race BBG/BP. The RIL population and its parents were genotyped with the 50 K diversity arrays technology (DArT) sequence system and simple sequence repeat (SSR) markers. A genetic map comprising 1150 markers was used to map the resistance loci. Four significant quantitative trait loci (QTLs) were detected on chromosomes 1BL, 2BC (centromere region), 5BL and 6BL. These QTLs, named Lr46, QLr.cim-2BC, QLr.cim-5BL and QLr.cim-6BL, respectively, explained 13.5-60.8%, 9.0-14.3%, 2.8-13.9%, and 11.6-29.4%, respectively, of leaf rust severity variation by the inclusive composite interval mapping method. All of these resistance loci were contributed by the resistant parent Bairds, except for QLr.cim-2BC, which came from susceptible parent Atred#1. Among these, the QTL on chromosome 1BL was the known pleiotropic APR gene Lr46, whereas QLr.cim-6BL, a consistently detected locus, should be a new leaf rust resistance locus in durum wheat. The mean leaf rust severity of RILs carrying all four QTLs ranged from 8.0 to 17.5%, whereas it ranged from 10.9 to 38.5% for three QTLs (Lr46 + 5BL + 6BL) derived from the resistant parent Bairds. Two RILs with four QTLs combinations can be used as sources of complex APR in durum wheat breeding.

A recently evolved hexose transporter variant confers resistance to multiple pathogens in wheat.

As there are numerous pathogen species that cause disease and limit yields of crops, such as wheat (Triticum aestivum), single genes that provide resistance to multiple pathogens are valuable in crop improvement. The mechanistic basis of multi-pathogen resistance is largely unknown. Here we use comparative genomics, mutagenesis and transformation to isolate the wheat Lr67 gene, which confers partial resistance to all three wheat rust pathogen species and powdery mildew. The Lr67 resistance gene encodes a predicted hexose transporter (LR67res) that differs from the susceptible form of the same protein (LR67sus) by two amino acids that are conserved in orthologous hexose transporters. Sugar uptake assays show that LR67sus, and related proteins encoded by homeoalleles, function as high-affinity glucose transporters. LR67res exerts a dominant-negative effect through heterodimerization with these functional transporters to reduce glucose uptake. Alterations in hexose transport in infected leaves may explain its ability to reduce the growth of multiple biotrophic pathogen species.

Identification and characterization of pleiotropic and co-located resistance loci to leaf rust and stripe rust in bread wheat cultivar Sujata.

KEY MESSAGE: Two new co-located resistance loci, QLr.cim - 1AS/QYr.cim - 1AS and QLr.cim - 7BL/YrSuj , in combination with Lr46 / Yr29 and Lr67/Yr46 , and a new leaf rust resistance quantitative trait loci, conferred high resistance to rusts in adult plant stage. The tall Indian bread wheat cultivar Sujata displays high and low infection types to leaf rust and stripe rust, respectively, at the seedling stage in greenhouse tests. It was also highly resistant to both rusts at adult plant stage in field trials in Mexico. The genetic basis of this resistance was investigated in a population of 148 F5 recombinant inbred lines (RILs) derived from the cross Avocet × Sujata. The parents and RIL population were characterized in field trials for resistance to leaf rust during 2011 at El Batán, and 2012 and 2013 at Ciudad Obregón, Mexico, and for stripe rust during 2011 and 2012 at Toluca, Mexico; they were also characterized three times for stripe rust at seedling stage in the greenhouse. The RILs were genotyped with diversity arrays technology and simple sequence repeat markers. The final genetic map was constructed with 673 polymorphic markers. Inclusive composite interval mapping analysis detected two new significant co-located resistance loci, QLr.cim-1AS/QYr.cim-1AS and QLr.cim-7BL/YrSuj, on chromosomes 1AS and 7BL, respectively. The chromosomal position of QLr.cim-7BL overlapped with the seedling stripe rust resistance gene, temporarily designated as YrSuj. Two previously reported pleiotropic adult plant resistance genes, Lr46/Yr29 and Lr67/Yr46, and a new leaf rust resistance quantitative trait loci derived from Avocet were also mapped in the population. The two new co-located resistance loci are expected to contribute to breeding durable rust resistance in wheat. Closely linked molecular markers can be used to transfer all four resistance loci simultaneously to modern wheat varieties.

Lr67/Yr46 confers adult plant resistance to stem rust and powdery mildew in wheat.

KEY MESSAGE: We demonstrate that Lr67/Yr46 has pleiotropic effect on stem rust and powdery mildew resistance and is associated with leaf tip necrosis. Genes are designated as Sr55, Pm46 and Ltn3 , respectively. Wheat (Triticum aestivum) accession RL6077, known to carry the pleiotropic slow rusting leaf and yellow rust resistance genes Lr67/Yr46 in Thatcher background, displayed significantly lower stem rust (P. graminis tritici; Pgt) and powdery mildew (Blumeria graminis tritici; Bgt) severities in Kenya and in Norway, respectively, compared to its recurrent parent Thatcher. We investigated the resistance of RL6077 to stem rust and powdery mildew using Avocet × RL6077 F6 recombinant inbred lines (RILs) derived from two photoperiod-insensitive F3 families segregating for Lr67/Yr46. Greenhouse seedling tests were conducted with Mexican Pgt race RTR. Field evaluations were conducted under artificially initiated stem rust epidemics with Pgt races RTR and TTKST (Ug99 + Sr24) at Ciudad Obregon (Mexico) and Njoro (Kenya) during 2010-2011; and under natural powdery mildew epiphytotic in Norway at Ås and Hamar during 2011 and 2012. In Mexico, a mean reduction of 41 % on stem rust severity was obtained for RILs carrying Lr67/Yr46, compared to RILs that lacked the gene, whereas in Kenya the difference was smaller (16 %) but significant. In Norway, leaf tip necrosis was associated with Lr67/Yr46 and RILs carrying Lr67/Yr46 showed a 20 % reduction in mean powdery mildew severity at both sites across the 2 years of evaluation. Our study demonstrates that Lr67/Yr46 confers partial resistance to stem rust and powdery mildew and is associated with leaf tip necrosis. The corresponding pleiotropic, or tightly linked, genes, designated as Sr55, Pm46, and Ltn3, can be utilized to provide broad-spectrum durable disease resistance in wheat.

Lr68: a new gene conferring slow rusting resistance to leaf rust in wheat.

The common wheat cultivar Parula possesses a high level of slow rusting, adult plant resistance (APR) to all three rust diseases of wheat. Previous mapping studies using an Avocet-YrA/Parula recombinant inbred line (RIL) population showed that APR to leaf rust (Puccinia triticina) in Parula is governed by at least three independent slow rusting resistance genes: Lr34 on 7DS, Lr46 on 1BL, and a previously unknown gene on 7BL. The use of field rust reaction and flanking markers identified two F(6) RILs, Arula1 and Arula2, from the above population that lacked Lr34 and Lr46 but carried the leaf rust resistance gene in 7BL, hereby designated Lr68. Arula1 and Arula2 were crossed with Apav, a highly susceptible line from the cross Avocet-YrA/Pavon 76, and 396 F(4)-derived F(5) RILs were developed for mapping Lr68. The RILs were phenotyped for leaf rust resistance for over 2 years in Ciudad Obregon, Mexico, with a mixture of P. triticina races MBJ/SP and MCJ/SP. Close genetic linkages with several DNA markers on 7BL were established using 367 RILs; Psy1-1 and gwm146 flanked Lr68 and were estimated at 0.5 and 0.6 cM, respectively. The relationship between Lr68 and the race-specific seedling resistance gene Lr14b, located in the same region and present in Parula, Arula1 and Arula2, was investigated by evaluating the RILs with Lr14b-avirulent P. triticina race TCT/QB in the greenhouse. Although Lr14b and Lr68 homozygous recombinants in repulsion were not identified in RILs, γ-irradiation-induced deletion stocks that lacked Lr68 but possessed Lr14b showed that Lr68 and Lr14b are different loci. Flanking DNA markers that are tightly linked to Lr68 in a wide array of genotypes can be utilized for selection of APR to leaf rust.

The emergence of Ug99 races of the stem rust fungus is a threat to world wheat production.

Race Ug99 of the fungus Puccinia graminis tritici that causes stem or black rust disease on wheat was first detected in Uganda in 1998. Seven races belonging to the Ug99 lineage are now known and have spread to various wheat-growing countries in the eastern African highlands, as well as Zimbabwe, South Africa, Sudan, Yemen, and Iran. Because of the susceptibility of 90% of the wheat varieties grown worldwide, the Ug99 group of races was recognized as a major threat to wheat production and food security. Its spread, either wind-mediated or human-aided, to other countries in Africa, Asia, and beyond is evident. Screening in Kenya and Ethiopia has identified a low frequency of resistant wheat varieties and breeding materials. Identification and transfer of new sources of race-specific resistance from various wheat relatives is underway to enhance the diversity of resistance. Although new Ug99-resistant varieties that yield more than current popular varieties are being released and promoted, major efforts are required to displace current Ug99 susceptible varieties with varieties that have diverse race-specific or durable resistance and mitigate the Ug99 threat.

New slow-rusting leaf rust and stripe rust resistance genes Lr67 and Yr46 in wheat are pleiotropic or closely linked.

The common wheat genotype 'RL6077' was believed to carry the gene Lr34/Yr18 that confers slow-rusting adult plant resistance (APR) to leaf rust and stripe rust but located to a different chromosome through inter-chromosomal reciprocal translocation. However, haplotyping using the cloned Lr34/Yr18 diagnostic marker and the complete sequencing of the gene indicated Lr34/Yr18 is absent in RL6077. We crossed RL6077 with the susceptible parent 'Avocet' and developed F(3), F(4) and F(6) populations from photoperiod-insensitive F(3) lines that were segregating for resistance to leaf rust and stripe rust. The populations were characterized for leaf rust resistance at two Mexican sites, Cd. Obregon during the 2008-2009 and 2009-2010 crop seasons, and El Batan during 2009, and for stripe rust resistance at Toluca, a third Mexican site, during 2009. The F(3) population was also evaluated for stripe rust resistance at Cobbitty, Australia, during 2009. Most lines had correlated responses to leaf rust and stripe rust, indicating that either the same gene, or closely linked genes, confers resistance to both diseases. Molecular mapping using microsatellites led to the identification of five markers (Xgwm165, Xgwm192, Xcfd71, Xbarc98 and Xcfd23) on chromosome 4DL that are associated with this gene(s), with the closest markers being located at 0.4 cM. In a parallel study in Canada using a Thatcher × RL6077 F(3) population, the same leaf rust resistance gene was designated as Lr67 and mapped to the same chromosomal region. The pleiotropic, or closely linked, gene derived from RL6077 that conferred stripe rust resistance in this study was designated as Yr46. The slow-rusting gene(s) Lr67/Yr46 can be utilized in combination with other slow-rusting genes to develop high levels of durable APR to leaf rust and stripe rust in wheat.

Gene-specific markers for the wheat gene Lr34/Yr18/Pm38 which confers resistance to multiple fungal pathogens.

The locus Lr34/Yr18/Pm38 confers partial and durable resistance against the devastating fungal pathogens leaf rust, stripe rust, and powdery mildew. In previous studies, this broad-spectrum resistance was shown to be controlled by a single gene which encodes a putative ATP-binding cassette transporter. Alleles of resistant and susceptible cultivars differed by only three sequence polymorphisms and the same resistance haplotype was found in the three independent breeding lineages of Lr34/Yr18/Pm38. Hence, we used these conserved sequence polymorphisms as templates to develop diagnostic molecular markers that will assist selection for durable multi-pathogen resistance in breeding programs. Five allele-specific markers (cssfr1-cssfr5) were developed based on a 3 bp deletion in exon 11 of the Lr34-gene, and one marker (cssfr6) was derived from a single nucleotide polymorphism in exon 12. Validation of reference genotypes, well characterized for the presence or absence of the Lr34/Yr18/Pm38 resistance locus, demonstrated perfect diagnostic values for the newly developed markers. By testing the new markers on a larger set of wheat cultivars, a third Lr34 haplotype, not described so far, was discovered in some European winter wheat and spelt material. Some cultivars with uncertain Lr34 status were re-assessed using the newly derived markers. Unambiguous identification of the Lr34 gene aided by the new markers has revealed that some wheat cultivars incorrectly postulated as having Lr34 may possess as yet uncharacterised loci for adult plant leaf and stripe rust resistance.

Association analysis of historical bread wheat germplasm using additive genetic covariance of relatives and population structure.

Linkage disequilibrium can be used for identifying associations between traits of interest and genetic markers. This study used mapped diversity array technology (DArT) markers to find associations with resistance to stem rust, leaf rust, yellow rust, and powdery mildew, plus grain yield in five historical wheat international multienvironment trials from the International Maize and Wheat Improvement Center (CIMMYT). Two linear mixed models were used to assess marker-trait associations incorporating information on population structure and covariance between relatives. An integrated map containing 813 DArT markers and 831 other markers was constructed. Several linkage disequilibrium clusters bearing multiple host plant resistance genes were found. Most of the associated markers were found in genomic regions where previous reports had found genes or quantitative trait loci (QTL) influencing the same traits, providing an independent validation of this approach. In addition, many new chromosome regions for disease resistance and grain yield were identified in the wheat genome. Phenotyping across up to 60 environments and years allowed modeling of genotype x environment interaction, thereby making possible the identification of markers contributing to both additive and additive x additive interaction effects of traits.