Identification of Sr67, a new gene for stem rust resistance in KU168-2 located close to the Sr13 locus in wheat.

KEY MESSAGE: Sr67 is a new stem rust resistance gene that represents a new resource for breeding stem rust resistant wheat cultivars Re-appearance of stem rust disease, caused by the fungal pathogen Puccinia graminis f. sp. tritici (Pgt), in different parts of Europe emphasized the need to develop wheat varieties with effective resistance to local Pgt populations and exotic threats. A Kyoto University wheat (Triticum aestivum L.) accession KU168-2 was reported to carry good resistance to leaf and stem rust. To identify the genomic region associated with the KU168-2 stem rust resistance, a genetic study was conducted using a doubled haploid (DH) population from the cross RL6071 × KU168-2. The DH population was phenotyped with three Pgt races (TTKSK, TPMKC, and QTHSF) and genotyped using the Illumina 90 K wheat SNP array. Linkage mapping showed the resistance to all three Pgt races was conferred by a single stem rust resistance (Sr) gene on chromosome arm 6AL, associated with Sr13. Presently, four Sr13 resistance alleles have been reported. Sr13 allele-specific KASP and STARP markers, and sequencing markers all showed null alleles in KU168-2. KU168-2 showed a unique combination of seedling infection types for five Pgt races (TTKSK, QTHSF, RCRSF, TMRTF, and TPMKC) compared to Sr13 alleles. The phenotypic uniqueness of the stem rust resistance gene in KU168-2 and null alleles for Sr13 allele-specific markers showed the resistance was conferred by a new gene, designated Sr67. Since Sr13 is less effective in hexaploid background, Sr67 will be a good source of stem rust resistance in bread wheat breeding programs.

Genetic mapping of the wheat leaf rust resistance gene Lr2a and its importance in Canadian wheat cultivars.

KEY MESSAGE: Leaf rust resistance gene Lr2a was located to chromosome arm 2DS in three mapping populations, which will facilitate map-based cloning and marker-assisted selection of Lr2a in wheat breeding programs. Incorporating effective leaf rust resistance (Lr) genes into high-yielding wheat cultivars has been an efficient method of disease control. One of the most widely used genes in Canada is the multi-allelic resistance gene Lr2, with alleles Lr2a, Lr2b, Lr2c, and Lr2d. The Lr2a allele confers complete resistance to a large portion of the Puccinia triticina (Pt) population in Canada. In this study, Lr2a was genetically mapped in two doubled haploid populations developed from the crosses Superb/BW278 and Superb/86ISMN 2137, and an F2 population developed from the cross Chinese Spring/RL6016. Seedlings were tested with the Lr2a avirulent Pt races 74-2 MGBJ (Superb/BW278) and 12-3 MBDS (Superb/86ISMN 2137 and Chinese Spring/RL6016) in greenhouse assays and were genotyped with 90K wheat Infinium SNP and kompetitive allele-specific PCR (KASP) markers. Lr2a was mapped to a collinear position on chromosome arm 2DS in all three populations, within a 1.00 cM genetic interval between KASP markers kwm1620 and kwm1623. This corresponded to a 305 kb genomic region of chromosome 2D in Chinese Spring RefSeq v2.1. The KASP marker kwh740 was predictive of Lr2a in all mapping populations. A panel of 260 wheats were tested with three Pt isolates, which revealed that Lr2a is common in Canadian wheats. The KASP markers kwh740 and kwm1584 were highly associated with resistance at the Lr2 locus, while kwm1622 was slightly less correlated. Genetic mapping of the leaf rust resistance gene Lr2a and DNA markers developed here will facilitate its use in wheat breeding programs.

Mapping pre-harvest sprouting resistance loci in AAC Innova × AAC Tenacious spring wheat population.

BACKGROUND: Pre-harvest sprouting (PHS) is a major problem for wheat production due to its direct detrimental effects on wheat yield, end-use quality and seed viability. Annually, PHS is estimated to cause > 1.0 billion USD in losses worldwide. Therefore, identifying PHS resistance quantitative trait loci (QTLs) is crucial to aid molecular breeding efforts to minimize losses. Thus, a doubled haploid mapping population derived from a cross between white-grained PHS susceptible cv AAC Innova and red-grained resistant cv AAC Tenacious was screened for PHS resistance in four environments and utilized for QTL mapping. RESULTS: Twenty-one PHS resistance QTLs, including seven major loci (on chromosomes 1A, 2B, 3A, 3B, 3D, and 7D), each explaining ≥10% phenotypic variation for PHS resistance, were identified. In every environment, at least one major QTL was identified. PHS resistance at most of these loci was contributed by AAC Tenacious except at two loci on chromosomes 3D and 7D where it was contributed by AAC Innova. Thirteen of the total twenty-one identified loci were located to chromosome positions where at least one QTL have been previously identified in other wheat genotype(s). The remaining eight QTLs are new which have been identified for the first time in this study. Pedigree analysis traced several known donors of PHS resistance in AAC Tenacious genealogy. Comparative analyses of the genetic intervals of identified QTLs with that of already identified and cloned PHS resistance gene intervals using IWGSC RefSeq v2.0 identified MFT-A1b (in QTL interval QPhs.lrdc-3A.1) and AGO802A (in QTL interval QPhs.lrdc-3A.2) on chromosome 3A, MFT-3B-1 (in QTL interval QPhs.lrdc-3B.1) on chromosome 3B, and AGO802D, HUB1, TaVp1-D1 (in QTL interval QPhs.lrdc-3D.1) and TaMyb10-D1 (in QTL interval QPhs.lrdc-3D.2) on chromosome 3D. These candidate genes are involved in embryo- and seed coat-imposed dormancy as well as in epigenetic control of dormancy. CONCLUSIONS: Our results revealed the complex PHS resistance genetics of AAC Tenacious and AAC Innova. AAC Tenacious possesses a great reservoir of important PHS resistance QTLs/genes supposed to be derived from different resources. The tracing of pedigrees of AAC Tenacious and other sources complements the validation of QTL analysis results. Finally, comparing our results with previous PHS studies in wheat, we have confirmed the position of several major PHS resistance QTLs and candidate genes.

Identification of loci for pre-harvest sprouting resistance in the highly dormant spring wheat RL4137.

KEY MESSAGE: Combination of RL4137 alleles at three QTLs on chromosomes 4A, 6B and 6D, and 'Roblin' allele at a novel QTL on chromosome 1D increases pre-harvest sprouting resistance in 'Roblin'/RL4137 doubled haploid population. Pre-harvest sprouting (PHS) significantly reduces wheat grain yield and quality. Therefore, identifying quantitative trait loci (QTL) for PHS resistance is key to facilitate marker-assisted breeding. To this end, we studied PHS in a population of 330 doubled haploid (DH) lines derived from 'Roblin'/RL4137. The parental and DH lines were examined for their PHS phenotype based on speed of germination index in five environments and genotyped using the wheat Infinium 90 K SNP array. A total of five QTLs were detected on linkage groups 1D, 4A.2, 6B.1, 6D and 7A over the five environments. The QTL QPhs.umb-4A on linkage group 4A.2 was the most consistent across all environments and explained 40-50% of phenotypic variation. The QTL on 1D is a novel QTL and explained 1.99-2.33% of phenotypic variation. The QTLs on 6B.1 and 6D each explained 3.09-4.33% and 1.62-2.45% of phenotypic variation, respectively. A combination of four stable QTLs on linkage groups 1D, 4A.2, 6B.1 and 6D greatly increased PHS resistance. Allelic effects for the QTLs QPhs.umb-4A, QPhs.umb-6B and QPhs.umb-6D were contributed by RL4137, whereas 'Roblin' contributed the resistant allele for QPhs.umb-1D. QPhs.umb-4A was required for strong dormancy in the 'Roblin'/RL4137 DH population, and the presence of QTLs QPhs.umb-1D, QPhs.umb-6B and QPhs.umb-6D incrementally increased dormancy; DH lines carrying all four QTLs are considerably more dormant than those carrying only QPhs.umb-4A or none of the four QTLs. Thus, the QTLs identified in this study have the potential to improve PHS resistance in spring wheat.

Evaluation of variant calling tools for large plant genome re-sequencing.

BACKGROUND: Discovering single nucleotide polymorphisms (SNPs) from agriculture crop genome sequences has been a widely used strategy for developing genetic markers for several applications including marker-assisted breeding, population diversity studies for eco-geographical adaption, genotyping crop germplasm collections, and others. Accurately detecting SNPs from large polyploid crop genomes such as wheat is crucial and challenging. A few variant calling methods have been previously developed but they show a low concordance between their variant calls. A gold standard of variant sets generated from one human individual sample was established for variant calling tool evaluations, however hitherto no gold standard of crop variant set is available for wheat use. The intent of this study was to evaluate seven SNP variant calling tools (FreeBayes, GATK, Platypus, Samtools/mpileup, SNVer, VarScan, VarDict) with the two most popular mapping tools (BWA-mem and Bowtie2) on wheat whole exome capture (WEC) re-sequencing data from allohexaploid wheat. RESULTS: We found the BWA-mem mapping tool had both a higher mapping rate and a higher accuracy rate than Bowtie2. With the same mapping quality (MQ) cutoff, BWA-mem detected more variant bases in mapping reads than Bowtie2. The reads preprocessed with quality trimming or duplicate removal did not significantly affect the final mapping performance in terms of mapped reads. Based on the concordance and receiver operating characteristic (ROC), the Samtools/mpileup variant calling tool with BWA-mem mapping of raw sequence reads outperformed other tests followed by FreeBayes and GATK in terms of specificity and sensitivity. VarDict and VarScan were the poorest performing variant calling tools with the wheat WEC sequence data. CONCLUSION: The BWA-mem and Samtools/mpileup pipeline, with no need to preprocess the raw read data before mapping onto the reference genome, was ascertained the optimum for SNP calling for the complex wheat genome re-sequencing. These results also provide useful guidelines for reliable variant identification from deep sequencing of other large polyploid crop genomes.

Genetic and transcriptional dissection of resistance to Claviceps purpurea in the durum wheat cultivar Greenshank.

KEY MESSAGE: Four QTL for ergot resistance (causal pathogen Claviceps purpurea) have been identified in the durum wheat cultivar Greenshank. Claviceps purpurea is a pathogen of grasses that infects flowers, replacing the seed with an ergot sclerotium. Ergot presents a significant problem to rye, barley and wheat, in particular hybrid seed production systems. In addition, there is evidence that the highly toxic alkaloids that accumulate within sclerotia can cross-contaminate otherwise healthy grain. Host resistance to C. purpurea is rare, few resistance loci having been identified. In this study, four ergot resistance loci are located on chromosomes 1B, 2A, 5A and 5B in the durum wheat cv. Greenshank. Ergot resistance was assessed through analysis of phenotypes associated with C. purpurea infection, namely the number of inoculated flowers that produced sclerotia, or resulted in ovary death but no sclerotia, the levels of honeydew produced, total sclerotia weight and average sclerotia weight and size per spike. Ergot testing was undertaken in Canada and the UK. A major effect QTL, QCp.aafc.DH-2A, was detected in both the Canadian and UK experiments and had a significant effect on honeydew production levels. QCp.aafc.DH-5B had the biggest influence on total sclerotia weight per spike. QCp.aafc.DH-1B was only detected in the Canadian experiments and QCp.aafc.DH-5A in the UK experiment. An RNASeq analysis, undertaken to identify wheat differentially expressed genes associated with different combinations of the four ergot resistance QTL, revealed a disproportionate number of DEGs locating to the QCp.aafc.DH-1B, QCp.aafc.DH-2A and QCp.aafc.DH-5B QTL intervals.

Genetic Characterization of Leaf and Stripe Rust Resistance in the Brazilian Wheat Cultivar Toropi.

Leaf and stripe rust are major threats to wheat production worldwide. The effective, multiple rust resistances present in the Brazilian cultivar Toropi makes it an excellent choice for a genetic study of rust resistance. Testing of DNA from different seed lots of Toropi with 2,194 polymorphic 90K iSelect single nucleotide polymorphism markers identified significant genetic divergence, with as much as 35% dissimilarity between seed lots. As a result, further work was conducted with a single plant line derived from Toropi variant Toropi-6.4. A double haploid population with 168 lines derived from the cross Toropi-6.4 × Thatcher was phenotyped over multiple years and locations in Canada, New Zealand, and Kenya, with a total of seven field trials undertaken for leaf rust and nine for stripe rust. Genotyping with the 90K iSelect array, simple sequence repeat and Kompetitive allele-specific polymerase chain reaction markers resulted in a genetic map of 3,043 cM, containing 1,208 nonredundant markers. Significant quantitative trait loci (QTL) derived from Toropi-6.4 were identified in multiple environments on chromosomes 1B (QLr.crc-1BL/QYr.crc-1BL), 3B (QLr.crc-3BS), 4B (QYr.crc-4BL), 5A (QLr.crc-5AL and QYr.crc-5AL), and 5D (QLr.crc-5DS). The QTL QLr.crc-1BL/QYr.crc-1BL colocated with the multi-rust resistance locus Lr46/Yr29, while the QTL QLr.crc-5DS located to the Lr78 locus previously found in a wheat backcross population derived from Toropi. Comparisons of QTL combinations showed QLr.crc-1BL to contribute a significantly enhanced leaf rust resistance when combined with QLr.crc-5AL or QLr.crc-5DS, more so than when QLr.crc-5AL and QLr.crc-5DS were combined. A strong additive effect was also seen when the stripe rust resistance QTL QYr.crc-1BL and QYr.crc-5AL were combined.

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

BACKGROUND: Research 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. RESULTS: QTL 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. CONCLUSIONS: The 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.

Quantitative Trait Loci Conferring Leaf Rust Resistance in Hexaploid Wheat.

Leaf rust, caused by the fungal pathogen Puccinia triticina, is a major threat to wheat production in many wheat-growing regions of the world. The introduction of leaf rust resistance genes into elite wheat germplasm is the preferred method of disease control, being environmentally friendly and crucial to sustained wheat production. Consequently, there is considerable value in identifying and characterizing new sources of leaf rust resistance. While many major, qualitative leaf rust resistance genes have been identified in wheat, a growing number of valuable sources of quantitative resistance have been reported. Here we review the progress made in the genetic identification of quantitative trait loci (QTL) for leaf rust resistance detected primarily in field analyses, i.e., adult plant resistance. Over the past 50 years, leaf rust resistance loci have been assigned to genomic locations through chromosome analyses and genetic mapping in biparental mapping populations, studies that represent 79 different wheat leaf rust resistance donor lines. In addition, seven association mapping studies have identified adult plant and seedling leaf rust resistance marker trait associations in over 4,000 wheat genotypes. Adult plant leaf rust resistance QTL have been found on all 21 chromosomes of hexaploid wheat, with the B genome carrying the greatest number of QTL. The group 2 chromosomes are also particularly rich in leaf rust resistance QTL. The A genome has the lowest number of QTL for leaf rust resistance. Copyright © 2018 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Highly predictive SNP markers for efficient selection of the wheat leaf rust resistance gene Lr16.

BACKGROUND: Lr16 is a widely deployed leaf rust resistance gene in wheat (Triticum aestivum L.) that is highly effective against the North American Puccinia triticina population when pyramided with the gene Lr34. Lr16 is a seedling leaf rust resistance gene conditioning an incompatible interaction with a distinct necrotic ring surrounding the uredinium. Lr16 was previously mapped to the telomeric region of the short arm of wheat chromosome 2B. The goals of this study were to develop numerous single nucleotide polymorphism (SNP) markers for the Lr16 region and identify diagnostic gene-specific SNP marker assays for marker-assisted selection (MAS). RESULTS: Forty-three SNP markers were developed and mapped on chromosome 2BS tightly linked with the resistance gene Lr16 across four mapping populations representing a total of 1528 gametes. Kompetitive Allele Specific PCR (KASP) assays were designed for all identified SNPs. Resistance gene analogs (RGAs) linked with the Lr16 locus were identified and RGA-based SNP markers were developed. The diagnostic potential of the SNPs co-segregating with Lr16 was evaluated in a diverse set of 133 cultivars and breeding lines. Six SNP markers were consistent with the Lr16 phenotype and are accurately predictive of Lr16 for all wheat lines/cultivars in the panel. CONCLUSIONS: Lr16 was mapped relative to SNP markers in four populations. Six SNP markers exhibited high quality clustering in the KASP assay and are suitable for MAS of Lr16 in wheat breeding programs.

Quantitative trait loci for resistance to stripe rust of wheat revealed using global field nurseries and opportunities for stacking resistance genes.

KEY MESSAGE: Quantitative trait loci controlling stripe rust resistance were identified in adapted Canadian spring wheat cultivars providing opportunity for breeders to stack loci using marker-assisted breeding. Stripe rust or yellow rust, caused by Puccinia striiformis Westend. f. sp. tritici Erikss., is a devastating disease of common wheat (Triticum aestivum L.) in many regions of the world. The objectives of this research were to identify and map quantitative trait loci (QTL) associated with stripe rust resistance in adapted Canadian spring wheat cultivars that are effective globally, and investigate opportunities for stacking resistance. Doubled haploid (DH) populations from the crosses Vesper/Lillian, Vesper/Stettler, Carberry/Vesper, Stettler/Red Fife and Carberry/AC Cadillac were phenotyped for stripe rust severity and infection response in field nurseries in Canada (Lethbridge and Swift Current), New Zealand (Lincoln), Mexico (Toluca) and Kenya (Njoro), and genotyped with SNP markers. Six QTL for stripe rust resistance in the population of Vesper/Lillian, five in Vesper/Stettler, seven in Stettler/Red Fife, four in Carberry/Vesper and nine in Carberry/AC Cadillac were identified. Lillian contributed stripe rust resistance QTL on chromosomes 4B, 5A, 6B and 7D, AC Cadillac on 2A, 2B, 3B and 5B, Carberry on 1A, 1B, 4A, 4B, 7A and 7D, Stettler on 1A, 2A, 3D, 4A, 5B and 6A, Red Fife on 2D, 3B and 4B, and Vesper on 1B, 2B and 7A. QTL on 1A, 1B, 2A, 2B, 3B, 4A, 4B, 5B, 7A and 7D were observed in multiple parents. The populations are compelling sources of recombination of many stripe rust resistance QTL for stacking disease resistance. Gene pyramiding should be possible with little chance of linkage drag of detrimental genes as the source parents were mostly adapted cultivars widely grown in Canada.

Genetic Mapping of Stem Rust Resistance to Puccinia graminis f. sp. tritici Race TRTTF in the Canadian Wheat Cultivar Harvest.

Stem rust, caused by Puccinia graminis f. sp. tritici, is a destructive disease of wheat that can be controlled by deploying effective stem rust resistance (Sr) genes. Highly virulent races of P. graminis f. sp. tritici in Africa have been detected and characterized. These include race TRTTF and the Ug99 group of races such as TTKSK. Several Canadian and U.S. spring wheat cultivars, including the widely grown Canadian cultivar 'Harvest', are resistant to TRTTF. However, the genetic basis of resistance to TRTTF in Canadian and U.S. spring wheat cultivars is unknown. The objectives of this study were to determine the number of Sr genes involved in TRTTF resistance in Harvest, genetically map the resistance with DNA markers, and use markers to assess the distribution of that resistance in a panel of Canadian cultivars. A doubled haploid (DH) population was produced from the cross LMPG-6S/Harvest. The DH population was tested with race TRTTF at the seedling stage. Of 92 DH progeny evaluated, 46 were resistant and 46 were susceptible which perfectly fit a 1:1 ratio indicating a single Sr gene was responsible for conferring resistance to TRTTF in Harvest. Mapping with single nucleotide polymorphism (SNP) and simple sequence repeat (SSR) markers placed the resistance gene distally on the chromosome 6AS genetic map, which corresponded to the location reported for Sr8. SSR marker gwm459 and 30 cosegregating SNP markers showed the closest linkage, mapping 2.2 cM proximal to the Sr gene. Gene Sr8a confers resistance to TRTTF and may account for the resistance in Harvest. Testing a panel of Canadian wheat cultivars with four SNP markers closely linked to resistance to TRTTF suggested that the resistance present in Harvest is present in many Canadian cultivars. Two of these SNP markers were also predictive of TRTTF resistance in a panel of 241 spring wheat lines from the United States, Canada, and Mexico.

The relationship of leaf rust resistance gene Lr13 and hybrid necrosis gene Ne2m on wheat chromosome 2BS.

KEY MESSAGE: Genetic and mutational analyses of wheat leaf rust resistance gene Lr13 and hybrid necrosis gene Ne2 m indicated that they are the same gene. Hybrid necrosis in wheat characterized by chlorosis and eventual necrosis of plant tissues in certain wheat hybrids is controlled by the interaction of complementary dominant genes Ne1 and Ne2 located on chromosome arms 5BL and 2BS, respectively. Multiple alleles at each locus can be identified by differences in necrotic phenotypes when varieties are crossed with a fixed accession of the other genotype. Some of at least five Ne2 alleles were described as s (strong), m (medium) and w (weak); alleles of Ne1 were similarly described. Ne2m causes moderate necrosis in hybrids with genotypes having Ne1s. Ne2 is located on chromosome arm 2BS in close proximity to Lr13. Most wheat lines with Ne2m carry Lr13, and all wheat lines with Lr13 appear to carry Ne2m. To further dissect the relationship between Lr13 and Ne2m, more than 350 crosses were made between cv. Spica (Triticum aestivum) or Kubanka (T. durum) carrying Ne1s and recombinant inbred lines or doubled haploid lines from three crosses segregating for Lr13. F1 plants from lines carrying Lr13 crossed with Spica (Ne1s) always showed progressive necrosis; those lacking Lr13 did not. Four wheat cultivars/lines carrying Lr13 were treated with the mutagen EMS. Thirty-five susceptible mutants were identified; eight were distinctly less glaucous and late maturing indicative of chromosome 2B or sub-chromosome loss. Hybrids of phenotypically normal Lr13 mutant plants crossed with Spica did not produce symptoms of hybrid necrosis. Thus, Lr13 and one particular Ne2m allele may be the same gene.

Genetics and mapping of seedling resistance to Ug99 stem rust in winter wheat cultivar Triumph 64 and differentiation of SrTmp, SrCad, and Sr42.

KEY MESSAGE: Resistance to Ug99 stem rust in Triumph 64 was conferred by SrTmp on chromosome arm 6DS and was mapped to the same position as SrCad and Sr42 , however, the three genes show functional differences. Stem rust, caused by Puccinia graminis f. sp. tritici (Pgt), is an important disease of wheat that can be controlled by effective stem rust resistance (Sr) genes. The emergence of virulent Pgt races in Africa, namely Ug99 and its variants, has stimulated the search for new Sr genes and genetic characterization of known sources of resistance. Triumph 64 is a winter wheat cultivar that carries gene SrTmp, which confers resistance to Ug99. The goals of this study were to genetically map SrTmp and examine its relationship with other Sr genes occupying a similar chromosome location. A doubled haploid (DH) population from the cross LMPG-6S/Triumph 64 was inoculated with Ug99 at the seedling stage. A single gene conditioning resistance to Ug99 segregated in the population. Genetic mapping with SSR markers placed SrTmp on chromosome arm 6DS in a region similar to SrCad and Sr42. SNP markers developed for SrCad were used to further map SrTmp and were also added to a genetic map of Sr42 using a DH population (LMPG-6S/Norin 40). Three SNP markers that co-segregated with SrTmp also co-segregated with SrCad and Sr42. The SNP markers showed no difference in the map locations of SrTmp, SrCad, and Sr42. Multi-race testing with DH lines from the Triumph 64 and Norin 40 populations and a recombinant inbred-line population from the cross LMPG-6S/AC Cadillac showed that SrTmp, SrCad, and Sr42 confer different spectra of resistance. Markers closely linked to SrTmp are suitable for marker-assisted breeding and germplasm development.

Genetic mapping of SrCad and SNP marker development for marker-assisted selection of Ug99 stem rust resistance in wheat.

KEY MESSAGE: New SNP markers that can be used for marker-assisted selection and map-based cloning saturate the chromosome region carrying SrCad , a wheat gene that confers resistance to Ug99 stem rust. Wheat stem rust, caused by Puccinia graminis f. sp. tritici, is a devastating disease of wheat worldwide. Development of cultivars with effective resistance has been the primary means to control this disease, but the appearance of new virulent strains such as Ug99 has rendered most wheat varieties vulnerable. The stem rust resistance gene SrCad located on chromosome arm 6DS has provided excellent resistance to various strains of Ug99 in field nurseries conducted in Njoro, Kenya since 2005. Three genetic populations were used to identify SNP markers closely linked to the SrCad locus. Of 220 SNP markers evaluated, 27 were found to be located within a 2 cM region surrounding SrCad. The diagnostic potential of these SNPs was evaluated in a diverse set of 50 wheat lines that were primarily of Canadian origin with known presence or absence of SrCad. Three SNP markers tightly linked proximally to SrCad and one SNP that co-segregated with SrCad were completely predictive of the presence or absence of SrCad. These markers also differentiated SrCad from Sr42 and SrTmp which are also located in the same region of chromosome arm 6DS. These markers should be useful in marker-assisted breeding to develop new wheat varieties containing SrCad-based resistance to Ug99 stem rust.

Lr70, a new gene for leaf rust resistance mapped in common wheat accession KU3198.

KEY MESSAGE: KU3198 is a common wheat accession that carries one novel leaf rust resistance (Lr) gene, Lr70 , and another Lr gene which is either novel, Lr52 or an allele of Lr52. Leaf rust, caused by Puccinia triticina Eriks. (Pt), is a broadly distributed and economically important disease of wheat. Deploying cultivars carrying effective leaf rust resistance (Lr) genes is a desirable method of disease control. KU3198 is a common wheat (Triticum aestivum L.) accession from the Kyoto collection that was highly resistant to Pt in Canada. An F2 population from the cross HY644/KU3198 showed segregation for two dominant Lr genes when tested with Pt race MBDS which was virulent on HY644. Multiple bulk segregant analysis (MBSA) was employed to find putative chromosome locations of these Lr genes using SSR markers that provided coverage of the genome. MBSA predicted that the Lr genes were located on chromosomes 5B and 5D. A doubled haploid population was generated from the cross of JBT05-714 (HY644*3/KU3198), a line carrying one of the Lr genes from KU3198, to Thatcher. This population segregated for a single Lr gene conferring resistance to Pt race MBDS, which was mapped to the terminal region of the short arm of chromosome 5B with SSR markers and given the temporary designation LrK1. One F3 family derived from the HY644/KU3198 F2 population that segregated only for the second Lr gene from KU3198 was identified. This family was treated as an F2-equivalent population and used for mapping the Lr gene, which was located to the terminal region of chromosome 5DS. As no other Lr gene has been mapped to 5DS, this gene is novel and has been designated as Lr70.

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

KEY MESSAGE: 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.

Development of a multiple bulked segregant analysis (MBSA) method used to locate a new stem rust resistance gene (Sr54) in the winter wheat cultivar Norin 40.

An important aspect of studying putative new genes in wheat is determining their position on the wheat genetic map. The primary difficulty in mapping genes is determining which chromosome carries the gene of interest. Several approaches have been developed to address this problem, each with advantages and disadvantages. Here we describe a new approach called multiple bulked segregant analysis (MBSA). A set of 423 simple sequence repeat (SSR) markers were selected based on profile simplicity, frequency of polymorphism, and distribution across the wheat genome. SSR primers were preloaded in 384-well PCR plates with each primer occupying 16 wells. In practice, 14 wells are reserved for "mini-bulks" that are equivalent to four gametes (e.g. two F(2) individuals) comprised of individuals from a segregated population that have a known homozygous genotype for the gene of interest. The remaining two wells are reserved for the parents of the population. Each well containing a mini-bulk can have one of three allele compositions for each SSR: only the allele from one parent, only the allele from the other parent, or both alleles. Simulation experiments were performed to determine the pattern of mini-bulk allele composition that would indicate putative linkage between the SSR in question and the gene of interest. As a test case, MBSA was employed to locate an unidentified stem rust resistance (Sr) gene in the winter wheat cultivar Norin 40. A doubled haploid (DH) population (n = 267) was produced from hybrids of the cross LMPG-6S/Norin 40. The DH population segregated for a single gene (χ (1:1) (2) = 0.093, p = 0.76) for resistance to Puccinia graminis f.sp. tritici race LCBN. Four resistant DH lines were included in each of the 14 mini-bulks for screening. The Sr gene was successfully located to the long arm of chromosome 2D using MBSA. Further mapping confirmed the chromosome location and revealed that the Sr gene was located in a linkage block that may represent an alien translocation. The new Sr gene was designated as Sr54.

Fine mapping and marker development for the wheat leaf rust resistance gene Lr32.

Wheat leaf rust is caused by the fungal pathogen Puccinia triticina and is one of the wheat diseases of concern globally. Among the known leaf rust resistance genes (Lr) genes, Lr32 is a broadly effective gene derived from the diploid species Aegilops tauschii coss. accession RL5497-1 and has been genetically mapped to chromosome arm 3DS. However, Lr32 resistance has not been utilized in current cultivars in part due to the lack of modern, predictive DNA markers. The goals of this study were to fine map the Lr32 region and develop SNP-based kompetitive allele-specific polymerase chain reaction markers. The genomic analysis was conducted by using doubled haploid and F2-derived mapping populations. For marker development, a 90K wheat chip array, 35K and 820K Axiom R SNPs, A. tauschii pseudomolecules v4.0 and International Wheat Genome Sequencing Consortium ReqSeq v2.1 reference genomes were used. Total 28 kompetitive allele-specific polymerase chain reaction and 2 simple sequence repeat markers were developed. The Lr32 region was fine mapped between kompetitive allele-specific polymerase chain reaction markers Kwh142 and Kwh355 that flanked 34-35 Mb of the diploid and hexaploid reference genomes. Leaf rust resistance mapped as a Mendelian trait that cosegregated with 20 markers, recombination restriction limited the further resolution of the Lr32 region. A total of 10-11 candidate genes associated with disease resistance were identified between the flanking regions on both reference genomes, with the majority belonging to the nucleotide-binding domain and leucine-rich repeat gene family. The validation analysis selected 2 kompetitive allele-specific polymerase chain reaction markers, Kwh147 and Kwh722, for marker-assisted selection. The presence of Lr32 along with other Lr genes such as Lr67 and Lr34 would increase the resistance in future wheat breeding lines and have a high impact on controlling wheat leaf rust.

Genetic mapping of leaf rust (Puccinia triticina Eriks) resistance genes in six Canadian spring wheat cultivars.

The Canada Western Red Spring wheat (Triticum aestivum L.) cultivars AAC Concord, AAC Prevail, CDC Hughes, Lillian, Glenlea, and elite line BW961 express a spectrum of resistance to leaf rust caused by Puccinia triticina Eriks. This study aimed to identify and map the leaf rust resistance of the cultivars using three doubled haploid populations, AAC Prevail/BW961 (PB), CDC Hughes/AAC Concord (HC), and Lillian/Glenlea (LG). The populations were evaluated for seedling resistance in the greenhouse and adult plant disease response in the field at Morden, MB for 3 years and genotyped with the 90K wheat Infinium iSelect SNP array. Genetic maps were constructed to perform QTL analysis on the seedling and field leaf rust data. A total of three field leaf rust resistance QTL segregated in the PB population, five in the HC, and six in the LG population. In the PB population, BW961 contributed two QTL on chromosomes 2DS and 7DS, and AAC Prevail contributed a QTL on 4AL consistent across trials. Of the five QTL in HC, AAC Concord contributed two QTL on 4AL and 7AL consistent across trials and a QTL on 3DL.1 that provided seedling resistance only. CDC Hughes contributed two QTL on 1DS and 3DL.2. Lillian contributed four QTL significant in at least two of the three trials on 2BS, 4AL, 5AL, and 7AL, and Glenlea two QTL on 4BL and 7BL. The 1DS QTL from CDC Hughes, the 2DS from BW961, the 4AL from the AAC Prevail, AAC Concord, and Lillian, and the 7AL from AAC Concord and Lillian conferred seedling leaf rust resistance. The QTL on 4AL corresponded with Lr30 and was the same across cultivars AAC Prevail, AAC Concord, and Lillian, whereas the 7AL corresponding with LrCen was coincident between AAC Concord and Lillian. The 7DS and 2DS QTL in BW961 corresponded with Lr34 and Lr2a, respectively, and the 1DS QTL in CDC Hughes with Lr21. The QTL identified on 5AL could represent a novel gene. The results of this study will widen our knowledge of leaf rust resistance genes in Canadian wheat and their utilization in resistance breeding.