The Coiled-Coil NLR Rph1, Confers Leaf Rust Resistance in Barley Cultivar Sudan.

Unraveling and exploiting mechanisms of disease resistance in cereal crops is currently limited by their large repeat-rich genomes and the lack of genetic recombination or cultivar (cv)-specific sequence information. We cloned the first leaf rust resistance gene Rph1 (Rph1 a) from cultivated barley (Hordeum vulgare) using "MutChromSeq," a recently developed molecular genomics tool for the rapid cloning of genes in plants. Marker-trait association in the CI 9214/Stirling doubled haploid population mapped Rph1 to the short arm of chromosome 2H in a physical region of 1.3 megabases relative to the barley cv Morex reference assembly. A sodium azide mutant population in cv Sudan was generated and 10 mutants were confirmed by progeny-testing. Flow-sorted 2H chromosomes from Sudan (wild type) and six of the mutants were sequenced and compared to identify candidate genes for the Rph1 locus. MutChromSeq identified a single gene candidate encoding a coiled-coil nucleotide binding site Leucine-rich repeat (NLR) receptor protein that was altered in three different mutants. Further Sanger sequencing confirmed all three mutations and identified an additional two independent mutations within the same candidate gene. Phylogenetic analysis determined that Rph1 clustered separately from all previously cloned NLRs from the Triticeae and displayed highest sequence similarity (89%) with a homolog of the Arabidopsis (Arabidopsis thaliana) disease resistance protein 1 protein in Triticum urartu In this study we determined the molecular basis for Rph1-mediated resistance in cultivated barley enabling varietal improvement through diagnostic marker design, gene editing, and gene stacking technologies.

Identification and characterization of a new stripe rust resistance gene Yr83 on rye chromosome 6R in wheat.

KEY MESSAGE: A physical map of Secale cereale chromosome 6R was constructed using deletion mapping, and a new stripe rust resistance gene Yr83 was mapped to the deletion bin of FL 0.73-1.00 of 6RL. Rye (Secale cereale L., RR) possesses valuable genes for wheat improvement. In the current study, we report a resistance gene conferring stripe rust resistance effective from seedling to adult plant stages located on chromosome 6R. This chromosome was derived from triticale line T-701 and also carries highly effective resistance to the cereal cyst nematode species Heterodera avenae Woll. A wheat-rye 6R(6D) disomic substitution line exhibited high levels of seedling resistance to Australian pathotypes of the stripe rust (Puccinia striiformis f. sp. tritici; Pst) pathogen and showed an even greater resistance to the Chinese Pst pathotypes in the field. Ten chromosome 6R deletion lines and five wheat-rye 6R translocation lines were developed earlier in the attempt to transfer the nematode resistance gene to wheat and used herein to map the stripe rust resistance gene. These lines were subsequently characterized by sequential multicolor fluorescence in situ hybridization (mc-FISH), genomic in situ hybridization (GISH), mc-GISH, PCR-based landmark unique gene (PLUG), and chromosome 6R-specific length amplified fragment sequencing (SLAF-Seq) marker analyses to physically map the stripe rust resistance gene. The new stripe rust resistance locus was located in a chromosomal bin with fraction length (FL) 0.73-1.00 on 6RL and was named Yr83. A wheat-rye translocation line T6RL (#5) carrying the stripe rust resistance gene will be useful as a new germplasm in breeding for resistance.

A strategy for identifying markers linked with stem rust resistance in wheat harbouring an alien chromosome introgression from a non-sequenced genome.

KEY MESSAGE: A set of molecular markers was developed for Sr26 from comparative genomic analysis. The comparative genomic approach also enabled the identification of a previously uncharacterised wheat chromosome that carried Sr26. Stem rust of wheat, a biotic stress caused by a fungal pathogen, continues to pose significant threats to wheat production. Considerable effort has been directed at surveillance and breeding approaches to minimize the impact of the widely virulent race of the stem rust pathogen (Puccinia graminis f. sp. tritici, Pgt) commonly known as Ug99 (TTKSK) and other races in its lineage. The stem rust resistance gene Sr26, derived from Thinopyrum ponticum, is an excellent example of the successful utilization of a gene from a wild relative of a crop plant and remains one of the few durable sources of resistance currently effective against all known field isolates of Pgt. We explored comparative genomic analysis of the nucleotide binding leucine rich repeat (NLR) genes of the diploid D genome and bread wheat genomes to target the Sr26 region from the non-sequenced Th. ponticum genome. A chromosomal interval harboring NLR genes in the distal end of homoeologous group 6 chromosomes was used to demarcate the Sr26 locus. A set of closely linked PCR-based molecular markers was developed for Sr26. Furthermore, the comparative analysis approach enabled the unambiguous identification of a previously uncharacterised wheat chromosome that carried Sr26 in an introgressed Th. ponticum segment and was validated by fluorescent and genomic in situ hybridisation (FISH/GISH) experiments. The genetic information generated from the target interval based on this study will benefit future related studies on group 6 chromosomes of wheat, including 6Dt from Aegilops tauschii, and chromosome 6Ae#1 from Th. ponticum.

Transfer of stem rust resistance gene SrB from Thinopyrum ponticum into wheat and development of a closely linked PCR-based marker.

KEY MESSAGE: We report transfer of a rust resistance gene named SrB, on the 6Ae#3 chromosome, to wheat by recombination with the 6Ae#1 segment carrying Sr26 and development of a linked marker. A stem rust resistance gene from a South African wheat W3757, temporarily named SrB, has been transferred onto chromosome 6A. Line W3757 is a 6Ae#3 (6D) substitution line in which the Thinopyrum ponticum chromosomes carry SrB. Crosses were made between W3757 and a T6AS·6AL-6Ae#1 recombinant line named WA-5 carrying the stem rust resistance gene Sr26 on a chromosome segment from another accession of Th. ponticum. The 6Ae#1 and 6Ae#3 chromosomes had previously been shown to pair at meiosis and were polymorphic for the distally located RFLP probes BCD001 and MWG798. A recombinant plant (Type A) was identified carrying a distal chromosome segment from the 6Ae#3 chromosome and a sub-terminal segment from the 6Ae#1 chromosome. Rust tests on the recombinant Type A showed the infection type for SrB. Segregation and linkage data combined with genomic in situ hybridization studies demonstrated that SrB had been transferred to wheat chromosome arm 6AL by recombination between the Thinopyrum chromosome segments. A recombinant positive for the 6Ae#1-6Ae#3 chromosome showed enhanced stem rust resistance compared to the 6Ae#3 addition line in repeated rust tests. A diagnostic PCR-based marker was developed for the 6Ae#3 chromosome segment on the Type A recombinant carrying SrB that distinguishes it from the Sr26-containing segment. A stem rust resistant line which combines SrB with Sr26 would be a great addition to the pool of resistant germplasm for wheat breeders to achieve more durable and effective control of stem rust because virulence has not been found for either of these two genes.

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.

Development of wheat-Aegilops speltoides recombinants and simple PCR-based markers for Sr32 and a new stem rust resistance gene on the 2S#1 chromosome.

KEY MESSAGE: Wheat- Aegilops speltoides recombinants carrying stem rust resistance genes Sr32 and SrAes1t effective against Ug99 and PCR markers for marker-assisted selection. Wild relatives of wheat are important resources for new rust resistance genes but underutilized because the valuable resistances are often linked to negative traits that prevent deployment of these genes in commercial wheats. Here, we report ph1b-induced recombinants with reduced alien chromatin derived from E.R. Sears' wheat-Aegilops speltoides 2D-2S#1 translocation line C82.2, which carries the widely effective stem rust resistance gene Sr32. Infection type assessments of the recombinants showed that the original translocation in fact carries two stem rust resistance genes, Sr32 on the short arm and a previously undescribed gene SrAes1t on the long arm of chromosome 2S#1. Recombinants with substantially shortened alien chromatin were produced for both genes, which confer resistance to stem rust races in the TTKSK (Ug99) lineage and representative races of all Australian stem rust lineages. Selected recombinants were back crossed into adapted Australian cultivars and PCR markers were developed to facilitate the incorporation of these genes into future wheat varieties. Our recombinants and those from several other labs now show that Sr32, Sr39, and SrAes7t on the short arm and Sr47 and SrAes1t on the long arm of 2S#1 form two linkage groups and at present no rust races are described that can distinguish these resistance specificities.

Single amino acid change alters specificity of the multi-allelic wheat stem rust resistance locus SR9.

Most rust resistance genes thus far isolated from wheat have a very limited number of functional alleles. Here, we report the isolation of most of the alleles at wheat stem rust resistance gene locus SR9. The seven previously reported resistance alleles (Sr9a, Sr9b, Sr9d, Sr9e, Sr9f, Sr9g, and Sr9h) are characterised using a synergistic strategy. Loss-of-function mutants and/or transgenic complementation are used to confirm Sr9b, two haplotypes of Sr9e (Sr9e_h1 and Sr9e_h2), Sr9g, and Sr9h. Each allele encodes a highly related nucleotide-binding site leucine-rich repeat (NB-LRR) type immune receptor, containing an unusual long LRR domain, that confers resistance to a unique spectrum of isolates of the wheat stem rust pathogen. The only SR9 protein effective against stem rust pathogen race TTKSK (Ug99), SR9H, differs from SR9B by a single amino acid. SR9B and SR9G resistance proteins are also distinguished by only a single amino acid. The SR9 allelic series found in the B subgenome are orthologs of wheat stem rust resistance gene Sr21 located in the A subgenome with around 85% identity in protein sequences. Together, our results show that functional diversification of allelic variants at the SR9 locus involves single and multiple amino acid changes that recognize isolates of wheat stem rust.

Rye-derived powdery mildew resistance gene Pm8 in wheat is suppressed by the Pm3 locus.

Genetic suppression of disease resistance is occasionally observed in hexaploid wheat or in its interspecific crosses. The phenotypic effects of genes moved to wheat from relatives with lower ploidy are often smaller than in the original sources, suggesting the presence of modifiers or partial inhibitors in wheat, especially dilution effects caused by possible variation at orthologous loci. However, there is little current understanding of the underlying genetics of suppression. The discovery of suppression in some wheat genotypes of the cereal rye chromosome 1RS-derived gene Pm8 for powdery mildew resistance offered an opportunity for analysis. A single gene for suppression was identified at or near the closely linked storage protein genes Gli-A1 and Glu-A3, which are also closely associated with the Pm3 locus on chromosome 1AS. The Pm3 locus is a complex of expressed alleles and pseudogenes embedded among Glu-A3 repeats. In the current report, we explain why earlier work indicated that the mildew suppressor was closely associated with specific Gli-A1 and Glu-A3 alleles, and predict that suppression of Pm8 involves translated gene products from the Pm3 locus.

A recombined Sr26 and Sr61 disease resistance gene stack in wheat encodes unrelated NLR genes.

The re-emergence of stem rust on wheat in Europe and Africa is reinforcing the ongoing need for durable resistance gene deployment. Here, we isolate from wheat, Sr26 and Sr61, with both genes independently introduced as alien chromosome introgressions from tall wheat grass (Thinopyrum ponticum). Mutational genomics and targeted exome capture identify Sr26 and Sr61 as separate single genes that encode unrelated (34.8%) nucleotide binding site leucine rich repeat proteins. Sr26 and Sr61 are each validated by transgenic complementation using endogenous and/or heterologous promoter sequences. Sr61 orthologs are absent from current Thinopyrum elongatum and wheat pan genome sequences, contrasting with Sr26 where homologues are present. Using gene-specific markers, we validate the presence of both genes on a single recombinant alien segment developed in wheat. The co-location of these genes on a small non-recombinogenic segment simplifies their deployment as a gene stack and potentially enhances their resistance durability.

Wheat stripe rust resistance genes Yr5 and Yr7 are allelic.

Stripe rust is one of the most destructive diseases of wheat. Breeding for resistance is the most economical and environmentally acceptable means to control stripe rust. Genetic studies on resistance sources are very important. Previous inheritance studies on Triticum aestivum subsp. spelta cv. album and wheat cultivar Lee showed that each possessed a single dominant gene for stripe rust resistance, i.e., Yr5 and Yr7, respectively. Both were located on the long arm of chromosome 2B, but due to the complexities caused by genetic background effects there was no clear evidence on the allelism or linkage status of these genes. Our study, involving an intercross of Avocet S near-isogenic lines possessing the genes, provided clear evidence for allelism or extremely close linkage of Yr5 and Yr7 based on phenotypic and molecular studies.