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The identification of R-genes using traditional map-based approaches is a long, laborious process, not to mention the time required for subsequent development of cultivars incorporating the new resistances. Breeders seek to reduce the length of breeding cycles, and researchers require new tools to accelerate discovery and understanding of mechanisms associated with durable resistance, especially adult plant resistance (APR). A new method for rapid generation advancement, known as ‘speed breeding’, significantly reduces the length of breeding cycles, provide increased recombination during line development and enable selection in early generations. The speed breeding protocol uses controlled temperature regimes and 24h light to accelerate plant growth and development. Phenotyping methods adapted for use in the speed breeding system permit year-round evaluation of APR to rust pathogens within 5 weeks from time of sowing. RNA sequencing (RNA-Seq) technology has revolutionized gene expression profiling in plants. We previously used RNAseq to identify novel transcripts and miRNAs associated with seedling resistance (Lr28) leading to identification of transcription factors and miRNA families (e.g. miR36, miR37 and miR39) involved in signalling and defense response (Kumar et al. J. Nuc. Acids 2014:570176). In this study we report the application of speed breeding and RNAseq technologies for the purpose of rapidly identifying transcripts and miRNA associated with APR. Wheat landraces harbouring novel sources of resistance were grown under speed breeding conditions and sampled for RNA at key growth stages, before and after inoculation, which enabled discovery of differentially expressed miRNAs. Our next steps are aimed at validating these genetic factors associated with APR in order to better understand the signalling pathways and deliver tools to assist the assembly of robust wheat cultivars for the future.
Wheat landrace PI 177906 has seedling and field resistance to Pgt races TTKSK and TTKST. From a cross between PI 177906 and LMPG-6, 138 doubled haploid (DH) lines and 144 recombinant inbred lines (RILs) were developed and tested for seedling resistance to Pgt race TTKSK. Goodness-of-fit tests from both populations indicated that two dominant genes in PI 177906 conditioned resistance to race TTKSK. Parents and the 138 DH lines were evaluated in the field in two experiments in Kenya; one in the main season and one in the off-season. The 90K wheat iSelect SNP genotyping platform was used to genotype the parents and DH lines and data were used to construct a genetic linkage map. Two loci for seedling resistance were mapped to chromosomes 2BL and 4BL. Two major QTL for field resistance mapped to the same regions, a 14.4 cM interval on 2BL and an 8.5 cM interval on 4BL. The QTL on 2BL and 4BL explained, respectively, 31.9-32.3% and 18.2-19.1% of the variation in the off-season and 28.3-30.4% and 5.4-6.5% of the variation in the main-season. Based on the mapping results, race specificity, and the seedling infection types, the resistance gene in 2BL could be Sr28, whereas the gene on chromosome 4BL could be novel. The mapping results will be verified in the RIL population using the flanking SNP markers in KASP assays.
High quality molecular markers that are closely linked, codominant, and high throughput are critical for developing varieties with durable rust resistance. We are using a combination of microsatellite, sequence tagged site, and Diversity Array Technology markers for haplotyping, pyramiding, and mapping stem rust resistance genes. The primary goal of our research team is to identify and optimize markers for previously characterized and novel stem rust resistance genes in wheat. The specific objectives are to: 1) optimize markers for previously characterized stem rust resistance genes to maximize efficiency of the breeding programs, 2) haplotype uncharacterized rust resistant genotypes to infer novelty and to plan new mapping experiments, 3) pyramid novel sources of rust resistance, and 4) map novel sources of rust resistance, including adult plant resistance. To date, we have evaluated 58 markers associated with 21 stem rust resistance genes and used 20 for haplotyping 318 wheat lines and varieties for 15 Ug99 effective resistance genes. This germplasm panel is also being DArT genotyped. For tetraploids, the pyramiding includes Sr2, Sr13 and Sr25 in the breeding line UC1113 which is a high yielding semi-dwarf durum variety with the high-grain protein content gene Gpc-B1 and the non-race specific stripe rust resistance gene Yr36. The Australian group is developing markers for the stem rust resistance genes Sr33 and Sr45 that come from Aegilops tauschii and are located on wheat chromosomes 1DS. Diagnostic, codominant markers for Sr25 and Sr26 have been developed and are being pyramided into CIMMYT breeding lines. Three new sources of race-specific resistance in CIMMYT-derived spring wheat have been mapped and are designated SrA, SrB, and SrC. SrA mapped on 3DL, SrB on 3BS and SrC on 5DL. These genes provided moderate levels of resistance to stem rust at the seedling stage and acceptable to moderate levels at the adult plant stage.
Rust fungi can cause devastating diseases in agriculture and are particularly important pathogens of wheat. We have been using the flax (Linum usitatissimum) and flax rust (Melampsora lini) model system to study disease resistance mechanisms to this important class of pathogens. Rust resistance in flax and other plants is mediated by the plant innate immunity system in which highly polymorphic resistance (R) proteins act as receptors that recognize specific avirulence (Avr) proteins produced by the pathogen. This race-specific resistance is characterised by Flor’s “gene-for-gene” model, first proposed based on the flax rust system. In gene-for-gene resistance, recognition between the R and Avr proteins initiates defense responses leading to host resistance to infection, including a localised necrosis or hypersensitive response. Nineteen different rust resistance genes have been cloned from flax, including 11 allelic variants of the L locus, which all encode cytosolic proteins with conserved nucleotide-binding (NB) and Leucine-rich repeat (LRR) domains. Four families of Avr genes, AvrL567, AvrM, AvrP123 and AvrP4, have been identified in the flax rust pathogen and all encode small secreted proteins. Rust Avr proteins are secreted from haustoria, specialized infection structures that penetrate the host cell wall, and are translocated across the host plasma membrane and into the host cytoplasm. These proteins are probably members of a suite of disease ‘effectors’ involved in manipulating host cell biology to facilitate infection, but have become targeted for recognition by the host immune system. As yet the mechanism of Avr protein transport is unknown, but could prove to be a useful target for novel disease control strategies. Recognition of at least two of these Avr proteins is based on direct interaction with the cytoplasmic NB-LRR R proteins. One interesting observation from the flax rust system is that all of the virulent rust strains retain intact copies of the Avr genes, but have altered their sequences sufficiently to escape recognition. Thus it may be possible to re-engineer R genes to recognise new Avr gene variants. We are currently identifying haustorially expressed secreted proteins from wheat stem rust as candidate Avr/effector proteins.