Characterisation of stem rust resistance genes effective against race Ug99 on wheat chromosome 7l

Abstract

Wheat is one the most important food crops in the world, grown on more acreage than any other. Puccinia graminis f. sp. tritici, the causal agent of wheat and barley stem rust, is responsible for major production losses around the world. The development of resistant cultivars is an effective way to manage the disease, but outbreaks can occur when new pathogen races overcome the existing resistances. This happened with Ug99 (TTKSK), a highly virulent race now spreading in Africa and the Middle East and dangerously threatening worldwide food security. Thus, constant efforts to find new sources of resistance must remain a priority. The hexaploid wheat cultivar Canthatch has long been known for carrying a suppressor of stem rust resistance on chromosome 7DL. This suppressor is now of particular interest, as the genes it inhibits were found to be strongly effective against Ug99. Because the 7DL suppressor seems to be common in wheat cultivars, cloning it would be the first step toward unlocking the masked resistance genes, as well as providing new information about the nature of action of a previously unstudied class of negative regulators of plant defence responses. This study first aimed to genetically map the 7DL suppressor. A F2:3 mapping population was developed from the cross between the susceptible line Columbus, thought to possess the 7DL suppressor, with the resistant line Columbus-NS, a near-isogenic line in which a mutated allele of the suppressor was inferred to have been transferred into the Columbus genetic background. The mapping population was phenotyped using two stem rust races, Ug99 and #313, for which results were similar, indicating a common resistance against both races. Molecular markers were found using two recently developed techniques: i) genotyping of nearly 9,000 putative single nucleotide polymorphisms (SNP) using the wheat 9k SNP chip and ii) genotyping by sequencing using the restriction-site associated DNA sequencing (RAD-Seq) method in association with bulked segregant analysis. This led to the rapid identification of thousands of markers putatively linked to the stem rust resistance in Columbus-NS. Surprisingly, genetic mapping revealed the resistance to be located on 7AL, not 7DL, indicating that it was due to a new resistance locus rather than a non-suppressor. This locus is most likely part of the resistance complex repressed by the 7DL suppressor. Although segregation ratios in the F2:3 population indicated monogenic segregation (1:2:1), phenotypic observations and genomic studies indicated the possible involvement of multiple genes in the resistance, notably on chromosome 6A. The 7AL locus seemed to be essential but not sufficient for full resistance. The resistance response was also investigated at a microscopic level, which showed the involvement of cell death during the rust penetration through the stoma, at two days post-inoculation. Transcriptome analysis using RNA-Seq revealed 353 genes differentially expressed between Columbus and two independent Columbus-NS lines. Notably, several genes coding for cysteine-rich receptor-like kinases were found to be good candidates for involvement in the defence response.