Rapid Cloning of Resistance Genes in Wheat

To defend against disease, plants have a repertoire of resistance (R) proteins that can detect the presence of pathogen effector proteins. Genes encoding these proteins, which typically have a nucleotide binding (NB) site domain and leucine-rich repeats (LRR), are interesting targets for recruiting new resistance from wild relatives into crops. The traditional way to identify genes is by map-based cloning, which is limited by recombination in the target area. As a complementary approach, we have developed a method based on mutational genomics and R gene enrichment sequencing (RenSeq). An accession with a confirmed single resistance gene is subjected to an EMS mutant screen. Genomic DNA from mutants that show loss of resistance as well as from the wild-type is subsequently enriched for NB-LRR type genes using a custom biotinylated oligonucleotide bait library followed by identification of candidate genes through bioinformatics pipeline. As a proof of concept, we re-cloned the previously cloned gene Sr33 from hexaploid wheat. From sequencing of six EMS mutants we unambiguously found a single candidate, which is identical to the real gene. We further cloned two stem rust resistance genes, Sr22 and Sr45, that confer resistance to several stem rust races including Ug99. In both cases, we identified a single, unambiguous candidate from six susceptible mutants, and for Sr22, we verified Sr22 function in transgenic wheat plants. Our method of using EMS mutants combined with RenSeq can efficiently clone NB-LRR type resistance genes. Due to the EMS based approach it relies on the presence of a single R gene in order to obtain susceptible mutants and it is limited to the gene space targeted by the bait library. The method, however, neither relies on positional fine mapping nor does it require a BAC library and therefore provides an excellent complementary approach to map-based cloning.