Targeting stem rust resistance genes Sr32 and Sr1644 for cloning by mutagenesis and sequence capture

Resistance offers the best means of control of the cereal rusts, but must be strategically deployed so as to avoid exposure of single major genes, which have faltered so many times in the past. The pyramiding of multiple effective resistance genes is a strategy that has proven effective in a number of wheat production areas around the world. However, the process of incorporating multiple resistance genes into a single cultivar using standard breeding techniques is time consuming, laborious, and hampered by the problem of linkage drag. If a suite of effective resistance genes could be efficiently cloned and transferred into wheat as a cassette, it would accelerate the development of durably resistant varieties without the problem of linkage drag. Toward this end, we have developed a resistance gene cloning technology based on resistance gene enrichment sequencing (RenSeq) of EMS-derived mutant R gene alleles. As a proof of concept test, we successfully ‘re’-cloned the already characterized gene Sr33 and are now targeting the cloning of eight other effective resistance genes. For the identification of susceptible mutants for the cloning of Sr32 from Aegilops speltoides, we screened 1,109 M₂ families with race TPMKC — as a surrogate for race TTKSK. Five susceptible M₂ mutants were confirmed by progeny testing. These mutants were also susceptible to race TTKSK. For the population involving Sr1644 from Ae. sharonensis, 1,649 M₂ families were screened, yielding 33 M₂ families that appeared to segregate for susceptibility. Thirteen of 33 families were confirmed as bona fide susceptible mutants by progeny tests in the M₃ generation. Identification of susceptible EMS mutants of Sr32 and Sr1644 suggests that the underlying resistance in these lines is conferred by single genes. We will report on progress to clone and characterize these genes using R gene exome capture and sequencing technology (RenSeq).