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Evolution of rust pathogens continues to pose challenges to global wheat production. Major resistance (R) genes, which encode proteins of the NBS-LRR (Nucleotide-binding site, leucine-rich repeat) family, have been a valuable resource for breeders to minimise yield losses from infection. Many wheat varieties harbor numerous R genes that could be identified and cloned in order to engineer more sustainable disease control. The advent of targeted gene enrichment and next-generation sequencing (NGS) has allowed rapid cloning of specific R genes, thus enhancing efforts to pyramid these genes and investigate their underlying resistance mechanisms. Several R genes present different phenotypes in certain genetic backgrounds, and cloning them would be an important step towards uncovering their interactions. Hybrid necrosis is one such phenotype observed in crosses of wheat genotypes involving the R gene Lr13 and complementary genes, Ne1 and Ne2, occurring in different allelic forms. It was recently concluded that Lr13 and an allele of Ne2 are actually the same gene based on genetic and mutational studies. The capability of Lr13 to confer both leaf rust resistance and hybrid necrosis cannot be answered without first cloning it. The lack of tightly linked markers coupled with the proximal 2BS chromosomal location of Lr13 does not make it easily amenable to map-based cloning. The NGS-based pipeline MutRenSeq (mutagenesis and R-gene enrichment sequencing) was used on EMS (Ethyl methanesulfonate) induced, susceptible Lr13 mutants along with support from comparative genomics to ascertain candidate gene sequences for Lr13, which are at advanced stages of screening and confirmation. Definite proof that a single gene is involved will only come with transformation studies when the cloned Lr13 candidate transformed into a susceptible line confers both a resistance phenotype in the transgenic line and a necrotic phenotype in the offspring of crosses between the transgenic line and a line possessing Ne1.
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).