cloning

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.