Dissection of a rapidly evolving wheat resistance gene cluster by long-read genome sequencing accelerated the cloning of Pm69.

Gene cloning in repeat-rich polyploid genomes remains challenging. Here, we describe a strategy for overcoming major bottlenecks in cloning of the powdery mildew resistance gene (R-gene) Pm69 derived from tetraploid wild emmer wheat. A conventional positional cloning approach was not effective owing to suppressed recombination. Chromosome sorting was compromised by insufficient purity. A Pm69 physical map, constructed by assembling Oxford Nanopore Technology (ONT) long-read genome sequences, revealed a rapidly evolving nucleotide-binding leucine-rich repeat (NLR) R-gene cluster with structural variations. A single candidate NLR was identified by anchoring RNA sequencing reads from susceptible mutants to ONT contigs and was validated by virus-induced gene silencing. Pm69 is likely a newly evolved NLR and was discovered in only one location across the wild emmer wheat distribution range in Israel. Pm69 was successfully introgressed into cultivated wheat, and a diagnostic molecular marker was used to accelerate its deployment and pyramiding with other R-genes.

A Post-Haustorial Defense Mechanism is Mediated by the Powdery Mildew Resistance Gene, PmG3M, Derived from Wild Emmer Wheat.

The destructive wheat powdery mildew disease is caused by the fungal pathogen Blumeria graminis f. sp. tritici (Bgt). PmG3M, derived from wild emmer wheat Triticum dicoccoides accession G305-3M, is a major gene providing a wide-spectrum resistance against Bgt. PmG3M was previously mapped to wheat chromosome 6B using an F6 recombinant inbred line (RIL) mapping population generated by crossing G305-3M with the susceptible T. durum wheat cultivar Langdon (LDN). In the current study, we aimed to explore the defense mechanisms conferred by PmG3M against Bgt. Histopathology of fungal development was characterized in artificially inoculated leaves of G305-3M, LDN, and homozygous RILs using fluorescence and light microscopy. G305-3M exhibited H2O2 accumulation typical of a hypersensitive response, which resulted in programmed cell death (PCD) in Bgt-penetrated epidermal cells, while LDN showed well-developed colonies without PCD. In addition, we observed a post-haustorial resistance mechanism that arrested the development of fungal feeding structures and pathogen growth in both G305-3M and resistant RIL, while LDN and a susceptible RIL displayed fully developed digitated haustoria and massive accumulation of fungal biomass. In contrast, both G305-3M and LDN exhibited callose deposition in attempt to prevent fungal invasion, supporting this as a mechanism of a basal defense response not associated with PmG3M resistance mechanism per se. The presented results shed light on the resistance mechanisms conferred by PmG3M against wheat powdery mildew.

Three previously characterized resistances to yellow rust are encoded by a single locus Wtk1.

The wild emmer wheat (Triticum turgidum ssp. dicoccoides; WEW) yellow (stripe) rust resistance genes Yr15, YrG303, and YrH52 were discovered in natural populations from different geographic locations. They all localize to chromosome 1B but were thought to be non-allelic based on differences in resistance response. We recently cloned Yr15 as a Wheat Tandem Kinase 1 (WTK1) and show here that these three resistance loci co-segregate in fine-mapping populations and share an identical full-length genomic sequence of functional Wtk1. Independent ethyl methanesulfonate (EMS)-mutagenized susceptible yrG303 and yrH52 lines carried single nucleotide mutations in Wtk1 that disrupted function. A comparison of the mutations for yr15, yrG303, and yrH52 mutants showed that while key conserved residues were intact, other conserved regions in critical kinase subdomains were frequently affected. Thus, we concluded that Yr15-, YrG303-, and YrH52-mediated resistances to yellow rust are encoded by a single locus, Wtk1. Introgression of Wtk1 into multiple genetic backgrounds resulted in variable phenotypic responses, confirming that Wtk1-mediated resistance is part of a complex immune response network. WEW natural populations subjected to natural selection and adaptation have potential to serve as a good source for evolutionary studies of different traits and multifaceted gene networks.