Temperature of elongation 59°C; Enzyme AciI; Fragment sizes: T. dicoccoides H52 = 266 bp, 148 bp; T. durum cv. Langdon = 414 bp. # Full description of yr15 mutants is provided in Klymiuk et al. (2018). * The first letter indicates the wild-type nucleotide, the number indicates its position relative to the ATG start codon, and the last letter shows the mutant nucleotide. The complete WTK1 coding regions of the above 19 mutants were sequenced; no additional mutations were detected. † The first letter indicates the wild-type amino acid, the number indicates its position relative to the start methionine, and the last letter shows the mutant amino acid.

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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 showed here that these three resistance loci co-segregate in fine-mapping populations and share identical full-length genomic sequence of functional Wtk1. Independent 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.HighlightWe demonstrate that Yr15, YrG303 and YrH52 resistances are encoded by the Wtk1 locus, but express variable resistance responses to yellow rust in a genetic background dependent manner.

Gene cloning in repeat-rich polyploid genomes remains challenging. Here we describe a strategy for overcoming major bottlenecks in the cloning of the powdery mildew (Pm) resistance gene (R-gene)Pm69derived from tetraploid wild emmer wheat (WEW). A conventional positional cloning approach encountered suppressed recombination due to structural variations, while chromosome sorting yielded an insufficient purity level. APm69physical map, constructed by assembling ONT long-read genome sequences, revealed a rapidly evolving nucleotide-binding leucine-rich repeat (NLR) R-gene cluster. A single candidate NLR was identified within this cluster by anchoring RNASeq reads of susceptible mutants to ONT contigs and was validated by the virus-induced gene silencing (VIGS) approach.Pm69, comprising Rx_N with RanGAP interaction sites, NB-ARC, and LRR domains, is probably a newly evolved NLR discovered only in one location across the WEW distribution range in the Fertile Crescent.Pm69was successfully introgressed into durum and bread wheat, and a diagnostic molecular marker could be used to accelerate its deployment and pyramiding with other resistance genes.