Abstract 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) Pm69 derived 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. A Pm69 physical 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. Pm69 was 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.

Long-read genome sequencing accelerated the cloning ofPm69by resolving the complexity of a rapidly evolving resistance gene cluster in wheat