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.

Crop Development Centre and Department of Plant Sciences University of Saskatchewan 51 Campus Drive Saskatoon SK S7N 5A8 Canada

Department of Crop Genomics and Bioinformatics China Agricultural University Beijing 100094 China

Institute of Evolution University of Haifa Mt Carmel Haifa 3498838 Israel; The Department of Evolutionary and Environmental Biology University of Haifa Mt Carmel Haifa 3498838 Israel

Institute of Evolution University of Haifa Mt Carmel Haifa 3498838 Israel; The Department of Evolutionary and Environmental Biology University of Haifa Mt Carmel Haifa 3498838 Israel; Crop Development Centre and Department of Plant Sciences University of Saskatchewan 51 Campus Drive Saskatoon SK S7N 5A8 Canada

Institute of Evolution University of Haifa Mt Carmel Haifa 3498838 Israel; The Department of Evolutionary and Environmental Biology University of Haifa Mt Carmel Haifa 3498838 Israel; Department of Vegetables and Field Crops Institute of Plant Sciences Agricultural Research Organization Volcani Center Rishon Lezion 7505101 Israel

Institute of Evolution University of Haifa Mt Carmel Haifa 3498838 Israel; The Department of Evolutionary and Environmental Biology University of Haifa Mt Carmel Haifa 3498838 Israel; The Institute of Plant Protection Sichuan Academy of Agricultural Sciences Chengdu 610066 China

Institute of Experimental Botany of the Czech Academy of Sciences Centre of the Region Haná for Biotechnological and Agricultural Research Šlechtitelů 31 779 00 Olomouc Czech Republic

Plant Pathology Department University of California Davis Davis CA 95616 USA

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