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Yinghui Li 
Zhen-Zhen Wei
Hanan Sela
Liubov Govta
Valentyna Klymiuk
Rajib Roychowdhury
Harmeet Singh Chawla
Jennifer Ens
Krystalee Wiebe
Valeria Bocharova
Roi Ben-David
Prerna B Pawar
Yuqi Zhang
Samidha Jaiwar
István Molnár
Jaroslav Doležel
Gitta Coaker
Curtis J Pozniak
Tzion Fahima 

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 was not effective due 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 RNASeq reads of susceptible mutants to ONT contigs and was validated by virus-induced gene silencing. Pm69 is likely a newly evolved NLR, which was discovered only in one location across the WEW 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.

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

Yinghui Li 
Zhen-Zhen Wei
Hanan Sela
Liubov Govta
Valentyna Klymiuk
Rajib Roychowdhury
Harmeet Singh Chawla
Jennifer Ens
Krystalee Wiebe
Valeria Bocharova
Roi Ben-David
Prerna B Pawar
Yuqi Zhang
Samidha Jaiwar
István Molnár
Jaroslav Doležel
Gitta Coaker
Curtis J Pozniak
Tzion Fahima 

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 the cloning of the powdery mildew (Pm) resistance gene (R-gene) Pm69 derived from tetraploid wild emmer wheat (WEW). A conventional positional cloning approach was not effective due 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 RNASeq reads of susceptible mutants to ONT contigs and was validated by virus-induced gene silencing. Pm69 is likely a newly evolved NLR, which was discovered only in one location across the WEW 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.

Scientific Publication
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