Long-read genome sequencing accelerated the cloning of Pm69 by resolving the complexity of a rapidly evolving resistance gene cluster in wheat

Li, Y.; Wei, Z.-Z.; Sela, H.; Govta, L.; Klymiuk, V.; Roychowdhury, R.; Chawla, H. S.; Ens, J.; Wiebe, K.; Bocharova, V.; Ben-David, R.; Pawar, P. B.; Jaiwar, S.; Molnar, I.; Dolezel, J.; Pozniak, C. J.; Fahima, T.

doi:10.1101/2022.10.14.512294

bioRxiv · 10.1101/2022.10.14.512294

Long-read genome sequencing accelerated the cloning of Pm69 by resolving the complexity of a rapidly evolving resistance gene cluster in wheat

Li, Y.·Wei, Z.-Z.·Sela, H.·Govta, L.·Klymiuk, V.·Roychowdhury, R.·Chawla, H. S.·Ens, J.·Wiebe, K.·Bocharova, V.·Ben-David, R.·Pawar, P. B.·Jaiwar, S.·Molnar, I.·Dolezel, J.·Pozniak, C. J.·Fahima, T.

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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.

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Li, Y., Wei, Z.-Z., Sela, H., Govta, L., Klymiuk, V., Roychowdhury, R., Chawla, H. S., Ens, J., Wiebe, K., Bocharova, V., Ben-David, R., Pawar, P. B., Jaiwar, S., Molnar, I., Dolezel, J., Pozniak, C. J., Fahima, T.. 2022-10-16. Long-read genome sequencing accelerated the cloning of Pm69 by resolving the complexity of a rapidly evolving resistance gene cluster in wheat. https://doi.org/10.1101/2022.10.14.512294

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