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Rapid cloning of disease-resistance genes in plants using mutagenesis and sequence capture


Wild relatives of domesticated crop species harbor multiple, diverse, disease resistance (R) genes that could be used to engineer sustainable disease control. However, breeding R genes into crop lines often requires long breeding timelines of 5–15 years to break linkage between R genes and deleterious alleles (linkage drag). Further, when R genes are bred one at a time into crop lines, the protection that they confer is often overcome within a few seasons by pathogen evolution1. If several cloned R genes were available, it would be possible to pyramid R genes2 in a crop, which might provide more durable resistance1. We describe a three-step method (MutRenSeq)-that combines chemical mutagenesis with exome capture and sequencing for rapid R gene cloning. We applied MutRenSeq to clone stem rust resistance genes Sr22 and Sr45 from hexaploid bread wheat. MutRenSeq can be applied to other commercially relevant crops and their relatives, including, for example, pea, bean, barley, oat, rye, rice and maize.

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Figure 1: Mutational genomics strategy for resistance gene cloning.
Figure 2: Cloning of Sr22 and Sr45 by sequencing EMS-induced susceptible mutants.
Figure 3: Stem rust infection phenotypes of Fielder and transgenic wheat lines carrying the Sr22 gene derived from Schomburgk.

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This research was supported by funds from the Gatsby Charitable Foundation, UK; Two Blades Foundation, USA; Biotechnology and Biological Sciences Research Council, UK; Borlaug Global Rust Initiative (BGRI) Durable Rust Resistance in Wheat (DRRW) Project (administered by Cornell University with a grant from the Bill & Melinda Gates Foundation and the UK Department for International Development); USDA-ARS National Plant Disease Recovery System; Grains Research and Development Corporation, Australia; and a fellowship to A.H. from Universiti Putra Malaysia (UPM), Malaysia. We are grateful to colleagues in The Sainsbury Laboratory and the Two Blades Foundation for helpful discussions. This research was supported in part by the NBI Computing infrastructure for Science (CiS) group and Dan MacLean's group by providing computational infrastructure.

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B.S., S.K.P., I.H.-P., K.W., M.N.R., G.Y., A.H., M.A., and H.B. performed experiments. B.S., S.K.P., E.S.L. and B.B.H.W. wrote the manuscript. B.S., S.K.P., K.W., J.D.G.J., E.S.L., and B.B.H.W. contributed to the design of the study.

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Correspondence to Evans S Lagudah or Brande B H Wulff.

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B.B.H.W., B.S., E.S.L., J.D.G.J., K.W., and S.K.P. have filed two patent applications based on this work (US patent application nos. 20150240233 and 62/200,894).

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Steuernagel, B., Periyannan, S., Hernández-Pinzón, I. et al. Rapid cloning of disease-resistance genes in plants using mutagenesis and sequence capture. Nat Biotechnol 34, 652–655 (2016).

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