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

Nature Biotechnology volume 34, pages 652–655 (2016)Cite this article

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Abstract

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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Acknowledgements

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.

Author information

Author notes

  1. Burkhard Steuernagel and Sambasivam K Periyannan: These authors contributed equally to this work.

Authors and Affiliations

  1. The Sainsbury Laboratory, Norwich, UK

    Burkhard Steuernagel, Inmaculada Hernández-Pinzón, Kamil Witek, Jonathan D G Jones & Brande B H Wulff

  2. John Innes Centre, Norwich, UK

    Burkhard Steuernagel, Guotai Yu, Asyraf Hatta & Brande B H Wulff

  3. Commonwealth Scientific and Industrial Research Organization (CSIRO), Agriculture Flagship, Canberra, NSW, Australia

    Sambasivam K Periyannan, Mick Ayliffe & Evans S Lagudah

  4. USDA-ARS Cereal Disease Laboratory and Department of Plant Pathology, University of Minnesota, St. Paul, Minnesota, USA

    Matthew N Rouse

  5. Department of Agriculture Technology, Universiti Putra Malaysia, Serdang, Malaysia

    Asyraf Hatta

  6. University of Sydney, Plant Breeding Institute, Cobbitty, NSW, Australia

    Harbans Bariana

Authors
  1. Burkhard Steuernagel
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Contributions

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.

Corresponding authors

Correspondence to Evans S Lagudah or Brande B H Wulff.

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Competing interests

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). https://doi.org/10.1038/nbt.3543

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