Letter | Published:

Rapid cloning of genes in hexaploid wheat using cultivar-specific long-range chromosome assembly

Nature Biotechnology volume 35, pages 793796 (2017) | Download Citation

Abstract

Cereal crops such as wheat and maize have large repeat-rich genomes that make cloning of individual genes challenging. Moreover, gene order and gene sequences often differ substantially between cultivars of the same crop species1,2,3,4. A major bottleneck for gene cloning in cereals is the generation of high-quality sequence information from a cultivar of interest. In order to accelerate gene cloning from any cropping line, we report 'targeted chromosome-based cloning via long-range assembly' (TACCA). TACCA combines lossless genome-complexity reduction via chromosome flow sorting with Chicago long-range linkage5 to assemble complex genomes. We applied TACCA to produce a high-quality (N50 of 9.76 Mb) de novo chromosome assembly of the wheat line CH Campala Lr22a in only 4 months. Using this assembly we cloned the broad-spectrum Lr22a leaf-rust resistance gene, using molecular marker information and ethyl methanesulfonate (EMS) mutants, and found that Lr22a encodes an intracellular immune receptor homologous to the Arabidopsis thaliana RPM1 protein.

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Acknowledgements

We are grateful to the staff at Dovetail Genomics for constructing the CH Campala Lr22a scaffolds. We thank M. Karafiátová for supervising chromosome 2D flow sorting and estimation of purity in flow sorted fractions, and Z. Dubská, R. Šperková and J. Weiserová for technical assistance. We also thank B. Senger and L. Luthi for assistance with field experiments and B. Keller for continuous support. This work was financed by an Ambizione fellowship of the Swiss National Science Foundation. J.V., H.Š., and J.D. were supported by the Ministry of Education, Youth and Sports of the Czech Republic (grant award LO1204 from the National Program of Sustainability I).

Author information

Author notes

    • Anupriya Kaur Thind
    •  & Thomas Wicker

    These authors contributed equally to this work.

Affiliations

  1. Department of Plant and Microbial Biology, University of Zurich, Zurich, Switzerland.

    • Anupriya Kaur Thind
    • , Thomas Wicker
    •  & Simon G Krattinger
  2. Institute of Experimental Botany, Centre of the Region Haná for Biotechnological and Agricultural Research, Olomouc, Czech Republic.

    • Hana Šimková
    • , Jan Vrána
    •  & Jaroslav Doležel
  3. Institute for Plant Production Sciences, Agroscope, Switzerland.

    • Dario Fossati
    • , Odile Moullet
    •  & Cécile Brabant

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Contributions

A.K.T., T.W., H.Š., J.D. and S.G.K. designed the experiments and wrote the manuscript, A.K.T., and S.G.K. performed phenotypic and molecular analyses, H.Š., J.V., and J.D. flow-sorted chromosome 2D and prepared high molecular weight (HMW) DNA, O.M., C.B., and D.F. developed the CH Campala Lr22a backcross line.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Simon G Krattinger.

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DOI

https://doi.org/10.1038/nbt.3877

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