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TILLING and Ecotilling for Rice

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Rice Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 956))

Abstract

Mutagenesis is frequently used to test gene function and to aid in crop improvement. Targeting Induced Local Lesions in Genomes (TILLING) is a reverse genetic strategy first developed to identify induced point mutations in Arabidopsis. This general strategy has since been applied to many plant and animal species. Here, we describe a protocol for high-throughput TILLING in rice. Gene segments are amplified using fluorescently tagged primers, and products are denatured and reannealed to form heteroduplexes between the mutated and wild-type sequences. These heteroduplexes are substrates for cleavage by single-strand-specific nucleases. Following cleavage, products are analyzed on denaturing polyacrylamide gels using the LI-COR DNA analyzer system. Several rice TILLING populations have been described, and a public mutation screening service is now available. The basic methods used for TILLING can be adapted for the discovery and cataloguing of natural nucleotide variation in populations, a strategy known as Ecotilling, which was first used to study genetic diversity among Arabidopsis ecotypes, and has since been applied to crop plants.

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Acknowledgments

The methods described here were developed with support from the National Science Foundation Plant Genome Research Program.

Author information

Authors and Affiliations

  1. Department of Chemistry, University of Akron, Akron, OH, USA

    Jennifer L. Cooper

  2. HHMI and Basic Sciences Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA

    Steven Henikoff

  3. UC Davis Genome Center, University of California, Davis, CA, USA

    Luca Comai

  4. Plant Breeding Unit, FAO/IAEA Agricultural & Biotechnology Laboratory, International Atomic Energy Agency, Vienna, Austria

    Bradley J. Till

Authors
  1. Jennifer L. Cooper
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  2. Steven Henikoff
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  3. Luca Comai
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  4. Bradley J. Till
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Corresponding author

Correspondence to Bradley J. Till .

Editor information

Editors and Affiliations

  1. Huck Institutes of Life Sciences, Department of Plant Pathology, Pennsylvania State University, Life Sciences Building 405C, University Park, 16802, Pennsylvania, USA

    Yinong Yang

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Cooper, J.L., Henikoff, S., Comai, L., Till, B.J. (2013). TILLING and Ecotilling for Rice. In: Yang, Y. (eds) Rice Protocols. Methods in Molecular Biology, vol 956. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-194-3_4

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  • DOI: https://doi.org/10.1007/978-1-62703-194-3_4

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-193-6

  • Online ISBN: 978-1-62703-194-3

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