TILLING and Ecotilling for Rice

  • Jennifer L. Cooper
  • Steven Henikoff
  • Luca Comai
  • Bradley J. TillEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 956)


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.

Key words

Enzymatic mismatch cleavage Mutation discovery Reverse genetics Nucleotide polymorphism Celery juice extract 



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


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Copyright information

© Springer Science+Business Media, LLC 2013

Authors and Affiliations

  • Jennifer L. Cooper
    • 1
  • Steven Henikoff
    • 2
  • Luca Comai
    • 3
  • Bradley J. Till
    • 4
    Email author
  1. 1.Department of ChemistryUniversity of AkronAkronUSA
  2. 2.HHMI and Basic Sciences DivisionFred Hutchinson Cancer Research CenterSeattleUSA
  3. 3.UC Davis Genome CenterUniversity of CaliforniaDavisUSA
  4. 4.Plant Breeding Unit, FAO/IAEA Agricultural & Biotechnology LaboratoryInternational Atomic Energy AgencyViennaAustria

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