Advertisement

TILLING and Ecotilling for Rice

  • Jennifer L. Cooper
  • Steven Henikoff
  • Luca Comai
  • Bradley J. TillEmail author
Protocol
Part of the Methods in Molecular Biology book series (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.

Key words

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

Notes

Acknowledgments

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

References

  1. 1.
    Ahloowalia BS, Maluszynski M, Nichterlein K (2004) Global impact of mutation-derived varieties. Euphytica 135:187–204CrossRefGoogle Scholar
  2. 2.
    Parry MAJ, Madgwick PJ, Bayon C, Tearall K, Hernandez-Lopez A, Baudo M, Rakszegi M, Hamada W, Al-Yassin A, Ouabbou H, Labhilili M, Phillips AL (2009) Mutation discovery for crop improvement. J Exp Bot 60:2817–2825PubMedCrossRefGoogle Scholar
  3. 3.
    McCallum CM, Comai L, Greene EA, Henikoff S (2000) Targeted screening for induced mutations. Nat Biotechnol 18:455–457PubMedCrossRefGoogle Scholar
  4. 4.
    Colbert T, Till BJ, Tompa R, Reynolds S, Steine MN, Yeung AT, McCallum CM, Comai L, Henikoff S (2001) High-throughput screening for induced point mutations. Plant Physiol 126:480–484PubMedCrossRefGoogle Scholar
  5. 5.
    Comai L, Young K, Till BJ, Reynolds SH, Greene EA, Codomo CA, Enns LC, Johnson JE, Burtner C, Odden AR, Henikoff S (2004) Efficient discovery of DNA polymorphisms in natural populations by Ecotilling. Plant J 37:778–786PubMedCrossRefGoogle Scholar
  6. 6.
    Till BJ, Afza R, Bado S, Huynh OA, Jankowicz-Cieslak J, Matijevic M, Mba C (2009) In: Shu QY (ed) Induced plant mutations in the genomics era. Food and Agriculture Organization of the United Nations, Rome, pp 237–239Google Scholar
  7. 7.
    Till BJ, Cooper J, Tai TH, Colowit P, Greene EA, Henikoff S, Comai L (2007) Discovery of chemically induced mutations in rice by TILLING. BMC Plant Biol 7:19PubMedCrossRefGoogle Scholar
  8. 8.
    Suzuki T, Eiguchi M, Kumamaru T, Satoh H, Matsusaka H, Moriguchi K, Nagato Y, Kurata N (2008) MNU-induced mutant pools and high performance TILLING enable finding of any gene mutation in rice. Mol Genet Genomics 279:213–223PubMedCrossRefGoogle Scholar
  9. 9.
    Sato Y, Shirasawa K, Takahashi Y, Nishimura M, Nishio T (2006) Mutant selection from progeny of gamma-ray-irradiated rice by DNA heteroduplex cleavage using Brassica petiole extract. Breeding Science 56:179–183CrossRefGoogle Scholar
  10. 10.
    Bruce M, Hess A, Bai J, Mauleon R, Diaz MG, Sugiyama N, Bordeos A, Wang GL, Leung H, Leach JE (2009) Detection of genomic deletions in rice using oligonucleotide microarrays. BMC Genomics 10:129PubMedCrossRefGoogle Scholar
  11. 11.
    Rios G, Naranjo MA, Iglesias DJ, Ruiz-Rivero O, Geraud M, Usach A, Talon M (2008) Characterization of hemizygous deletions in citrus using array-comparative genomic hybridization and microsynteny comparisons with the poplar genome. BMC Genomics 9:381PubMedCrossRefGoogle Scholar
  12. 12.
    Li X, Lassner M, Zhang YL (2002) Deleteagene: a fast neutron deletion mutagenesis-based gene knockout system for plants. Comp Funct Genomics 3:158–160PubMedCrossRefGoogle Scholar
  13. 13.
    Rogers C, Wen J, Chen R, Oldroyd G (2009) Deletion based reverse genetics in Medicago truncatula. Plant Physiol.Google Scholar
  14. 14.
    Till BJ, Zerr T, Comai L, Henikoff S (2006) A protocol for TILLING and Ecotilling in plants and animals. Nat Protoc 1:2465–2477PubMedCrossRefGoogle Scholar
  15. 15.
    Till BJ, Colbert T, Codomo C, Enns L, Johnson J, Reynolds SH, Henikoff JG, Greene EA, Steine MN, Comai L, Henikoff S (2006) High-throughput TILLING for Arabidopsis. Methods Mol Biol 323:127–135PubMedGoogle Scholar
  16. 16.
    Till BJ, Zerr T, Bowers E, Greene EA, Comai L, Henikoff S (2006) High-throughput discovery of rare human nucleotide polymorphisms by Ecotilling. Nucleic Acids Res 34:e99PubMedCrossRefGoogle Scholar
  17. 17.
    Gilchrist EJ, Haughn GW, Ying CC, Otto SP, Zhuang J, Cheung D, Hamberger B, Aboutorabi F, Kalynyak T, Johnson L, Bohlmann J, Ellis BE, Douglas CJ, Cronk QC (2006) Use of Ecotilling as an efficient SNP discovery tool to survey genetic variation in wild populations of Populus trichocarpa. Mol Ecol 15:1367–1378PubMedCrossRefGoogle Scholar
  18. 18.
    Nieto C, Piron F, Dalmais M, Marco CF, Moriones E, Gomez-Guillamon ML, Truniger V, Gomez P, Garcia-Mas J, Aranda MA, Bendahmane A (2007) EcoTILLING for the identification of allelic variants of melon eIF4E, a factor that controls virus susceptibility. BMC Plant Biol 7:34PubMedCrossRefGoogle Scholar
  19. 19.
    Weil CF (2009) TILLING in grass species. Plant Physiol 149:158–164PubMedCrossRefGoogle Scholar
  20. 20.
    Raghavan C, Naredo MEB, Wang HH, Atienza G, Liu B, Qiu FL, McNally KL, Leung H (2007) Rapid method for detecting SNPs on agarose gels and its application in candidate gene mapping. Mol Breeding 19:87–101CrossRefGoogle Scholar
  21. 21.
    Till BJ, Burtner C, Comai L, Henikoff S (2004) Mismatch cleavage by single-strand specific nucleases. Nucleic Acids Res 32:2632–2641PubMedCrossRefGoogle Scholar
  22. 22.
    Wienholds E, van Eeden F, Kosters M, Mudde J, Plasterk RH, Cuppen E (2003) Efficient target-selected mutagenesis in zebrafish. Genome Res 13:2700–2707PubMedCrossRefGoogle Scholar
  23. 23.
    Winkler S, Schwabedissen A, Backasch D, Bokel C, Seidel C, Bonisch S, Furthauer M, Kuhrs A, Cobreros L, Brand M, Gonzalez-Gaitan M (2005) Target-selected mutant screen by TILLING in Drosophila. Genome Res 15:718–723PubMedCrossRefGoogle Scholar
  24. 24.
    CODDLe and PARSESNP Input Form. (Accessed September 16, 2009, at http://www.proweb.org/input/)Google Scholar
  25. 25.
    Till BJ, Reynolds SH, Greene EA, Codomo CA, Enns LC, Johnson JE, Burtner C, Odden AR, Young K, Taylor NE, Henikoff JG, Comai L, Henikoff S (2003) Large-scale discovery of induced point mutations with high-throughput TILLING. Genome Res 13:524–530PubMedCrossRefGoogle Scholar
  26. 26.
    Cooper JL, Greene EA, Till BJ, Codomo CA, Wakimoto BT, Henikoff S (2008) Retention of induced mutations in a Drosophila reverse-genetic resource. Genetics 180:661–667PubMedCrossRefGoogle Scholar
  27. 27.
    Triques K, Piednoir E, Dalmais M, Schmidt J, Le Signor C, Sharkey M, Caboche M, Sturbois B, Bendahmane A (2008) Mutation detection using ENDO1: application to disease diagnostics in humans and TILLING and Eco-TILLING in plants. BMC Mol Biol 9:42PubMedCrossRefGoogle Scholar
  28. 28.
    Elias R, Till BJ, Mba C, Al-Safadi B (2009) Optimizing TILLING and Ecotilling techniques for potato (Solanum tuberosum L). BMC Res Notes 2:141PubMedCrossRefGoogle Scholar
  29. 29.
    Till BJ, Reynolds SH, Weil C, Springer N, Burtner C, Young K, Bowers E, Codomo CA, Enns LC, Odden AR, Greene EA, Comai L, Henikoff S (2004) Discovery of induced point mutations in maize genes by TILLING. BMC Plant Biol 4:12PubMedCrossRefGoogle Scholar
  30. 30.
    Cooper JL, Till BJ, Laport RG, Darlow MC, Kleffner JM, Jamai A, El-Mellouki T, Liu S, Ritchie R, Nielsen N, Bilyeu KD, Meksem K, Comai L, Henikoff S (2008) TILLING to detect induced mutations in soybean. BMC Plant Biol 8:9PubMedCrossRefGoogle Scholar
  31. 31.
    Greene EA, Codomo CA, Taylor NE, Henikoff JG, Till BJ, Reynolds SH, Enns LC, Burtner C, Johnson JE, Odden AR, Comai L, Henikoff S (2003) Spectrum of chemically induced mutations from a large-scale reverse-genetic screen in Arabidopsis. Genetics 164:731–740PubMedGoogle Scholar
  32. 32.
    Draper BW, McCallum CM, Stout JL, Slade AJ, Moens CB (2004) A high-throughput method for identifying N-ethyl-N-nitrosourea (ENU)-induced point mutations in zebrafish. Methods Cell Biol 77:91–112PubMedCrossRefGoogle Scholar
  33. 33.
    Till BJ, Colbert T, Tompa R, Enns LC, Codomo CA, Johnson JE, Reynolds SH, Henikoff JG, Greene EA, Steine MN, Comai L, Henikoff S (2003) High-throughput TILLING for functional genomics. Methods Mol Biol 236:205–220PubMedGoogle Scholar

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

Personalised recommendations