Molecular Breeding

, Volume 19, Issue 2, pp 87–101

Rapid method for detecting SNPs on agarose gels and its application in candidate gene mapping

  • Chitra Raghavan
  • Ma. Elizabeth B. Naredo
  • Hehe Wang
  • Genelou Atienza
  • Bin Liu
  • Fulin Qiu
  • Kenneth L. McNally
  • Hei Leung


TILLING (Targeting Induced Local Lesions IN Genomes) exploits the fact that CEL I endonuclease cleaves heteroduplexes at positions of single nucleotide or small indel mismatches. To detect single nucleotide polymorphisms (SNPs) across a population, DNA pools are created and a target locus under query is PCR-amplified and subjected to heteroduplex formation, followed by CEL I cleavage. Currently, the common method used to detect cleaved products is by polyacrylamide gel electrophoresis using a high-throughput genotyping platform. Exact SNPs are then determined by sequencing. We sought to simplify the detection of CEL I-cleaved products on conventional agarose gels to make the technique more accessible to collaborating partners in developing countries where access to instrumentation could be limiting. Here, we used a panel of stress-related genes to evaluate SNP detection across 48 rice genotypes by contrasting them individually against IR64 and Nipponbare. SNP detection calls corresponded perfectly with those obtained from the Li-Cor genotypers. We were able to detect SNPs in pools of eight DNA templates, suggesting that the agarose gel system could be used to screen for SNPs with comparable throughput as that of the Li-Cor genotypers and showed that the throughput can be increased by analyzing larger amplicons (∼3 kb). The agarose method offers a significant advantage by alleviating the need for labeled primers. We further demonstrated that the agarose method can be effectively used in gene mapping, an application particularly useful for parental lines with low levels of polymorphism. The lower cost and simplicity of the technique make it possible for broader applications of SNP-based markers for germplasm characterization and mapping studies.


TILLING/EcoTILLING SNP detection CEL I Agarose Mapping Rice 



Targeting induced local lesions IN genomes


Single nucleotide polymorphism


Trehalose 6-phosphate phosphatase


Actin depolymerizing factor


Recombinant inbred lines


  1. Bannwarth S, Procaccio V, Paquis-Flucklinger V (2005) Surveyortrade mark nuclease: a new strategy for a rapid identification of heteroplasmic mitochondrial DNA mutations in patients with respiratory chain defects. Hum Mutat 25:575–582PubMedCrossRefGoogle Scholar
  2. Borevitz JO, Liang D, Plouffe D, Chang HS, Zhu T, Weigel D, Berry CC, Winzeler E, Chory J (2003) Large-scale identification of single-feature polymorphisms in complex genomes. Genome Res 13:513–523PubMedCrossRefGoogle Scholar
  3. Caldwell DG, McCallum N, Shaw P, Muehlbauer GJ, Marshall DF, Waugh R (2004) A structured mutant population for forward and reverse genetics in Barley (Hordeum vulgare L.). Plant J 40:143–150PubMedCrossRefGoogle Scholar
  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. 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. European Plant Science Organization (2005) European plant science: a field of opportunities. J Exp Bot 56:1699–1709CrossRefGoogle Scholar
  7. Fulton TM, Chunwongse J, Tanksley SD (1995) Microprep protocol for extraction of DNA from tomato and other herbaceous plants. Plant Mol Biol Rep 13:207–209Google Scholar
  8. Gilchrist EJ, Haughn GW (2005) TILLING without a plough: a new method with applications for reverse genetics. Curr Opin Plant Biol 8:211–215PubMedCrossRefGoogle Scholar
  9. Greber B, Tandara H, Lehrach H, Himmelbauer H (2005) Comparison of PCR-based mutation detection methods and application for identification of mouse Sult1a1 mutant embryonic stem cell clones using pooled templates. Hum Mutat 25:483–490PubMedCrossRefGoogle Scholar
  10. Henikoff S, Comai L (2003) Single-nucleotide mutations for plant functional genomics. Annu Rev Plant Biol 54:375–401PubMedCrossRefGoogle Scholar
  11. Henikoff S, Till BJ, Comai L (2004) TILLING. Traditional mutagenesis meets functional genomics. Plant Physiol 135:630–636PubMedCrossRefGoogle Scholar
  12. International Rice Genome Sequencing Project (2005) The map-based sequence of the rice genome. Nature 436:793–800CrossRefGoogle Scholar
  13. Liu B, Zhang S, Zhu X, Yang Q, Wu S, Mei M, Mauleon R, Leach J, Mew T, Leung H (2004) Candidate defense genes as predictors of quantitative blast resistance in rice. Mol Plant Microbe Interact 17:1146–1152PubMedCrossRefGoogle Scholar
  14. McCallum CM, Comai L, Greene EA, Henikoff S (2000) Targeting induced local lesions IN genomes (TILLING) for plant functional genomics. Plant Physiol 123:439–442PubMedCrossRefGoogle Scholar
  15. Oleykowski CA, Bronson Mullins CR, Godwin AK, Yeung AT (1998) Mutation detection using a novel plant endonuclease. Nucleic Acids Res 26:4597–4602PubMedCrossRefGoogle Scholar
  16. Perry JA, Wang TL, Welham TJ, Gardner S, Pike JM, Yoshida S, Parniske M (2003) A TILLING reverse genetics tool and a web-accessible collection of mutants of the legume Lotus japonicus. Plant Physiol 131:866–871PubMedCrossRefGoogle Scholar
  17. Rafalski A (2002) Applications of single nucleotide polymorphisms in crop genetics. Curr Opin Plant Biol 5:94–100PubMedCrossRefGoogle Scholar
  18. Ruano G, Kidd KK (1992) Modeling of heteroduplex formation during PCR from mixtures of DNA templates. PCR Methods Appl 2:112–116PubMedGoogle Scholar
  19. Slade AJ, Fuerstenberg SI, Loeffler D, Steine MN, Facciotti D (2005) A reverse genetic, nontransgenic approach to wheat crop improvement by TILLING. Nat Biotechnol 23:75–81PubMedCrossRefGoogle Scholar
  20. Sokurenko EV, Tchesnokova V, Yeung AT, Oleykowski CA, Trintchina E, Hughes KT, Rashid RA, Brint JM, Moseley SL, Lory S (2001) Detection of simple mutations and polymorphisms in large genomic regions. Nucleic Acids Res 29:e111PubMedCrossRefGoogle Scholar
  21. Suzuki T, Eiguchi M, Satoh H, Kumamaru T, Kurata N (2005) A modified TILLING system for rice mutant screening. Rice Genet Newsl 22:85–87Google Scholar
  22. Syvanen AC (2001) Accessing genetic variation: genotyping single nucleotide polymorphisms. Nat Rev Genet 2:930–942PubMedCrossRefGoogle Scholar
  23. Till BJ, Burtner C, Comai L, Henikoff S (2004) Mismatch cleavage by single-strand specific nucleases. Nucleic Acids Res 32:2632–2641PubMedCrossRefGoogle Scholar
  24. 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
  25. Wright SI, Bi IV, Schroeder SG, Yamasaki M, Doebley JF, McMullen MD, Gaut BS (2005) The effects of artificial selection on the maize genome. Science 308:1310–1314PubMedCrossRefGoogle Scholar
  26. Wu J, Wu C, Lei C, Baraoidan M, Boredos A, Madamba RS, Ramos-Pamplona M, Mauleon R, Portugal A, Ulat V, Bruskiewich R, Wang GL, Leach JE, Khush G, Leung H (2005) Chemical- and irradiation-induced mutants of indica rice IR64 for forward and reverse genetics. Plant Mol Biol 59:85–97PubMedCrossRefGoogle Scholar
  27. Yamanaka S, Nakamura I, Watanabe KN, Sato Y (2004) Identification of SNPs in the waxy gene among glutinous rice cultivars and their evolutionary significance during the domestication process of rice. Theor Appl Genet 108:1200–1204PubMedCrossRefGoogle Scholar
  28. Yang W, Bai X, Kabelka E, Eaton C, Kamoun S, van der Knaap E, David F (2004) Discovery of single nucleotide polymorphisms in Lycopersicon esculentum by computer aided analysis of expressed sequence tags. Mol Breed 14:21CrossRefGoogle Scholar
  29. Yeung AT, Hattangadi D, Blakesley L, Nicolas E (2005) Enzymatic mutation detection technologies. Biotechniques 38:749–758PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2006

Authors and Affiliations

  • Chitra Raghavan
    • 1
  • Ma. Elizabeth B. Naredo
    • 2
  • Hehe Wang
    • 1
  • Genelou Atienza
    • 2
  • Bin Liu
    • 1
    • 3
  • Fulin Qiu
    • 1
  • Kenneth L. McNally
    • 2
  • Hei Leung
    • 1
  1. 1.Entomology and Plant Pathology DivisionInternational Rice Research Institute, DAPOMetro ManilaPhilippines
  2. 2.Genetic Resources CenterInternational Rice Research Institute, DAPOMetro ManilaPhilippines
  3. 3.Rice Research InstituteGuangdong Academy of Agricultural SciencesGuangzhouPeople’s Republic of China

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