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
OriginalPaper
  • 591 Downloads

Abstract

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.

Keywords

TILLING/EcoTILLING SNP detection CEL I Agarose Mapping Rice 

Abbreviations

TILLING

Targeting induced local lesions IN genomes

SNP

Single nucleotide polymorphism

TPP

Trehalose 6-phosphate phosphatase

ADF

Actin depolymerizing factor

RILs

Recombinant inbred lines

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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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