Discovery of chemically induced mutations in rice by TILLING

BMC Plant Biol. 2007 Apr 11;7:19. doi: 10.1186/1471-2229-7-19.

Abstract

Background: Rice is both a food source for a majority of the world's population and an important model system. Available functional genomics resources include targeted insertion mutagenesis and transgenic tools. While these can be powerful, a non-transgenic, unbiased targeted mutagenesis method that can generate a range of allele types would add considerably to the analysis of the rice genome. TILLING (Targeting Induced Local Lesions in Genomes), a general reverse genetic technique that combines traditional mutagenesis with high throughput methods for mutation discovery, is such a method.

Results: To apply TILLING to rice, we developed two mutagenized rice populations. One population was developed by treatment with the chemical mutagen ethyl methanesulphonate (EMS), and the other with a combination of sodium azide plus methyl-nitrosourea (Az-MNU). To find induced mutations, target regions of 0.7-1.5 kilobases were PCR amplified using gene specific primers labeled with fluorescent dyes. Heteroduplexes were formed through denaturation and annealing of PCR products, mismatches digested with a crude preparation of CEL I nuclease and cleaved fragments visualized using denaturing polyacrylamide gel electrophoresis. In 10 target genes screened, we identified 27 nucleotide changes in the EMS-treated population and 30 in the Az-MNU population.

Conclusion: We estimate that the density of induced mutations is two- to threefold higher than previously reported rice populations (about 1/300 kb). By comparison to other plants used in public TILLING services, we conclude that the populations described here would be suitable for use in a large scale TILLING project.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Agriculture*
  • DNA, Plant / genetics
  • DNA, Plant / isolation & purification
  • Ethyl Methanesulfonate / pharmacology*
  • Methylnitrosourea / pharmacology*
  • Mutagenesis
  • Mutagens / pharmacology
  • Mutation*
  • Oryza / drug effects
  • Oryza / genetics*
  • Sodium Azide / pharmacology*

Substances

  • DNA, Plant
  • Mutagens
  • Methylnitrosourea
  • Sodium Azide
  • Ethyl Methanesulfonate