TILLING

The technology, known as TILLING (Targeting Induced Local Lesions IN Genomes) is a 'reverse genetics' process as developed by Colbert et. al. 2001. The method relies on the ability of a special enzyme to detect mismatches in normal and mutant DNA strands when they are annealed. Seed was treated with ethylmethanesulphonate (EMS) to generate a population of plants with random point mutations. By selectively pooling the DNA and amplifying with fluorescently labelled primers, mismatched heteroduplexes were generated between wild type and mutant DNA. Heteroduplexes were incubated with the plant endonuclease CEL I, (cleaves heteroduplex mismatched sites) and the resultant products visualised on a ABI3730. Subsequent analysis of the individual plant DNA from the pool DNA identified the plant bearing the mutation.

RevGenUK has an EMS mutagenised population of plants for:

  • Lotus japonicus 'Gifu' and 'MG20'
  • Medicago truncatula 'jemalong'
  • Brassica rapa 'R-o-18'

The TILLING process explained

When a mutation is detected, the individual DNA samples are sequenced to identify the specific plant carrying the mutation The cleavage products are run on a sequencer and the fluorescently labelled traces analysed CEL I preferentially cleaves at sites of heteroduplex mismatches mutant amplification products form mismatched heteroduplexes DNA samples are pooled and amplified with fluorescent primers DNA is individually extracted and seed stored M1 plants self-fertilise and M2 seed harvested and sown seeds are chemically mutagenised to produce a population with random point mutations A plant, seed and mutant database will be developed as a web-accessible information source

  1. M1 Mutagenesis
  2. M2 plants
  3. DNA extraction
  4. Database
  5. Amplification
  6. Heteroduplex formation
  7. Cleavage by CEL I
  8. Cleavage products
  9. Mutant identification