Tilling Explained

Initial situation

Plants

In addition to the 'classical' goals of plant breeding there will be an increased demand for healthier, higher quality and more diverse food, while agricultural production needs to become more environmentally friendly. Thus, the need of rapid establishment of new crop varieties with improved or novel characteristics is dramatically increasing. This immense challenge can be met by making efficient use of the increasing knowledge of the genetic basis of plant function gained through functional plant genome research. This area of research is characterized by the most rapid advances in the identification of gene functions in model plant species, which results in a rapid accumulation of lead gene information that awaits utilization in crops for the support of plant breeding programs. An important first step towards the use of this rapidly and massively accumulating knowledge is the test for the function of the corresponding genes in the relevant crops. The most immediate approach to determine the role of a gene and its importance is the creation and identification of mutants with decreased or increased activity of the genes of interest. The technology platform that will be implemented through the proposed project is based on the TILLING methodology. It provides the most efficient means for gene function testing in crops and for the identification of natural or induced diversity of crop genes.

TILLING (Targeting Induced Local Lesions in Genomes)

The term TILLING relates to a technique developed for the detection of mutations in genes (DNA segemnts in general). The basic principle involves the isolation of DNA from individuals of a mutant population (usually created through chemical mutagenesis using EMS)

TILLING workflow

  • Creation of a mutant population (usually through chemical mutagenesis using EMS).
  • DNA preparation and creation of DNA pools.
  • PCR amplification of a region of interest including heteroduplex formation.
  • Treatment of double stranded DNA fragments with CelI endonuclease (Oleykowski et al. 1998, Nucleic Acids Res. 26: 4597-4602). Purification and denaturation.
  • Detection of cleavage products by separation of the DNA fragments usually on a sequencer / fragment analyser and detection of the mutation through appearance of shortened DNA fragments.
  • Identification of the mutant individual through TILLING reactions of individual DNA samples.
  • Sequencing of mutant PCR product.