Efficient identification of Arabidopsis knock-out mutants using DNA-arraysof transposon flanking sequences

Mutants obtained by insertional mutagenesis are widely used for determining gene-phenotype relationships. In Arabidopsis thaliana, several populations mutagenized either by T-DNA or transposon insertion are available for scre ening for knockout mutants in genes of interest. We have so far screened ou r Arabidopsis population mutagenized with the Zea mays transposon En-1/Spm for insertion mutations in 718 genes, using PCR on DNA pools. Although succ essful, this common approach is too time consuming for use in systematic sc reening of all 25498 predicted genes of the Arabidopsis genome. We therefor e investigated the use of DNA arrays for the direct identification of mutan ts in our population. All transposon-flanking regions from individual plant s are amplified by PCR and subsequently spotted at high density onto nylon membranes. A single hybridization experiment with a gene-specific probe the n allows one to identify candidate mutant plants. The efficiency of each se parate step was determined and optimized. Screening of filters representing 2880 plants for insertions in 144 genes and subsequent investigation of so me of the potential insertion mutants suggest that an overall screening eff iciency of 50% is attained.