Figure 2.  Identification of Phytophthora isolates with gene specific point mutations using the TILLING strategy. A, zoospores are treated with EMS or ENU. Single colonies are arrayed in 384-well microtiter plates and grown for long-term storage and DNA extraction. B, genomic DNA is extracted, pooled, and a 1000-bp target amplified with PCR. C, PCR products are heated and re-annealed to form hetero- and homoduplexes, treated with the mismatch endonuclease CEL I, and the resulting fragments resolved on a denaturing polyacrylamide gel. The CEL I enzyme cuts 3’ of mismatched DNA and novel fragments are present in pools containing mutant isolates. Positive pools are then analyzed to identify the isolate carrying the mutation. BACK