Fig. 1.

Flow chart for constructing mutant populations with double-EMS mutagenesis. The conventional process of EMS mutagenesis (upper panel) was described by Saito et al. (2011). In the following steps, bulked M₃ (M₃M₀) seeds were mutagenized with 1.0% EMS and M₃M₁ seeds were sown in the greenhouse to harvest M₄M₂ seeds from single M₃M₁ plants. Ten M₄M₂ seeds were sown and grown as an M₄M₂ line and M₅M₃ seeds were harvested from the same M₄M₂ lines as a bulked seed. Mutant DNA was extracted from each M₄M₂ line (10 plants per line) and pooled as 8-fold DNA for TILLING screening.