High-throughput TILLING of Arabidopsis. Seeds are mutagenized with EMS, which causes G/C-to-A/T point mutations. To avoid ambiguities caused by chimerism of mutant plants in the first (M 1) generation, they are self-fertilized, andM 2 progeny from single seed descent are used for screening. Tissue is collected from each M 2 plant, and DNA is extracted. Plants are self-fertilized, and theM 3 seed is collected and shipped to theArabidopsis Biological Resource Center for distribution. For screening, DNAs are pooled eightfold to maximize the efficiency of mutation detection. PCR is performed using 5′-end-labeled gene-specific primers to target the desired locus, and heteroduplexes are formed by heating and cooling the PCR products. CEL I nuclease is used to cleave at base mismatches, and products representing induced mutations are visualized with denaturing polyacrylamide gel electrophoresis. SeeColbert et al. (2001) and Till et al. (2003) for details.
