Discovery of chemically induced mutations in rice by TILLING

Background: Rice is both a food source for a majority of the world's population and an important model system. Available functional genomics resources include targeted insertion mutagenesis and transgenic tools. While these can be powerful, a non- transgenic, unbiased targeted mutagenesis method that can generate a range of allele types would add considerably to the analysis of the rice genome. TILLING ( Targeting Induced Local Lesions in Genomes), a general reverse genetic technique that combines traditional mutagenesis with high throughput methods for mutation discovery, is such a method. Results: To apply TILLING to rice, we developed two mutagenized rice populations. One population was developed by treatment with the chemical mutagen ethyl methanesulphonate ( EMS), and the other with a combination of sodium azide plus methyl- nitrosourea (Az- MNU). To find induced mutations, target regions of 0.7 - 1.5 kilobases were PCR amplified using gene specific primers labeled with fluorescent dyes. Heteroduplexes were formed through denaturation and annealing of PCR products, mismatches digested with a crude preparation of CEL I nuclease and cleaved fragments visualized using denaturing polyacrylamide gel electrophoresis. In 10 target genes screened, we identified 27 nucleotide changes in the EMS- treated population and 30 in the AzMNU population. Conclusion: We estimate that the density of induced mutations is two- to threefold higher than previously reported rice populations ( about 1/300 kb). By comparison to other plants used in public TILLING services, we conclude that the populations described here would be suitable for use in a large scale TILLING project.