TILLING and Ecotilling for Rice

Mutagenesis is frequently used to test gene function and to aid in crop improvement. Targeting Induced Local Lesions in Genomes (TILLING) is a reverse genetic strategy first developed to identify induced point mutations in Arabidopsis . This general strategy has since been applied to many plant and animal species. Here, we describe a protocol for high-throughput TILLING in rice. Gene segments are amplified using fluorescently tagged primers, and products are denatured and reannealed to form heteroduplexes between the mutated and wild-type sequences. These heteroduplexes are substrates for cleavage by single-strand-specific nucleases. Following cleavage, products are analyzed on denaturing polyacrylamide gels using the LI-COR DNA analyzer system. Several rice TILLING populations have been described, and a public mutation screening service is now available. The basic methods used for TILLING can be adapted for the discovery and cataloguing of natural nucleotide variation in populations, a strategy known as Ecotilling, which was first used to study genetic diversity among Arabidopsis ecotypes, and has since been applied to crop plants.