title: TILLING and ecotilling for rice.
abstract: 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.
authors.affiliation: Department of Chemistry, University of Akron, Akron, OH, USA.
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Cooper
authors.forename: Steven
authors.initials: S
authors.lastname: Henikoff
authors.forename: Luca
authors.initials: L
authors.lastname: Comai
authors.forename: Bradley J
authors.initials: BJ
authors.lastname: Till
mesh_terms: D017360:Arabidopsis / Q000235:genetics; D060146:Ecotype; D014644:Genetic Variation; D018745:Genome, Plant; D016296:Mutagenesis; D012275:Oryza / Q000235:genetics*