Proceedings of the XLVI Italian Society of Agricultural Genetics - SIGA Annual Congress

Giardini Naxos, Italy - 18/21 September, 2002

ISBN 88-900622-3-1

Oral Communication Abstract - S4i

TILLING, a general tool for reverse genetics and for the discovery of polymorphisms 

Comai L., Till B.J., Reynolds S.H., Enns L., Codomo C., Johnson J.E., Young K., Henikoff J.G., Greene E.A., Henikoff S.

Department of Botany, University of Washington, Seattle, Washington 98195and Basic Sciences Division, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109

gene function, genotyping, heteroduplex cleavage, reverse genetics, Arabidopsis

The recent availability of plant genomic sequences emphasizes the increasing need to develop tools for determining gene function. We describe high-throughput TILLING (Targeting Induced Local Lesions IN Genomes), a strategy for obtaining allelic series of mutations that can be applied to almost any organism. For TILLING, mutations are induced by treatment with a chemical mutagen. DNAs are prepared from individuals and arrayed in pools for initial screening. These pools become templates for PCR using primers that amplify a region of interest. Heteroduplexes are formed between wild-type and mutant fragments in the pool by denaturing and reannealing PCR products. These heteroduplexes are the substrate for cleavage by the nuclease CEL I. After digestion, the resulting products are visualized using standard fluorescent sequencing slab gel electrophoresis. Positive pools are then rescreened as individual DNAs, thus identifying the mutant plant and the approximate position of the mutation along the sequence. This positional information increases the efficiency of sequence analysis, as heterozygous mutations may be otherwise difficult to identify. High-throughput TILLING is available as a service to the Arabidopsis community through the Arabidopsis TILLING Project (ATP, http://tilling.fhcrc.org:9366/). Since its establishment on September 2002, ATP has produced over 800 sequenced mutations in about 90 targeted genes. ATP is also working in collaboration with the International Rice Research Institute to establish TILLING in rice. To facilitate additional groups establishing similar services for other plant and animal species, we have developed our screening methodologies and informatics tools to be both general and easily exportable. The methodology developed for TILLING can also be applied to the discovery and genotyping of natural variation in populations, providing a low cost alternative to full DNA sequencing.We have demonstrated its applicability to genotyping by surveying the haplotypic make up of 192 Arabidopsis ecotypes at selected loci. The technique was very efficient for discovery of 1 to multiple SNPs and small indels in one kb segment of genome.