Large-Scale Discovery of Induced Point Mutations With High-Throughput TILLING

Abstract

TILLING (Targeting Induced Local Lesions in Genomes) is a general reverse-genetic strategy that provides an allelic series of induced point mutations in genes of interest. High-throughput TILLING allows the rapid and low-cost discovery of induced point mutations in populations of chemically mutagenized individuals. As chemical mutagenesis is widely applicable and mutation detection for TILLING is dependent only on sufficient yield of PCR products, TILLING can be applied to most organisms. We have developed TILLING as a service to the Arabidopsis community known as the Arabidopsis TILLING Project (ATP). Our goal is to rapidly deliver allelic series of ethylmethanesulfonate-induced mutations in target 1-kb loci requested by the international research community. In the first year of public operation, ATP has discovered, sequenced, and delivered >1000 mutations in >100 genes ordered by Arabidopsis researchers. The tools and methodologies described here can be adapted to create similar facilities for other organisms.

Zebrafish: A model system for the study of eye genetics

“…Targeting induced local lesions in genomes (TILLING), a method originally developed for plant mutagenesis screens, has been used successfully to identify genetic lesions in specific genes of interest (McCallum et al, 2000;Wienholds et al, 2002Wienholds et al, , 2003Henikoff et al, 2004;Till et al, 2004). Although providing the opportunity to screen for mutations in virtually any gene, TILLING and similar approaches are very labor intensive, require largescale ENU mutagenesis combined with PCR-based assays to identify fish carrying lesions in the genes of interest, followed by the recovery of the alleles in subsequent generations.…”

A protocol for TILLING and Ecotilling in plants and animals

“…The first production-scale TILLING service to be offered was the Arabidopsis TILLING Project (ATP; http://tilling.fhcrc.org:9366/). Several computational tools were adapted to provide a completely web-based system for target selection, primer design and evaluation of sequence-verified mutations 16 . The service began in August 2001 and after 5 years of operation had delivered more than 6,700 mutations to the Arabidopsis community (http://tilling.fhcrc.org:9366/arab/status.html).…”

“…Approximately 90% of orders supplied by users are successful, with occasional failures usually attributable to primer design. ATP was used as a model to create maize (http://genome.purdue.edu/ maizetilling/) 4 and Drosophila (Fly-TILL, http://tilling.fhcrc. org:9366/fly) TILLING services.…”

“…TIL-LING is a reverse genetic strategy that combines traditional mutagenesis with high-throughput discovery of single-nucleotide changes 2,3 . The method is general and, following its original application to the model plant Arabidopsis thaliana, has been applied to a variety of plant and animal species including maize, lotus, barley, wheat, Drosophila and zebrafish [4][5][6][7][8][9] .…”

Differentially expressed genes during malting and correlation with malting quality phenotypes in barley (Hordeum vulgare L.)

“…A candidate gene (CxpI) and the relative expression level polymorphism both mapped to a region containing a QTL for diastatic power on chromosome 3H (Potokina et al 2006). Another potential approach for validating the function of genes identiWed in this study would be to knock down gene expression using RNA interference or TILLING (McCallum et al 2000;Till et al 2003). TILLING lines developed in malting barley cultivars are available (Caldwell et al 2004).…”