Submitted to: Current Genomics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: April 28, 2008
Publication Date: June 4, 2008
Citation: Barkley, N.L., Wang, M.L. 2008. Application of TILLING and EcoTILLING as Reverse Genetic Approaches to Elucidate the Function of Genes in Plants and Animals. Current Genomics 9:212-226. Interpretive Summary: In the last 13 years, the technology for obtaiing the basic chemical components or building blocks of DNA has rapidly improved so that researchers can quickly unravel the underlying genetic code. The building blocks (A,C,G, and T) or sequence of bases in genes are currently publicly available for many model plant and animal species. Therefore, the focus has now shifted from determining the sequence of a gene to evaluating the function of each gene. Understanding the role of each gene in a genome can allow scientists to pinpoint disease and improve germplasm. Determining the function of a gene requires collecting empirical data from the species of interest. TILLING (Target Induced Local Lesions In Genomes) is a method to facilitate a scientist's task of elucidating gene function. In this method, one would would induce mutations in a species of interest using a chemical mutagen, identify samples that have an induced mutation in a gene of interest in a high throughput manner, and subsequently, use the mutants discovered from the technique to ultimately gain insight on the role of a gene. EcoTILLING is similar to TILLING; however, the focus of this method is to identify naturally occurring variants in a gene of interest instead of induced mutations. Both of these methods have proven to be highly effective in plant and animal species. This review details how to perform TILLING and EcoTILLING, provides an overview of the current literature, advantages and disadvantages of these methods, and discusses technological challenges.
Technical Abstract: With the fairly recent advent of inexpensive, rapid sequencing technologies that continue to improve sequencing efficiency and accuracy, many species of animals, plants, and microbes have annotated genomic information publicly available. The focus on genomics has thus been shifting from the collection of whole sequenced genomes to the study of functional genomics. Reverse genetic approaches have been used for many years to advance from sequence data to the resulting phenotype in an effort to deduce the function of a gene in the species of interest. Many of the currently used approaches (RNAi, gene knockout, site-directed mutagenesis, transposon tagging) rely on the creation of transgenic material, the development of which is not always feasible for many plant or animal species. TILLING is a non-transgenic reverse genetics approach that is applicable to all animal and plant species which can be mutagenized, regardless of its mating/pollinating system, ploidy level, or genome size. This approach requires prior DNA sequence information and takes advantage of a mismatch endonuclease to locate and detect induced mutations. Ultimately, it can provide an allelic series of silent, missense, nonsense, and splice site mutations to examine the effect of various mutations in a gene. TILLING has proven to be a practical, efficient, and an effective approach for functional genome studies in numerous plant and animal species. EcoTILLING, which is a variant of TILLING, examines natural genetic variation in populations and has been successfully utilized in animals and plants to discover SNPs including rare ones. In this review, TILLING and EcoTILLING techniques, beneficial applications and limitations from plant and animal studies are discussed.