TILLING without a plough: a new method with applications for reverse genetics

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TILLING (Targeting Induced Local Lesions IN Genomes) is a powerful reverse genetic technique that employs a mismatch-specific endonuclease to detect induced or natural DNA polymorphisms. Its advantages over other reverse genetic techniques include its applicability to virtually any organism, its facility for high-throughput and its independence of genome size, reproductive system or generation time. TILLING is currently being used for the detection of both induced and natural variation in several plant species.

Introduction

Genome sequencing projects have been completed for several plant species and many more plant genome projects are underway. To exploit the potential of this resource, researchers have shifted their attention to a variety of functional genomics tools that can help decipher the functions of the thousands of newly identified genes.

Genetic mutation is a powerful tool that establishes a direct link between the biochemical function of a gene product and its role in vivo. For decades, genes have been identified and the function of their products determined by isolating and studying mutants that are defective in a specific process of interest (i.e. by forward genetics). With the advent of molecular biology, several distinct techniques have been developed to generate or identify mutations in cloned genes to determine the function of these genes (reverse genetics). In plants, the most commonly used reverse genetic approaches are posttranscriptional gene silencing (PTGS) [1] and insertional mutagenesis [2]. Such methods have several disadvantages, however, that limit their use for functional genomics. PTGS is labour-intensive, can give ambiguous results, and is unsuitable for isolating mutants that have lethal or sterile phenotypes. For insertional mutagenesis, the frequency of mutations per genome is typically low, necessitating the screening of large numbers of plants in a mutagenised population to identify mutations in any given gene. In addition, the mutant alleles are likely to result in a complete loss-of-function for the gene product which, if the effect is lethal or detrimental, might limit the analysis that can be done.

The use of chemical mutagens represents an alternative method of mutagenesis that has none of the drawbacks of the methods mentioned above. Ethylmethane sulfonate (EMS) has been the most commonly used mutagen in plants and induces large numbers of recessive mutations per genome [3]. Other alkylating agents such as ethylnitrosourea (ENU) have also been used effectively. Induced point mutations can create a diverse range of alleles for genetic analysis. In Arabidopsis, five percent of EMS-induced mutations in targeted coding regions result in premature termination of the gene product, whereas fifty percent result in missense mutations that alter the amino-acid sequence of the encoded protein [4]. Other advantages of EMS mutagenesis are that the mutations generated are randomly distributed in the genome, and that a high degree of mutational saturation can be achieved without excessive collateral DNA damage. Despite the clear advantages of EMS mutagenesis, until recently, it has not been useful as a tool for reverse genetics because of the lack of high-throughput techniques for detecting point mutations.

Section snippets

The TILLING technique

TILLING (Targeting Induced Local Lesions IN Genomes) allows the identification of single-base-pair (bp) allelic variation in a target gene in a high-throughput manner [5••]. It has several advantages over other techniques used to detect single-bp polymorphisms. Other gel-based assays, such as single-strand conformation polymorphism (SSCP) and denaturing gradient gel electrophoresis (DGGE), do not pinpoint the location or the type of polymorphism present in the DNA fragment [6]. Techniques that

TILLING projects in different species

Several TILLING reverse genetics projects are now underway in diverse plant species, including Arabidopsis [5••], lotus [14], maize [15] and Brassica oleracea (EJ Gilchrist, I Parkin, GW Haughn, unpublished), and in some animal species [16, 17, 18, 19]. Further discussion of the latter is beyond the scope of this review.

In 2001, Till and colleagues [5••, 20, 21] established the Arabidopsis TILLING Project (ATP) as a joint effort between the Comai Laboratory at the University of Washington and

Ecotilling

TILLING technology holds much potential for examining natural as well as induced variation. Many of the species for which complete genome sequence data are available, including the tree Populus trichocarpa, are not amenable to classical mutagenesis and genetic analysis. For these organisms, the identification of natural genetic variants can provide much information about gene function, and can also be useful for association mapping and linkage disequilibrium analysis. Even in species for which

Conclusions

TILLING is a technique that adds significantly to the arsenal of reverse genetics tools that are available to researchers wanting to capitalise on the information being provided by genome-sequencing projects. It is efficient and cost-effective, and both mutagenised and natural populations of any organism can be screened. As no reverse genetics technique developed to date is ideal for all purposes, TILLING complements other techniques well and, in the absence of site-directed mutagenesis in

References and recommended reading

Papers of particular interest, published within the annual period of review, have been highlighted as:

  • • of special interest

  • •• of outstanding interest

Acknowledgements

We wish to thank Steve Henikoff, Luca Comai and Quentin Cronk for their support. We are grateful to Brad Till for his support and for helpful comments on this manuscript. Funding for our laboratory comes from the UBC Biotechnology Laboratory and from the Genome Canada through Genome Prairie's FGAS Project and the Genome BC's Forestry Genomics Project.

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