T argeting I nduced L ocal L esions IN G enomes (TILLING) for Plant Functional Genomics

Abstract

One of the most important breakthroughs in the history of genetics was the discovery that mutations can be induced (Muller, 1930; Stadler, 1932). The high frequency with which ionizing radiation and certain chemicals can cause genes to mutate made it possible to perform genetic studies that were not feasible when only spontaneous mutations were available. As a result, much of our understanding of genetics of higher organisms is based upon studies utilizing induced mutations for analyzing gene function. Alkylating agents, which yield predominantly point mutations, have been especially valuable, since the resulting altered and truncated protein products help to precisely map gene and protein function. Because of the high mutational density and the great utility of point mutations, traditional chemical mutagenesis methods have continued to be popular in phenotypic screens despite the development of other mutagenic tools such as transposon mobilization (Bingham et al., 1981). With the recent expansion of sequence databanks, locus-to-phenotype reverse genetic strategies have become an increasingly popular alternative to phenotypic screens for functional analysis. Sequence information alone may be sufficient to consider a gene to be of interest, because sequence comparison tools that detect protein sequence similarity to previously studied genes often allow a related function to be inferred. Hypotheses concerning gene function that are generated in this way must be confirmed empirically. Experimental determination of gene function is desirable in other situations as well, for example, when a genetic interval has been associated with a phenotype of interest. In such cases, the functions of genes in an interval can be inferred by using reverse genetic methods. Routine reverse genetics (Scherer and Davis, 1979) has been an important factor in the popularity of baker's yeast over the past two decades, and the RNAi technique (Fire et al., 1998) now provides Caenorhabditis elegansinvestigators with a routine knockout method that has enjoyed huge popularity over the past year (Sharp, 1999). In most other eukaryotes, however, the situation remains unsatisfactory.