Gene Identification: Reverse Genetics

The number of sequenced genes whose function remains unknown continues to climb with the continuing decrease in the cost of genome sequencing. Comparative genetics and bioinformatics have been invaluable in investigating the function of the genes that have been sequenced, but the elucidation of gene function in planta remains a huge challenge. Many gene functions have been defined through the use of forward genetics, where a phenotype is identified and used to clone the gene responsible. However, in most instances, genes of known sequence are not associated with a phenotype. This is particularly true in non-model species where forward genetics can be more challenging due to genetic redundancy. Reverse genetics is a powerful tool that can be used to identify the phenotype that results from disruption of a specific sequenced gene, even with no prior knowledge of its function. Several approaches have been developed in plants that have led to the production of resources including collections of T-DNA insertion mutants, RNAigenerated mutants, and populations carrying point mutations that can be detected by TILLING, direct sequencing or high resolution melting analysis (Table 4.1). These reverse genetics resources allow for the identification of mutations in candidate genes and subsequent phenotypic analysis of these mutants. In addition, new advances in technology and reduction in technical costs may soon make it practical to use whole genome sequencing or gene targeting on a routine basis to identify or generate mutations in specific genes in a variety of different plant species. This chapter will present the current status and promising prospects for the future of reverse genetics in plants.