Transposon Resources for Forward and Reverse Genetics in Maize

The maize geneticists toolkit includes an impressive set of strategies for creating mutations that facilitate identifying genes based on phenotypes (forward genetics) and/or assigning phenotypes to genes identified by sequence (reverse genetics). Key to both forward and reverse genetics strategies are methods for construction and efficient molecular analysis of large, mutagenized maize populations that ideally contain mutations in all genes. Hence, as the technologies for high-throughput phenotype analysis of maize populations advance apace with DNA sequencing and genotyping technologies, the conventional distinction between forward and reverse genetics is likely to blur. Strategies for comprehensive mutagenesis of maize genes include TILLING (Till et al., 2004); RNAi (McGinnis et al., 2007); and transposon insertional mutagenesis, the focus of this chapter. These three approaches have complementary strengths and weaknesses with differences in relative cost per gene, precision, genetic background limitations, scalability, accessibility and relative coverage of the maize genome. While insertional mutagenesis is the most venerable of these technologies, resources based on mutations caused by defined DNA insertions are likely to have an enduring importance in functional genomics for several practical reasons: 1) compared to other types of mutations (e.g. point mutations) insertions are relatively easy to identify and map in the genome using conventional or high-throughput sequencing technologies, 2) large insertions are highly effective in causing significant disruptions of gene function (e.g. null mutations), and 3) the resulting loss-of-function mutations are genetically stable and typically recessive. Recessive, loss of function mutations are an important reference point for functional analysis of a gene.

Various DNA elements including random T-DNA insertions introduced by transformation (Alonso et al., 2003), engineered transposons (Muskett et al., 2003; Kolesnik et al., 2004; Raizada et al., 2003), as well as native transposons (Yamazaki et al., 2001) have been employed for large scale insertional mutagenesis of plant genomes. For maize, transposon-based resources are currently favored for several reasons: 1) the relative inefficiency of methods for transformation of maize limits production of large numbers of T-DNA lines; 2) maize is a pre-eminent model for transposon genetics with multiple genetically well-characterized transposon families; and 3) because maize is more easily out-crossed than self-pollinating species such as Arabidopsis and rice, plant populations containing large numbers of independent transpositions are comparatively easy to construct. The so-called “cut and paste” DNA transposons that have been the most favored for genomic resource development in maize include the Ac/Ds (Cowperthwaite et al., 2002; Kolkman et al., 2005) and Robertson's Mutator (Bensen et al., 1995, May et al., 2003; McCarty et al., 2005) systems. Each of these systems has well-characterized mechanisms enabling genetic control of transposon mobility in the genome. These transposon systems differ in properties that affect their suitability for functional genomics applications including: 1) copy number of active elements in the genome, 2) relative bias for insertion into gene sequences, and 3) propensity for transposition to linked sites.