A modified Enhancer-Inhibitor transposon system was used to generate a series of mutant lines by single-seed descent such that multiple I insertions occurred per plant. The distribution of original insertions in the population was assessed by isolating transposon-flanking DNA, and a database of insertion sites was created. Approximately threequarters of the identified insertion sites show similarity to sequences stored in public databases, which demonstrates the power of this regimen of insertional mutagenesis. To isolate insertions in specific genes, we developed threedimensional pooling and polymerase chain reaction strategies that we then validated by identifying mutants for the regulator genes APETALA1 and SHOOT MERISTEMLESS. The system then was used to identify inserts in a class of uncharacterized genes involved in lipid biosynthesis; one such insertion conferred a fiddlehead mutant phenotype.
INTRODUCTIONOver the past two decades, Arabidopsis has become a model organism for research in plant physiology, development, biochemistry, and pathogenesis. Recent developments in Arabidopsis genomics research include the construction of a nearly complete physical map of the genome based on yeast artificial chromosomes (Choi et al., 1995;Schmidt et al., 1996Schmidt et al., , 1997 Zachgo et al., 1996;Camilleri et al., 1998), bacterial artificial chromosomes (BACs; Mozo et al., 1998), P1 clones (Liu et al., 1995Kaneko et al., 1998), the systematic sequencing of expressed sequence tags (ESTs; Höfte et al., 1993;Newman et al., 1994), and coordinated multinational sequencing efforts (Goodman et al., 1995;Bevan et al., 1998).The large amount of sequence information generated by Arabidopsis genome and EST sequencing projects will provide the basis for systematic studies of gene function and eventually will allow for unlimited comparisons with the yeast (Mewes et al., 1997) and Caenorhabditis ( C. elegans Sequencing Consortium, 1998) genomes. Such cross-kingdom analyses might reveal those genes that are necessary for the development and maintenance of eukaryotic cells and those that are associated with functions unique to either plants or animals. An intriguing finding to have emerged already from these studies is that half of the Arabidopsis ESTs bear no significant similarity to sequences from the previously established databases (Rounsley et al., 1996).Over the past decade, insertional mutagenesis has proven to be one of the most efficient ways of isolating and identifying genes via the traditional approaches of "forward" genetics (Feldmann, 1991;Koncz et al., 1992;Aarts et al., 1993;Jones et al., 1994;Okuley et al., 1994;Azpiroz-Leehan and Feldmann, 1997). In light of the ongoing accumulation of ever more DNA sequences of unknown function, insertional mutagenesis now offers the means for determining the function of newly elaborated sequences by a process of "reverse" genetics. The strategy begins with the broad insertional mutagenesis of the genome through the use of T-DNA or transposons followed by the identification o...