DLA-Based Strategies for Cloning Insertion Mutants: Cloning the gl4 Locus of Maize Using Mu Transposon Tagged Alleles

Digestion–ligation–amplification (DLA), a novel adaptor-mediated PCR-based method that uses a single-stranded oligo as the adaptor, was developed to overcome difficulties of amplifying unknown sequences flanking known DNA sequences in large genomes. DLA specifically overcomes the problems associated with existing methods for amplifying genomic sequences flanking Mu transposons, including high levels of nonspecific amplification. Two DLA-based strategies, MuClone and DLA-454, were developed to isolate Mu-tagged alleles. MuClone allows for the amplification of subsets of the numerous Mu transposons in the genome, using unique three-nucleotide tags at the 3′ ends of primers, simplifying the identification of flanking sequences that cosegregate with mutant phenotypes caused by Mu insertions. DLA-454, which combines DLA with 454 pyrosequencing, permits the efficient cloning of genes for which multiple independent insertion alleles are available without the need to develop segregating populations. The utility of each approach was validated by independently cloning the gl4 (glossy4) gene. Mutants of gl4 lack the normal accumulation of epicuticular waxes. The gl4 gene is a homolog of the Arabidopsis CUT1 gene, which encodes a condensing enzyme involved in the synthesis of very-long-chain fatty acids, which are precursors of epicuticular waxes.INSERTIONAL mutagenesis is widely used in functional genomics. For example, insertion mutants obtained via T-DNA in Arabidopsis (Alonso et al. 2003) and rice (Sallaud et al. 2004) and via transposons in maize (Brutnell 2002; Brutnell and Conrad 2003; May et al. 2003; McCarty et al. 2005; Settles et al. 2007), rice (Kolesnik et al. 2004; Miyao et al. 2003; Kumar et al. 2005), and Arabidopsis (Speulman et al. 1999) have been used for both forward and reverse genetics. In both situations it is necessary to identify sequences flanking the insertional mutagen. For example, the availability of sequence-indexed collections of T-DNA insertion mutants (Alonso et al. 2003) has greatly facilitated the functional analysis of Arabidopsis. Such reverse genetic resources are generated by creating large numbers of independent insertion events and then identifying and sequencing the DNA flanking the insertional mutagen. To be cost effective such flanking sequences are typically amplified using one of several available “genome-walking” strategies (Shyamala and Ames 1989; Alonso et al. 2003; O''Malley et al. 2007; Vandenbussche et al. 2008; Uren et al. 2009).Similarly, once mutant phenotypes have been identified following forward genetic screens, the challenge in cloning the affected gene is to identify the specific genic sequences that flank causative insertions. Insertional mutagensis is typically more productive if multiple copies of the insertional mutagens are present. The Mutator (Mu) transposon of maize has been widely used for forward genetics because of its high copy number and transposition activity (Benito and Walbot 1997). This high copy number can, however, complicate the identification of the specific insertion responsible for a mutant phenotype. Traditionally, identifying a gene sequence that had been tagged by an insertion involved genomic DNA blotting using multiple wild-type and mutant siblings to identify a DNA fragment that contained the insertion and that cosegregated with the mutant phenotype (James et al. 1995). However, both DNA blotting and subsequent postblotting gene isolation steps were laborious, time-consuming, and often unpredictable.Here, we report two strategies, MuClone and DLA-454, for cloning mutant alleles derived from insertional mutagenesis. Both strategies are based on an adaptation of a novel highly specific and efficient genome-walking method, digestion–ligation–amplification (DLA) that uses a single-stranded oligo as the adaptor instead of the partially double-stranded adaptors used in other methods. MuClone, a cost-efficient strategy, adds unique three-nucleotide tags to the 3′ ends of the common adaptor primer so subsets of high-copy Mu transposons can be separately amplified in a manner analogous to AFLP technology (Yunis et al. 1991). It is then possible to identify which copy of the transposon cosegregates with the mutant allele in the cosegregating population. DLA-454 combines DLA with 454 pyrosequencing to amplify and sequence multiple independent alleles of a gene to be cloned. Analysis of the resulting Mu flanking sequences (MFSs) identifies the target gene. To illustrate the applicability of the MuClone and DLA-454 strategies, each was used to independently clone the glossy4 (gl4) gene. The maize gl4 is a homolog of the Arabidopsis CUT1 gene involved in epicuticular wax accumulation.