Site - Selected Transposon Mutagenesis at the hcf 706 Locus in Maize

The High chlorophyll fluorescencel06 (HcflO6) gene in maize is required for chloroplast membrane biogenesis, and the hcf706muml allele is caused by the insertion of a Robertson‘s Mutator Mul element into the promoter of the gene. Seedlings homozygous for hcfl06-muml are pale green and die 3 weeks after germination, but only in the presence of Mutator activity conferred by active, autonomous Mu regulatory transposons elsewhere in the genome. When Mutator activity is lost, the mutant phenotype is suppressed, and homozygous plants have an almost wild-type phenotype. To isolate derivative alleles at the hcflO6 locus that no longer require Mutator activity for phenotypic expression, we have developed a method for site-selected transposon mutagenesis in maize. This procedure, first described for Caenorhabditis elegans and Drosophila, involves using polymerase chain reaction (PCR) to screen pools of individuals for insertions and deletions in genes of known sequence. Pools of seedlings segregating for the progenitor allele hcf708muml were screened by PCR for insertions and deletions associated with Robertson’s Mutator. In a 360-bp target region, two new insertions and one deletion were identified in only 700 Mu-active gametes screened. One of the insertions was in the progenitor hcfl06-muml allele and the other was in the wild-type allele, but all three new alleles were found to have breakpoints at the Same nucleotide in the first intron. Unlike the hcfl06-muml progenitor allele, the deletion and one of the insertions conferred pale green seedling lethal phenotypes in the absence of Mutator activity. However, the second insertion had a weak, viable phenotype under these conditions. Although the sample size was small, our results suggest that this procedure can be used to rapidly identify transposon insertions into known genes in a single generation. Null derivative alleles,can then be isolated in a second generation using the insertion as a starting point. This two-step procedure represents a powerful and simple way to “knock out” maize genes identified by sequence alone by using only a few thousand progeny from a simple cross.