TREGED: A new strategy for inducing deletions in plant genomes

Abstract

We have created a DNA construct, TREGED (transposon-and recombinase-mediated genome deletion), that will automatically induce deletions in plant genomes. TREGED contains the maizeAc/Ds transposon, the yeast R-RS site-specific recombination system, the bacterialtetR repression systems, a novel artificial superintron, and the marker genesGUS andLc. The novelty of TREGED is that only one cross is required to trigger a sequence of events leading to deletion and, simultaneously, to a color assay to detect the deletion. Crossing is done to introduceAc transposase which activatesDs transposition from TREGED to a nearby chromosome region.Ds transposition, in turn, activates recombination between an engineeredRS site on TREGED and anRS site on the transposedDs fragment, thus deleting the genome segment between TREGED andDs. The recombination event also deletesLc orGUS and part oftetR, which triggers expression ofGUS orLc color genes for an upstream or downstream deletion respectively. Each TREGED insertion site will produce multiple kinds of deletions identifiable by inspecting a single F₁ plant and its progeny for colored tissue. The color markers can also be used to differentiate between deletion and other more rare events such as translocation and inversion. We anticipate TREGED will greatly simplify the steps required to obtain useful deletions—eventually allowing the creation of detailed deletion libraries. Such libraries will be particularly useful for anlaysis of gene and chromatin function in plant species with large genomes.