Use of the transposable element Ac/Ds in conjunction with Spm/dSpm for gene tagging allows extensive genome coverage with a limited number of starter lines: functional analysis of a four-element system in Arabidopsis thaliana

Abstract

We have developed a novel four-element based gene tagging system in Arabidopsis to minimize the number of starter lines required to generate genome-wide insertions for saturation mutagenesis. In this system, the non-autonomous cassette, Ds(dSpm), comprises of both Ds and dSpm elements cloned one within the other along with appropriate selection markers to allow efficient monitoring of excision and re-integration of the transposons. Trans-activation of the outer borders (Ds) and selection against the negative selection marker (iaaH) linked to the cassette ensures unlinked spread of the Ds(dSpm) cassette from the initial site of integration of the T-DNA. This creates several launch pads within the genome from where the internal element (dSpm) can be subsequently mobilized to generate secondary insertions. In this study, starting from a single T-DNA integration we could spread the Ds(dSpm) cassette to 11 different locations over all the five chromosomes of Arabidopsis. The frequency of unlinked Ds transpositions in the F2 generation varied between 0.05 and 3.35%. Three of these lines were then deployed to trans-activate the internal dSpm element which led to the selection of 29 dSpm insertions. The study conclusively shows the feasibility of deploying Ds and the dSpm elements in a single construct for insertional mutagenesis.