Ac/Ds-transposon activation tagging in poplar: a powerful tool for gene discovery

Rapid improvements in the development of new sequencing technologies have led to the availability of genome sequences of more than 300 organisms today. Thanks to bioinformatic analyses, prediction of gene models and protein-coding transcripts has become feasible. Various reverse and forward genetics strategies have been followed to determine the functions of these gene models and regulatory sequences. Using T-DNA or transposons as tags, significant progress has been made by using "Knock-in" approaches ("gain-of-function" or "activation tagging") in different plant species but not in perennial plants species, e.g. long-lived trees. Here, large scale gene tagging resources are still lacking.

The novel strategy of first genotyping and then phenotyping a tagging population as proposed here is, in particular, applicable for long-lived, difficult to transform plant species. We could demonstrate the power of the ATDs transposon approach and the simplicity to induce ATDs transposition in vitro. Since a transposon is able to pass chromosomal boundaries, only very few primary transposon-carrying transgenic lines are required for the establishment of large transposon tagging populations. In contrast to T-DNA-based activation tagging, which is plagued by a lack of transformation efficiency and its time consuming nature, this for the first time, makes it feasible one day to tag (similarly to Arabidopsis) every gene within a perennial plant genome.

We describe the first application of an inducible transposon-based activation tagging system for a perennial plant species, as example a poplar hybrid (P. tremula L. × P. tremuloides Michx.). Four activation-tagged populations comprising a total of 12,083 individuals derived from 23 independent "Activation Tagging Ds" (ATDs) transgenic lines were produced and phenotyped. To date, 29 putative variants have been isolated and new ATDs genomic positions were successfully determined for 24 of those. Sequences obtained were blasted against the publicly available genome sequence of P. trichocarpa v2.0 (Phytozome v7.0; http://www.phytozome.net/poplar) revealing possible transcripts for 17 variants.In a second approach, 300 randomly selected individuals without any obvious phenotypic alterations were screened for ATDs excision. For one third of those transposition of ATDs was confirmed and in about 5% of these cases genes were tagged. Conclusions: The novel strategy of first genotyping and then phenotyping a tagging population as proposed here is, in particular, applicable for long-lived, difficult to transform plant species. We could demonstrate the power of the ATDs transposon approach and the simplicity to induce ATDs transposition in vitro. Since a transposon is able to pass chromosomal boundaries, only very few primary transposon-carrying transgenic lines are required for the establishment of large transposon tagging populations. In contrast to T-DNA-based activation tagging, which is plagued by a lack of transformation efficiency and its time consuming nature, this for the first time, makes it feasible one day to tag (similarly to Arabidopsis) every gene within a perennial plant genome.