IN INOCULANTS Nodulaid - 17th International Nitrogen Fixation ...

17 th International Congress on Nitrogen Fixation
Fremantle, Western Australia
27 November – 1 December 2011
Title Establishment of retrotransposon-mutagenized population of the model legume Lotus
japonicus and high throughput, deep sequencing-based insertion site identification.
Authors Dorian Fabian Urbański, Anna Małolepszy, Stig Uggerhøj Andersen, Jens Stougaard
Poster Board Number 36
Centre for Carbohydrate Recognition and Signalling, Department of Molecular Biology,
Aarhus University, 8000 Aarhus C, Denmark
Targeted gene knockout by homologues recombination is not yet a standard technique for reverse genetics in
plants. As an alternative we have taken advantage of inducible de-repression of Lotus retrotransposon 1
(LORE1), a member of small family belonging to the Ty3-Gypsy class of LTR retro-elements. LORE1 has a
unique feature of being transcriptionally active in the male gametophyte 4, 5 , producing a small number of new
insertions at random sites only during transition to a new generation. We are currently using a founder line of
Lotus japonicus, with active LORE1 to generate a large, mutagenized population. For robust identification of
novel insertion sites we are using a 2D pooling strategy and taking advantage of the known LORE1 LTR
sequence to simultaneously amplify LORE1 flanking sequence tags (FSTs) in dozens of pooled plants. Illumina
deep sequencing technology, an extremely high degree of multiplexing and custom-designed bioinformatics‟
pipeline led us to establishing high throughput detection method for robust identification of those insertions.
Study on a test set of 3744 plants, enabled identification of app. 8000 novel insertions with an average of 2,4 per
plant. Novel LORE1 copies do not show tendency to cluster and are evenly distributed along Lotus
chromosomes. Moreover, genes were preferentially targeted and insertions in exons were 5.7 times more
frequent than in intergenic regions (P