Minisymposium 9: Legume Biology

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Abs # M0902: Tnt1 tagging in the model legume Medicago truncatula

Presenter:	Tadege, Million       Contact Presenter
Authors	Tadege, Million  (A)   Tu, Haidi  (A)   Hartwell, Miki  (A)   Gallaway, Janie  (A)   Wen, Jiangqi S (A)   Ratet, Pascal  (B)   Mysore, Kirankumar S (A)
Affiliations:	(A): Samuel Roberts Noble Foundation (B): Institut des Sciences du Vegetal, CNRS

With concerted international genome sequencing project, aiming at completion of the gene-space by 2007, and an extensive EST collection, Medicago truncatula has become a fast developing model for legume functional biology. Many functional genomics tools including transcript and metabolite profiling, as well as EMS and fastneutron mutant populations are being developed, but insertional mutants are not available. Large-scale T-DNA tagging in M. truncatula poses a challenge because fast and efficient in planta transformation strategy is not available, and the genome size is approximately four to five times larger than that of Arabidopsis thaliana. It is estimated that more than 500,000 tagged lines would be required to achieve near-saturation mutagenesis in M. truncatula (~500 Mb genome) using T-DNA. An attractive alternative is to use retrotransposons as mutagens which encode functions required for their own replication and transposition, and move in the genome by a 'copy and paste' mechanism. Retrotransposition involves an RNA intermediate in which the retroelement is first transcribed into an mRNA and reverse transcribed into a cDNA which then inserts into new location. We resorted to the use of a tobacco retrotransposon (Tnt1) that can insert into multiple positions per line which partly offsets the requirement for large scale transformation. We show that Tnt1 is very active and transposes into, on average, 20 positions during M. truncatula tissue culture. Mutations induced by Tnt1 insertion are stable during seed-to-seed generation. In most cases the multiple insertions per line are independent from each other and can be separated by crossing for further genetic analyses. We use one transgenic line harbouring Tnt1 as the source of explants for in vitro reactivation of Tnt1 transposition to develop a large collection of Tnt1 tagged M. truncatula mutants. In an initial international collaborative forward genetics screening effort of our 922 lines, we identified visible mutants in 25 distinct classes that we looked for including nodulation, mycorrhizal colonization, vegetative development, and flowering time. We show a very high mutation frequency of approximately 30 percent visible phenotype in the R1 progenies demonstrating that Tnt1 tagging is a very efficient system for genome-wide saturation mutagenesis in M. truncatula. Collaborative efforts are underway to generate a sizable number of tagged mutant lines and develop a flanking sequence database.