Insertional mutagenesis of Brachypodium distachyon using Tnt1 retrotransposable element.

Brachypodium distachyon is an annual C3 grass used as a monocot model system for functional genomics research. Insertional mutagenesis is a powerful tool for both forward and reverse genetics studies. In this study, we explored the possibility of using tobacco retrotransposon Tnt1 to create a transposon-based insertion mutant population in B. distachyon. We developed transgenic B. distachyon plants expressing Tnt1 (R0) and in the subsequent regenerants (R1) we observed that Tnt1 actively transposed during somatic embryogenesis generating an average of 6.37 insertions per line in a population of 19 independent R1 regenerant plants analyzed. In seed derived progeny of R1 plants, Tnt1 segregated in a Mendelian ratio of 3:1 and no new Tnt1 transposition was observed. A total of 126 flanking sequence tags (FSTs) were recovered from R0 and R1 lines analyzed. FST analyses showed a uniform pattern of insertion in all the chromosomes (1 to 5) without any preference for a particular region of the chromosome. Considering the average length of a gene transcript as 3.37 kb, we estimated that 29,613 lines are required to achieve a 90% possibility of tagging a given gene in the B. distachyon genome using the Tnt1 based mutagenesis approach. Our results show the possibility of using Tnt1 to achieve near-saturation mutagenesis in B. distachyon that will aid in functional genomics studies of other C3 grasses.