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SUMMARY   Brachypodium distachyon  is an annual C3 grass used as a monocot model system in 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 the 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 the analyzed R0 and R1 lines. Analysis of the FSTs showed a uniform pattern of insertion in all the chromosomes (1–5) without any preference for a particular chromosome region. Considering the average length of a gene transcript to be 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 , which will aid in functional genomics studies of other C3 grasses.

Insertional mutagenesis of Brachypodium distachyon using the Tnt1 retrotransposable element