Large‐scale insertional mutagenesis using the Tnt1 retrotransposon in the model legume Medicago truncatula

Abstract

SummaryMedicago truncatula is a fast-emerging model for the study of legume functional biology. We used the tobacco retrotransposon Tnt1 to tag the Medicago genome and generated over 7600 independent lines representing an estimated 190 000 insertion events. Tnt1 inserted on average at 25 different locations per genome during tissue culture, and insertions were stable during subsequent generations in soil. Analysis of 2461 Tnt1 flanking sequence tags (FSTs) revealed that Tnt1 appears to prefer gene-rich regions. The proportion of Tnt1 insertion in coding sequences was 34.1%, compared to the expected 15.9% if random insertions were to occur. However, Tnt1 showed neither unique target site specificity nor strong insertion hot spots, although some genes were more frequently tagged than others. Forward-genetic screening of 3237 R 1 lines resulted in identification of visible mutant phenotypes in approximately 30% of the regenerated lines. Tagging efficiency appears to be high, as all of the 20 mutants examined so far were found to be tagged. Taking the properties of Tnt1 into account and assuming 1.7 kb for the average M. truncatula gene size, we estimate that approximately 14 000-16 000 lines would be sufficient for 90% gene tagging coverage in M. truncatula. This is in contrast to more than 500 000 lines required to achieve the same saturation level using T-DNA tagging. Our data demonstrate that Tnt1 is an efficient insertional mutagen in M. truncatula, and could be a primary choice for other plant species with large genomes.

The Small GTPase ROP10 of Medicago truncatula Is Required for Both Tip Growth of Root Hairs and Nod Factor-Induced Root Hair Deformation

“…To further ascertain the role of ROP10, we obtained a homozygous Tnt1 line of the ROP10 gene (NF2968, designated rop10; Supplemental Figure 2) from a Tnt1 retrotransposon-tagged mutant population of M. truncatula (Tadege et al, 2008). The Tnt1 insertion was inserted in the second intron at position 459 bp downstream from the translation initiation codon of ROP10 in the rop10 mutant, which was confirmed by PCR (Supplemental Figures 2A and 2B).…”

Opposing control by transcription factors MYB61 and MYB3 Increases Freezing Tolerance by relieving C-repeat Binding Factor suppression

“…Transgenic plants (T3 generation) were used for phenotyping. The Tnt1 insertional mutant lines were screened from the Noble Foundation using a reverse genetic PCR approach: NF9694, cbf4-1; NF7967, cbf4-2; NF13136, myb3-1; NF19047, myb3-2; and NF14253, myb61 (Tadege et al, 2008;Cheng et al, 2014).…”

NODULE ROOT and COCHLEATA Maintain Nodule Development and Are Legume Orthologs of Arabidopsis BLADE-ON-PETIOLE Genes

“…We identified four lines (tnk507, NF2717, NF4445, and NF5894) characterized by altered nodule development during screens of M. truncatula Tnt1 transposon mutant collections (d 'Erfurth et al, 2003;Tadege et al, 2008). In these mutant lines, the nodule can develop one or multiple roots in an apical position on the fully developed nodule ( Figures 1A and 1B); thus, the noot mutants can switch from a nodule to a root developmental program.…”

“…NF4445 was obtained by reverse genetics screening using pools of genomic DNA from the Noble Foundation Tnt1 mutant collection (Tadege et al, 2008). All these lines are derived from the M. truncatula R108-1 ecotype.…”