An efficient reverse genetics platform in the model legume Medicago truncatula

Abstract

SummaryMedicago truncatula is one of the model species for legume studies. In an effort to develop legume genetics resources, > 21 700 Tnt1 retrotransposon insertion lines have been generated.To facilitate fast-growing needs in functional genomics, two reverse genetics approaches have been established: web-based database searching and PCR-based reverse screening. More than 840 genes have been reverse screened using the PCR-based approach over the past 6 yr to identify mutants in these genes. Overall, c. 84% (705 genes) success rate was achieved in identifying mutants with at least one Tnt1 insertion, of which c. 50% (358 genes) had three or more alleles.To demonstrate the utility of the two reverse genetics platforms, two mutant alleles were isolated for each of the two floral homeotic MADS-box genes, MtPISTILATA and MtAGAMOUS. Molecular and genetic analyses indicate that Tnt1 insertions in exons of both genes are responsible for the defects in floral organ development.In summary, we have developed two efficient reverse genetics platforms to facilitate functional characterization of M. truncatula genes.

The Root Hair “Infectome” of Medicago truncatula Uncovers Changes in Cell Cycle Genes and Reveals a Requirement for Auxin Signaling in Rhizobial Infection

“…Tnt1 retrotransposon insertions in ARF16a were screened using a nested PCR approach (Cheng et al, , 2014. Subsequent genotyping in the R2 generation progeny was performed using primers ARF16_geno_F2 and Tnt1F for the arf16a-1 allele and ARF16_geno_F2 and Tnt1R for the arf16a-2 and arf16a-3 alleles (Supplemental Table 1, Primers).…”

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

“…To further identify the function of CBF4 in cold acclimation, we screened a Tnt1 insertion mutant collection (Cheng et al, 2014). Two allelic mutants were identified, cbf4-1 and cbf4-2, with insertions located in the coding sequence (Supplemental Fig.…”

“…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).…”

A Lipid-Anchored NAC Transcription Factor Is Translocated into the Nucleus and Activates Glyoxalase I Expression during Drought Stress

“…To further determine the function of MfNACsa in drought stress, we evaluated two homozygous Tnt1 insertion mutants of NACsa identified from a Tnt1 retrotransposon-tagged mutant population of M. truncatula (Cheng et al, 2014), NF5250 (designated nacsa-1) and NF9803 (designated nacsa-2), with insertions located in the first intron and third exon, respectively, via PCR (Supplemental Figures 8C and 8D, i). RT-PCR verified that the expression of MtNACsa was impaired (Supplemental Figure 8D, ii).…”

“…The Tnt1 insertion mutant lines, NF5250 (nacsa-1 mutant), NF9803 (nacsa-2 mutant), NF15130 (apt1 mutant), NF20885 (glyI-1 mutant), and NF10049 (glyI-2 mutant) were screened from a Tnt1 retrotransposontagged mutant population of M. truncatula (Tadege et al, 2008;Cheng et al, 2014).…”