Fig. 1: Isolation of tobacco mutants harbouring TALEN-induced deletions of the mitochondrial nad9 locus.
From: Targeted knockout of a conserved plant mitochondrial gene by genome editing
a, Targeted search for nad9 deletion mutants. Due to the presence of NUMTs, even homochondriomic nad9 deletion mutants will yield a nad9 amplification product in genotyping PCRs. Therefore, semiquantitative ratiometric PCR was used to screen for deletion mutants using an rrn18 amplicon as internal standard. In mitochondrial nad9 deletion mutants, the relative PCR product intensities shift from dominance of the nad9 amplicon in the wild type (WT) towards dominance of the rrn18 amplicon in nad9 deletion mutants (lanes indicated with star). Lanes 1–9 represent nine regenerated shoots subjected to primary screening for nad9 deletions; lanes 10–17 are PCR assays of shoots derived from an additional regeneration round of a single identified deletion event (line Δnad9-01). M, DNA size marker. b, PCR genotyping of plants from all 31 identified Δnad9 lines (after backcrossing with the wild type as pollen donor). Numbers below the gel indicate the intensity of the nad9 signal relative to the rrn18 signal. H2O, buffer control. c, Expression analysis of plants from all 31 Δnad9 lines. atp9 and nad9 were co-amplified by RT–PCR in a single reaction. See text and Methods for details. The experiments in a and b were repeated independently at least once (with related but not identical plant material); the experiments in c were repeated once independently with similar results.
