Extended Data Fig. 10: Targeted identification of nad9 deletions by PCR and amplicon sequencing.
From: Targeted knockout of a conserved plant mitochondrial gene by genome editing
(a) The region around nad9 was amplified by long-range PCR with primers oJF1369 and oJF1374 (binding sites depicted in panel c; for sequences see Supplementary Table 4) from total genomic DNA of all nad9 deletion lines (TALEN-free individuals; cf. Extended Data Fig. 4). Expected amplicon sizes are 5,742 bp for the wild-type mitochondrial DNA and 1,938 bp for the sequenced line Δnad9-29 (cf. Fig. 2). A PCR product smaller than the wild-type amplicon will appear only in those nad9 deletion lines, where sequences upstream and downstream of nad9 were directly fused by MMR (putative ‘clean’ deletions), but not in lines where the sequences upstream and downstream of nad9 have undergone MMR with sequences elsewhere in the genome. Only lines Δnad9-10, Δnad9-29 and Δnad9-30 yield smaller PCR products. Note that the large 5,742 bp wild-type amplicon is not amplified in all lines, presumably because it is close to the upper size limit of standard PCRs. M, DNA size marker; WT, wild type control; H2O, buffer control (without DNA template); JF1006-30, TALEN control line (containing the wild-type nad9 locus). (b) Alignment of the sites of microhomology-mediated recombination involved in generating the nad9 deletion in line Δnad9-10. The breakpoint sequences in the nad9 region are depicted in magenta, the microhomology is indicated in green. Sequencing of the Δnad9-10 PCR product revealed that the MMR event occurred in an imperfect 18 bp repeat (mismatches indicated as lowercase letters). (c) Deletion in the mitochondrial genome of mutant line Δnad9-10. The deletion of 3,014 bp (marked in red) encompasses the nad9 locus and the downstream trnP-UGG gene (removing the genomic sequence between nucleotide positions 334,622 and 337,639). The linear physical map (not drawn to scale) shows the binding sites of the two primers (horizontal arrows) employed for PCR screening and the two sites that engaged in MMR (dotted vertical lines). See text for details. The experiments presented here in (a) were repeated twice independently with similar results (as technical replicates).
