Fig. 6: Abundant presence of promiscuous nad9 copies in the nucleus (NUMTs).
From: Targeted knockout of a conserved plant mitochondrial gene by genome editing
Taking advantage of the absence of a mitochondrial nad9 locus in our nad9 deletion mutants, nuclear nad9 sequences could be selectively amplified from total DNA (of line Δnad9-29). The PCR product was cloned and 200 randomly picked bacterial clones were sequenced, yielding 16 different DNA sequences. While the consensus sequence of the NUMT alleles is identical to the mitochondrial nad9 sequence, only one NUMT allele is identical to the mitochondrial nad9 copy (thus probably originating from a recent transfer event). The sequence from positions 335,675 to 336,791 (in the reference mitochondrial genome; NC_006581.1) was analysed. The nucleotide positions of the nad9 5′UTR are 335,988–336,110, the positions of the nad9 coding sequence are 336,111–336,683 and the positions of the nad9 3′UTR are 336,684–336,736. a, Partial alignment of the 16 identified NUMT sequences with the mitochondrial reference sequence (WT) around the nad9 start codon. Nucleotide positions in the NUMTs that are identical to the mitochondrial nad9 sequence are indicated by green cells, deviating positions are marked in magenta (with the mutated nucleotide indicated in the cell). When two different mutations are found at a given position, the second mutation is shown in yellow. For a full account of the sequence deviations from the mitochondrial nad9, see Supplementary Table 2. b, An NUMT predating the speciation event that produced N. tabacum. The mitochondrial nad9 sequence of N. tomentosiformis, one of the diploid progenitor species of the allotetraploid species N. tabacum, was determined and aligned to that of the nad9 locus of N. tabacum (whose mitochondrial DNA originates from the other diploid progenitor species, N. sylvestris). N. tomentosiformis exhibits two diagnostic sequence differences: the SNP G-335,722-T and the deletion ΔCATCAAAG (positions 335,735–335,742), marked in magenta. The corresponding N. tabacum nucleotides are marked in cyan. Of the 16 detected NUMT alleles, 1 shows the two N. tomentosiformis signature mutations, indicating that this mitochondrial sequence was transferred to the nucleus before the formation of N. tabacum. The sequence of the respective NUMT allele is shown. Note that it harbours an additional mutation (G-335,746-A; with the A marked in green, and the G marked in bold and black).
