Tobacco twin and single spots result from genome instability, chromosome breaks, and translocations

Somatic recombination can have profound consequences including clonal evolution. In plants, however, the contribution of homologous recombination (HR) to genomic instability remains unclear. The semidominant sulfur mutant of tobacco, named for its yellow foliage resulting from a chlorophyll defect, spontaneously develops green and white twin spots that have been attributed to mitotic crossovers. To test this hypothesis, we sequenced DNA from twin spots and surrounding tissues in su/+ F1 hybrids, expecting somatic crossovers to result in reciprocal chromosome arm exchange in the paired sectors. Contrary to expectations, 20 of 22 twin spots exhibited aneuploidy or reciprocal translocations between non-homologous chromosomes. One twin spot showed a pattern consistent with a reciprocal crossover. Single white or green spots were [~]10X more frequent than twin spots. Among 26 single spots, 18 resulted from terminal arm deletion, 2 from aneuploidy, and 4 were associated with translocations. Deletion and reciprocal exchange breakpoints clustered near the centromere. Together, these findings indicate that nearly all spots arise from deletions, translocations, and other rearrangements likely mediated by chromosome missegregation or nonhomologous repair rather than by HR. These rearrangements altered the ratio of mutant to wild-type alleles and consequently chlorophyll levels. We conclude that mitotic recombination between homologous chromosomes is rare, and that genome instability is the dominant driver of somatic chimerism in this system.