Instable Tabacco Genome as Indicated by Frequent Large Deletions

The gene N in tobacco encodes resistance against TMV. A high throughput method was developed to screen for loss of function mutants of the N gene. First, the avirulence gene (p50) in TMV was transformed into a susceptible tobacco genotype; then transgenic plant was selfed to obtain homozygous plant. The transgenic plant was crossed with a resistant genotype (with homozygous resistance gene N). All hybrids contain one copy of the resistance gene N and its avirulence gene p50. The presence of both the resistance gene and its avirulence gene in the hybrid causes program cell death, and the seedling die two weeks after germination. The loss of function mutation of either the N gene or the avirulence gene leads to the survival of the seedling. Approximately 8 million hybrid seeds were screened and 1,456 survivals with loss of resistance were identified, with a mutation rate of 1/5,500 for the loss of function mutation for the N gene. Of the 1,456 mutants, 9 and 4 mutants were caused by point mutations and small deletions, respectively, while none was caused by sequence exchanges. The other 1,443 mutants had deletions of the entire N gene and up to several megabases of flanking sequences. The breakpoints vary between different mutants, but several hot spots of deletions were identified probably due to regions prone to double strand break (DSB). Interestingly, similar mutation rates were found in other three loci investigated in tobacco, likely also due to large deletions. Our data suggest that the tobacco genome is very instable, and genome rearrangement plays much more important roles in genome evolution than point mutations and small insertions/deletions.