Comparison of Sequence Changes in Rice Regenerants and Transformants

Rice is one of the world’s most important crops. The rice research community uses insertional mutants extensively for studying gene function. Approximately half a million flanking sequence tags from rice insertional mutant libraries are publicly available. However, the relationship between genotype (disrupted/activated gene) and phenotype is very low. We used high-throughput sequencing to analyze three non-transformed regenerants and four dwarf tillering mutants among the T-DNA transformants produced by the Taiwan Rice Insertional Mutant (TRIM) resource. We developed stringent criteria to identify small insertions/deletions (indels) and single nucleotide polymorphisms (SNPs) in the genomes of these lines. We also used Targeting Induced Local Lesions in Genomes and capillary sequencing to reveal sequence changes among several transformants in the TRIM population. We identified chromosome doublings, SNPs, small indels, and large deletions in these lines. Some of these sequence changes disrupted genes and resulted in mutant phenotypes. The mutation rates of the transformants were ~10-fold higher than those of the regenerants. Among the eight dwarf and one seedling lethal mutants we analyzed, all phenotypes were caused by DNA sequence changes other than those resulting from T-DNA integration. These data suggest that, in addition to tissue culture-induced somaclonal variation, the process of Agrobacterium-mediated transformation may induce sequence changes and may therefore be inherently mutagenic.