Genome-wide profiling of genetic variation in Agrobacterium-transformed rice plants

Agrobacterium-mediated transformation has been widely used in producing transgenic plants, and was recently used to generate "transgene-clean" targeted genomic modifications coupled with the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas9) system. Although tremendous variation in morphological and agronomic traits, such as plant height, seed fertility, and grain size, was observed in transgenic plants, the underlying mechanisms are not yet well understood, and the types and frequency of genetic variation in transformed plants have not been fully disclosed. To reveal the genome-wide variation in transformed plants, we sequenced the genomes of five independent T₀ rice plants using next-generation sequencing (NGS) techniques. Bioinformatics analyses followed by experimental validation revealed the following: (1) in addition to transfer-DNA (T-DNA) insertions, three transformed plants carried heritable plasmid backbone DNA of variable sizes (855-5216 bp) and in different configurations with the T-DNA insertions (linked or apart); (2) each transgenic plant contained an estimated 338-1774 independent genetic variations (single nucleotide variations (SNVs) or small insertion/deletions); and (3) 2-6 new Tos17 insertions were detected in each transformed plant, but no other transposable elements or bacterial genomic DNA.