Genome‐wide analysis of Agrobacterium T‐DNA integration sites in the Arabidopsis genome generated under non‐selective conditions

Previous work from numerous laboratories has suggested that integration of Agrobacterium tumefaciens T-DNA into the plant genome occurs preferentially in promoter or transcriptionally active regions. However, all of these studies were conducted on plants recovered from selective conditions requiring the expression of transgenes. The conclusions of these studies may therefore have been biased because of the selection of transformants. In this study, we investigated T-DNA integration sites in the Arabidopsis genome by analyzing T-DNA/plant DNA junctions generated under non-selective conditions. We found a relatively high frequency of T-DNA insertions in heterochromatic regions, including centromeres, telomeres and rDNA repeats. These T-DNA insertion regions are disfavored under selective conditions. The frequency with which T-DNA insertions mapped to exon, intron, 5' upstream and 3' downstream regions closely resembled their respective proportions in the Arabidopsis genome. Transcriptional profiling indicated that expression levels of T-DNA pre-integration target sites recovered using selective conditions were significantly higher than those of random Arabidopsis sequences, whereas expression levels of genomic sequences targeted by T-DNA under non-selective conditions were similar to those of random Arabidopsis sequences. T-DNA target sites identified using non-selective conditions did not correlate with DNA methylation status, suggesting that T-DNA integration occurs without regard to DNA methylation. Our results indicate that T-DNA integration may occur more randomly than previously indicated, and that selection pressure may shift the recovery of T-DNA insertions into gene-rich or transcriptionally active regions of chromatin.