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The integration of T-DNA in plant genomes is widely used for basic research and agriculture. The high heterogeneity in the number of integration events per genome, their configuration, and their impact on genome integrity highlight the critical need to detect the genomic locations of T-DNA insertions and their associated chromosomal rearrangements, and the great challenge in doing so. Here, we present 4SEE, a circular chromosome conformation capture (4C)-based method for robust, rapid, and cost-efficient detection of the entire scope of T-DNA locations. Moreover, by measuring the chromosomal architecture of the plant genome flanking the T-DNA insertions, 4SEE outlines their associated complex chromosomal aberrations. Applying 4SEE to a collection of confirmed T-DNA lines revealed previously unmapped T-DNA insertions and chromosomal rearrangements such as inversions and translocations. Uncovering such events in a feasible, robust, and cost-effective manner by 4SEE in any plant of interest has implications for accurate annotation and phenotypic characterization of T-DNA insertion mutants and transgene expression in basic science applications as well as for plant biotechnology.

The Position and Complex Genomic Architecture of Plant T-DNA Insertions Revealed by 4SEE