The position and complex genomic architecture of plant T-DNA insertions revealed by 4C

The integration of T-DNA to plant genomes is widely used for basic research and agriculture. High heterogeneity in the number of integration events per genome, their configuration and impact on genome integrity highlight both, the critical need and great challenge to detect the genomic locations of T-DNA insertions and their associated chromosomal rearrangements. Here we present ‘4SEE’, a circular chromosome conformation capture (4C) based method for robust, rapid and cost-efficient detection of the entire scope T-DNA locations. Moreover, by measuring the chromosomal architecture at 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 feasible, robust and cost-effective manners by 4SEE in any plant of interest have 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. 4C-seq was performed on WT, SALK_011436, SALK_009469, SALK_041474, SALK_064627, SALK_005512 from the SALK stock, and CS68932 from the Arabidopsis Biological Resource Center (ABRC) were used in this study. There are 118 samples, as follows: three endogenous genes (two replicas each), four SALK lines in a gradient of template amounts: six samples of SALK_063720 and SALK_064627, five samples of SALK_011436 and seven samples of SALK_005512. The remaining SALK lines have 1 sample each. Seventy-two files are on the ADF viewpoint (CS68932), with a gradient of template amounts and reads. The remaining 14 samples are seven viewpoints measured in WT or CS68932 plant, which map the chromosomal rearrangements.