Abstract
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.
Highlights
The development of efficient methods for introducing foreign DNA into plant genomes by Agrobacterium tumefaciens mediated transformation prompted its widespread application in biotechnology and basic science [1]
The inverse PCR primers are positioned adjacent to the ligation junction so that short (>60 bp) reads include the viewpoint and the associating region
We present 4SEE, which combines the application of 4C-seq in plants with a computational approach and guidelines to detect the genomic positions of T-DNA insertions together with their associated chromosomal rearrangements at high genomic resolution
Summary
The development of efficient methods for introducing foreign DNA into plant genomes by Agrobacterium tumefaciens mediated transformation prompted its widespread application in biotechnology and basic science [1]. The random nature of genomic integration, which is a major drawback for agricultural biotechnology, has been utilized to generate powerful resources for genetic studies by large-scale T-DNA-based mutagenesis in the model plant Arabidopsis thaliana [2,3,4]. The use of Agrobacterium mediated transformation is expected to increase with the developing capability of targeting T-DNA to specific genomic locations by contemporary genome editing technologies such as CRISPR/Cas or the use of Agrobacterium to deliver genome editing reagents to the plant cells [1,5]. The widespread use of T-DNA-based mutagenesis and genomic engineering prompted the development of methods to map its integration sites and their associated chromosomal abnormalities. From Southern blotting, cytology, and genetic mapping [10] to genomic and molecular approaches, have increased the resolution and throughput of detection capability
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
