abstract
scientists have developed many approaches based on pcr or next-generation sequencing to localize and characterize integrated t-dnas in transgenic plants generated by agrobacterium tumefaciens -mediated t-dna transfer. however, none of these methods has the robust ability to handle all transgenic plants with diversified t-dna patterns. utilizing the valuable information in the whole-genome sequencing data of transgenic plants, we have developed a comprehensive approach (t-loc) to localize and characterize t-dna integration sites (tiss). we evaluated the performance of t-loc on genome sequencing data from 48 transgenic rice ( oryza sativa ) plants that provide real and unbiased resources of t-dna integration patterns. t-loc discovered 75 full tiss and reported a diversified pattern of t-dna integration: the ideal single-copy t-dna between two borders, multiple-copy of t-dnas in tandem or inverted repeats, truncated partial t-dnas with or without the selection hygromycin gene, the inclusion of t-dna backbone, the integration at the genome repeat region, and the concatenation of multiple ideal or partial t-dnas. in addition, we reported that dna fragments from the two a. tumefaciens plasmids can be fused with t-dna and integrated into the plant genome. besides, t-loc characterizes the genomic changes at tiss, including deletion, duplication, accurate repair, and chromosomal rearrangement. moreover, we validated the robustness of t-loc using pcr, sanger sequencing, and nanopore sequencing. in summary, t-loc is a robust approach to studying the tiss independent of the integration pattern and can recover all types of tiss in transgenic plants.
plant physiology, if="8.005
https://pubmed.ncbi.nlm.nih.gov/35640125/
