T-LOC: A comprehensive tool to localize and characterize T-DNA integration sites

Abstract

Abstracts 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 T-DNA integration sites independent of the integration pattern and can recover all types of TISs in transgenic plants.

MeGT2.6 increases cellulose synthesis and active gibberellin content to promote cell enlargement in cassava

“…The mature shoots were cut off and transferred to MS medium (MS + 50 mg/l carbenicillin +10 mg/l hygromycin) at 28°C for screening (Nyaboga et al., 2014; Wang, Lu, et al., 2022; Zhou et al., 2020). The OE lines were identified by PCR and T‐LOC re‐sequencing (Li et al., 2022) and the gene‐editing lines ( c lines) were tested by using the Hi‐TOM programme to determine the editing model and frequency (Wang et al., 2018). The above lines were cultured in pots for 120 days in a greenhouse with 12 h light/12 h dark at 28 °C.…”

“…T-LOC re-sequencing and Hi-TOM sequencing T-LOC re-sequencing were used a comprehensive approach (T-LOC) to localise and characterise T-DNA integration sites (TISs) by following Li's experimental method from Li et al (2022).…”

Iterative gene integration mediated by 26S rDNA and non-homologous end joining for the efficient production of lycopene in Yarrowia lipolytica

“…After purifying the PCR products, the libraries were analyzed for size distribution using the Agilent 2100 Bioanalyzer and quantified by real-time PCR (3 nM). Finally, the paired-end DNA-seq sequencing library was sequenced with the Illumina NovaSeq system (Li et al 2022 ; Sun et al 2019 ).…”