Characterization of integration sites and transfer DNA structures in Agrobacterium-mediated transgenic events of maize inbred B104

Abstract

In maize, the community-standard transformant line B104 is a useful model for dissecting features of transfer DNA (T-DNA) integration due to its compatibility with Agrobacterium-mediated transformation and the availability of its genome sequence. Knowledge of transgene integration sites permits the analysis of the genomic environment that governs the strength of gene expression and phenotypic effects due to the disruption of an endogenous gene or regulatory element. In this study, we optimized a fusion primer and nested integrated PCR (FPNI-PCR) technique for T-DNA detection in maize to characterize the integration sites of 89 T-DNA insertions in 81 transformant lines. T-DNA insertions preferentially occurred in gene-rich regions and regions distant from centromeres. Integration junctions with and without microhomologous sequences as well as junctions with de novo sequences were detected. Sequence analysis of integration junctions indicated that T-DNA was incorporated via the error prone repair pathways of non-homologous (predominantly) and microhomology-mediated (minor) end-joining. This report provides a quantitative assessment of Agrobacterium-mediated T-DNA integration in maize, with respect to insertion site features, the genomic distribution of T-DNA incorporation, and mechanisms of integration. It also demonstrates the utility of the FPNI-PCR technique which can be adapted to any species of interest.

Genomic consequences associated with Agrobacterium‐mediated transformation of plants

“…These small deletions frequently facilitate microhomologies at the T‐DNA/genome junctions, which are small sequences 1–8 bp long shared between both the T‐DNA and the site of insertion in the genome (Figure 2a). These have been remarked upon on numerous occasions (e.g., Forsbach et al., 2003; Gheysen et al., 1991; Gong et al., 2021; Holsters et al., 1983; Kim et al., 2003; Kleinboelting et al., 2015; Kralemann et al., 2022; Mayerhofer et al., 1991; Neelakandan et al., 2023) and occur in 40–50% of T‐DNA/genome junctions, with a slightly higher proportion of LB junctions having microhomology compared to RB junctions (Forsbach et al., 2003; Gong et al., 2021; Kleinboelting et al., 2015). In a large transgenic Arabidopsis population, it was found that 45% of T‐DNA/genome junctions had microhomology of at least 1 bp, 38% 2–5 bp, and 7% 6 bp or more (Kleinboelting et al., 2015).…”

“…A further 40–60% of T‐DNA/genome junctions have short filler sequences between the T‐DNA and the genomic sequence (Forsbach et al., 2003; Gong et al., 2021; Gorbunova & Levy, 1997; Kleinboelting et al., 2015; Kralemann et al., 2022; Mayerhofer et al., 1991; Neelakandan et al., 2023; van Kregten et al., 2016; Windels et al., 2003). In the previously mentioned large transgenic Arabidopsis population, almost 70% of T‐DNA integrations contained at least one T‐DNA/genome junction with filler sequence.…”