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

Authors: NEELAKANDAN, ANJANASREE - Iowa State University; KABAHUMA, MERCY - Iowa State University; YANG, QIN - North Carolina State University; Lopez, Miriam; WISSER, RANDALL - University Of Delaware; Balint-Kurti, Peter; Lauter, Nicholas

Submitted to: G3, Genes/Genomes/Genetics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/31/2023
Publication Date: 10/1/2023
Citation: Neelakandan, A.K., Kabahuma, M., Yang, Q., Lopez, M.D., Wisser, R., Balint Kurti, P.J., Lauter, N.C. 2023. Characterization of integration sites and transfer DNA structures in Agrobacterium-mediated transgenic events of maize inbred B104. G3, Genes/Genomes/Genetics. 13(10):jkad166. https://doi.org/10.1093/g3journal/jkad166.
DOI: https://doi.org/10.1093/g3journal/jkad166

Interpretive Summary: Transformation of crop plants means stable insertion of novel DNA sequences into the genome. This is usually accomplished by specific manipulations of the bacterium Agrobacterium tumefaciens. Here we looked at exactly how DNA became inserted into the maize genome: Where in the genome it went in, how much DNA was transferred and whether there were complex duplications and rearrangements associated with insertion. We showed that insertions seemed to occur most frequently at some distance from the centromere and in or close to genes.

Technical 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 nonhomologous (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 the mechanisms of integration. It also demonstrates the utility of the FPNI-PCR technique, which can be adapted to any species of interest.