Characterization of integration sites and transfer DNA structures in Agrobacterium-mediated transgenic events of maize inbred B104
G3: Genes | Genomes | Genetics
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.
This article was originally published in G3: Genes | Genomes | Genetics. The version of record is available at: https://doi.org/10.1093/g3journal/jkad166. Published by Oxford University Press on behalf of The Genetics Society of America 2023. This work is written by (a) US Government employee(s) and is in the public domain in the US.
maize, T-DNA, transformation, nonhomologous end-joining, integration, Agrobacterium, genetic engineering, disease resistance, Plant Genetics and Genomics
Anjanasree K Neelakandan, Mercy Kabahuma, Qin Yang, Miriam Lopez, Randall J Wisser, Peter Balint-Kurti, Nick Lauter, Characterization of integration sites and transfer DNA structures in Agrobacterium-mediated transgenic events of maize inbred B104, G3 Genes|Genomes|Genetics, Volume 13, Issue 10, October 2023, jkad166, https://doi.org/10.1093/g3journal/jkad166