Characterization of integration sites and transfer DNA structures in Agrobacterium -mediated transgenic events of maize inbred B104
2023
Neelakandan, Anjanasree | Kabahuma, Mercy | Yang, Qin | Lopez, Miriam | Wisser, Randall, J. | Balint-Kurti, Peter | Lauter, Nick | Iowa State University (ISU) | North Carolina State University [Raleigh] (NC State) ; University of North Carolina System (UNC) | Northwest A & F University | USDA-ARS : Agricultural Research Service | Écophysiologie des Plantes sous Stress environnementaux (LEPSE) ; Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Institut Agro Montpellier ; Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro) | University of Delaware [Newark] | US National Science Foundation (Award #1127076) with additional support from the USDA-ARS project #5030-21000-067
International audience
显示更多 [+] 显示较少 [-]英语. 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.
显示更多 [+] 显示较少 [-]