Efficient identification of genomic insertions and surrounding regions in two transgenic maize events using third-generation single-molecule nanopore sequencing technology

The increasing development of new genetically modified organisms underscores the critical need for comprehensive safety assessments, emphasizing the significance of molecular evidence such as gene integration, copy numbers, and adjacent sequences. In this study, the maize nitrate-efficient utilization gene ZmNRT1.1 A was introduced into maize variety y822 using transgenic technology, producing transgenic maize events ND4401 and ND4403 with enhanced tolerance to low nitrogen stress. Southern hybridization confirmed that the exogenous T-DNA was singly inserted in both maize transformation events, ND4401 and ND4403. This study utilized third-generation sequencing technology-nanopore single-molecule sequencing-to perform molecular characterization of the integration events. It successfully determined the exogenous gene insertion sites and flanking sequences in ND4401 and ND4403. Comparative analysis with the control group facilitated the preliminary identification of the integration sites of the exogenous T-DNA fragments in these transgenic maize events. Based on the obtained flanking sequences, specific PCR primers were designed for different transformation events. The insertion site for ND4401 was pinpointed in the non-coding region of chromosome 5, and for ND4403, in the non-coding region of chromosome 3. Utilizing the sequencing results, the study developed specific detection primers for the maize transformation events, establishing a precise method for detecting newly created transgenic maize events, which will contribute to subsequent safety assessments.