Amidst the escalating challenges posed by global climate degradation and an ever-increasing population, a primary concern for nations worldwide is ensuring food security (Santos et al.20). Since commercial cultivation of genetically modified crops began in 1996, the area dedicated to their cultivation has shown a consistent increase. From 1996 to 2016, the cultivation area evolved from 1.7 million hectares to an impressive 190.4 million hectares, providing substantial economic, societal, and ecological benefits (James13). In recent years, the strategic incorporation of superior exogenous genes into diverse crops through genetic engineering has proven to be an efficient method for breeding superior cultivars (Qaim18).
The introduction of genetically modified crops has invariably placed their safety under extensive scrutiny and discussion (Domingo et al.6). In response, numerous countries worldwide have implemented strict legal frameworks and established dedicated regulatory bodies to oversee the deployment of genetically modified crops (Falkner et al.7). The careful selection of genetically modified varieties for commercial cultivation comes from a rigorous selection process among many transformation events, subjecting them to thorough scrutiny and safety assessments from production to regulatory approval (Bradford et al3.). In the context of genetically modified crop screening and safety evaluation, molecular evidence that proves the integration of exogenous genes into the recipient genome is essential. Submission of comprehensive molecular evidence and information, including sequence details of inserted exogenous genes, copy numbers, insertion sites, and adjacent sequences, is imperative for compliance with established standards (Li et al.15).
During the integration of exogenous genes into the recipient genome, the insertion of plasmid DNA can cause mutations or breaks in the native genes of the recipient plant genome (Wilson et al28.). This may lead to gene silencing or activation, potentially forming new proteins or deactivating existing ones, resulting in unexpected changes that affect the quality and safety of genetically modified crops (Latham et al14.). These changes require the submission of detailed evidence to safety assessment authorities for thorough examination and approval (Cellini et al5.). Currently, traditional methods to characterize exogenous gene integration sites mainly use PCR technology, including techniques such as Genome Walking (GW), Thermal Asymmetric Interlaced PCR (TAIL-PCR), Inverse PCR (I-PCR), and T-linker PCR (Shu et al22.). These methods have effectively been used to examine the molecular features of integration sites in various genetically modified crops like Arabidopsis (Tan et al.25), maize (Spalinskas et al24.; Liu et al16.), rice (Fraiture et al8.), and tomato (Yang et al.32). While these techniques can identify the number of copies of inserted exogenous genes and the presence of plasmid backbones, they have limitations. The accuracy of these methods can be affected by factors such as primer selection, particularly in cases of complex genome rearrangements at the insertion site (Holst-Jensen et al12.). Furthermore, when transgenic elements are similar to sequences in the recipient genome, the effectiveness of traditional methods can be reduced, making it difficult to identify insertion sites and nearby sequences using PCR-based methods (Yang et al.31).
In recent years, DNA sequencing technology has evolved rapidly, marked by significant increases in sequencing capacity along with a decrease in costs. This evolution has made sequencing technology more accessible and widespread (Satam et al.21). A significant advancement in this field is represented by third-generation sequencing technologies, such as PacBio’s SMRT and Oxford Nanopore Technologies’ nanopore single-molecule sequencing. The primary advantage of these technologies is their ability to produce long reads, which are crucial for the comprehensive mapping and characterization of complex genomic regions (Lu et al.17). These long reads not only enhance the depth and breadth of genome sequencing but also improve the accuracy of detecting and characterizing the insertion sites of exogenous genes in transgenic plants (Heather and Chain11). Studies show that deep sequencing can comprehensively cover complex genomes (Wang et al.27), laying the groundwork for precise whole-genome research (Ajay et al1.). Different from Southern hybridization, PCR technology, and previous generations of sequencing, the key feature of third-generation sequencing is its ability to sequence single molecules, removing the need for PCR amplification during sequencing (Van Dijk et al.26). This method ensures high standardization, reliable repeatability, and superior accuracy, effectively and accurately detailing the insertion of exogenous genes, changes in the recipient genome, and the detection of plasmid scaffolds (Goodwin et al.10). Despite the vast, complex, and highly repetitive nature of the maize genome, the use of this technology in detailing the molecular characteristics of transgenic maize is still limited, presenting significant challenges (Cade et al.4).
In a previous study, two separate transgenic maize transformation events, ND4401 and ND4403, were created using the ZmNRT1.1 A nitrate gene, which is recognized for its ability to withstand low nitrogen conditions. The goal was to provide strong molecular evidence to deeply investigate these two events, aiming to develop new transgenic maize varieties that can tolerate low nitrogen levels. To enhance the safety evaluation of these events, third-generation nanopore single-molecule sequencing technology was used to accurately determine the insertion sites and surrounding sequences of the exogenous genes in the ND4401 and ND4403 events. From the sequences obtained near the exogenous genes, specific PCR primers were crafted for each transformation event and used to detect these transgenic maize events. This study highlights the effectiveness of nanopore single-molecule sequencing technology in identifying molecular characteristics in transgenic plants.
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- Source: https://www.nature.com/articles/s41598-024-83403-6