Beyond traditional GMOs: a comprehensive approach to identify unknown cisgenic rice events

We investigated unauthorized glyphosate-tolerant rice plants from fields where no genetically modified herbicide-resistant varieties have regulatory approval. The unusual herbicide tolerance phenotype suggested potential unauthorized genetic modification, necessitating comprehensive molecular characterization. We employed integrated analytical approaches: PCR screening for transgenic elements, quantitative polymerase chain reaction for copy number determination, Illumina whole-genome sequencing, and PacBio long-read sequencing. Bioinformatic analysis identified integration sites and insertion structures. Gene-specific and event-specific detection assays were developed following international regulatory standards. We identified six independent cisgenic rice events, each containing 4-9 tandem copies of a mutated rice OsEPSPS gene conferring glyphosate tolerance. Each event exhibited unique chromosomal integration sites, distinct flanking sequences, and complex tandem repeat structures. The mutations were absent from natural rice germplasm databases (3K RG), confirming intentional genetic modification. Detection assays achieved 0.05-0.1% limits of detection, meeting international performance standards. This study reveals critical gaps in current genetically modified organism monitoring systems that fail to detect cisgenic products. Our findings demonstrate that unauthorized cisgenic crops can evade conventional regulatory oversight, challenging biosafety management and international trade under process-based frameworks. This work underscores the urgent need to transition from element-based detection to comprehensive genomic approaches, providing essential methodologies for detecting new breeding technique products and maintaining regulatory oversight.