An integrated strategy involving high‐throughput sequencing to characterize an unknown GM wheat event in Canada

Abstract

SummaryGlyphosate‐resistant wheat plants were discovered in southern Alberta in 2017, representing an unauthorized GM release in Canada. The Canadian Food Inspection Agency undertook a series of experiments to characterize and identify this unknown GM wheat, as well as to develop and validate construct‐specific and event‐specific qPCR assays. Results of PCR‐based assays and Sanger sequencing indicated the presence of CaMV 35S promoter (p35S), Rice Actin 1 intron (RactInt1), CP4‐EPSPS gene and nopaline synthase terminator (tNOS) elements in the unknown GM wheat. Genome walking and bead capture strategies, combined with high‐throughput sequencing, were used to identify the 5′ and 3′ wheat junctions and the subsequent mapping of the insert to chromosome 3B of the wheat genome. A probable transformation vector, pMON25497, was recognized, and further testing identified the unknown GM wheat as MON71200 event, one of two events obtained with the pMON25497 vector. The two construct‐specific assays targeted the junctions of the RactInt1 and the CP4‐EPSPS elements and the CP4‐EPSPS and tNOS elements, while the event‐specific assay was located at the 3′ junction into the wheat genome. Both construct‐specific and event‐specific assays had limits of detection of 0.10% of MON71200 in a seed pool. As expected, the two construct‐specific assays cross‐reacted with other wheat and corn events containing the same elements in the same order. No cross‐reactivity was observed for the event‐specific assay. The integrated strategy employed in this study can serve as a model for other cases when facing similar challenges involving unknown GM events.