Standardization of PCR and sequencing-based methods for the detection of the presence of CP4 EPSPS gene in Zea mays

Abstract

With the rise in planting of transgenic cultivars in Brazil as well as worldwide, the demand for legal regulations has increased. The transgenic event MON88017 is often found in maize cultivars marketed in Brazil. The event contains the CP4 EPSPS and cry3Bb1 genes, which encode tolerance to the herbicide glyphosate and resistance to caterpillars, respectively. Globally, methodologies to track transgenic events are mandatory. The objective of this study was to standardize a method based on qualitative PCR and sequencing for detection of the CP4 EPSPS gene in Zea mays. DNA was extracted from three commercial strains of transgenic maize, containing the MON88017 event, as well as conventional maize. Primers were designed for partial detection of the zein and CP4 EPSPS genes. PCR reactions were performed for detection of partial regions of CP4 EPSPS and Zein (as endogenous marker of Z. mays) genes. The three transgenic maize inbred lines tested positive for zein and CP4 EPSPS, and the two conventional strains tested negative for CP4 EPSPS and positive for the Zein gene. To confirm the presence of the genic regions, PCR products were sequenced and showed 100% identity with sequences of Zein and CP4 EPSPS genes deposited in GenBank. Thus, the results of this study suggest the applicability of an ‘in-house’ method for the qualitative detection of CP4 EPSPS in genetically modified maize cultivars.