An Integrated Strategy for Analyzing the Complete Complex Integrated Structure of Maize MON810 and Identification of an SNP in External Insertion Sequences

Abstract

Genetically modified maize (Zea mays L.) MON810 was approved for importation into China for feed use in 2004; however, the localization data concerning exogenous insertion sequences, which confer insect resistance, have been questionable. MON810 maize plants discovered in northeastern China were used to analyze the molecular characteristics of the exogenous insertion. Using PacBio-HiFi sequencing and PCR assays, we found the insertion was located in chromosome 8, and there was a CaMV35S promoter, hsp70 intron, and insecticide gene cry1Ab, except for genome sequence insertion in the MON810 event. Importantly, the 5′ and 3′ flanking sequences were located in the region of 55869747–55879326 and 68416240–68419152 on chromosome 5, respectively. The results of this study correct previous results on the genomic localization of the insertion structure for the MON810 event. We also found a single-nucleotide polymorphism (SNP) in the hsp70 intron, which is most likely the first SNP found in a transgenic insertion sequence. PCR amplification in conjunction with Sanger sequencing, allele-specific PCR (AS-PCR), and blocker displacement amplification (BDA) assays were all effective at detecting the base variance. The integrated strategy used in this study can serve as a model for other cases when facing similar challenges involving partially characterized genetic modification events or SNPs.

Keywords

• MON810
• Pacbio-Hifi
• single-nucleotide polymorphism
• allele-specific PCR
• blocker displacement amplification