Molecular Characterization Of Genetically Modified Maize Events

Molecular characterization is a basic component of the safety assessment and monitoring of genetically modified(GM)crops.Molecular characteristics including insertion site,flanking sequence,and copy number of GM events have to be revealed comprehensively in a case-by-case manner.Various polymerase chain reaction(PCR)-based molecular methods have been developed for such purposes,each with its unique merits and demerits,and none of them alone proves to be satisfied enough to completely reveal the molecular characteristics of a given GM event,particularly in cases where the host genome is complicated or the insertion is mixed with unintended rearrangements.The recent development of next-generation sequencing(NGS)provides a potential alternative to molecular characterize GM crops with a much higher resolution than PCR based approaches.Nevertheless,although NGS has been successfully applied to characterize GM crops such as GM soybean and GM rice,its usages in GM maize characterization is impeded due to the complex maize genome.In this study,we applied both PCR based and NGS based approaches to reveal the molecular characteristics(mainly the insertion site and the flanking sequence)of two GM maize events that were developed in China,IE09S034 and 12-5.The former is an insect-resistant GM maize,and the latter is an herbicide tolerant and insect resistant GM maize.The tested PCR approaches included thermal asymmetric interlaced PCR(Tail-PCR)and inverse PCR(I-PCR),while NGS using Illumina HiSeq X Ten platform was used as well.Our results indicated that although Tail-PCR and I-PCR have been reported to be effective in many cases,they,however,failed to reveal the insertion site and flanking sequence of these two GM events.NGS did not work for 12-5 event,and the actual insertion site of the insertion was not determined because the identified flanking sequence had a very large number of homologous fragments in maize genome.However,NGS combined with de novo assembly of the exogenous fragments identified precisely the flanking sequence and insertion site at 3' of the IE09S034 event,which were not identified by Tail-PCR or Inverse-PCR.The NGS identified 3' insertion site and PCR and subsequent Sanger sequencing of these PCR products confirmed flanking sequence of IE09S034 event.It is noteworthy that NGS also identified unintended exogenous elements in IE09S034 event including a 536 bp fragment of kanamycin gene and several backbone fragments,all were confirmed by PCR amplification and Sanger sequencing of these PCR products.Our results demonstrated the applicability of NGS in the molecular characterization of GM maize event,and at the same time,pointed out that the new NGS platforms with longer sequencing readers,such as PacBio or Nanopore could provide with the best solutions to molecular characterization of GM maize events.Finally,the output of this study provides important genetic evidence for the safety assessment and monitoring of IE09S034 maize event,and a good start point for future studies on molecular characterization of GM crops using NGS approaches.