Molecular Characterization Of Transgenic Maize GM11019 Based On High-throughput Sequencing Technology

Molecular characterization of transgenic crops mainly includes exogenous DNA(T-DNA)integration sites,flanking sequence,exogenous sequence integrity,copy number,and whether the vector backbone sequence is present.Accurate molecular characterization information plays an important role in safety assessment,regulation,traceability and labeling of GM crops.In this study,the complete and detailed molecular characterization information of this transformant material was obtained and experimentally verified based on a self-developed bioinformatics data analysis method for molecular characterization of transgenic crops,using Illumina sequencing platform and Pac Bio sequencing platform for genome resequencing,respectively.The main research contents and results are as follows:1.Information was obtained on the molecular characterization of the transgenic maize GM11019.Genomic DNA of GM11019 was extracted,Illumina and Pac Bio libraries were constructed,and whole genome resequencing was performed.The molecular characterization information of the transformant was successfully identified using a self-developed bioinformatics data analysis method by combining information from the vector sequence,maize genome and transgenic exogenous frangment database,etc.The results showed that the exogenous DNA fragment was integrated into the recipient genome by passing forwarding insertion at 162,402,905 bp ? 162,402,952 bp of chromosome 2 of the recipient genome with a single copy number of insertion.The novel deletion of 49 bp was described in the maize genome,the deletion of 719 bp and the missing 79 bp at the LB and RB ends of the expected integrated sequence.An unintended insertion fragment of5,281 bp was also identified,which was later found to be the residual sequence of the vector backbone.2.A technical system for analyzing the molecular characterization of genetically modified crops based on the combination of high-throughput sequencing technology and experimental validation was developed.Taking the GM11019 transformation event as an example,a bioinformatics method was developed that is applicable to Illumina second-generation sequencing data and Pac Bio third-generation sequencing data.Sanger sequencing verified the insertion site and flanking sequences of this transformant material,and Illumina sequencing of the Long-PCR target product verified the full-length sequence of the exogenous insertion and its sequence variation.This technical system provides a novel and reliable method for the identification of molecular characterization of complex transformants with incomplete background information of exogenous insertion sequences,deletions or insertions of large fragments.3.The advantages and disadvantages of second and third-generation sequencing technologies in the analysis of molecular characterization of transgenic crops were compared and analyzed.Illumina second-generation sequencing has short read length,uneven coverage,complex data analysis process and it is difficult to obtain all the molecular characterization information.Pac Bio third-generation sequencing technology has a high base error rate,but with its long reads,high throughput,uniform coverage and simple data analysis,it can accurately identify the integration sites of exogenous insertion sequences in one step when the vector backbone sequence is unknown.This demonstrates that Pac Bio sequencing technology has technical advantages in resolving large fragment deletions and insertions,especially in complex T-DNA insertion cases.In summary,the results of this research show that the application of high-throughput sequencing technology enables rapid,accurate and efficient identification of all molecular characterizations compared with conventional methods,providing a new technical means for the analysis of molecular characterization of genetically modified crops,while technically supporting the sound and healthy development of transgenic technology in agriculture in China.