Application Of NGS For Molecular Characterization Of Biotech Crops

With the rapid development of sequencing technology,Next Generation Sequencing(NGS)is currently the mainstream sequencing technology in the field of nucleic acid sequence analysis,and is widely used in various omics of studies.NGS is a high-throughput,wide-coverage,rapid and efficient whole-genome resequencing method,providing a new choice for molecular characterization of biotech crops.Molecular characterization are comprehensive and accurate descriptions of changes in the genome of biotech crops and their impact.In this study,biotech rice was used as experimental material to construct a data analysis system for the analysis of molecular characterization of biotech crops.The main research contents and results are as follows:1.An NGS analysis method was developed for molecular characterization of biotech crops.The insect-resistant rice Kefeng 2 was used as the research material,and the NGS technology was used to establish a preliminary data analysis system for the analysis of molecular characterization of biotech crops.The analysis results are consistent with molecular characterization of Kefeng 2 rice obtained by hiTAIL-PCR method,which proves the feasibility and reliability of the analysis system.At the same time,the analysis system provides a relatively simple strategy for molecular characterization,that is,only using NGS to analyze the insertion site,and using PCR amplification to obtain the full-length sequence of exogenous DNA at the insertion site.For the relatively short fragment of exogenous DNA,this method is simple and applicable.2.Molecular characterization of T1C-19 and T2A-1 rice were obtained,and molecular characterization analysis system based on NGS was further improved.For the complexity of the exogenous fragment structure of two materials,a personalized analysis method was adopted.Through analysis of the sequencing data and prediction of copy number of the exogenous fragments,molecular characterization of two materials were obtained: T1C-19 rice exogenous insert was two tandem repeats of T-DNA in the same direction.The structure was verified by PCR.The partial sequence of ubiquitin gene of T2A-1 rice was reverse complemented,and the structure was verified by PacBio third generation sequencing.Therefore,the analysis system provides another molecular characterization analysis strategy,that is,molecular characterization information was obtained by NGS,PCR amplification and third generation sequencing was used to verify it.This strategy provided a reference method for solving the problem of exogenous DNA tandem repetition.In addition,the homozygosity and heterozygosity of single crop can be determined directly by sequencing data,which can provide service for genetic breeding and variety identification of biotechnology crops.In summary,the research results of this paper show that molecular characterization analysis system of biotechnology crops based on NGS technology overcomes the deficiencies of traditional analysis methods and is of great significance in the approval,supervision,traceability and identification of biotech crops.It also provides technical support for the development of biotech crops component detection technology.