Molecular Characterization Of Transgenic Rice Based On Pair-end Resequencing

DNA recombination has been wildly used in modern agricultural production.More than 400 transgenic plant events have been approved and commercialized in globe with the area of 185.1 million hectares at the end of 2016.However,the concerns on safety issues of biotech products,especially for transgenic crops,still exist in the public.Therefore,many countries have emplemented strict risk assessments and regulations on biotech crops.In general,the description of the molecular characterization of biotech crops is the primary step,including the exogenous DNA expression vector,exogenous DNA integration locus and flanking sequences,size and structure of the exogenous DNA insert,copy number of the insert,any partial DNA residual of the vector or carrier,the stability of the insert,etc.In this study,we focused on developing an algorithm and corresponging tools to depcit molecular characterization of biotech crops based on pair-end(including mate-pair)resequencing.The results confirmed the applicaibility of developed algorithm: 1)the insertion site and flanking sequences,insert copy number,entire inserted DNA sequences and plasmid backbone sequences residuals of transgenic rice G6H1 with single plasmid transformation;2)the insertion site and flanking sequences,insert copy number and plasmid backbone sequences residuals of transgenic rice 114-7-2 with two plasmids co-transformation.The advantages were shown as below: 1)this strategy and algorithm can be used for different sequencing data,for example mate-pair and pair-end sequencing data;2)this method can be used to solve complicated exogenous DNA integration data,for example multi-insertion site and multi-copy gene rearragment have been identified in 114-7-2 sequencing data;3)the algorithm is well built,the computationally efficient and the experimental validation process is simple and easy-to-use.In order to clearly character the complex integration of exogenous gene,such as multi-copy gene insertion or muilt-inserted long DNA,we proposed a new method for long target DNA fragments capture strategy combined with single molecular sequencing technology.Up to date,the primary long fragment DNA library and targets capture protocol has been established,and the sequencing and data analysis will be performed in future.