AUTHOR=Peng Cheng , Mei Yingting , Ding Lin , Wang Xiaofu , Chen Xiaoyun , Wang Junmin , Xu Junfeng 

TITLE=Using Combined Methods of Genetic Mapping and Nanopore-Based Sequencing Technology to Analyze the Insertion Positions of G10evo-EPSPS and Cry1Ab/Cry2Aj Transgenes in Maize

JOURNAL=Frontiers in Plant Science

VOLUME=Volume 12 - 2021

YEAR=2021

URL=https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2021.690951

DOI=10.3389/fpls.2021.690951

ISSN=1664-462X

ABSTRACT=The insertion position of the exogenous fragment sequence in a genetically modified organism (GMO) is important for the safety assessment and labeling of GMOs. SK12-5 is a newly developed transgenic maize line transformed with two trait genes (G10evo-EPSPS, Cry1Ab/ Cry2Aj) that was recently approved for commercial use in China. Here we tried to determine the insertion position of the exogenous fragment for SK12-5. The transgene-host LB and RB integration junctions were obtained from SK12-5 genomic DNA by using Thermal Asymmetric Interlaced PCR (TAIL-PCR) and next-generation Illumina sequencing technology. However, a BLASTanalysis revealed that the flanking sequences in maize genome are unspecific, and that the insertion position is located in a repetitive sequence area in the maize genome. To locate the fine-scale insertion position in SK12-5, we combined methods of genetic mapping and nanopore-based sequencing technology. From a classical bulked segregant analysis (BSA), the insertion position in SK12-5 was mapped to Bin9.03 of chromosome 9 between the simple sequence repeat (SSR) markers umc2337 and umc1743 (26,822,048 – 100,724,531 bp). The nanopore sequencing results uncovered 10 reads for which one end was mapped to the vector and the other end to the maize genome. These observations indicated that the exogenous T-DNA fragments were putatively integrated at the position of chromosome 9 from 82329568 to 82379296 in transgenic maize SK12-5. This study is helpful for the safety assessment of the novel transgenic maize SK12-5, and shows that the combined method of genetic mapping and nanopore-based sequencing technology will be a useful approach for identifying the insertion positions of transgenic sequences in other GM plants with relatively large and complex genomes.