Cargando…

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

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 [i.e., G10evo-5-enolpyrul-shikimate-3-phosphate synthase...

Descripción completa

Detalles Bibliográficos
Autores principales: Peng, Cheng, Mei, Yingting, Ding, Lin, Wang, Xiaofu, Chen, Xiaoyun, Wang, Junmin, Xu, Junfeng
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8358107/
https://www.ncbi.nlm.nih.gov/pubmed/34394143
http://dx.doi.org/10.3389/fpls.2021.690951
_version_ 1783737267569819648
author Peng, Cheng
Mei, Yingting
Ding, Lin
Wang, Xiaofu
Chen, Xiaoyun
Wang, Junmin
Xu, Junfeng
author_facet Peng, Cheng
Mei, Yingting
Ding, Lin
Wang, Xiaofu
Chen, Xiaoyun
Wang, Junmin
Xu, Junfeng
author_sort Peng, Cheng
collection PubMed
description 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 [i.e., G10evo-5-enolpyrul-shikimate-3-phosphate synthase (EPSPS) and Cry1Ab/Cry2Aj] that was recently approved for commercial use in China. In this study, we tried to determine the insertion position of the exogenous fragment for SK12-5. The transgene–host left border and right border integration junctions were obtained from SK12-5 genomic DNA by using the thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR) and next-generation Illumina sequencing technology. However, a Basic Local Alignment Search Tool (BLAST) analysis revealed that the flanking sequences in the 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 the methods of genetic mapping and nanopore-based sequencing technology. From a classical bulked-segregant analysis (BSA), the insertion position in SK12-5 was mapped onto 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 onto the vector and the other end was mapped onto the maize genome. These observations indicated that the exogenous T-DNA fragments were putatively integrated at the position from 82,329,568 to 82,379,296 bp of chromosome 9 in the 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 the 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.
format Online
Article
Text
id pubmed-8358107
institution National Center for Biotechnology Information
language English
publishDate 2021
publisher Frontiers Media S.A.
record_format MEDLINE/PubMed
spelling pubmed-83581072021-08-13 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 Peng, Cheng Mei, Yingting Ding, Lin Wang, Xiaofu Chen, Xiaoyun Wang, Junmin Xu, Junfeng Front Plant Sci Plant Science 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 [i.e., G10evo-5-enolpyrul-shikimate-3-phosphate synthase (EPSPS) and Cry1Ab/Cry2Aj] that was recently approved for commercial use in China. In this study, we tried to determine the insertion position of the exogenous fragment for SK12-5. The transgene–host left border and right border integration junctions were obtained from SK12-5 genomic DNA by using the thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR) and next-generation Illumina sequencing technology. However, a Basic Local Alignment Search Tool (BLAST) analysis revealed that the flanking sequences in the 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 the methods of genetic mapping and nanopore-based sequencing technology. From a classical bulked-segregant analysis (BSA), the insertion position in SK12-5 was mapped onto 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 onto the vector and the other end was mapped onto the maize genome. These observations indicated that the exogenous T-DNA fragments were putatively integrated at the position from 82,329,568 to 82,379,296 bp of chromosome 9 in the 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 the 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. Frontiers Media S.A. 2021-07-29 /pmc/articles/PMC8358107/ /pubmed/34394143 http://dx.doi.org/10.3389/fpls.2021.690951 Text en Copyright © 2021 Peng, Mei, Ding, Wang, Chen, Wang and Xu. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Plant Science
Peng, Cheng
Mei, Yingting
Ding, Lin
Wang, Xiaofu
Chen, Xiaoyun
Wang, Junmin
Xu, Junfeng
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
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
title_full 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
title_fullStr 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
title_full_unstemmed 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
title_short 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
title_sort 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
topic Plant Science
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8358107/
https://www.ncbi.nlm.nih.gov/pubmed/34394143
http://dx.doi.org/10.3389/fpls.2021.690951
work_keys_str_mv AT pengcheng usingcombinedmethodsofgeneticmappingandnanoporebasedsequencingtechnologytoanalyzetheinsertionpositionsofg10evoepspsandcry1abcry2ajtransgenesinmaize
AT meiyingting usingcombinedmethodsofgeneticmappingandnanoporebasedsequencingtechnologytoanalyzetheinsertionpositionsofg10evoepspsandcry1abcry2ajtransgenesinmaize
AT dinglin usingcombinedmethodsofgeneticmappingandnanoporebasedsequencingtechnologytoanalyzetheinsertionpositionsofg10evoepspsandcry1abcry2ajtransgenesinmaize
AT wangxiaofu usingcombinedmethodsofgeneticmappingandnanoporebasedsequencingtechnologytoanalyzetheinsertionpositionsofg10evoepspsandcry1abcry2ajtransgenesinmaize
AT chenxiaoyun usingcombinedmethodsofgeneticmappingandnanoporebasedsequencingtechnologytoanalyzetheinsertionpositionsofg10evoepspsandcry1abcry2ajtransgenesinmaize
AT wangjunmin usingcombinedmethodsofgeneticmappingandnanoporebasedsequencingtechnologytoanalyzetheinsertionpositionsofg10evoepspsandcry1abcry2ajtransgenesinmaize
AT xujunfeng usingcombinedmethodsofgeneticmappingandnanoporebasedsequencingtechnologytoanalyzetheinsertionpositionsofg10evoepspsandcry1abcry2ajtransgenesinmaize