Efficient identification of genomic insertions and surrounding regions in two transgenic maize events using third-generation single-molecule nanopore sequencing technology
Abstract The increasing development of new genetically modified organisms underscores the critical need for comprehensive safety assessments, emphasizing the significance of molecular evidence such as gene integration, copy numbers, and adjacent sequences. In this study, the maize nitrate-efficient...
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Nature Portfolio
2024-12-01
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| Online Access: | https://doi.org/10.1038/s41598-024-83403-6 |
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| author | Qing Liu Qi Wang Lihua Ning Ziqi Chen Chuang Zhang Yang Liu Buxuan Qian Jia Guo Yuejia Yin |
| author_facet | Qing Liu Qi Wang Lihua Ning Ziqi Chen Chuang Zhang Yang Liu Buxuan Qian Jia Guo Yuejia Yin |
| author_sort | Qing Liu |
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| description | Abstract The increasing development of new genetically modified organisms underscores the critical need for comprehensive safety assessments, emphasizing the significance of molecular evidence such as gene integration, copy numbers, and adjacent sequences. In this study, the maize nitrate-efficient utilization gene ZmNRT1.1 A was introduced into maize variety y822 using transgenic technology, producing transgenic maize events ND4401 and ND4403 with enhanced tolerance to low nitrogen stress. Southern hybridization confirmed that the exogenous T-DNA was singly inserted in both maize transformation events, ND4401 and ND4403. This study utilized third-generation sequencing technology—nanopore single-molecule sequencing—to perform molecular characterization of the integration events. It successfully determined the exogenous gene insertion sites and flanking sequences in ND4401 and ND4403. Comparative analysis with the control group facilitated the preliminary identification of the integration sites of the exogenous T-DNA fragments in these transgenic maize events. Based on the obtained flanking sequences, specific PCR primers were designed for different transformation events. The insertion site for ND4401 was pinpointed in the non-coding region of chromosome 5, and for ND4403, in the non-coding region of chromosome 3. Utilizing the sequencing results, the study developed specific detection primers for the maize transformation events, establishing a precise method for detecting newly created transgenic maize events, which will contribute to subsequent safety assessments. |
| format | Article |
| id | doaj-art-a83d484edf184efe976274a1fdf515cf |
| institution | Kabale University |
| issn | 2045-2322 |
| language | English |
| publishDate | 2024-12-01 |
| publisher | Nature Portfolio |
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| series | Scientific Reports |
| spelling | doaj-art-a83d484edf184efe976274a1fdf515cf2025-01-05T12:28:19ZengNature PortfolioScientific Reports2045-23222024-12-011411910.1038/s41598-024-83403-6Efficient identification of genomic insertions and surrounding regions in two transgenic maize events using third-generation single-molecule nanopore sequencing technologyQing Liu0Qi Wang1Lihua Ning2Ziqi Chen3Chuang Zhang4Yang Liu5Buxuan Qian6Jia Guo7Yuejia Yin8Institute of Agricultural Biotechnology/Institute of Agricultural Quality Standard and Testing Technology, Jilin Academy of Agricultural Sciences (Northeast Innovation Center of Agricultural Science and Technology in China)Institute of Agricultural Biotechnology/Institute of Agricultural Quality Standard and Testing Technology, Jilin Academy of Agricultural Sciences (Northeast Innovation Center of Agricultural Science and Technology in China)Jiangsu Provincia Key Laboratory of Agrobiology, Jiangsu Academy of Agricultural SciencesInstitute of Agricultural Biotechnology/Institute of Agricultural Quality Standard and Testing Technology, Jilin Academy of Agricultural Sciences (Northeast Innovation Center of Agricultural Science and Technology in China)Institute of Agricultural Biotechnology/Institute of Agricultural Quality Standard and Testing Technology, Jilin Academy of Agricultural Sciences (Northeast Innovation Center of Agricultural Science and Technology in China)Institute of Agricultural Biotechnology/Institute of Agricultural Quality Standard and Testing Technology, Jilin Academy of Agricultural Sciences (Northeast Innovation Center of Agricultural Science and Technology in China)Institute of Agricultural Biotechnology/Institute of Agricultural Quality Standard and Testing Technology, Jilin Academy of Agricultural Sciences (Northeast Innovation Center of Agricultural Science and Technology in China)Institute of Agricultural Biotechnology/Institute of Agricultural Quality Standard and Testing Technology, Jilin Academy of Agricultural Sciences (Northeast Innovation Center of Agricultural Science and Technology in China)Institute of Agricultural Biotechnology/Institute of Agricultural Quality Standard and Testing Technology, Jilin Academy of Agricultural Sciences (Northeast Innovation Center of Agricultural Science and Technology in China)Abstract The increasing development of new genetically modified organisms underscores the critical need for comprehensive safety assessments, emphasizing the significance of molecular evidence such as gene integration, copy numbers, and adjacent sequences. In this study, the maize nitrate-efficient utilization gene ZmNRT1.1 A was introduced into maize variety y822 using transgenic technology, producing transgenic maize events ND4401 and ND4403 with enhanced tolerance to low nitrogen stress. Southern hybridization confirmed that the exogenous T-DNA was singly inserted in both maize transformation events, ND4401 and ND4403. This study utilized third-generation sequencing technology—nanopore single-molecule sequencing—to perform molecular characterization of the integration events. It successfully determined the exogenous gene insertion sites and flanking sequences in ND4401 and ND4403. Comparative analysis with the control group facilitated the preliminary identification of the integration sites of the exogenous T-DNA fragments in these transgenic maize events. Based on the obtained flanking sequences, specific PCR primers were designed for different transformation events. The insertion site for ND4401 was pinpointed in the non-coding region of chromosome 5, and for ND4403, in the non-coding region of chromosome 3. Utilizing the sequencing results, the study developed specific detection primers for the maize transformation events, establishing a precise method for detecting newly created transgenic maize events, which will contribute to subsequent safety assessments.https://doi.org/10.1038/s41598-024-83403-6Maize (Zea mays L.)TransgenicNanopore single-molecule sequencingExogenous gene insertion sites |
| spellingShingle | Qing Liu Qi Wang Lihua Ning Ziqi Chen Chuang Zhang Yang Liu Buxuan Qian Jia Guo Yuejia Yin Efficient identification of genomic insertions and surrounding regions in two transgenic maize events using third-generation single-molecule nanopore sequencing technology Scientific Reports Maize (Zea mays L.) Transgenic Nanopore single-molecule sequencing Exogenous gene insertion sites |
| title | Efficient identification of genomic insertions and surrounding regions in two transgenic maize events using third-generation single-molecule nanopore sequencing technology |
| title_full | Efficient identification of genomic insertions and surrounding regions in two transgenic maize events using third-generation single-molecule nanopore sequencing technology |
| title_fullStr | Efficient identification of genomic insertions and surrounding regions in two transgenic maize events using third-generation single-molecule nanopore sequencing technology |
| title_full_unstemmed | Efficient identification of genomic insertions and surrounding regions in two transgenic maize events using third-generation single-molecule nanopore sequencing technology |
| title_short | Efficient identification of genomic insertions and surrounding regions in two transgenic maize events using third-generation single-molecule nanopore sequencing technology |
| title_sort | efficient identification of genomic insertions and surrounding regions in two transgenic maize events using third generation single molecule nanopore sequencing technology |
| topic | Maize (Zea mays L.) Transgenic Nanopore single-molecule sequencing Exogenous gene insertion sites |
| url | https://doi.org/10.1038/s41598-024-83403-6 |
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