Flanking Sequence and Copy-Number Analysis of Transformation Events by Integrating Next-Generation Sequencing Technology with Southern Blot Hybridization

• Published in: Plant breeding and biotechnology pp. 269 - 281
• Main Authors: 친양, 우희종, 신공식, 임명호, 조현석, 이성곤
• Format: Journal Article
• Language: English
• Published: 한국육종학회 01.12.2017
• Subjects: 농학
• ISSN: 2287-9358; 2287-9366
• DOI: 10.9787/PBB.2017.5.4.269

With the continual development of genetically modified (GM) crops, it has become necessary to develop detailed and effective molecular characterization methods to select candidate events from a large pool of transformation events. Relative to traditional molecular analysis methods such as the polymerase chain reaction (PCR) and Southern blot hybridization, next generation sequencing (NGS) technology for whole-genome sequencing of complex crop genomes had proven comparatively useful for in-depth molecular characterization. In this study, four transformation events, including one in Bacillus thuringiensis (Bt)-resistant rice, one in resveratrol-producing rice, and two in beta-carotene-enhanced soybeans, were selected for molecular characterization. To merge NGS analysis and Southern blot-hybridization results, we confirmed the transgene insertion sites, insertion construction, and insertion numbers of these four transformation events. In addition, the read-coverage depth assessed by NGS analysis for inserted genes might provide consistent results in terms of inserted T-DNA numbers in case of complex insertion structures and highly duplicated donor genomes; however, PCR-based methods can produce incorrect conclusions. Our combined method provides an effective and complete analytical approach for whole-genome visual inspection of transformation events that require biosafety assessment. KCI Citation Count: 0