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

• 발행기관: 한국육종학회
• 저자명: Yang Qin Hee-Jong Woo Kong-Sik Shin Myung-Ho Lim Hyun-Suk Cho Seong-Kon Lee
• 간행물 정보: 『Plant breeding and biotechnology』Vol.5 No.4, 269~281쪽, 전체 13쪽
• 주제분류: 농수해양 > 기타농수해양
• 파일형태: PDF
• 발행일자: 2017.12.01

영문 초록

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.

목차

INTRODUCTION

MATERIALS AND METHODS

RESULTS

DISCUSSION

ACKNOWLEDGEMENTS

REFERENCES

키워드

T-DNA flanking sequence copy number NGS Southern blot hybridization Read coverage