Jilin Provincial Key Laboratory of Agricultural Biotechnology, Jilin Academy of Agricultural Sciences, Changchun, 130033, China.
School of Life Science, Jilin Normal University, Siping, 136000, China.
Transgenic Res. 2021 Feb;30(1):1-9. doi: 10.1007/s11248-020-00225-8. Epub 2021 Jan 4.
Genomic insertions and flanking regions of transgenes in host genomes constitute a critical component of precise molecular characterization and event-specific detection, which are required in the development and assessment for regulatory approval of genetically modified (GM) crops. Previously, we reported three transgenic soybean events harboring the inverted repeats of the soybean mosaic virus NIb (nuclear inclusion b) gene, exhibiting significantly enhanced resistance to multiple Potyvirus strains. To facilitate safety assessment and event-specific detection, we identified the transgene insertion sites and flanking sequences of the events L120, L122, and L123 using whole-genome sequencing. More than 14.48 Gb sequence data (13 × coverage) were generated using the Illumina HiSeq Xten platform for each event. The sequence reads corresponding to boundaries of inserted T-DNA, and associated native flanking sequences were identified by bioinformatic comparison with the soybean reference genome (Wm82.a2.v1) and the transformation vector sequence. The results indicated that two T-DNA insertions occurred in L120, on Chr07 and Chr13, while L122 and L123 showed single insertions, on Chr02 and Chr06, respectively. Based on the flanking sequences of the inserted T-DNA, the event-specific detection for each event was established using specific PCR primers, and PCR amplification followed by sequencing of PCR products further confirmed the putative insertion loci and flanking regions in the transgenic lines. Our results demonstrate the efficacy and robustness of whole-genome sequencing in identifying the genomic insertions and flanking regions in GM crops. Moreover, the characterization of insertion loci and the establishment of event-specific detection will facilitate the application and development of broad-spectrum virus-resistant transgenic soybean cultivars.
基因组插入和转基因侧翼区域是精确分子特征描述和事件特异性检测的关键组成部分，这是转基因（GM）作物开发和监管审批所必需的。此前，我们报道了三个含有大豆花叶病毒 NIb（核包含体 b）基因反向重复的转基因大豆事件，表现出对多种马铃薯 Y 病毒株系的显著增强抗性。为了便于进行安全性评估和事件特异性检测，我们使用全基因组测序鉴定了事件 L120、L122 和 L123 的转基因插入位点和侧翼序列。每个事件使用 Illumina HiSeq Xten 平台生成了超过 14.48 Gb 的序列数据（13×覆盖度）。通过与大豆参考基因组（Wm82.a2.v1）和转化载体序列的生物信息学比较，确定了与插入 T-DNA 边界和相关天然侧翼序列相对应的序列读段。结果表明，L120 中的两个 T-DNA 插入发生在 Chr07 和 Chr13，而 L122 和 L123 分别在 Chr02 和 Chr06 中显示出单个插入。基于插入 T-DNA 的侧翼序列，使用特异性 PCR 引物建立了每个事件的特异性检测方法，随后对 PCR 产物进行 PCR 扩增和测序进一步证实了转基因系中假定的插入位点和侧翼区域。我们的结果证明了全基因组测序在鉴定 GM 作物基因组插入和侧翼区域方面的有效性和稳健性。此外，插入位点的特征描述和事件特异性检测的建立将有助于广谱抗病毒转基因大豆品种的应用和开发。
