Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing, China.
PLoS One. 2020 Apr 10;15(4):e0226455. doi: 10.1371/journal.pone.0226455. eCollection 2020.
Safety assessment of genetically modified (GM) crops is crucial at the product-development phase before GM crops are placed on the market. Determining characteristics of sequences flanking exogenous insertion sequences is essential for the safety assessment and marketing of transgenic crops. In this study, we used genome walking and whole-genome sequencing (WGS) to identify the flanking sequence characteristics of the SbSNAC1 transgenic drought-tolerant maize line "SbSNAC1-382", but both of the two methods failed. Then, we constructed a genomic fosmid library of the transgenic maize line, which contained 4.18×105 clones with an average insertion fragment of 35 kb, covering 5.85 times the maize genome. Subsequently, three positive clones were screened by pairs of specific primers, and one of the three positive clones was sequenced by using single-molecule real-time (SMRT) sequencing technology. More than 1.95 Gb sequence data (~105× coverage) for the sequenced clone were generated. The junction reads mapped to the boundaries of T-DNA, and the flanking sequences in the transgenic line were identified by comparing all sequencing reads with the maize reference genome and the sequence of the transgenic vector. Furthermore, the putative insertion loci and flanking sequences were confirmed by PCR amplification and Sanger sequencing. The results indicated that two copies of the exogenous T-DNA fragments were inserted at the same genomic site, and the exogenous T-DNA fragments were integrated at the position of Chromosome 5 from 177155650 to 177155696 in the transgenic line 382. In this study, we demonstrated the successful application of the SMRT technology for the characterization of genomic insertion and flanking sequences.
转基因作物的安全性评估在产品开发阶段至关重要，在转基因作物投放市场之前。确定外源插入序列侧翼序列的特征对于转基因作物的安全性评估和营销至关重要。在本研究中，我们使用基因组步移和全基因组测序（WGS）来鉴定抗旱转基因玉米品系“ SbSNAC1-382”的侧翼序列特征，但这两种方法都失败了。然后，我们构建了转基因玉米品系的基因组fosmid 文库，该文库包含 4.18×105个克隆，平均插入片段为 35kb，覆盖玉米基因组的 5.85 倍。随后，通过两对特异性引物筛选出三个阳性克隆，并用单分子实时（SMRT）测序技术对其中一个阳性克隆进行测序。该测序克隆产生了超过 1.95 Gb 的序列数据（~105×覆盖度）。连接读取映射到 T-DNA 的边界，通过将所有测序读取与玉米参考基因组和转基因载体序列进行比较，鉴定了转基因系中的侧翼序列。此外，通过 PCR 扩增和 Sanger 测序证实了潜在的插入位点和侧翼序列。结果表明，两个外源 T-DNA 片段插入到相同的基因组位点，外源 T-DNA 片段整合到 382 号转基因系第 5 号染色体上 177155650 到 177155696 的位置。在本研究中，我们证明了 SMRT 技术成功应用于基因组插入和侧翼序列的特征描述。
