Identification of genomic insertion and flanking sequences of the transgenic drought-tolerant maize line “SbSNAC1-382” using the single-molecule real-time (SMRT) sequencing method

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（GM）作物上市前的产品开发阶段，对其开展安全评估至关重要。明确外源插入序列的侧翼区域特征，是转基因作物安全评估与商业化推广的核心环节。本研究采用基因组步移与全基因组测序（WGS）技术，对转SbSNAC1基因抗旱玉米株系"SbSNAC1-382"的侧翼序列特征进行鉴定，但两种方法均未获得有效结果。随后，我们构建了该转基因玉米株系的基因组黏粒文库，该文库包含4.18×105个克隆，平均插入片段长度为35 kb，覆盖度达玉米基因组的5.85倍。继而通过多对特异性引物筛选得到3个阳性克隆，并采用单分子实时（SMRT）测序技术对其中1个阳性克隆进行测序，共获得超过1.95 Gb的测序数据（覆盖度约105×）。将比对到T-DNA边界的连接读段与玉米参考基因组及转基因载体序列进行比对，成功鉴定出该转基因株系的侧翼序列。此外，通过聚合酶链式反应（PCR）扩增与桑格测序，验证了推定的插入位点与侧翼序列。结果表明，2个外源T-DNA片段插入至同一基因组位点，且该外源片段整合于转基因株系382的5号染色体177155650至177155696位置。本研究证实了单分子实时测序技术可有效用于基因组插入位点与侧翼序列的特征鉴定。