Identification of Genomic Insertion and Flanking Sequences of the Transgenic Drought-tolerant Maize Line "SbSNAC1-382" using the Single Molecular Real-Time (SMRT) Sequencing Method

Abstract

Safety assessment of genetically modified (GM) crops is crucial in the phase of product development before the GM crops are put on the market. Characteristics of flanking sequences of exogenous insertion sequences are essential for the safety assessment and marking of transgenic crops. In this study, we used the methods of genome walking and whole genome sequencing (WGS) to identify the flanking sequence characteristics of a SbSNAC1 transgenic drought-tolerant maize line "SbSNAC1-382", but both of the methods failed. Then, we constructed a genomic fosmid library of the transgenic maize line, which contained 4.18x105 clones with an average insertion fragment of 35 kb, covering 5.85 times of 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 the Single Molecule Real-Time (SMRT) sequencing technology. More than 1.95 Gb sequence data ([~]105 x coverage) for the sequenced clone was 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 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 in the same genomic site. And the exogenous T-DNA fragments were integrated at the position of Chromosome 5: 177155650 to 177155696 in the transgenic line 382. Herein, we have demonstrated the successful application of the SMRT technology for the characterization of genomic insertion and flanking sequences.