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

Abstract

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 4.18×10 5 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 (~10 5 × 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.

Wristwatch PCR: A Versatile and Efficient Genome Walking Strategy

“…Genome walking relies on genome library screening or the PCR. However, the construction and screening of genomic DNA libraries are cumbersome and labor-intensive ( Li et al, 2015 ; Zeng et al, 2020 ). Hence, PCR-based methods are currently promising as they are considered simple and rapid.…”

Protocol to access unknown flanking DNA sequences using Wristwatch-PCR for genome-walking

“… 2 , 3 As the advancement of molecular biology, genome-walking relying on PCR has been promising given its simplicity and clarity. 4 , 5 According to the involved rationales, the available genome-walking strategies can generally be classified into three types: 1) inverse PCR 6 ; 2) cleavage-ligation-mediated PCR 7 , 8 ; and 3) randomly primed PCR. 9 , 10 , 11 …”

Analysis of the Candidate Genes and Underlying Molecular Mechanism of P198, an RNAi-Related Dwarf and Sterile Line

“…Among these methods, thermal asymmetric interleaved PCR (TAIL-PCR) is a practical approach to locating T-DNA insertion sites in transgenic plants, such as transgenic Arabidopsis thaliana [ 23 , 24 ], Zea mays [ 25 ], Oryza sativa ( O. sativa ) [ 26 ], Nicotiana tabacum [ 27 ], and Populus plants [ 28 ]. However, these PCR-based methods are time-consuming and cannot accurately identify all T-DNA insertion sites, especially those in exceptionally complex genomes [ 29 , 30 ]. As sequencing technology has advanced, next-generation sequencing (NGS) and single-molecule real-time (SMRT) sequencing have been successfully applied in transgenic plants to identify T-DNA insertion sites [ 31 , 32 , 33 , 34 , 35 , 36 ].…”