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.

Tingru Zeng,Yongxiang Li,Yanchun Song,Yu Li,Xuyang Liu,Dengfeng Zhang,Tianyu Wang,Chunhui Li,Yunsu Shi,Ruslan Kalendar

doi:10.1371/journal.pone.0226455

Tingru Zeng, Yongxiang Li + Show 8 more

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https://doi.org/10.1371/journal.pone.0226455

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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 4.18×105 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.

Highlights

Since genetically modified (GM) crops were first introduced in the US in the mid-1990s, they have become widely adopted by growers of many countries in the world [1]
By aligning with the maize genome sequence of B73 on Maize GDB and the T-DNA sequence, it was showed that the fragment was made up of 932 bp of non-insert DNA and 295 bp of insert DNA
Detailed molecular characteristics of flanking sequences of insertions play an important role in the safety assessment of genetically modified crops [30]

Summary

Introduction

Since genetically modified (GM) crops were first introduced in the US in the mid-1990s, they have become widely adopted by growers of many countries in the world [1]. Extensive testing and comprehensive analyses of transgenic lines with excellent objective traits are necessary for biosafety assessment before the lines may be approved and entering the market. Among these methods, molecular characterization of GM crops at the chromosomal level, including insertion sequences, sites, copy numbers and flanking sequences, is essential for the safety assessment and specific detection of GM crops [3, 4]. Identifying T-DNA flanking sequences of GM crops and developing specific detection methods are useful for breeding programs and important for bio-risk management to ensure food, feed and environmental safety [5, 6]

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