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

9Safety assessment of genetically modified (GM) crops is crucial in the phase of 10 product development before the GM crops are put on the market. Characteristics of 11 flanking sequences of exogenous insertion sequences are essential for the safety 12 assessment and marking of transgenic crops. In this study, we used the methods of 13 genome walking and whole genome sequencing (WGS) to identify the flanking 14 sequence characteristics of a SbSNAC1 transgenic drought-tolerant maize line 15 "SbSNAC1-382", but both of the methods failed. Then, we constructed a genomic 16 fosmid library of the transgenic maize line, which contained 4.18×10 5 clones with an 17 average insertion fragment of 35 kb, covering 5.85 times of the maize genome. 18 Subsequently, three positive clones were screened by pairs of specific primers and 19 one of the three positive clones was sequenced by using the Single Molecule 20 Real-Time (SMRT) sequencing technology. More than 1.95 Gb sequence data (~10 5  21 coverage) for the sequenced clone was generated. The junction reads mapped to the 22 boundaries of T-DNA and the flanking sequences in the transgenic line were 2 23identified by comparing all sequencing reads with the maize reference genome and 24 the sequence of transgenic vector. Furthermore, the putative insertion loci and 25 flanking sequences were confirmed by PCR amplification and Sanger sequencing. 26 The results indicated that two copies of the exogenous T-DNA fragments were 27 inserted in the same genomic site. And the exogenous T-DNA fragments were 28 integrated at the position of Chromosome 5: 177155650 to 177155696 in the 29 transgenic line 382. Herein, we have demonstrated the successful application of the 30 SMRT technology for the characterization of genomic insertion and flanking 31 sequences. 32 Keywords: transgenic maize, flanking sequence, fosmid library, SMRT 33 sequencing 34 Introduction 35Since genetically modified (GM) crops were first introduced in the U. S. in the 36 mid-1990s, they have become widely adopted by growers of many countries in the 37 world [1]. In 2017 alone, 189.8 million hectares of GM crops were planted worldwide 38 [2]. It is an international consensus that GM crops could be commercialized after they 39 are proven to be safe. As a result, extensive testing and comprehensive analyses of 40 transgenic lines with excellent objective traits are necessary for biosafety assessment 41 before being approved and entering into market. Among these, molecular 42 characterization of GM crops at the chromosomal level including insertion sequences, 43 sites, copy numbers and flanking sequences is essential for the safety assessment and 44 specific detection of GM crops [3, 4]. Furthermore, identification of T-DNA flanking 3 45 sequences of GM crops and the development of specific detection methods are useful 46 for breeding program, and important for bio-risk management to ensure food, feed 47 and environmental safety [5, 6]. 48 Traditionally, exogenous fragments flanking sequences of transgenic plants a...