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Acta Agronomica Sinica ›› 2020, Vol. 46 ›› Issue (11): 1703-1710.doi: 10.3724/SP.J.1006.2020.02002

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

Molecular characterization identification by genome sequencing of transgenic glyphosate-tolerant rice G2-7

MA Shuo1(), JIAO Yue2(), YANG Jiang-Tao1, WANG Xu-Jing1,*(), WANG Zhi-Xing1,*()   

  1. 1 Biotechnology Research Institute, Chinese Academy of Agricultural Sciences / MARA Key Laboratory on Safety Assessment Molecular of Agri-GMO, Beijing 100081, China
    2 Development Center for Science and Technology / MARA, Beijing 100122, China
  • Received:2020-01-14 Accepted:2020-06-02 Online:2020-11-12 Published:2020-06-22
  • Contact: Xu-Jing WANG,Zhi-Xing WANG E-mail:mashuo0801@163.com;jiaoyue@agri.gov.cn;wangxujing@caas.cn;wangcotton@126.com
  • Supported by:
    This study was supported by the National Major Project for Developing New GM Crops(2016ZX08010-003)

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Abstract:

Molecular characterization, such as copy number and flanking sequence of foreign DNA fragment insertion site, is the important identity information, provided during safety assessment of genetic modified crop. In this study, the T-DNA insertion site, copy number and flanking sequences were identified in transgenic glyphosate-tolerant rice G2-7 based on whole genome sequencing in combination bioinformatics analysis method. 47.13 Gb clean sequence data for G2-7 was generated on Illumina NovaSeq 6000 platform. The junction reads mapped to boundaries of T-DNA and flanking sequences in G2-7 were identified by comparing with sequence of transformation vector and rice reference genome. The results showed that exogenous T-DNA fragments was integrated in the position of Chr. 1 36,189,491-36,189,507 with a single copy, 16 bp rice genome sequence was deleted at the insertion site and no insertion of vector backbone. 375 bp and 353 bp flanking host DNA sequence of 5′-end and 3′-end of the insertion DNA fragment were also obtained, respectively. The putative insertion location and flanking sequences were further confirmed by PCR amplification and Sanger sequencing. The results not only provided data support for safety assessment and event specific detection, but also demonstrated that WGS was an effective technique for identifying molecular characterization in rice.

Key words: genome sequencing, transgenic rice, molecular characterization, copy number, flanking sequence

Table 1

High throughput sequencing data quality control statistics"

样品名称
Sample		 原始数据量
Raw bases		 原始读序量
Raw reads		 有效数据量
Clean bases		 有效读序量Clean reads Q20
(%)		 Q30
(%)		 GC含量
GC content (%)
ZH11 41,196,003,900 274,646,422 41,155,766,400 274,371,776 96.22 90.36 43.04
ZH11-p 41,266,820,400 275,112,136 41,228,215,500 274,854,770 97.73 93.69 42.93
G2-7 47,246,859,300 314,990,174 47,125,680,000 314,171,200 96.58 91.15 44.27

Fig. 1

Analysis of the binding site between the inserted fragments in G2-7 and the receptor genome (comparison results of some binding region sequences)"

Fig. 2

Visualization of sequencing data and plasmid DNA comparison results"

Fig. 3

PCR verification of reads aligned to backbone"

Fig. 4

Analysis of integration site and flanking sequence of insert DNA fragment in receptor genome A: analysis of flanking sequence and integration site of G2-7; B: sketch map of insert DNA integration in G2-7."

Fig. 5

Agarose gel electrophoresis and sequence alignment results A: amplification of rice transformant G2-7 flanking sequence; 1: G2-7-5F/5R; 2: ZH11-5F/5R; 3: G2-7-3F/3R; 4: ZH11-3F/3R. B: G2-7 5′ end sequence comparison and verification. C: G2-7 5′ end sequence comparison and verification."

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