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Acta Agronomica Sinica ›› 2022, Vol. 48 ›› Issue (7): 1843-1850.doi: 10.3724/SP.J.1006.2022.13043

• RESEARCH NOTES • Previous Articles    

Molecular characterization of transgenic maize GM11061 based on high-throughput sequencing technology

YANG Ying-Xia1(), ZHANG Guan1,2, WANG Meng-Meng1,2, LU Guo-Qing1, WANG Qian1, CHEN Rui1,*()   

  1. 1Institute of Germplasm Resources and Biotechnology, Tianjin Academy of Agricultural Sciences, Tianjin 300381, China
    2Tianjin Key Laboratory of Food Biotechnology / School of Biotechnology and Food Science, Tianjin University of Commerce, Tianjin 300134, China
  • Received:2021-06-03 Accepted:2021-11-30 Online:2022-07-12 Published:2021-12-27
  • Contact: CHEN Rui E-mail:yingxiayang@126.com;chenrui_taas@126.com

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Abstract:

Insertion site, integrated sequences, and copy number information of exogenous DNA integrations in host genome are key steps in the safety evaluation of genetically modified plants. Traditional identification techniques for molecular characteristics of transgenic plants are complicated, laborious, inefficient and limited. Here, we reported the molecular characterization of one new transgenic maize event GM11061 via a whole genome (paired-end) sequencing approach and laboratory-developed bioinformatics methods. The results showed there was only one copy of the exogenous DNA inserted, which was located within the region of 198,621,571-198,621,620 bp on chromosome 5 in GM11061 genome, without the vector skeleton sequence. Its upstream and downstream binding sites were validated using conventional PCR and sanger sequencing. Gradient analysis of sequencing data revealed that the lowest 5× resequencing raw data could be used to identify the integration site. The study confirmed that the whole genome resequencing approach combined with bioinformatics methods could achieve simple, rapid, and accurate identification of plant molecular characteristics. These results are helpful to the basic research of functional genomics and provide technical support for the safety evaluation of transgenic plants.

Key words: high throughput sequencing, transgenic maize, molecular characterization

Fig. 1

Pipeline for the molecular characterization of transgenic maize using the WGS method and the schematic diagram of the filtered clean reads"

Table 1

Results of the plasmid sequence compared with maize reference genome"

查询序列Query 目标序列
Subject		 一致性
百分比
Identity
(%)		 区域长度
Alignment length		 错配数Mismatch Gap数目
Gap
opening		 查询序列
起始位点Query
start site		 查询序列
终止位点Query
end site		 目标序列
起始位点
Subject
start site		 目标序列
终止位点
Subject
end site		 E-value Bit score
GM11061 NC_024463.2 99.950 1993 0 1 6861 8853 84,635,058 84,633,067 0 3674
GM11061 NC_024463.2 89.778 225 12 4 8189 8405 84,633,511 84,633,290 4.30E-71 278
GM11061 NC_024463.2 89.778 225 12 4 8409 8630 84,633,731 84,633,515 4.30E-71 278
GM11061 NC_024466.2 99.876 804 1 0 5135 5938 174,182,636 174,181,833 0 1480

Fig. 2

Sequence alignments of LB and RB junction-region reads for maize transgenic GM11061 A: the flanking sequences of the 5' end of the T-DNA, red and black nucleotide sequences represent the maize genome and the T-DNA sequence, respectively; B: the flanking sequences of the 3' end of the T-DNA, black and green nucleotide sequences represent the T-DNA and maize genome sequence, respectively."

Fig. 3

IGV analysis of transgenic maize GM11061 Pink boxes indicate the junction regions between the T-DNA border and maize genome."

Fig. 4

Experimental verification of maize transgenic GM11061 insertion site and flanking sequence A: the position of the primer pair designed for the upstream and downstream of the T-DNA insertion site. B: the PCR amplification result. Lanes 1 and 3 are transgenic corn GM11061, respectively; lanes 2 and 4 are non-transgenic receptor materials, respectively. C: Sanger sequencing peaks map of the upstream PCR amplification products; D: Sanger sequencing peaks map of the downstream PCR amplification products."

Table 2

Result of the contigs assembled by megahit compared with GM11061_Molecular_Characteristics.fa"

查询序列Query 目标序列Subject 一致性
百分比Identity (%)		 区域长度
Alignment length		 错配数Mismatch Gap数目
Gap
opening		 查询序列
起始位点Query
start site		 查询序列
终止位点Query
end site		 目标序列
起始位点Subject
start site		 目标序列
终止位点
Subject
end site		 E-value Bit score
k141_0 T-DNA 100 408 0 0 1 408 6313 6720 0 754
k141_0 T-DNA 100 266 0 0 1 266 6214 5949 2.61E-142 492
k141_0 T-DNA 99.306 144 1 0 265 408 10,099 10,242 8.01E-73 261
k141_3 T-DNA 100 437 0 0 1 437 10,102 10,538 0 808
k141_3 T-DNA 100 253 0 0 1 253 6580 6832 6.25E-135 468
k141_1 T-DNA 100 141 0 0 1 141 8713 8853 8.31E-73 261
k141_5 T-DNA 99.876 804 1 0 428 1231 5135 5938 0.00E+00 1480
k141_7 T-DNA 100 1993 0 0 1 1993 8853 6861 0 3681
k141_7 T-DNA 89.778 225 12 4 224 445 8405 8189 4.61E-77 278
k141_7 T-DNA 89.778 225 12 4 449 665 8630 8409 4.61E-77 278
k141_6 T-DNA 100 1530 0 0 1 1530 10,242 8713 0 2826
k141_6 T-DNA 99.306 144 1 0 1 144 6720 6577 3.08E-72 261
k141_9 T-DNA 99.761 4602 1 1 141 4732 155 4756 0.00E+00 8429
k141_9 T-DNA 100 498 0 0 4471 4968 4756 5253 0 920

Fig. 5

Sequence similarity between contigs assembled by megahit and T-DNA"

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