|Table of Contents|

Efficient Identification of T-DNA Insertions and Qualitative Analysis in Two Virus-resistant Transgenic Soybean Events(PDF)

《大豆科学》[ISSN:1000-9841/CN:23-1227/S]

Issue:
2020年01期
Page:
23-29
Research Field:
Publishing date:

Info

Title:
Efficient Identification of T-DNA Insertions and Qualitative Analysis in Two Virus-resistant Transgenic Soybean Events
Author(s):
LIU Dong-bo1; 2;  XING Guo-jie2;  ZHAO Qian-qian2;  YANG Jing2;  NIU Lu2;  YANG Xiang-dong2;  ZHONG Xiao-fang2
(1.School of Life Science, Jilin Normal University, Siping 136000, China; 2.Agricultural Biotechnology Institute, Jilin Academy of Agricultural Sciences/Jilin Provincial Key Laboratory of Agricultural Biotechnology, Changchun 130033, China)
Keywords:
Insertion site;  Flanking sequence;  Whole genome sequencing;  Virus-resistant transgenic soybean;  Time-specific detection
PACS:
-
DOI:
10.11861/j.issn.1000-9841.2020.01.0023
Abstract:
Genomic insertions and flanking regions of transgenes in host genomes constitute a critical component of precise molecular characterization and event-specific detection, which are required in development and assessment for regulatory approval of genetically modified (GM) crops. Previously, we reported two transgenic soybean events, that were L13 and L104, harboring the reverted repeats of soybean mosaic virus P3 gene, exhibiting significantly enhanced and broad-spectrum resistance to soybean mosaic virus, soybean mosaic necrosis virus, watermelon mosaic virus. To facilitate safety assessment and event-specific detection establishment, we identified the transgenic insertion sites and flanking sequences of the event L13 and L104 using whole genome sequencing(WGS) through Illumina Xten platform combined with bioinformatics analysis and PCR method. More than 12.13 Gb sequence data (11 coverage) were generated of each event through WGS. The sequence reads corresponding to boundaries of inserted T-DNA and associated native flanking sequences were identified by comparison with soybean reference genome(Wm82.a2.v1) and sequence of the transformation vector. Inserted T-DNAs were mapped on Chromosome 04 and 07 for the events L13 and L104, respectively. The putative insertion loci and flanking regions were further confirmed by PCR amplification and sequencing. The T-DNAs of the transgenic event L13 were two copies inserted in Chr04:46747946 site. And the T-DNA of the L104 was inserted in Chr11:30461895 with one copy. Based on the flanking sequences of the inserted T-DNA, the event-specific detection for each event was established with specific PCR primers. Our results demonstrate the efficacy and robustness of whole genome sequencing in identifying the T-DNA insertions and flanking regions of GM crops. Moreover, characterization of insertion loci and establishment of event-specific detection will facilitate the application and development of broad-spectrum virus-resistant transgenic soybean cultivars.

References:

［1］Latham J R, Wilson A K, Steinbrecher R A. The mutational consequences of plant transformation［J］. Journal of Biomedicine and Biotechnollogy, 2006(2): 25376.［2］ Nacry P, Camilleri C, Courtial B, et al. Major chromosomal rearrangements induced by T-DNA transformation in Arabidopsis［J］. Genetics, 1998, 149(2): 641-650.［3］ Wilson A K, Latham J R, Steinbrecher R A. Transformation-induced mutations in transgenic plants: Analysis and biosafety implications［J］. Biotechnolnoly and Genetic Engineering Reviews, 2006, 23(1):209-238.［4］ Windels P, Sylvie D B, Erik V B, et al. T-DNA integration in Arabidopsis chromosomes. Presence and origin of filler DNA sequences［J］. Plant Physiology, 2003, 133(4): 2061-2068.［5］ Codex Alimentarius Commission. Guide line for the conduct of food safety assessment of foods derived from recombinant-DNA plants［S］. FAO/WHO Joint Publications, 2003:1-14. 〖HJ1.5mm〗［6］ European Food Safety Authority Panel on Genetically Modified Organisms. Guidance on the environmental risk assessment of genetically modified plants［J］. European Food Safety Authority Journal,2010, 8(11):1879. ［7］ Kok E J, Pedersen J, Onori R, et al. Plants with stacked genetically modified events: To assess or not to assess［J］. Trends Biotechnology, 2014, 32(2): 70-73. ［8］ Ochman H, Gerber A S, Hartl D L. Genetic applications of an inverse polymerase chain reaction［J］. Genetics, 1998, 120(3): 621-623.［9］ Liu Y G, Mitsukawa N, Oosumi T, et al. Efficient isolation and mapping of Arabidopsis thaliana T-DNA insert junctions by thermal asymmetric interlaced PCR［J］. Plant Journal, 8(3):457-463. ［10］O′Malley R C, Ecker J R. Linking genotype to phenotype using the Arabidopsis unimutant collection［J］. Plant Journal, 61(6): 928-940.［11］Ji J B, Braam J. Restriction site extension PCR: A novel method for high-throughput characterization of tagged DNA fragments and genome walking［J］. PLoS ONE, 2010, 5: e10577.［12］Spalinskas R, Bulcke M V D, Eede G V D, et al. LT-RADE: An efficient user-friendly genome walking method applied to the molecular characterization of the insertion site of genetically modified maize MON810 and rice LLRICE62［J］. Food Analytical Method, 2013, 6(2): 705-713.［13］Babekova R, Funk T, Pecoraro S, et al. Development of an event-specific real-time PCR detection method for the transgenic Bt rice line KMD1［J］. European Food Research and Technology, 2009, 228(5): 707-716.［14］Milcamps A, Rabe S, Cade R, et al. Validity assessment of the detection method of maize event Bt10 through investigation of its molecular structure［J］. Journal of Agricural and Food Chemistry, 2009, 57(8):3156-3163. ［15］Wang J, Wang W, Li R, et al. The diploid genome sequence of an Asian individual［J］. Nature, 2008, 456(7218): 60-65.［16］Ajay S S, Parker S C, Abaan H O, et al. Accurate and comprehensive sequencing of personal genomes［J］. Genome Research, 2011, 21(9): 1498-1505. ［17］Lepage E, Zampini E, Boyle B, et al. Time and cost-efficient identification of T-DNA insertion sites through targeted genomic sequencing［J］. PLoS One, 2013, 8(8): e70912.［18］Inagaki S, Henry I M, Lieberman M C, et al. High-throughput analysis of T-DNA location and structure using sequence capture［J］. Plos One, 2015, 10(10): e0139672.［19］Wahler D, Schauser L, Bendiek J, et al. Next-generation sequencing as a tool for detailed molecular characterisation of genomic insertions and flanking regions in genetically modified plants: A pilot study using a rice event unauthorised in the EU［J］. Food Analytical Methods, 2013, 6(6): 1718-1727.［20］Park D, Kim D, Jang G, et al. Efficiency to discovery transgenic loci in GM rice using next generation sequencing whole genome re-sequencing［J］. Genomics & Informatics, 2015, 13(3):81. ［21］Williams-Carrier R, Stiffler N, Belcher S, et al. Use of illumina sequencing to identify transposon insertions underlying mutant phenotypes in high-copy mutator lines of maize［J］. The Plant Journal, 2010, 63(1):167.［22］Kovalic D. The use of next generation sequencing and junction sequence analysis bioinformatics to achieve molecular characterization of crops improved through modern biotechnology［J］. Plant Genome, 2012, 5(3):149-163.［23］Guo B, Yong G, Hong H, et al. Identification of genomic insertion and flanking sequence of G2-EPSPS and GAT transgenes in soybean using whole genomes equencing method［J］. Frontiers in Plant Science, 2016, 7: 1009.［24］Yang X D, Niu L, Zhang W, et al. RNAi-mediated SMV P3 cistron silencing confers significantly enhanced resistance to multiple Potyvirus strains and isolates in transgenic soybean［J］. Plant Cell Reports, 2018, 37(1): 103-114.［25］Porebski S, Bailey L G, Baum B R. Modification of a CTAB DNA extraction protocol for plants containing high polysaccharide and polyphenol components［J］. Plant Molecular Biology Reporter, 1997, 15(1): 8-15.［26］马阔, 仲晓芳, 牛陆, 等. 耐盐转基因大豆FA8015旁侧序列分离及定性PCR检测［J］. 大豆科学, 2018, 37（6）:854-859.（Ma K, Zhong X F, NIU L, et al. Sequence separation and qualitative PCR detection of salt-tolerant transgenic soybean FA8015［J］. Soybean Science, 2018, 37(6): 854-859.）［27］仲晓芳, 杨静, 贺红利, 等. 基于基因组重测序的高含量油酸转基因大豆T-DNA旁侧序列分析及事件特异性PCR检测［J］. 农业生物技术学报, 2018, 26（12）:2017-2026.（Zhong X F, Yang J, He H L, et al. Sequence analysis and event specific PCR detection of high content oleic acid transgenic soybean T-DNA based on genomic resequencing［J］. Journal of Agricultural Biotechnology, 2018, 26(12): 2017-2026.）［28］Langmead B and alzberg S L. Fast gapped-read alignment with Bowtie 2［J］. Nature Methods, 2012, 9:357-359.［29］Yang L T, Wang C M, Jensen A H, et al. Characterization of GM events by insert knowledge adapted re-sequencing approaches［J］. Scientific Reports, 2013, 3:2839. ［30］Campbell P J, Stephens P J, Pleasance E D, et al. Identification of somatically acquired rearrangements in cancer using genome-wide massively parallel paired-end sequencing［J］. Nature Genetics, 40(6):722-729. ［31］Hormozdiari F, Hajirasouliha I, Mcpherson A, et al. Simultaneous structural variation discovery among multiple paired-end sequenced genomes［J］. Genome Research, 2011, 21(12):2203-2212. ［32］DuBose A J, Lichtenstein S T, Narisu N, et al. Use of microarray hybrid capture and next-generation sequencing to identify the anatomy of a transgene［J］. Nucleic Acids Research, 2013, 41(6):e70. ［33］Kovalic D, Garnaat C, Guo L, et al. The use of next generation sequencing and junction sequence analysis bioinformatics to achieve molecular characterization of crops improved through modern biotechnology［J］. The Plant Genome, 2012, 5(3): 149-163.［34］Urbanski D F, Malolepszy A, Stougaard J, et al. Genome wide LORE1 retrotransposon mutagenesis and high-throughput insertion detection in Lotus japonicus［J］. The Plant Journal, 2012, 69(4):731-741.［35］Daniela W, Leif S, Joachim B, et al.Next-generation sequencing as a tool for detailed molecular characterization of genomic insertions and flanking regions in genetically modified plants: A pilot study using a rice event unauthorized in the EU［J］. Food Analytical Method, 2013, 6(6):1718-1727. ［36］Pauwels K, DeKeersmaecker S C J, DeSchrijver A, et al.Next-generation sequencing as a tool for the molecular characterization and risk assessment of genetically modified plants: Add value or not?［J］. Trends in Food Science and Technology, 2015, 45(2):319-326.

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Last Update: 2020-03-17