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Stability of the MON 810 transgene in maize

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Abstract

We analysed the DNA variability of the transgene insert and its flanking regions in maize MON 810 commercial varieties. Southern analysis demonstrates that breeding, since the initial transformation event more than 10 years ago, has not resulted in any rearrangements. A detailed analysis on the DNA variability at the nucleotide level, using DNA mismatch endonuclease assays, showed the lack of polymorphisms in the transgene insert. We conclude that the mutation rate of the transgene is not significantly different from that observed in the maize endogenous genes. Six SNPs were observed in the 5′flanking region, corresponding to a Zeon1 retrotransposon long terminal repeat. All six SNPs are more than 500 bp upstream of the point of insertion of the transgene and do not affect the reliability of the established PCR-based transgene detection and quantification methods. The mutation rate of the flanking region is similar to that expected for a maize repetitive sequence. We detected low levels of cytosine methylation in leaves of different transgenic varieties, with no significant differences on comparing different transgenic varieties, and minor differences in cytosine methylation when comparing leaves at different developmental stages. There was also a reduction in cryIAb mRNA accumulation during leaf development.

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Acknowledgments

This work was funded by the European collaborative project Co-Extra (Co-Existence and Traceability of Genetically Modified Organisms in the European Supply Chain; http://www.coextra.eu/; VI Framework Programme), the Centre CONSOLIDER on Agrigenomics and the Xarxa de Referencia en Biotecnologia of the Generalitat de Catalunya. We thank Drs. Josep M. Casacuberta and Michael A. Phillips for their comments on the manuscript.

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Authors and Affiliations

  1. Molecular Genetics Department, Centre for Research in Agricultural Genomics CRAG (CSIC-IRTA-UAB), Jordi Girona 18-24, 08034, Barcelona, Spain

    Jose Luis La Paz, Pere Puigdomènech & Carlos M. Vicient

  2. Institute for Agricultural and Food Technology, University of Girona, Campus Montilivi, EPS-1, 17071, Girona, Spain

    Maria Pla

  3. Unit Technology and Food, Institute for Agricultural and Fisheries Research, Burg. Van Gansberghelaan 115, 9820, Merelbeke, Belgium

    Nina Papazova

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  1. Jose Luis La Paz
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  2. Maria Pla
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  3. Nina Papazova
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  4. Pere Puigdomènech
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  5. Carlos M. Vicient
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Corresponding author

Correspondence to Carlos M. Vicient.

Electronic supplementary material

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Online Resource 1

SNP detection electropherograms. Representative examples of capillary micro-electropherograms of heteroduplex-DNA after CelI digestion. X-axis, fragment size (bp); Y-axis, fluorescence intensity. The first and last peaks (15 and 1,500 bp) correspond to the mw markers. a Mismatch C/G control, corresponding to a heteroduplex fragment of 612 bp with a single C/G SNP. b Example of a heteroduplex DNA digestion corresponding to PCR products with no polymorphisms. c Electropherogram corresponding to region R1, where multiple peaks indicate the presence of polymorphisms. (DOC 106 kb)

Online Resource 2

Sequence alignment of the 5′flanking region in maize MON 810 varieties. Alignment of the R1 region, comprising the 5′flanking region of the transgene, corresponding to a Zeon1 LTR copy, and the entire P-35S. Polymorphic sites are highlighted in green. Sequences were deposited in GeneBank under the accession numbers FN706511 to FN706516 (DOC 132 kb)

Online Resource 3

Methylation status of the transgene in leaves of maize MON 810 varieties. Average percentage of different types of methylated sites in leaves at the V7 stage of seven maize MON 810 varieties. Means (% methylation) and SD of at least 5 independent replicates are shown. (DOC 87 kb)

Online Resource 4

Statistical significance of the methylation levels comparing maize MON 810 varieties, transgene regions and type of methylation sites. Significances have been calculated using Student’s t-test. Significant differences are highlighted in red (P < 0.05). (DOC 480 kb)

Online Resource 5

Dot plot representation of the distribution of methylated sites in the transgene in leaves of different MON 810 varieties. The region analysed is indicated at the top of each graph. Each row represents an independent sequenced clone; the maize variety is indicated on the left and the replica number in brackets. Closed circles indicate methylated sites and open circles, unmethylated; red circles are CpG sites, blue circles, CpNpG sites and green circles are CpNpN sites. (DOC 124 kb)

Online Resource 6

Methylation status of the transgene in leaves of maize MON 810 varieties at different developmental stages. Average percentage of different types of methylated sites in leaves at the V3, V7, R3 and R6 stages of two maize MON 810 varieties. Means (% methylation) and SD of 5 independent replicates are shown. (DOC 118 kb)

Online Resource 7

Dot plot representation of the distribution of methylated sites in the transgene in leaves of two MON 810 varieties and at four developmental stages. The region analysed is indicated at the top of each graph. Each row represents an independent sequenced clone; the maize variety is indicated on the left and the replica number in brackets. V3, V7, R3 and R6 are the developmental stages. Closed circles indicate methylated sites and open circles, unmethylated; red circles are CpG sites, blue circles, CpNpG sites and green circles are CpNpN sites. (DOC 191 kb)

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Paz, J.L.L., Pla, M., Papazova, N. et al. Stability of the MON 810 transgene in maize. Plant Mol Biol 74, 563–571 (2010). https://doi.org/10.1007/s11103-010-9696-2

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  • DOI: https://doi.org/10.1007/s11103-010-9696-2

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