Plant Molecular Biology

, Volume 74, Issue 6, pp 563–571 | Cite as

Stability of the MON 810 transgene in maize

  • Jose Luis La Paz
  • Maria Pla
  • Nina Papazova
  • Pere Puigdomènech
  • Carlos M. Vicient


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.


Genetically modified organism MON 810 maize Single nucleotide polymorphisms DNA methylation 



This work was funded by the European collaborative project Co-Extra (Co-Existence and Traceability of Genetically Modified Organisms in the European Supply Chain;; 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.

Supplementary material

11103_2010_9696_MOESM1_ESM.doc (106 kb)
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)
11103_2010_9696_MOESM2_ESM.doc (132 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)
11103_2010_9696_MOESM3_ESM.doc (87 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)
11103_2010_9696_MOESM4_ESM.doc (480 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)
11103_2010_9696_MOESM5_ESM.doc (124 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)
11103_2010_9696_MOESM6_ESM.doc (118 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)
11103_2010_9696_MOESM7_ESM.doc (191 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)


  1. Clark RM, Tavaré S, Doebley J (2005) Estimating a nucleotide substitution rate for maize from polymorphism at a major domestication locus. Mol Biol Evol 22:2304–2312CrossRefPubMedGoogle Scholar
  2. Delannay X, La Vallee BJ, Proksch RK, Fuchs RL, Sims SR, Greenplate JT, Marrone PG, Dodson RB, Augustine JJ, Layton JG, Fischhoff DA (1989) Field performance of transgenic tomato plants expressing the Bacillus thuringiensis var kurstaki insect control protein. Bio/Technology 7:1265–1269Google Scholar
  3. Dorlhac de Borne F, Vincentz M, Chupeau Y, Vaucheret H (1994) Co-suppression of nitrate reductase host genes and transgenes in transgenic tobacco plants. Mol Gen Genom 243:613–621Google Scholar
  4. Duan X, Li X, Xue Q, Abo-el-Saad M, Xu D, Wu R (1996) Transgenic rice plants harboring an introduced potato proteinase inhibitor II gene are insect resistant. Nat Biotech 14:494–498CrossRefGoogle Scholar
  5. Fearing PL, Brown D, Vlachos D, Meghji M, Privalle L (1997) Quantitative analysis of Cry1A (b) expression in Bt maize plants, and silage and stability of expression over successive generations. Mol Breed 3:169–176CrossRefGoogle Scholar
  6. Gruntman E, Qi Y, Slotkin RK, Roeder T, Martienssen RA, Sachidanandam R (2008) Kismeth: analyzer of plant methylation states through bisulfite sequencing. BMC Bioinf 9:371CrossRefGoogle Scholar
  7. Hernández M, Pla M, Esteve T, Prat S, Puigdomenech P, Ferrando A (2003) A specific real-time quantitative PCR detection system for event MON 810 in maize YieldGard based on the 3′-transgene integration sequence. Transg Res 12:179–189CrossRefGoogle Scholar
  8. Holck A, Vaïtilingom M, Didierjean L, Rudi K (2002) 5′-Nuclease PCR for quantitative event-specific detection of the genetically modified MON 810 MaisGard maize. Eur Food Res Tech 214:449–453CrossRefGoogle Scholar
  9. Holst-Jensen A, De Loose M, van den Eede G (2006) Coherence between legal requirements and approaches for detection of genetically modified organisms (GMOs) and their derived products. J Agric Food Chem 19:2799–2809CrossRefGoogle Scholar
  10. Kohli A, Griffiths S, Palacios N, Twyman RM, Vain P, Laurie DA, Christou P (1999) Molecular characterization of transforming plasmid rearrangements in transgenic rice reveals a recombination hotspot in the CaMV 35S promoter and confirms the predominance of microhomology mediated recombination. Plant J 17:591–601CrossRefPubMedGoogle Scholar
  11. La Paz JL, Vicient CM, Puigdomènech P, Pla M (2010) Characterization of polyadenylated cryIA(b) transcripts in maize MON 810 commercial varieties. Anal Bioanal Chem 396:2125–2133CrossRefPubMedGoogle Scholar
  12. Lippman Z, May B, Yordan C, Singer T, Martienssen R (2003) Distinct mechanisms determine transposon inheritance and methylation via small interfering RNA and histone modification. PLoS Biol 1:e67CrossRefPubMedGoogle Scholar
  13. Makarevitch I, Stupar RM, Iniguez AL, Haun WJ, Barbazuk WB, Kaeppler SM, Springer NM (2007) Natural variation for alleles under epigenetic control by the maize chromomethylase zmet2. Genetics 77:749–760CrossRefGoogle Scholar
  14. Mette MF, Aufsatz W, van der Winden J, Matzke MA, Matzke AJ (2000) Transcriptional silencing and promoter methylation triggered by double-stranded RNA. EMBO J 19:5194–5201CrossRefPubMedGoogle Scholar
  15. Meyer P, Linn F, Heidmann I, Meyer H, Niedenhof I, Saedler H (1992) Endogenous and environmental factors influence 35S promoter methylation of a maize A1 gene construct in transgenic petunia and its colour phenotype. Mol Gen Genom 231:345–352Google Scholar
  16. Müller AJ, Mendel RR, Schiemann J, Simoens C, Inzé D (1987) High meiotic stability of a foreign gene introduced into tobacco by Agrobacterium-mediated transformation. Mol Gen Genom 207:171–175Google Scholar
  17. Nguyen HT, Jehle JA (2007) Quantitative analysis of the seasonal and tissue specific expression of Cry1Ab in transgenic maize MON 810. J Plant Dis Protec 114:82–87Google Scholar
  18. Ogasawara T, Chikagawa Y, Arakawa F, Nozaki A, Itoh Y, Sasaki K, Umetsu H, Watanabe T, Akiyama H, Maitani T, Toyoda M, Kamada H, Goda Y, Ozeki Y (2005) Frequency of mutations of the transgene, which might result in the loss of the glyphosate-tolerant phenotype, was lowered in roundup ready soybeans. J Health Sci 51:197–201CrossRefGoogle Scholar
  19. Padgette SR, Kolacz KH, Delannay X, Re DB, Re DB, LaVallee BJ, Tinius CN, Rhodes WK, Otero YI, Barry GF, Eichholtz DA, Peschke VM, Nida DL, Taylor NB, Kishore GM (1995) Development, identification, and characterization of a glyphosate-tolerant soybean line. Crop Sci 35:1451–1461CrossRefGoogle Scholar
  20. Rabinowicz PD (2003) Constructing gene enriched genomic libraries using methylation filtration technology. Methods Mol Biol 236:21–36PubMedGoogle Scholar
  21. Register JC 3rd, Peterson DJ, Bell PJ, Bullock WP, Evans IJ, Frame B, Greenland AJ, Higgs NS, Jepson I, Jiao S, Lewnau CJ, Sillick JM, Wilson HM (1994) Structure and function of selectable and non-selectable transgenes in maize after introduction by particle bombardment. Plant Mol Biol 25:951–961CrossRefPubMedGoogle Scholar
  22. Rosati A, Bogani P, Santarlasci A, Buiatti M (2008) Characterisation of 3′ transgene insertion site and derived mRNAs in MON 810 YieldGard maize. Plant Mol Biol 67:271–281CrossRefPubMedGoogle Scholar
  23. Ruiz-Garcia L, Cervera MT, Martínez Zapater JM (2005) DNA methylation increases throughout development. Planta 222:301–306CrossRefPubMedGoogle Scholar
  24. Sunilkumar G, Mohr L, Lopata-Finch E, Emani C, Rathore KS (2002) Developmental and tissue-specific expression of CaMV 35S promoter in cotton as revealed by GFP. Plant Mol Biol 50:463–474CrossRefPubMedGoogle Scholar
  25. Van Rie J, Jansens S, Höfte H, Degheele D, Van Mellaert H (1989) Specificity of Bacillus thuringiensis delta-endotoxins. Importance of specific receptors on the brush border membrane of the mid-gut of target insects. Eur J Bioch 186:239–247CrossRefGoogle Scholar
  26. Vigouroux Y, Jaqueth JS, Matsuoka Y, Smith OS, Beavis WD, Smith JS, Doebley J (2002) Rate and pattern of mutation at microsatellite loci in maize. Mol Biol Evol 19:1251–1260PubMedGoogle Scholar
  27. Widmer F, Seidler RJ, Donegan KK, Reed GL (1997) Quantification of transgenic plant marker gene persistence in the field. Mol Ecol 6:1–7CrossRefGoogle Scholar
  28. Williamson JD, Hirsch-Wyncott ME, Larkins BA, Gelvin SB (1989) Differential accumulation of a transcript driven by the CaMV 35S promoter in transgenic tobacco. Plant Physiol 90:1570–1576CrossRefPubMedGoogle Scholar
  29. Yakovlev A, Khafizova M, Abdullaev Z, Loukinov D, Kondratyev A (2010) Epigenetic regulation of caspase-3 gene expression in rat brain development. Gene 450:103–108CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2010

Authors and Affiliations

  • Jose Luis La Paz
    • 1
  • Maria Pla
    • 2
  • Nina Papazova
    • 3
  • Pere Puigdomènech
    • 1
  • Carlos M. Vicient
    • 1
  1. 1.Molecular Genetics DepartmentCentre for Research in Agricultural Genomics CRAG (CSIC-IRTA-UAB)BarcelonaSpain
  2. 2.Institute for Agricultural and Food TechnologyUniversity of GironaGironaSpain
  3. 3.Unit Technology and Food, Institute for Agricultural and Fisheries ResearchMerelbekeBelgium

Personalised recommendations