European Food Research and Technology

, Volume 244, Issue 8, pp 1473–1485 | Cite as

Genetic and epigenetic characterization of the cry1Ab coding region and its 3′ flanking genomic region in MON810 maize using next-generation sequencing

  • Sina-Elisabeth Ben Ali
  • Alexandra Schamann
  • Stefanie Dobrovolny
  • Alexander Indra
  • Sarah Zanon Agapito-Tenfen
  • Rupert Hochegger
  • Alexander G. Haslberger
  • Christian Brandes
Original Paper


MON810 maize was first commercialized in 1997 and it is one of the most marketed genetically modified crops worldwide. Although MON810 maize has been studied extensively, its genetic stability and epigenetics have not been studied very well. We used next-generation sequencing to investigate the genetics and epigenetics of the cry1Ab coding region and its 3′ flanking genomic region in three different MON810 maize varieties. Genetic characterization of the cry1Ab coding region allowed us to identify and quantify several sequence variants. Samples from seeds containing a stacked MON810 event had more variants than MON810 single event varieties. Specifically, position 71 of the analyzed region varied in 15 of 600 samples tested and thus appears to be a mutational hotspot. In addition, position 71 varied at very different frequencies in the samples. Epigenetic analysis revealed a low degree of methylation, making it difficult to associate the coding region variants with methylation status. In conclusion, the variation in the coding region is either due to the increased age of the seeds from the tested maize varieties, which is known to increase the mutation rate, or due to the presence of a second (non-functional) cry1Ab fragment in the genome of the MON810 maize variety.


Genetic stability GMO MON810 Amplicon sequencing SNP Bt maize Methylation Bisulfite sequencing 



We would like to express our gratitude to all three anonymous reviewers for their helpful and detailed comments. Material costs for this work were partially supported by the Hochschuljubiläumsstiftung (HJST) of the city of Vienna.

Compliance with ethical standards

Conflict of interest

All the authors declare that they have no competing interest.

Ethics requirements

This article does not contain any studies with human or animal subjects.


  1. 1.
    ISAAA (2016) Global Status of Commercialized Biotech/GM Crops: 2016. ISAAA. Accessed 26 Sept 2017
  2. 2.
    Monsanto (2017) Safety Assessment of YieldGard Insect-Protected Corn Event MON 810. Monsanto, Accessed 30 Oct 2017
  3. 3.
    Trtikova M, Wikmark OG, Zemp N, Widmer A, Hilbeck A (2015) Transgene expression and Bt protein content in transgenic Bt maize (MON810) under optimal and stressful environmental conditions. PLoS One 10(4):e0123011. CrossRefGoogle Scholar
  4. 4.
    Armstrong CL, Green CE, Phillips RL (1991) Development and availability of germplasm with high Type II culture formation response. Maize Genet Cooper Newsl 65:92–93Google Scholar
  5. 5.
    Haslberger AG (2003) Codex guidelines for GM foods include the analysis of unintended effects. Nat Biotech 21(7):739–741CrossRefGoogle Scholar
  6. 6.
    Lowe BA, Shiva Prakash N, Way M, Mann MT, Spencer TM, Boddupalli RS (2009) Enhanced single copy integration events in corn via particle bombardment using low quantities of DNA. Transgenic Res 18(6):831–840. CrossRefGoogle Scholar
  7. 7.
    Yin Z, Plader W, Malepszy S (2004) Transgene inheritance in plants. J Appl Genet 45(2):127–144Google Scholar
  8. 8.
    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 Cell Mol Biol 17(6):591–601CrossRefGoogle Scholar
  9. 9.
    Schnell J, Steele M, Bean J, Neuspiel M, Girard C, Dormann N, Pearson C, Savoie A, Bourbonnière L, Macdonald P (2015) A comparative analysis of insertional effects in genetically engineered plants: considerations for pre-market assessments. Transgenic Res 24(1):1–17. CrossRefGoogle Scholar
  10. 10.
    Hernandez M, Pla M, Esteve T, Prat S, Puigdomenech P, Ferrando A (2003) A specific real-time quantitative PCR detection system for event MON810 in maize YieldGard based on the 3′-transgene integration sequence. Transgenic Res 12(2):179–189CrossRefGoogle Scholar
  11. 11.
    Rosati A, Bogani P, Santarlasci A, Buiatti M (2008) Characterisation of 3′transgene insertion site and derived mRNAs in MON810 yield gard maize. Plant Mol Biol. Google Scholar
  12. 12.
    Aguilera M, Querci M, Pastor S, Bellocchi G, Milcamps A, Van den Eede G (2009) Assessing copy number of MON 810 integrations in commercial seed maize varieties by 5′ event-specific real-time PCR validated method coupled to 2-∆∆CT analysis. Food Anal Methods 2(1):73–79. CrossRefGoogle Scholar
  13. 13.
    Corbisier P, Bhat S, Partis L, Xie VR, Emslie KR (2010) Absolute quantification of genetically modified MON810 maize (Zea mays L.) by digital polymerase chain reaction. Anal Bioanal Chem 396(6):2143–2150. CrossRefGoogle Scholar
  14. 14.
    Privalle LS, Chen J, Clapper G, Hunst P, Spiegelhalter F, Zhong CX (2012) Development of an agricultural biotechnology crop product: testing from discovery to commercialization. J Agric Food Chem 60(41):10179–10187. CrossRefGoogle Scholar
  15. 15.
    EFSA (2011) Scientific Opinion on Guidance for risk assessment for food and feed from genetically modified plants. EFSA J. Google Scholar
  16. 16.
    Guttikonda SK, Marri P, Mammadov J, Ye L, Soe K, Richey K, Cruse J, Zhuang M, Gao Z, Evans C, Rounsley S, Kumpatla SP (2016) Molecular characterization of transgenic events using next generation sequencing approach. PLoS One 11(2):e0149515. CrossRefGoogle Scholar
  17. 17.
    Ben Ali SE, Madi ZE, Hochegger R, Quist D, Prewein B, Haslberger AG, Brandes C (2014) Mutation scanning in a single and a stacked genetically modified (GM) event by real-time PCR and high resolution melting (HRM) analysis. Int J Mol Sci 15(11):19898–19923. CrossRefGoogle Scholar
  18. 18.
    Neumann G, Brandes C, Joachimsthaler A, Hochegger R (2011) Assessment of the genetic stability of GMOs with a detailed examination of MON810 using Scorpion probes. Eur Food Res Technol 233(1):19–30. CrossRefGoogle Scholar
  19. 19.
    Illumina (2015) Highly targeted resequencing of regions of interest. Accessed 20 Jul 2017
  20. 20.
    Sassa A, Kanemaru Y, Kamoshita N, Honma M, Yasui M (2016) Mutagenic consequences of cytosine alterations site-specifically embedded in the human genome. Genes Environ 38(1):17. CrossRefGoogle Scholar
  21. 21.
    Kurdyukov S, Bullock M (2016) DNA methylation analysis: choosing the right method. Biology 5(1):3. CrossRefGoogle Scholar
  22. 22.
  23. 23.
    Liu N, Chen H (2010) An accurate and rapid PCR-based zygosity testing method for genetically modified maize. Mol Plant Breed 7(3):619–623Google Scholar
  24. 24.
    Druml B, Cichna-Markl M (2014) High resolution melting (HRM) analysis of DNA—its role and potential in food analysis. Food Chem 158:245–254. CrossRefGoogle Scholar
  25. 25.
    Madi ZE, Brandes C, Neumann G, Quist D, Ruppitsch W, Hochegger R (2013) Evaluation of Adh1 alleles and transgenic soybean seeds using Scorpion PCR and HRM analysis. Eur Food Res Technol 237(2):125–135. CrossRefGoogle Scholar
  26. 26.
    Castan M, Ben Ali S-E, Hochegger R, Ruppitsch W, Haslberger AG, Brandes C (2017) Analysis of the genetic stability of event NK603 in stacked corn varieties using high-resolution melting (HRM) analysis and Sanger sequencing. Eur Food Res Technol 243(3):353–365. CrossRefGoogle Scholar
  27. 27.
    Illumina (2013) 16S metagenomic sequencing library preparation. Accessed 22 Jun 2017
  28. 28.
  29. 29.
    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(1):371. CrossRefGoogle Scholar
  30. 30.
    Wang J, Wang C, Long Y, Hopkins C, Kurup S, Liu K, King GJ, Meng J (2011) Universal endogenous gene controls for bisulphite conversion in analysis of plant DNA methylation. Plant Methods 7:39–39. CrossRefGoogle Scholar
  31. 31.
    Griffiths A, Gelbart W, Miller J (1999) Modern genetic analysis. W. H. Freeman, New YorkGoogle Scholar
  32. 32.
    Zhang D, Corlet A, Fouilloux S (2008) Impact of genetic structures on haploid genome-based quantification of genetically modified DNA: theoretical considerations, experimental data in MON 810 maize kernels (Zea mays L.) and some practical applications. Transgenic Res 17(3):393–402. CrossRefGoogle Scholar
  33. 33.
    la Paz JL, Pla M, Papazova N, Puigdomènech P, Vicient CM (2010) Stability of the MON 810 transgene in maize. Plant Mol Biol 74(6):563–571. CrossRefGoogle Scholar
  34. 34.
    Aguilera M, Querci M, Balla B, Prospero A, Ermolli M, Van den Eede G (2008) A qualitative approach for the assessment of the genetic stability of the MON 810 trait in commercial seed maize varieties. Food Anal Methods 1(4):252–258. CrossRefGoogle Scholar
  35. 35.
    la Paz JL, Pla M, Centeno E, Vicient CM, Puigdomènech P (2014) The use of massive sequencing to detect differences between immature embryos of MON810 and a comparable non-GM maize variety. PLoS One. Google Scholar
  36. 36.
    Waminal NE, Ryu KH, Choi S-H, Kim HH (2013) Randomly detected genetically modified (GM) maize (Zea mays L.) near a transport route revealed a fragile 45S rDNA phenotype. PLoS One 8(9):e74060. CrossRefGoogle Scholar
  37. 37.
    Singh CK, Ojha A, Kamle S, Kachru DN (2007) Assessment of cry1Ab transgene cassette in commercial Bt corn MON810: gene, event, construct and GMO specific concurrent characterization. Protocol Exch. Google Scholar
  38. 38.
    Zhang R, Yin Y, Zhang Y, Li K, Zhu H, Gong Q, Wang J, Hu X, Li N (2012) Molecular characterization of transgene integration by next-generation sequencing in transgenic cattle. PLoS One 7(11):e50348. CrossRefGoogle Scholar
  39. 39.
    Chen SL, Lee W, Hottes AK, Shapiro L, McAdams HH (2004) Codon usage between genomes is constrained by genome-wide mutational processes. Proc Natl Acad Sci USA 101(10):3480–3485. CrossRefGoogle Scholar
  40. 40.
    Angov E (2011) Codon usage: nature’s roadmap to expression and folding of proteins. Biotechnol J 6(6):650–659. CrossRefGoogle Scholar
  41. 41.
    van der Salm T, Bosch D, Honée G, Feng L, Munsterman E, Bakker P, Stiekema WJ, Visser B (1994) Insect resistance of transgenic plants that express modified Bacillus thuringiensis cryIA(b) and cryIC genes: a resistance management strategy. Plant Mol Biol 26(1):51–59. CrossRefGoogle Scholar
  42. 42.
    Latham JR, Love M, Hilbeck A (2017) The distinct properties of natural and GM cry insecticidal proteins. Biotechnol Genet Eng Rev 33(1):62–96. CrossRefGoogle Scholar
  43. 43.
    Khan MS, Musatafa G, Nazir S, Joyia FA (2016) Applied molecular biotechnology: the next generation of genetic engineering. Plant molecular biotechnology: applications of transgenics. Taylor & Francis Group, New YorkCrossRefGoogle Scholar
  44. 44.
    Liu H, He R, Zhang H, Huang Y, Tian M, Zhang J (2010) Analysis of synonymous codon usage in Zea mays. Mol Biol Rep 37(2):677–684. CrossRefGoogle Scholar
  45. 45.
    Giroux MJ, Shaw J, Barry G, Cobb BG, Greene T, Okita T, Hannah LC (1996) A single mutation that increases maize seed weight. Proc Natl Acad Sci 93(12):5824–5829CrossRefGoogle Scholar
  46. 46.
    D’Amato F (1997) Role of somatic mutations in the evolution of higher plants. Caryologia 50(1):1–15. CrossRefGoogle Scholar
  47. 47.
    Agapito-Tenfen SZ, Wickson F (2017) Challenges for transgene detection in landraces and wild relatives: learning from 15 years of debate over GM maize in Mexico. Biodivers Conserv. Google Scholar
  48. 48.
    Vilperte V, Agapito-Tenfen SZ, Wikmark O-G, Nodari RO (2016) Levels of DNA methylation and transcript accumulation in leaves of transgenic maize varieties. Environ Sci Eur 28(1):29. CrossRefGoogle Scholar
  49. 49.
    Rocca E, Andersen F (2017) How biological background assumptions influence scientific risk evaluation of stacked genetically modified plants: an analysis of research hypotheses and argumentations. Life Sci Soc Policy 13(1):11. CrossRefGoogle Scholar
  50. 50.
    Sun XQ, Li DH, Xue JY, Yang SH, Zhang YM, Li MM, Hang YY (2016) Insertion DNA accelerates meiotic interchromosomal recombination in Arabidopsis thaliana. Mol Biol Evol 33(8):2044–2053. CrossRefGoogle Scholar
  51. 51.
    Ayarpadikannan S, Kim H-S (2014) The impact of transposable elements in genome evolution and genetic instability and their implications in various diseases. Genom Inf 12(3):98–104. CrossRefGoogle Scholar
  52. 52.
    Bennetzen JL (2005) Transposable elements, gene creation and genome rearrangement in flowering plants. Curr Opin Genet Dev 15(6):621–627. CrossRefGoogle Scholar
  53. 53.
    Mehrotra S, Goyal V (2014) Repetitive sequences in plant nuclear DNA: types, distribution, evolution and function. Genom Proteom Bio 12(4):164–171. CrossRefGoogle Scholar
  54. 54.
    Weber N, Halpin C, Hannah LC, Jez JM, Kough J, Parrott W (2012) Editor’s choice: crop genome plasticity and its relevance to food and feed safety of genetically engineered breeding stacks. Plant Physiol 160(4):1842–1853. CrossRefGoogle Scholar
  55. 55.
    Buiatti M, Christou P, Pastore G (2013) The application of GMOs in agriculture and in food production for a better nutrition: two different scientific points of view. Genes Nutr 8(3):255–270. CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Sina-Elisabeth Ben Ali
    • 1
    • 2
  • Alexandra Schamann
    • 1
    • 2
  • Stefanie Dobrovolny
    • 1
    • 3
  • Alexander Indra
    • 4
  • Sarah Zanon Agapito-Tenfen
    • 5
  • Rupert Hochegger
    • 1
  • Alexander G. Haslberger
    • 2
  • Christian Brandes
    • 1
  1. 1.Institute for Food SafetyAustrian Agency for Health and Food SafetyViennaAustria
  2. 2.Department of Nutritional SciencesUniversity of ViennaViennaAustria
  3. 3.Department of Analytical ChemistryUniversity of ViennaViennaAustria
  4. 4.Institute for Medical Microbiology and HygieneAustrian Agency for Health and Food SafetyViennaAustria
  5. 5.Genøk-Centre for BiosafetyTromsøNorway

Personalised recommendations