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

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Abstract

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.

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References

  1. ISAAA (2016) Global Status of Commercialized Biotech/GM Crops: 2016. ISAAA. http://www.isaaa.org/purchasepublications/itemdescription.asp?ItemType=BRIEFS&Control=IB052-2016. Accessed 26 Sept 2017

  2. Monsanto (2017) Safety Assessment of YieldGard Insect-Protected Corn Event MON 810. Monsanto, https://monsanto.com/company/media/statements/mon810-bans/. Accessed 30 Oct 2017

  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. https://doi.org/10.1371/journal.pone.0123011

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Armstrong CL, Green CE, Phillips RL (1991) Development and availability of germplasm with high Type II culture formation response. Maize Genet Cooper Newsl 65:92–93

    Google Scholar 

  5. Haslberger AG (2003) Codex guidelines for GM foods include the analysis of unintended effects. Nat Biotech 21(7):739–741

    Article  CAS  Google Scholar 

  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. https://doi.org/10.1007/s11248-009-9265-0

    Article  CAS  PubMed  Google Scholar 

  7. Yin Z, Plader W, Malepszy S (2004) Transgene inheritance in plants. J Appl Genet 45(2):127–144

    PubMed  Google Scholar 

  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):591–601

    Article  CAS  Google Scholar 

  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. https://doi.org/10.1007/s11248-014-9843-7

    Article  CAS  PubMed  Google Scholar 

  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):179–189

    Article  CAS  PubMed  Google Scholar 

  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. https://doi.org/10.1007/s11103-008-9315-7

    Article  PubMed  Google Scholar 

  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. https://doi.org/10.1007/s12161-008-9036-1

    Article  Google Scholar 

  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. https://doi.org/10.1007/s00216-009-3200-3

    Article  CAS  PubMed  Google Scholar 

  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. https://doi.org/10.1021/jf302706e

    Article  CAS  PubMed  Google Scholar 

  15. EFSA (2011) Scientific Opinion on Guidance for risk assessment for food and feed from genetically modified plants. EFSA J. https://doi.org/10.2903/j.efsa.2011.2150

    Article  Google Scholar 

  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. https://doi.org/10.1371/journal.pone.0149515

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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. https://doi.org/10.3390/ijms151119898

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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. https://doi.org/10.1007/s00217-011-1487-8

    Article  CAS  Google Scholar 

  19. Illumina (2015) Highly targeted resequencing of regions of interest. https://www.illumina.com/techniques/sequencing/dna-sequencing/targeted-resequencing/amplicon-sequencing.html. Accessed 20 Jul 2017

  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. https://doi.org/10.1186/s41021-016-0045-9

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Kurdyukov S, Bullock M (2016) DNA methylation analysis: choosing the right method. Biology 5(1):3. https://doi.org/10.3390/biology5010003

    Article  CAS  PubMed Central  Google Scholar 

  22. ThermoFisherScientific Qubit® 3.0 Fluorometer. https://www.thermofisher.com/at/en/home/industrial/spectroscopy-elemental-isotope-analysis/molecular-spectroscopy/fluorometers/qubit/qubit-fluorometer.html. Accessed 22 Jun 2017

  23. Liu N, Chen H (2010) An accurate and rapid PCR-based zygosity testing method for genetically modified maize. Mol Plant Breed 7(3):619–623

    Google Scholar 

  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. https://doi.org/10.1016/j.foodchem.2014.02.111

    Article  CAS  PubMed  Google Scholar 

  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. https://doi.org/10.1007/s00217-013-1969-y

    Article  CAS  Google Scholar 

  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. https://doi.org/10.1007/s00217-016-2749-2

    Article  CAS  Google Scholar 

  27. Illumina (2013) 16S metagenomic sequencing library preparation. https://support.illumina.com/downloads/16s_metagenomic_sequencing_library_preparation.html. Accessed 22 Jun 2017

  28. Illumina (2016) Illumina adapter sequences. https://support.illumina.com/content/dam/illumina-support/documents/documentation/chemistry_documentation/experiment-design/illumina-adapter-sequences_1000000002694-01.pdf. Accessed 28 Jul 2017

  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. https://doi.org/10.1186/1471-2105-9-371

    Article  CAS  Google Scholar 

  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. https://doi.org/10.1186/1746-4811-7-39

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Griffiths A, Gelbart W, Miller J (1999) Modern genetic analysis. W. H. Freeman, New York

    Google Scholar 

  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. https://doi.org/10.1007/s11248-007-9114-y

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  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. https://doi.org/10.1007/s12161-008-9035-2

    Article  Google Scholar 

  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. https://doi.org/10.1371/journal.pone.0100895

    Article  PubMed  PubMed Central  Google Scholar 

  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. https://doi.org/10.1371/journal.pone.0074060

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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. https://doi.org/10.1038/nprot.2007.440

    Article  Google Scholar 

  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. https://doi.org/10.1371/journal.pone.0050348

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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. https://doi.org/10.1073/pnas.0307827100

    Article  CAS  PubMed  Google Scholar 

  40. Angov E (2011) Codon usage: nature’s roadmap to expression and folding of proteins. Biotechnol J 6(6):650–659. https://doi.org/10.1002/biot.201000332

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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. https://doi.org/10.1007/bf00039519

    Article  PubMed  Google Scholar 

  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. https://doi.org/10.1080/02648725.2017.1357295

    Article  CAS  PubMed  Google Scholar 

  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 York

    Book  Google Scholar 

  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. https://doi.org/10.1007/s11033-009-9521-7

    Article  CAS  PubMed  Google Scholar 

  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):5824–5829

    Article  CAS  PubMed  Google Scholar 

  46. D’Amato F (1997) Role of somatic mutations in the evolution of higher plants. Caryologia 50(1):1–15. https://doi.org/10.1080/00087114.1997.10797380

    Article  Google Scholar 

  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. https://doi.org/10.1007/s10531-017-1471-0

    Article  Google Scholar 

  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. https://doi.org/10.1186/s12302-016-0097-2

    Article  PubMed  PubMed Central  Google Scholar 

  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. https://doi.org/10.1186/s40504-017-0057-7

    Article  PubMed  PubMed Central  Google Scholar 

  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. https://doi.org/10.1093/molbev/msw087

    Article  CAS  PubMed  Google Scholar 

  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. https://doi.org/10.5808/GI.2014.12.3.98

    Article  Google Scholar 

  52. Bennetzen JL (2005) Transposable elements, gene creation and genome rearrangement in flowering plants. Curr Opin Genet Dev 15(6):621–627. https://doi.org/10.1016/j.gde.2005.09.010

    Article  CAS  PubMed  Google Scholar 

  53. Mehrotra S, Goyal V (2014) Repetitive sequences in plant nuclear DNA: types, distribution, evolution and function. Genom Proteom Bio 12(4):164–171. https://doi.org/10.1016/j.gpb.2014.07.003

    Article  Google Scholar 

  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. https://doi.org/10.1104/pp.112.204271

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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. https://doi.org/10.1007/s12263-012-0316-4

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

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.

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Correspondence to Christian Brandes.

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Ben Ali, SE., Schamann, A., Dobrovolny, S. et al. Genetic and epigenetic characterization of the cry1Ab coding region and its 3′ flanking genomic region in MON810 maize using next-generation sequencing. Eur Food Res Technol 244, 1473–1485 (2018). https://doi.org/10.1007/s00217-018-3062-z

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