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Levels of DNA methylation and transcript accumulation in leaves of transgenic maize varieties

View Article: PubMed Central - PubMed

ABSTRACT

Background: Prior to their release in the environment, transgenic crops are examined for their health and environmental safety. In addition, transgene expression needs to be consistent in order to express the introduced trait (e.g. insecticidal and/or herbicide tolerance). Moreover, data on expression levels for GM events are usually required for approval, but these are rarely disclosed or they are considered insufficient. On the other hand, biosafety regulators do not consider epigenetic regulation (e.g. DNA methylation, ncRNAs and histone modifications), which are broadly known to affect gene expression, within their risk assessment analyses. Here we report the results of a DNA methylation (bisulfite sequencing) and transgene transcript accumulation (RT-qPCR) analysis of four Bt-expressing single transgenic maize hybrids, under different genetic backgrounds, and a stacked transgenic hybrid expressing both insecticidal and herbicide tolerance traits.

Results: Our results showed differences in cytosine methylation levels in the FMV promoter and cry2Ab2 transgene of the four Bt-expressing hybrid varieties. The comparison between single and stacked hybrids under the same genetic background showed differences in the 35S promoter sequence. The results of transgene transcript accumulation levels showed differences in both cry1A.105 and cry2Ab2 transgenes among the four Bt-expressing hybrid varieties. The comparison between single and stacked hybrids showed difference for the cry2Ab2 transgene only.

Conclusions: Overall, our results show differences in DNA methylation patterns in all varieties, as well as in transgene transcript accumulation levels. Although the detection of changes in DNA methylation and transgenic accumulation levels does not present a safety issue per se, it demonstrates the need for additional studies that focus on detecting possible safety implications of such changes.

Electronic supplementary material: The online version of this article (doi:10.1186/s12302-016-0097-2) contains supplementary material, which is available to authorized users.

No MeSH data available.


Cytosine methylation levels among MON-89Ø34-3 maize varieties in four regions of the transgenes. The levels of cytosine methylation were measured in CG, CHG and CHH residues. a FMV promoter region; bcry2Ab2 transgene region; c 35S promoter region; dcry1A.105 transgene region. Vertical bars indicate standard errors. Means followed by different letters in the same cytosine type are significantly different according to the Kruskal–Wallis test (p < 0.05). Exact p values for the statistical test of each of the cytosine comparisons are shown on the right upper corner
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Fig2: Cytosine methylation levels among MON-89Ø34-3 maize varieties in four regions of the transgenes. The levels of cytosine methylation were measured in CG, CHG and CHH residues. a FMV promoter region; bcry2Ab2 transgene region; c 35S promoter region; dcry1A.105 transgene region. Vertical bars indicate standard errors. Means followed by different letters in the same cytosine type are significantly different according to the Kruskal–Wallis test (p < 0.05). Exact p values for the statistical test of each of the cytosine comparisons are shown on the right upper corner

Mentions: The first experiment setup analysed the transgenic regions among four different commercial available varieties (DKB350, AG9045, AG8041 and DKB240) containing the MON-89Ø34-3 event under different genetic backgrounds. The analysis of the FMV promoter region showed significant differences between the varieties for the CG cytosine type, according to the Kruskal–Wallis test for non-parametric data (p = 0.007), while there was no statistical difference in methylation patters among CHG and CHH types. The AG8041 variety showed 2.08% of methylated CG, DKB240 showed 1.04% and DKB350 and AG9045 showed no methylation in this region (Fig. 2a).Fig. 2


Levels of DNA methylation and transcript accumulation in leaves of transgenic maize varieties
Cytosine methylation levels among MON-89Ø34-3 maize varieties in four regions of the transgenes. The levels of cytosine methylation were measured in CG, CHG and CHH residues. a FMV promoter region; bcry2Ab2 transgene region; c 35S promoter region; dcry1A.105 transgene region. Vertical bars indicate standard errors. Means followed by different letters in the same cytosine type are significantly different according to the Kruskal–Wallis test (p < 0.05). Exact p values for the statistical test of each of the cytosine comparisons are shown on the right upper corner
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC5120055&req=5

Fig2: Cytosine methylation levels among MON-89Ø34-3 maize varieties in four regions of the transgenes. The levels of cytosine methylation were measured in CG, CHG and CHH residues. a FMV promoter region; bcry2Ab2 transgene region; c 35S promoter region; dcry1A.105 transgene region. Vertical bars indicate standard errors. Means followed by different letters in the same cytosine type are significantly different according to the Kruskal–Wallis test (p < 0.05). Exact p values for the statistical test of each of the cytosine comparisons are shown on the right upper corner
Mentions: The first experiment setup analysed the transgenic regions among four different commercial available varieties (DKB350, AG9045, AG8041 and DKB240) containing the MON-89Ø34-3 event under different genetic backgrounds. The analysis of the FMV promoter region showed significant differences between the varieties for the CG cytosine type, according to the Kruskal–Wallis test for non-parametric data (p = 0.007), while there was no statistical difference in methylation patters among CHG and CHH types. The AG8041 variety showed 2.08% of methylated CG, DKB240 showed 1.04% and DKB350 and AG9045 showed no methylation in this region (Fig. 2a).Fig. 2

View Article: PubMed Central - PubMed

ABSTRACT

Background: Prior to their release in the environment, transgenic crops are examined for their health and environmental safety. In addition, transgene expression needs to be consistent in order to express the introduced trait (e.g. insecticidal and/or herbicide tolerance). Moreover, data on expression levels for GM events are usually required for approval, but these are rarely disclosed or they are considered insufficient. On the other hand, biosafety regulators do not consider epigenetic regulation (e.g. DNA methylation, ncRNAs and histone modifications), which are broadly known to affect gene expression, within their risk assessment analyses. Here we report the results of a DNA methylation (bisulfite sequencing) and transgene transcript accumulation (RT-qPCR) analysis of four Bt-expressing single transgenic maize hybrids, under different genetic backgrounds, and a stacked transgenic hybrid expressing both insecticidal and herbicide tolerance traits.

Results: Our results showed differences in cytosine methylation levels in the FMV promoter and cry2Ab2 transgene of the four Bt-expressing hybrid varieties. The comparison between single and stacked hybrids under the same genetic background showed differences in the 35S promoter sequence. The results of transgene transcript accumulation levels showed differences in both cry1A.105 and cry2Ab2 transgenes among the four Bt-expressing hybrid varieties. The comparison between single and stacked hybrids showed difference for the cry2Ab2 transgene only.

Conclusions: Overall, our results show differences in DNA methylation patterns in all varieties, as well as in transgene transcript accumulation levels. Although the detection of changes in DNA methylation and transgenic accumulation levels does not present a safety issue per se, it demonstrates the need for additional studies that focus on detecting possible safety implications of such changes.

Electronic supplementary material: The online version of this article (doi:10.1186/s12302-016-0097-2) contains supplementary material, which is available to authorized users.

No MeSH data available.