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DNA hypomethylation in 5-azacytidine-induced early-flowering lines of flax

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Abstract

HPLC analysis was used to examine the cytosine methylation of total DNA extracted from four early-flowering lines that were induced by treating germinating seeds of flax (Linum usitatissimum) with the DNA demethylating agent 5-azacytidine. In the normal lines that gave rise to the induced early-flowering lines, flowering usually begins approximately 50 days after sowing. The early-flowering lines flower 7–13 days earlier than normal. The normal level of cytosine methylation was approximately 14% of the cytosines and 2.7% of the nucleosides. In the early-flowering lines, these levels were 6.2% lower than normal in DNA from the terminal leaf clusters of 14-day-old seedlings and 9.7% lower than normal in DNA from the cotyledons and immature shoot buds of 4-day-old seedlings. This hypomethylation was seen in lines that were five to nine generations beyond the treatment generation. The level of hypomethylation was similar in three of the four early-flowering lines, but was not as low in the fourth line, which flowers early but not quite as early as the other three lines. Unexpectedly, the degree of hypomethylation seen in segregant lines, derived by selecting for the early-flowering phenotype in the F2 and F3 generations of out-crosses, was similar to that seen in the early-flowering lines. Analysis of the methylation levels in segregating generations of out-crosses between early-flowering and normal lines demonstrated a decrease in methylation level during the selection of early-flowering segregants. The results suggest an association between hypomethylation and the early-flowering phenotype, and that the hypomethylated regions may not be randomly distributed throughout the genome of the early-flowering lines.

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Acknowledgements

Thanks are extended to Dr. D. Goussev, for translation of the article by Vanyshin and Belozerskii. The research was made possible by two Natural Sciences and Engineering Research Council of Canada Undergraduate Student Research Awards (S.M.S. and J.C.L.B.) and a Discovery Grant (M.A.F.), and by infrastructure funded by the Canadian Foundation for Innovation, the Ontario Innovation Trust, Wilfrid Laurier University, and Varian Canada.

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  1. Department of Biology, Wilfrid Laurier University, 75 University Ave. West, Waterloo, ON, N2L 3C5, Canada

    M. A. Fieldes, S. M. Schaeffer & J. C. L. Brown

  2. Department of Chemistry, Wilfrid Laurier University, Waterloo, ON, N2L 3C5, Canada

    M. J. Krech

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  1. M. A. Fieldes
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  2. S. M. Schaeffer
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  3. M. J. Krech
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Correspondence to M. A. Fieldes.

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Communicated by C. Möllers

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Fieldes, M.A., Schaeffer, S.M., Krech, M.J. et al. DNA hypomethylation in 5-azacytidine-induced early-flowering lines of flax. Theor Appl Genet 111, 136–149 (2005). https://doi.org/10.1007/s00122-005-2005-9

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