Abstract
Plant meiosis involves complex and dynamic processes that occur within the space inside the nucleus. Direct inspection of meiotic chromosomes by fluorescence microscopy has been used to investigate many of these processes. In particular, optical sectioning microscopy of fluorescence in situ hybridization (FISH)-stained nuclei provides three-dimensional spatial information about the organization and distribution of specific sequences and chromosomal loci within the nucleus. Here we provide a fully detailed three-dimensional (3D) acrylamide FISH method for the analysis of plant meiotic nuclei. Several examples illustrate the versatility of this technique for the investigation of meiotic telomere dynamics in maize, Arabidopsis, and oat. Additional examples of 3D FISH include chromosome painting in a maize chromosome-addition line of oat and telomere FISH with maize nuclei from plants expressing a fluorescently tagged fusion protein, histone H2B-mCherry.
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Acknowledgments
We would like to thank A.B. Thistle and D.L. Vera for critical reading of the manuscript and insightful comments and T.E. Clemente for providing the histone H2B-mCherry line of maize. This work was supported by a Women in Science Math and Engineering Fellowship to ESH, an American Heart Association predoctoral fellowship to SPM (AHA, Greater Southeast Affiliate, number 0715487B), a CRC-planning grant to HWB (2008), and a National Science Foundation grant to HWB (NSF IOS-1025954).
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Howe, E.S., Murphy, S.P., Bass, H.W. (2013). Three-Dimensional Acrylamide Fluorescence In Situ Hybridization for Plant Cells. In: Pawlowski, W., Grelon, M., Armstrong, S. (eds) Plant Meiosis. Methods in Molecular Biology, vol 990. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-333-6_6
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DOI: https://doi.org/10.1007/978-1-62703-333-6_6
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