Abstract
The compactness of plant chromosomes and the structure of the plant cell wall and cytoplasm provide a great obstacle to fluorescence in situ hybridization (FISH) for single-copy or low-copy DNA sequences. Consequently, many new methods for improving spatial resolution via chromosomal stretching have been employed to overcome this technical challenge. In this article, a technique for extracting cell-wall free nuclei at mitotic interphase, then using these nuclei to prepare extended DNA fibers (EDFs) by the method of a receding interface, whereby slide-mounted chromatin produces EDFs in concert with gravity-assisted buffer flow, was adopted as a result of the low frequency of EDF damage produced by this procedure. To examine the quality of these EDFs, we used single-copy gene encoding S-locus receptor kinase and multi-copy 5S rDNA (ribosomal DNA) as probes. The resulting EDFs proved suitable for high-resolution FISH mapping for repetitive DNA sequences, and the localization of a single-copy locus.
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Notes
All the images in this paper were captured with an epifluorescence microscope using a 100× oil immersion objective.
Abbreviations
- FISH:
-
Fluorescence in situ hybridization
- PCR:
-
Polymerase chain reaction
- 2× SSC:
-
Sodium chloride-sodium citrate buffer (300 mM sodium chloride, 30 mM sodium citrate, pH 7.0)
- PBS:
-
Phosphate buffered saline (150 mM sodium chloride, 2 mM potassium chloride, 10 mM sodium hydrogen phosphate, 2 mM potassium dihydrogen phosphate, pH 7.4)
- DAPI:
-
4′,6′-Diamidino-2-phenylindole
- SRK:
-
S-locus receptor kinase
- SLG:
-
S-locus glycoprotein
- EDFs:
-
Extended DNA fibers
- APES:
-
3-Aminopropyltriethoxysilane
- 1× TAE:
-
Tris-acetate EDTA (40 mM Tris acetate, 1 mM EDTA, pH 8.0)
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Acknowledgments
Special thanks are due to Dr. Yujin Zhang for useful suggestions. This research was sponsored by the Fundamental Research Funds for the Central Universities (Grant No. XDJK2009C109), Science Foundation for Young Scholars College of Agronomy and Biotechnology of Southwest University, and Chinese National Science Foundation (Grant No. 30971849).
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Communicated by F. Brandizzi.
K. Yang and H. Zhang have the equal contributions for the paper.
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Yang, K., Zhang, H., Converse, R. et al. Fluorescence in situ hybridization on plant extended chromatin DNA fibers for single-copy and repetitive DNA sequences. Plant Cell Rep 30, 1779–1786 (2011). https://doi.org/10.1007/s00299-011-1086-y
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DOI: https://doi.org/10.1007/s00299-011-1086-y