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Plant Cell Reports

, 30:1779 | Cite as

Fluorescence in situ hybridization on plant extended chromatin DNA fibers for single-copy and repetitive DNA sequences

  • Kun Yang
  • Hecui Zhang
  • Richard Converse
  • Yong Wang
  • Xiaoying Rong
  • Zhigang Wu
  • Bing Luo
  • Liyan Xue
  • Li Jian
  • Liquan Zhu
  • Xiaojia Wang
Original Paper

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.

Keywords

Extended DNA fibers Receding interface FISH Single-copy DNA Multi-copy DNA sequences 

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)

Notes

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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Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Kun Yang
    • 1
  • Hecui Zhang
    • 1
  • Richard Converse
    • 2
  • Yong Wang
    • 1
  • Xiaoying Rong
    • 1
  • Zhigang Wu
    • 1
  • Bing Luo
    • 1
  • Liyan Xue
    • 1
  • Li Jian
    • 1
  • Liquan Zhu
    • 1
  • Xiaojia Wang
    • 3
  1. 1.College of Agronomy and BiotechnologySouthwest UniversityChongqingChina
  2. 2.Department of BiologyUniversity of CincinnatiCincinnatiUSA
  3. 3.College of Horticulture and Landscape ArchitectureSouthwest UniversityChongqingChina

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