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


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.


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



Fluorescence in situ hybridization


Polymerase chain reaction

2× SSC

Sodium chloride-sodium citrate buffer (300 mM sodium chloride, 30 mM sodium citrate, pH 7.0)


Phosphate buffered saline (150 mM sodium chloride, 2 mM potassium chloride, 10 mM sodium hydrogen phosphate, 2 mM potassium dihydrogen phosphate, pH 7.4)




S-locus receptor kinase


S-locus glycoprotein


Extended DNA fibers



1× TAE

Tris-acetate EDTA (40 mM Tris acetate, 1 mM EDTA, pH 8.0)



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).


  1. Armstrong SJ, Fransz P, Marshall DF, Jones GH (1998) Physical mapping of DNA repetitive sequences to mitotic and meiotic chromosomes of Brassica oleracea var. alboglabra by fluorescence in situ hybridization. Heredity 81:666–673CrossRefGoogle Scholar
  2. Boyes DC, Nasrallah JB (1993) Physical linkage of the SLG and SRK genes at the self-incompatibility locus of Brassica oleracea. Mol Gen Genet 236:369–373PubMedCrossRefGoogle Scholar
  3. Chen SY, Jin WZ, Wang MY, Zhang F, Zhou J, Jia QJ, Wu YR, Liu FY, Wu P (2003) Distribution and characterization of over 1000 T-DNA tags in rice genome. Plant J 36:105–113PubMedCrossRefGoogle Scholar
  4. Cheng Z, Buell CR, Wing RA, Gu M, Jiang J (2001) Toward a cytological characterization of the rice genome. Genome Res 11:2133–2141PubMedCrossRefGoogle Scholar
  5. de Jong HJ, Fransz P, Zabel P (1999) High-resolution FISH in plants—techniques and applications. Trends Plant Sci 4:258–263CrossRefGoogle Scholar
  6. Elaine CH, Guy CB, Gareth HJ, Michael JK, Graham JK, Erik PK, Carol DR, Graham RT, Joana JoanaGV, Vicente G, Susan JA (2002) Integration of the cytogenetic and genetic linkage maps of Brassica oleracea. Genetics 161:1225–1234Google Scholar
  7. FISH guides: DNA fibers. Accessed 19 Oct 2010
  8. Fransz PF, Alonso-Blanco C, Liharska TB, Peeters AJM, Zbel P, de Jong JH (1996) High-resolution physical mapping in Arobidopsis thaliana and tomato by fluorescence in situ hybridization to extended DNA fibers. Plant J 9:421–430PubMedCrossRefGoogle Scholar
  9. Fransz PF, de Jong JH, Endo TR (1998) Preparation of extended DNA fibers for high-resolution mapping by fluorescence in situ hybridization (FISH). Plant Mol Biol Man G5:1–18Google Scholar
  10. Henegariu O, Heerema NA, Wright LL, Bray-Ward P, Ward DC, Vance GH (2001) Improvements in cytogenetic slide preparation: controlled chromosome spreading, chemical aging and gradual denaturing. Cytometry 43(2):101–109PubMedCrossRefGoogle Scholar
  11. Heng HHQ, Squire J, Tsui LC (1992) High-resolution mapping of mammalian genes by in situ hybridization to free chromatin. Proc Natl Acad Sci USA 89:9509–9513PubMedCrossRefGoogle Scholar
  12. Jiang J, GillB S (1994) Nonisotopic in situ hybridization and plant genome mapping: the first 10 years. Genome 37:7l7–7l725CrossRefGoogle Scholar
  13. Jiang J, Hulbert SH, Gill BS, Ward DC (1996) Interphase fluorescence in situ hybridization mapping: a physical mapping strategy for plant species with large complex genomes. Mol Gen Genet 252:497–502PubMedCrossRefGoogle Scholar
  14. Kamisugi Y, Nakayama S, O’Neil CM, Mathias RJ, Trick M, Fukui K (1998) Visualization of the Brassica self-incompatibility S-locus on identified oilseed rape chromosomes. Plant Mol Biol 38:1081–1087PubMedCrossRefGoogle Scholar
  15. Khrustaleva LI, Kik C (2001) Localization of single-copy T-DNA insertion in transgenic shallots (Allium cepa) by using ultra-sensitive FISH with tyramide signal amplification. Plant J 25(6):699–706PubMedCrossRefGoogle Scholar
  16. Kondo K, Honda Y, Tanaka R (1996) Chromosome marking in Dendranthema japonica var. wakasaense and its closely related species by fluorescence in situ hybridization using rDNA probe. La Kromosomo II-81:2785–2791Google Scholar
  17. Koo DH, Jiang J (2009) Super-stretched pachytene chromosomes for fluorescence in situ hybridization mapping and immunodetection of DNA methylation. Plant J 59:509–516PubMedCrossRefGoogle Scholar
  18. Langer-Safer PR, Levine M, Ward DC (1982) Immunological method for mapping genes on Drosophila polytene chromosomes. Proc Natl Acad Sci USA 79:4381–4385PubMedCrossRefGoogle Scholar
  19. Lavania UC, Yamamoto M, Mukai Y (2003) Extended chromatin and DNA fibers from active plant nuclei for high-resolution fish. J Histochem Cytochemistry 51(10):1249–1253CrossRefGoogle Scholar
  20. Lichter P, Tang CJ, Call K, Hermanson G, Evans GA, Housman D, Ward DC (1990) High-resolution mapping of human chromosome 11 by in situ hybridization with cosmid clones. Science 247:65–69CrossRefGoogle Scholar
  21. Lim KB, de Jong H, Yang TJ, Park JY, Kwon SJ, Kim JS, Lim MH, Kim JA, Jin M, Jin YM, Kim SH, Lim YP, Bang JW, Kim HI, Park BS (2005) Characterization of rDNAs and tandem repeats in the heterochromatin of Brassica rapa. Mol Cells 19(3):436–444PubMedGoogle Scholar
  22. Martin R, Busch W, Herrmann RG, Wanner G (1994) Efficient preparation of plant chromosomes for high-resolution scanning electron microscopy. Chromosom Res 2:411–415CrossRefGoogle Scholar
  23. Ohmido N, Kijima K, Hirose T, de Jong H, Fukui K (1999) Recent advances in the physical mapping of genes by fluorescence in situ hybridization (FISH) of rice. Application NoteGoogle Scholar
  24. Pedersen C, Linde-Laursen I (1995) The relationship between physical and genetic distances at the Hor1 and Hor2 loci of barley estimated by 2-color fluorescent in situ hybridization. Theor Appl Genet 1995(91):941–946Google Scholar
  25. Snowdon RJ, Köhler A, Köhler W, Friedt W (1999) FISH-ing for new rapeseed lines: the application of molecular cytogenetic techniques to Brassica breeding. “New Horizons for an old Crop”. In: Proceedings of the international rapeseed congress, Canberra, AustraliaGoogle Scholar
  26. Snowdon RJ, Friedt W, Köhler A, Köhler W (2000) Molecular cytogenetic localisation and characterisation of 5S and 25S rDNA loci for chromosome identification in oilseed rape (Brassica napus L.). Ann Bot 86:201–204CrossRefGoogle Scholar
  27. Valárik M, BartoŠ J, Kovářová P, Kubaláková M, de Jong JH, Doležel J (2004) High-resolution FISH on super-stretched flow-sorted plant chromosomes. Plant J 37:940–950PubMedCrossRefGoogle Scholar
  28. Wang M, Duell T, Gray JW, Weier HUG (1996) High sensitivity, high resolution physical mapping by fluorescence in situ hybridization on to individual straightened DNA molecules. Bioimaging 4:73–83CrossRefGoogle Scholar
  29. Wang Y, Zhu LQ, Rong XY, Chen XD, Tang ZL, Wang XJ (2009) 5S rDNA was localized on chromosome 2 in Brassica oleracea. Sci Agric Sin 42(12):4294–4300Google Scholar
  30. Yamashita K, Takatori Y, Tashiro Y (2005) Chromosomal location of a pollen fertility-restoring gene, Rf, for CMS in Japanese bunching onion (Allium fistulosum L.) possessing the cytoplasm of A. galanthum Kar. et Kir. revealed by genomic in situ hybridization. Theor Appl Genet 111:15–22PubMedCrossRefGoogle Scholar
  31. Yang K, Qi HY, Zhu LQ (2006) Localization of S genes on extended dna fibers (EDFs) in Brassica oleracea by high-resolution FISH. Acta Genet Sin 33(3):277–284PubMedCrossRefGoogle Scholar
  32. Zhong XB, de Jong JH, Zabel P (1996) Preparation of tomato meiotic pachytene and mitotic metaphase chromosomes suitable for fluorescence in situ hybridization (FISH). Chromosom Res 4(1):24–28CrossRefGoogle Scholar
  33. Zhong XB, Fransz PF, Wennekes J, van Kammen A, de Jong JH, Zabe P (1998) Fluorescence in situ hybridization to pachytene chromosomes and extended DNA fibres in plants. Acta Genet Sin 25(2):142–149Google Scholar
  34. Ziolkowski PA, Sadowski J (2002) FISH-mapping of rDNAs and Arabidopsis BACs on pachytene complements of selected Brassicas. Genome 45:189–197PubMedCrossRefGoogle Scholar

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

Personalised recommendations