Skip to main content

Advertisement

Log in

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

  • Original Paper
  • Published:
Plant Cell Reports Aims and scope Submit manuscript

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

Notes

  1. 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)

References

  • 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 81:666–673

    Article  CAS  Google Scholar 

  • Boyes DC, Nasrallah JB (1993) Physical linkage of the SLG and SRK genes at the self-incompatibility locus of Brassica oleracea. Mol Gen Genet 236:369–373

    Article  PubMed  CAS  Google Scholar 

  • 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 36:105–113

    Article  PubMed  CAS  Google Scholar 

  • Cheng Z, Buell CR, Wing RA, Gu M, Jiang J (2001) Toward a cytological characterization of the rice genome. Genome Res 11:2133–2141

    Article  PubMed  CAS  Google Scholar 

  • de Jong HJ, Fransz P, Zabel P (1999) High-resolution FISH in plants—techniques and applications. Trends Plant Sci 4:258–263

    Article  Google Scholar 

  • 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 161:1225–1234

    Google Scholar 

  • FISH guides: DNA fibers. http://medicine.yale.edu/labs/henegariu/tavi/fi19.html. Accessed 19 Oct 2010

  • 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 9:421–430

    Article  PubMed  CAS  Google Scholar 

  • 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 G5:1–18

    Google Scholar 

  • 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):101–109

    Article  PubMed  CAS  Google Scholar 

  • 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 89:9509–9513

    Article  PubMed  CAS  Google Scholar 

  • Jiang J, GillB S (1994) Nonisotopic in situ hybridization and plant genome mapping: the first 10 years. Genome 37:7l7–7l725

    Article  Google Scholar 

  • 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 252:497–502

    Article  PubMed  CAS  Google Scholar 

  • 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 38:1081–1087

    Article  PubMed  CAS  Google Scholar 

  • 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):699–706

    Article  PubMed  CAS  Google Scholar 

  • 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-81:2785–2791

    Google Scholar 

  • Koo DH, Jiang J (2009) Super-stretched pachytene chromosomes for fluorescence in situ hybridization mapping and immunodetection of DNA methylation. Plant J 59:509–516

    Article  PubMed  CAS  Google Scholar 

  • Langer-Safer PR, Levine M, Ward DC (1982) Immunological method for mapping genes on Drosophila polytene chromosomes. Proc Natl Acad Sci USA 79:4381–4385

    Article  PubMed  CAS  Google Scholar 

  • 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):1249–1253

    Article  CAS  Google Scholar 

  • 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 247:65–69

    Article  Google Scholar 

  • 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):436–444

    PubMed  CAS  Google Scholar 

  • Martin R, Busch W, Herrmann RG, Wanner G (1994) Efficient preparation of plant chromosomes for high-resolution scanning electron microscopy. Chromosom Res 2:411–415

    Article  CAS  Google Scholar 

  • 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 Note

  • 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):941–946

    Google Scholar 

  • 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, Australia

  • 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 86:201–204

    Article  CAS  Google Scholar 

  • 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 37:940–950

    Article  PubMed  Google Scholar 

  • 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 4:73–83

    Article  CAS  Google Scholar 

  • 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):4294–4300

    CAS  Google Scholar 

  • 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 111:15–22

    Article  PubMed  CAS  Google Scholar 

  • 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):277–284

    Article  PubMed  CAS  Google Scholar 

  • 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):24–28

    Article  CAS  Google Scholar 

  • 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):142–149

    CAS  Google Scholar 

  • Ziolkowski PA, Sadowski J (2002) FISH-mapping of rDNAs and Arabidopsis BACs on pachytene complements of selected Brassicas. Genome 45:189–197

    Article  PubMed  CAS  Google Scholar 

Download references

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).

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Liquan Zhu.

Additional information

Communicated by F. Brandizzi.

K. Yang and H. Zhang have the equal contributions for the paper.

Rights and permissions

Reprints and permissions

About this article

Cite this article

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

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00299-011-1086-y

Keywords

Navigation