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High-resolution mapping on pachytene chromosomes and extended DNA fibres by fluorescencein-situ hybridisation

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Abstract

This article describes two protocols for high-resolution physical mapping of DNA sequences in tomato using fluorescencein situ hybridisation (FISH). The first technique involves FISH to spread chromosomes from pollen mother cells at pachytene and proves to be an excellent method for assigning DNA sequences to chromosome regions at a resolution of up to a few hundred kilobase. An even higher resolution was obtained for extended DNA fibre, prepared from interphase nuclei and used as hybridising component. This technique permits strong enhancement of physical map resolution to values of a few kilobase. The power of both methods simultaneously applied for the same material was demonstrated with the combination of the telomeric repeat and the tomato specific telomere-associated repeat TGR1 as example.

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Abbreviations

DAPI:

diamidino-phenyl-indole

EDTA:

ethylene diamine tetraacetate

FISH:

fluorescencein-situ hybridisation

FITC:

fluorescein isothiocyanate

SDS:

sodium dodecyl sulphate

SSC:

standard saline citrate

TGR:

tomato genomic repeat

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Authors and Affiliations

  1. Department of Molecular Biology, Wageningen Agricultural University, The Netherlands

    Xiao-bo Zhong, Paul F. Fransz & Ab van Kammen

  2. Department of Genetics, Wageningen Agricultural University, Dreijenlaan 2, 6703 HA, Wageningen, The Netherlands

    Jannie Wennekes-van Eden, Pim Zabel & J. Hans de Jong

Authors
  1. Xiao-bo Zhong
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  2. Paul F. Fransz
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  3. Jannie Wennekes-van Eden
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  4. Pim Zabel
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  5. Ab van Kammen
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  6. J. Hans de Jong
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Correspondence to J. Hans de Jong.

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Zhong, Xb., Fransz, P.F., Wennekes-van Eden, J. et al. High-resolution mapping on pachytene chromosomes and extended DNA fibres by fluorescencein-situ hybridisation. Plant Mol Biol Rep 14, 232–242 (1996). https://doi.org/10.1007/BF02671658

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