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Rapid and reliable screening of a tomato YAC library exclusively based on PCR

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Abstract

An improved procedure is presented to select clones from a tomato yeast artificial chromosome (YAC) library. The procedure is based exlcusively on the polymerase chain reaction (PCR). We combined DNA from approximately 36,000 YAC clones in pools containing 96-single YAC clones from one master plate and further in super pools representing 10 master plates. This pooling strategy allows the selection of single YAC clones homologous to a target sequence after three rounds of PCR using super pools, single pools, and single YAC clones as a template. Single YAC clones were spheroplasted prior to the third PCR round in order to omit the conventional radioactive colony hybridization step. To date, we applied this PCR-based selection strategy to isolate clones homologousto ten different sequence-tagged sites (STS) that are linked to genes targeted for map-based cloning. The selection of YAC clones can be readily accomplished within three days. The PCR-based screening strategy is easy to set up and contributes to a further acceleration of the construction of YAC contigs.

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Author notes

  1. Klaus Pillen

    Present address: Institute of Agronomy & Plant Breeding, University of Bonn, Karzenburgweg 5, 53115, Bonn, Germany

Authors and Affiliations

  1. Dept. of Plant Breeding & Biometry, Cornell University, 255 Emerson Hall, 14853, Ithaca, NY, USA

    Klaus Pillen, Kevin B. Alpert & Steven D. Tanksley

  2. Dept. of Horticultural Sciences, Texas A&M University, 77843, College Station, TX, USA

    James J. Giovannoni

  3. Institute of Plant Genetics and Crop Plant Research, Corrensstr. 3, D-06466, Gatersleben, Germany

    Martin W. Ganal

Authors
  1. Klaus Pillen
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  2. Kevin B. Alpert
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  3. James J. Giovannoni
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  4. Martin W. Ganal
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  5. Steven D. Tanksley
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Pillen, K., Alpert, K.B., Giovannoni, J.J. et al. Rapid and reliable screening of a tomato YAC library exclusively based on PCR. Plant Mol Biol Rep 14, 58–67 (1996). https://doi.org/10.1007/BF02671903

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