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Construction of a barley (Hordeum vulgare L.) YAC library and isolation of a Hor1-specific clone

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Abstract

We have constructed an EcoRI-based YAC (yeast artificial chromosome) library from barley (Hordeum vulgare L. cv. Franka) using the vector pYAC4. The library consists of approximately 18 000 recombinant YACs with insert sizes ranging between 100 and 1000 kb (average of 160 kb) corresponding to 50% of the barley genome. Size fractionation after ligation resulted in an increased average insert size (av. 370 kb) but also in a substantial decrease in cloning efficiency. Less than 1% of the colonies showed homology to a plastome-specific probe; approximately 50% of the colonies displayed a signal with a dispersed, highly repetitive barley-specific probe. Using a primer combination deduced from the sequence of a member of the small Hor1 gene family coding for the C-hordein storage proteins, the library was screened by polymerase chain reaction and subsequently by the colony hybridization technique. A single YAC, designated Y66C11, with a 120 kb insert was isolated. This DNA fragment represents a coherent stretch from the terminal part of the Hor1 gene region as judged from the correspondence of the restriction patterns between Y66C11 DNA and barley DNA after hybridization with the Hor1-specific probe. Restriction with the isoschizomeric enzymes HpaII/MspI suggests a high degree of methylation of the Hor1 region in mesophyll cells but not in YAC-derived (yeast) DNA.

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Authors and Affiliations

  1. Botanisches Institut der Ludwig-Maximilians-Universität Munchen, Menzinger Strasse 67, D-80638, München, Germany

    Michael Kleine & Reinhold G. Herrmann

  2. Lehrstuhl für Pflanzenbau und Pflanzenzüchtung, Weihenstephan, D-85350, Freising, Germany

    Wolfgang Michalek & Christian Jung

  3. Institut für Resistenzgenetik der Biologischen Bundesanstalt, D-85461, Grünbach, Germany

    Andreas Graner

Authors
  1. Michael Kleine
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  2. Wolfgang Michalek
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  3. Andreas Graner
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  4. Reinhold G. Herrmann
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  5. Christian Jung
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Communicated by R Hagemann

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Kleine, M., Michalek, W., Graner, A. et al. Construction of a barley (Hordeum vulgare L.) YAC library and isolation of a Hor1-specific clone. Molec. Gen. Genet. 240, 265–272 (1993). https://doi.org/10.1007/BF00277065

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  • DOI: https://doi.org/10.1007/BF00277065

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