Theoretical and Applied Genetics

, Volume 86, Issue 5, pp 629–636 | Cite as

Microdissection and microcloning of the barley (Hordeum vulgare L.) chromosome 1HS

  • J. Schondelmaier
  • R. Martin
  • A. Jahoor
  • A. Houben
  • A. Graner
  • H. -U. Koop
  • R. G. Herrmann
  • C. Jung
Article
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Accepted:

Abstract

We have applied a refined microdissection procedure to create a plasmid library of the barley (Hordeum vulgare L.) chromosome arm 1HS. The technical improvements involved include synchronization of meristematic root tissue, a metaphase drop-spread technique, paraffin protection of the collection drop to avoid evaporation, and a motorized and programmable microscope stage. Thirteen readily-discernible telocentric chromosomes have been excised from metaphases of synchronized root-tip mitoses. After lysis in a collection drop (2 nl), the DNA was purified, restricted withRsaI, ligated into a vector containing universal sequencing primers, and amplified by the polymerase chain reaction. Finally, the amplified DNA was cloned into a standard plasmid vector. The size of the library was estimated to be approximately 44,000 recombinant plasmids, of which approximately 13% can be utilized for RFLP analysis. Tandem repetitive probes could be rapidly excluded from further analysis after colony hybridization with labelled total barley DNA. Analysis of 552 recombinant plasmids established that: (1) the insert sizes ranged between 70 and 1150 bp with a mean of 250 bp, (2) approximately 60% of the clones contained highly repetitive sequences, and (3) all single- or low-copy probes tested originate from chromosome 1HS. Four probes were genetically mapped, using an interspecificH. vulgare xH. spontaneum F2 population. One of these probes was found to be closely linked to theMla locus conferring mildew resistance.

Key words

Hordeum vulgare Microdissection Microcloning Chromosome specific library RFLP 
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Copyright information

© Springer-Verlag 1993

Authors and Affiliations

  • J. Schondelmaier
    • 2
  • R. Martin
    • 1
  • A. Jahoor
    • 2
  • A. Houben
    • 4
  • A. Graner
    • 3
  • H. -U. Koop
    • 1
  • R. G. Herrmann
    • 1
  • C. Jung
    • 1
  1. 1.Institute of Botany, Ludwig-Maximilians-University MunichMunich 19Germany
  2. 2.Institute for Plant Breeding, Technical University MunichFreising-WeihenstephanGermany
  3. 3.Institute for Resistance Genetics, Federal Biological Research Center for Agriculture and ForestryGrünbachGermany
  4. 4.Institute of Plant Genetics and Crop Plant ResearchGaterslebenGermany

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