Plant Genome II Conference
Town & Country Conference Center, San Diego, CA, January, 1994.
PG-II: CHROMOSOME-SPECIFIC DNA LIBRARIES; PLANT CHROMOSOMES AND GENES
AT HIGH RESOLUTION IN SITU
CHROMOSOME-SPECIFIC DNA LIBRARIES; PLANT CHROMOSOMES AND GENES
AT HIGH RESOLUTION IN SITU.
Reinhold G. Herrmann, Botanisches Institut der
Ludwig-Maximilians-Universitat, D-80638 Munchen, Germany
Modern genome analysis and plant breeding concepts rest on
dense linkage maps based on RFLPs. A high marker saturation,
i.e., 1 cM or less, is not readily attainable with customary
techniques involving random selection of recombinant DNA for
RFLPs from shot-gun libraries. It can be substantially
facilitated by directly cloning DNA from individual chromosomes
or chromosome segments, either by metaphase chromosome
flow-sorting or microdissection via glass needles or laser
techniques. In collaboration of our institute with institutes in
Weihenstephan and Grunbach, we have worked out a refined
microdissection procedure using monosomic, telosomic or
ditelosomic addition lines of barley and wheat. The technique
has been improved significantly by the use of (i) mitotic
metaphase spreads, (ii) synchronized meristematic root tissue,
(iii) short fixation times, (iv) a novel drop-spread technique,
(v) specific vectors, (vi) mineral oil rather than a moist
chamber to prevent evaporation from the collection drop, and
(vii) an instrumental set-up that allows to select suitable
metaphases, to store coordinates of their positions, and to
address sequentially specific metaphases or the collection drop
automatically. Chromatin and its DNA are handled in 1 - 2 nl.
The amplified DNA is finally inserted into a conventional vector
for cloning. Chromosome and subchromosmal libraries of up to
50.000 recombinant plasmids can be obtained from a few
chromosomes, of which in barley 10 - 15% can be utilized for RFLP
analysis. - Modern high resolution field emission scanning
electron microscopy in combination with various detection systems
has now reached the "nanometer scale" of resolution. This
approach and improved light microscopic techniques for
hybridizing fluoresceing probes in situ provide access to
chromosomal fine structure at all stages of the cell cycle and
offer unique perspectives to bridge the gap between the
macromolecule DNA and the cytological range. - Supported by the
Bundesministerium for Forschung und Technologie.
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