Plant Genome II Conference
Town & Country Conference Center, San Diego, CA, January, 1994.
PG-II: CONSTRUCTION AND CHARACTERISANON OF A LARGE INSERT YAC LIBRARY
OF ARABIDOPSIS THALIANA
CONSTRUCTION AND CHARACTERISANON OF A LARGE INSERT YAC LIBRARY
OF ARABIDOPSIS THALIANA.
David Bouchez, Laboratoire de Biologie Cellulaire INRA, 78026
Versailles Cedex, France. In collaboration with: CEPH, 27 rue
Juliette Dodu 75010 Paris; Laboratoire de Physiologie Vegetale
Metabolique, ERA CNRS 799, B&t 430, 91405 Orsay Cedex; IBMP, 12
rue du General Zimer 67084 Strasbourg Cedex; URA CNRS 360, 63177
Aubieres Cedex.
We have constructed a genomic library of Arabidopsis
thaliana (ecotype Columbia) in Yeast Artificial Chromosomes.
High molecular weight DNA was prepared from protoplasts inserted
in agarose plugs. The DNA was partially digested with EcoR1,
sized by CHEF PFGE, ligated to pYAC4 arms, sized again by CHEF
PFGE and used to transform yeast strain ABI1380. Ura+ clones
were selected and screened for Trp+/Ade- before incorporation
into the library. The library presently contains 1150 clones (12
microliter plates). Given the proportion of cpDNA/mtDNA (about
20%), this represents about four genomic equivalents. The size
of the library should increase in the near future, with a goal of
ten genome equivalents. The size of all the clones has been
established by CHEF pulsed field gel electrophoresis, transfer
and hybridization to a vector probe. This should facilitate the
use of the library in mapping/walking experiments. The mean
insert size is about 4.50 kilobases. We have set up a
three-dimensional pooling strategy to screen the library by PCF-
80 PCR reactions on pools of 36 or 48 clones are necessary to
urlivocally assign markers to YAC clone(s). From our first
results with 20 different pairs of primers, at least 80% of the
genome seems to be present in the library.
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