Construction of chromosome-specific DNA libraries covering the whole genome of field bean (vicia faba L.)

Recombinant DNA libraries were constructed for seven chromosome types isolated from two translocation lines of field bean (Vicia faba L.) with reconstructed karyotypes. The chromosomes were selected so that the set of libraries covers the whole V. faba genome more than once. Individual chromosome ty...

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Bibliographic Details
Published in:Chromosome research 1996-11, Vol.4 (7), p.531-539
Main Authors: Macas, J, Gualberti, G, Nouzova, M, Samec, P, Lucretti, S, Dolezel, J
Format: Article
Language:English
Subjects:
Chromosome Mapping
chromosome morphology
Chromosomes - genetics
Cloning, Molecular
DNA Primers - chemistry
DNA, Plant - genetics
DNA, Recombinant
DNA, Satellite - genetics
Electrophoresis, Agar Gel
Fabaceae - genetics
Flow Cytometry
Gene Dosage
Gene Library
microsatellite repeats
Plants, Medicinal
Polymerase Chain Reaction
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Summary:Recombinant DNA libraries were constructed for seven chromosome types isolated from two translocation lines of field bean (Vicia faba L.) with reconstructed karyotypes. The chromosomes were selected so that the set of libraries covers the whole V. faba genome more than once. Individual chromosome types were highly purified by flow sorting, and their DNA was amplified by degenerate oligonucleotide-primed (DOP) polymerase chain reaction (PCR) and cloned into a plasmid vector. The choice of restriction site present in PCR primer and refinement of cloning protocol resulted in high cloning efficiency and allowed generation of libraries consisting of about 10(5) clones from 250 or 1000 sorted chromosomes. The insert size ranged between 50 and 2200 bp and the mean length estimated in individual libraries varied between 310 and 487 bp. Hybridization of cloned fragments with labelled genomic DNA showed that about 60% of inserts represented unique or low-copy sequences. The suitability of the libraries for genome mapping was demonstrated by isolation of clones containing microsatellite motifs.
ISSN:0967-3849
1573-6849
DOI:10.1007/bf02261781