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.) wi th reconstructed karyotypes. The chromosomes were selected so that the set of libraries covers the whole V. faba genome more than once. Indi vidual chromosome types were highly purified by flow sorting, and thei r DNA was amplified by degenerate oligonucleotide-primed (DOP) polymer ase chain reaction (PCR) and cloned into a plasmid vector. The choice of restriction site present in pen primer and refinement of cloning pr otocol resulted in high cloning efficiency and allowed generation of l ibraries consisting of about 10(6) clones from 250 or 1000 sorted chro mosomes. The insert size ranged between 50 and 2200 bp and the mean le ngth estimated in individual libraries varied between 310 and 487 bp. Hybridization of cloned fragments with labelled genomic DNA showed tha t about 60% of inserts represented unique or low-copy sequences. The s uitability of the libraries for genome mapping was demonstrated by iso lation of clones containing microsatellite motifs.