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

Abstract

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.

Faba Bean

“…For the large chromosome 1, for which trisomics could not be obtained so far, a linkage map has been generated with an average 8 cM map interval . The integration of genetic and physical map of faba bean is further improved by techniques of flow sorting (Dolezel and Lucretti, 1995;Macas et al, 1996) and in situ hybridization (Fuchs and Schubert, 1995). Even more, flow sorting of V. faba chromosomes, due to their large size, has become a model for plant chromosome sorting in general.…”

Flow cytogenetics and plant genome mapping

“…Arumuganathan et al (1994) developed eleven RFLP markers from tomato chromosome 2. Macas et al (1996) and Koblı´z› kova¤ et al (1998) showed that libraries constructed from £ow-sorted chromosomes contain microsatellite sequences, and that the libraries can be e⁄ciently enriched for these loci to facilitate their screening for microsatellite markers. The utility of DNA libraries depends on the size of inserts, genome coverage, and contamination with unwanted sequences.…”

“…Following the same approach, Macas et al (1996) were the ¢rst to prepare a complete set of chromosome-speci¢c libraries covering the whole genome of the ¢eld bean. So far, the ¢eld bean is the only plant species whose genome is available in the form of chromosome-speci¢c DNA libraries.…”

Cytometry and plant sciences: A personal retrospective

“…ISAC member Kathiravetpillai Arumuganathan was one of the first to attempt chromosome sorting from the crops tomato and maize (44,45), and ISAC member Nigel Miller provided flow expertise in similar work by using wheat chromosomes (46). ISAC members Sergio Lucretti and Jaroslav Dolezel and their coworkers have made some of the greatest progress in this area, through devising methods for isolation of large numbers of intact chromosomes from seedlings of crop species, including wheat, pea, maize, barley, chickpea, and broad bean, and for flow sorting of many, and in some cases all, pairs of chromosomes (47)(48)(49)(50)(51)(52)(53)(54)(55). For some species, this requires the use of specific genotypes carrying multiple defined chromosomal translocations that have the effect of producing karyotypes within which all chromosome pairs are of different sizes and therefore can be sorted (47).…”