Chromosome Research

, Volume 4, Issue 7, pp 531–539 | Cite as

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

  • J. MacasEmail author
  • G. Gualberti
  • M. Nouzová
  • P. Samec
  • S. Lucretti
  • J. Doležel


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 wholeV. faba genome more than once. Individual chromosome types were highly purified by flow sorting, and their DNA was amplified by degenerate oligonucleotideprimed (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 106 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 pb. 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.

Key words

chromosome flow sorting degenerate oligonucleotide-primed polymerase chain reaction chromosome-specific DNA libraries genome mapping Vicia faba 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Arumuganathan K, Martin GB, Telenius H, Tanksley SD, Earle ED (1994) Chromosome 2-specific DNA clones from flow-sorted chromosomes of tomato.Mol Gen Genet 242: 551–558.Google Scholar
  2. Ausbel FM, Brent R, Kingston RE et al. (1991)Current Protocols in Molecular Biology. New York: John Wiley & Sons.Google Scholar
  3. Bennett MD, Leitch I (1995) Nuclear DNA amounts in angiosperms.Ann Bot 76: 113–176.Google Scholar
  4. Breneman JW, Ramsey MJ, Lee DA, Eveleth GG, Minkler JL, Tucker JD (1993) The development of chromosome-specific composite DNA probes for the mouse and their application to chromosome painting.Chromosoma 102: 591–598.Google Scholar
  5. Crowe JS, Cooper HJ, Smith MA, Sims MJ, Parker D, Gewert D (1991) Improved cloning efficiency of polymerase chain reaction (PCR) products after proteinase K digestion.Nucleic Acids Res 19: 184.Google Scholar
  6. Doležel J, Lucretti S (1995) High-resolution flow karyotyping and chromosome sorting inVicia faba lines with standard and reconstructed karyotypes.Theor Appl Genet 90: 797–802.Google Scholar
  7. Doležel J, Binarová P, Lucretti S (1989) Analysis of nuclear DNA content in plant cells by flow cytometry.Biol Plant 31: 113–120.Google Scholar
  8. Doležel J, Sgorbati S, Lucretti S (1992a) Comparison of three DNA fluorochromes for flow cytometric estimation of nuclear DNA content in plants.Physiol Plant 85: 625–631.Google Scholar
  9. Doležel J, Čihalíková J, Lucretti S (1992b) A high-yield procedure for isolation of metaphase chromosomes from root tips ofVicia faba L.Planta 188: 93–98.Google Scholar
  10. Doležel J, Lucretti S, Schubert I (1994) Plant chromosome analysis and sorting by flow cytometry.Crit Rev Plant Sci 13: 275–309.Google Scholar
  11. Doležel J, Lucretti S, Macas J. (1995) Flow cytometric analysis and sorting of plant chromosomes. In: Brandham PE, Bennett MD, eds.Kew Chromosome Conference IV. Kew: Royal Botanic Gardens, pp 185–200.Google Scholar
  12. Fuchs J, Schubert I (1995) Localization of seed protein genes on metaphase chromosomes ofVicia faba via fluorescencein situ hybridization.Chrom Res 3: 94–100.Google Scholar
  13. Gray JW, Cram LS. (1990) Flow karyotyping and chromosome sorting. In: Melamed MR, Lindmo T, Mendelsohn ML, eds.Flow Cytometry and Sorting. 2nd edn, New York: Wiley-Liss, pp 503–529.Google Scholar
  14. Guan XY, Meltzer PS, Trent JM (1994) Rapid generation of whole chromosome painting probes (WCPs) by chromosome microdissection.Genomics 22: 101–107.Google Scholar
  15. Hoebee B, Destoppelaar JM, Suijkerbuijk RF, Monard S (1994) Isolation of rat chromosome-specific paint probes by bivariate flow sorting followed by degenerate oligonucleotide primed PCR.Cytogenet Cell Genet 66: 277–282.Google Scholar
  16. Jung V, Pestka SB, Pestka S (1993) Cloning of polymerase chain reaction-generated DNA containing terminal restriction endonuclease recognition sites. In: Wu R, ed.Methods in Enzymology. San Diego: Academic Press, pp 357–362.Google Scholar
  17. Langford CF, Telenius H, Carter NP, Miller NGA, Tucker EM (1992) Chromosome painting using chromosome-specific probes from flow-sorted pig chromosomes.Cytogenet Cell Genet 61: 221–223.Google Scholar
  18. Lucretti S, Doležel J, Schubert I, Fuchs J (1993) Flow karyotyping and sorting ofVicia faba chromosomes.Theor Appl Genet 85: 665–672.Google Scholar
  19. Macas J, Doležel J, Lucretti S et al. (1993a) Localization of seed protein genes on flow-sorted field bean chromosomes.Chrom Res 1: 107–115.Google Scholar
  20. Macas J, Weschke W, Baumlein H et al. (1993b) Localization of vicilin genes via polymerase chain reaction on microisolated field bean chromosomes.Plant J 3: 883–886.Google Scholar
  21. McCormick MK, Buckler A, Bruno W et al. (1993) Construction and characterization of a YAC library with a low frequency of chimeric clones from flow-sorted human chromosome 9.Genomics 18: 553–558.Google Scholar
  22. Pich U, Houben A, Fuchs J, Meister A, Schubert I (1994) Utility of DNA amplified by degenerate oligonucleotide-primed PCR (DOP-PCR) from the total genome and defined chromosomal regions of field bean.Mol Gen Genet 243: 173–177.Google Scholar
  23. Riess O, Siedlaczck I, Kredtke S, Melmer G, Epplen JT, Deaven LL (1994) Characterization of a human chromosome-4 flow-sorted cosmid library.Cytogenet Cell Genet 65: 238–242.Google Scholar
  24. Riquet J, Milan D, Woloszyn N et al. (1995) A linkage map with microsatellites isolated from swine flow-sorted chromosome 11.Mammalian Genome 6: 623–628.Google Scholar
  25. Sanger F, Nicklen D, Coulson AR (1977) DNA sequencing with chain terminating inhibitors.Proc Natl Acad Sci USA 74: 5463–5467.Google Scholar
  26. Schubert I, Rieger R, Michaelis A (1986) Structural and numerical manipulation of theVicia faba karyotype: results and perspectives.Biol Zentbl 105: 9–17.Google Scholar
  27. Telenius H, Carter NP, Bebb CE, Nordenskjold M, Ponder BAJ, Tunnacliffe A (1992) Degenerate oligonucleotide-primed PCR: general amplification of target DNA by a single degenerate primer.Genomics 13: 718–725.Google Scholar
  28. Torres AM, Weeden NF, Martin A (1993) Linkage among isozyme, RFLP and RAPD markers inVicia faba.Theor Appl Genet 85: 937–945.Google Scholar
  29. Van Dilla MA, Deaven LL (1990) Construction of gene libraries for each human chromosome.Cytometry 11: 208–218.Google Scholar
  30. Wang ML, Leitch AR, Schwarzacher T, Heslop-Harrison JS, Moore G (1992) Construction of a chromosome-enrichedHpaII library from flow-sorted wheat chromosomes.Nucleic Acids Res 20: 1897–1901.Google Scholar
  31. Yakura K, Tanifuji S (1983) Molecular cloning and restriction analysis of EcoRI-fragments ofVicia faba rDNA.Plant Cell Physiol 24: 1327–1330.Google Scholar

Copyright information

© Rapid Science Publishers 1996

Authors and Affiliations

  • J. Macas
    • 1
    Email author
  • G. Gualberti
    • 1
  • M. Nouzová
    • 1
  • P. Samec
    • 1
  • S. Lucretti
    • 2
  • J. Doležel
    • 3
  1. 1.Institute of Plant Molecular BiologyČeské BudějoviceCzech Republic
  2. 2.Biotechnology and Agriculture DivisionENEA, CR CasacciaRomeItaly
  3. 3.Institute of Experimental BotanyDe Montfort University Norman Borlaug Centre for Plant Science ResearchOlomoucCzech Republic

Personalised recommendations