Theoretical and Applied Genetics

, Volume 91, Issue 6–7, pp 928–935 | Cite as

The molecular cytogenetics of Vigna unguiculata (L.) Walp: the physical organization and characterization of 18s-5.8s-25s rRNA genes, 5s rRNA genes, telomere-like sequences, and a family of centromeric repetitive DNA sequences

  • I. Galasso
  • T. Schmidt
  • D. Pignone
  • J. S. Heslop-Harrison
Article

Abstract

A knowledge of genome organization is important for understanding how genomes function and evolve, and provide information likely to be useful in plant breeding programmes involving hybridization and genetic manipulation. Molecular techniques, including in situ hybridization, molecular cloning and DNA sequencing, are proving valuable tools to investigate the structure, organization, and diversity of chromosomes in agricultural crops. Heterologous labelled 18 s-5.8 s-25 s (pTa71) and 5 s rDNAs (pTa794) were used for in situ hybridization on Vigna unguiculata (L.) Walp. chromosomes. Hybridization with 18 s-5.8 s-25 s rRNA gene probes occurred at the same chromosomal sites which were positive to the CMA fluorochrome. Silver staining of nucleolar-organizing regions indicated that all the rDNA sites detected using the 18 s-5.8 s-25 s rRNA gene probe possessed active genes. Degenerate telomeric repeats gave hybridization signals at the telomeres of most chromosomes and no intercalary sites were detected at metaphase; the sequences appear to have no preferential distribution in interphase nuclei. A repetitive DraI family from V. unguiculata was cloned (pVuKB1) and characterized. The DraI repeat is 488 nucleotides long, AT rich (74%), and hybridized on all chromosomes in the centromeric areas. The presence of this sequence family was investigated by Southern hybridization in different Vigna species and other Leguminoseae. It was only detected in V. unguiculata, and hence represents a species-specific DNA sequence.

Key words

rDNA sites Centromeric repetitive DNA Telomere In situ hybridization Southern hybridization Ag-NOR Cowpea Physical maps 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Bennett MD, Smith JB (1976) Nuclear DNA amounts in angiosperms. Phil Trans R Soc Lond B 274:227–274Google Scholar
  2. Bennett MD, Smith JB, Heslop-Harrison JS (1982) Nuclear DNA amounts in angiosperms. Proc R Soc Lond B 216:179–199Google Scholar
  3. Bloom SE, Goodpasture C (1976) An improved technique for selective silver staining of nucleolar organizer regions in human chromosomes. Hum Genet 34:199–206PubMedGoogle Scholar
  4. Castilho A, Heslop-Harrison JS (1995) Physical mapping of 5 s and 18 s-25 s rDNA and repetitive DNA sequences in Aegilops umbellulata. Genome (in press)Google Scholar
  5. Charlesworth B, Sniegowski P, Stephan W (1994) The evolutionary dynamics of repetitive DNA in eukaryotes. Nature 371:215–220CrossRefPubMedGoogle Scholar
  6. Chen HK, Mok MC, Shanmugasundaram S, Mok DWS (1989) Interspecific hybridization between Vigna radiata (L.) Wilczek and V. glabrescens. Theor Appl Genet 78:641–647Google Scholar
  7. Dellaporta SL, Wood J, Hicks JB (1983) A plant DNA minipreparation: version II. Plant Mol Biol Rep 1:19–21Google Scholar
  8. Devos KM, Atkinson MD, Chinoy CN, Francis HA, Harcourt RL, Koebner RMD, Liu CJ, Masojc P, Xie DX, Gale MD (1993 a) Chromosomal rearrangements in the rye genome relative to that of wheat. Theor Appl Genet 85:673–680Google Scholar
  9. Devos KM, Millan T, Gale MD (1993 b) Comparative RFLP maps of the homoeologous group-2 chromosomes of wheat, rye and barley. Theor Appl Genet 85:784–792Google Scholar
  10. Fatokun CA, Singh BB (1987) Interspecific hybridization between Vigna pubescens and V. unguiculata (L.) Walp. through embryo rescue. Plant Cell, Tissue Org Cult 9:229–233Google Scholar
  11. Fatokun CA, Danesh D, Young ND, Steward EL (1993) Molecular taxonomic relationships in the genus Vigna based on RFLP analysis. Theor Appl Genet 86:97–104Google Scholar
  12. Friebe B, Mukai Y, Gill BS, Cauderon Y (1992) C-banding and in situ hybridization analyses of Agropyron intermedium, a partial wheat x Ag. intermedium amphiploid, and six derived chromosome addition lines. Theor Appl Genet 84:899–905Google Scholar
  13. Fuchs J, Brandes A, Schubert I (1995) Telomere sequence localization and karyotype evolution in higher plants. Plant Syst Evol (in press)Google Scholar
  14. Galasso I, Pignone D, Perrino P (1992) Cytotaxonomic studies in Vigna. I. General technique and Vigna unguiculata C-banding. Caryologia 45:155–161Google Scholar
  15. Galasso I, Pignone D, Perrino P (1993) Cytotaxonomic studies in Vigna. II. Heterochromatin characterization in Vigna unguiculata and three related wild species. Caryologia 46:275–282Google Scholar
  16. Ganal MW, Lapitan NLV, Tanksley SD (1991) Macrostructure of the tomato telomeres. Plant Cell 3:87–94Google Scholar
  17. Gerlach WL, Bedbrook JR (1979) Cloning and characterization of ribosomal RNA genes from wheat and barley. Nucleic Acids Res 7:1869–1885PubMedGoogle Scholar
  18. Gerlach WL, Dyer TA (1980) Sequence organization of the repeating units in the nucleus of wheat which contain 5 s rRNA genes. Nucleic Acids Res 8:4851–4855Google Scholar
  19. Harrison GE, Heslop-Harrison JS (1995) Centromeric repetitive DNA in the genus Brassica. Theor Appl Genet 90:157–165Google Scholar
  20. Heslop-Harrison JS (1991) The molecular cytogenetics of plants. J Cell Sci 100:15–21Google Scholar
  21. Heslop-Harrison JS, Schwarzacher T (1991) The ultrastructure of Arabidopsis thaliana chromosomes. In: Schweizer D, Peuker K, Loidl J (eds) 4th Int Conf Arabidopsis Research. University of Vienna, Vienna, p 3Google Scholar
  22. Heslop-Harrison JS, Schwarzacher T (1993) Molecular cytogenetics — biology and applications in plant breeding. Chromosomes Today 11:191–198Google Scholar
  23. Heslop-Harrison JS, Schwarzacher T, Anamtawat-Jonsson K, Leitch AR, Shi M, Leitch IJ (1991) In situ hybridization with automated chromosome denaturation. Technique 3:109–116Google Scholar
  24. Ijdo JM, Wells RA, Baldini A, Reeders ST (1991) Improved telomere detection using a telomere-repeat probe (TTTAGGG)n by PCR. Nucleic Acids Res 19:4780Google Scholar
  25. Kamm A, Schmidt T, Heslop-Harrison JS (1994) Molecular and physical organization of highly repetitive undermethylated DNA from Pennisetum glaucum. Mol Gen Genet 244:420–425Google Scholar
  26. Kamm A, Galasso I, Schmidt T, Heslop-Harrison JS (1995) Analysis of a repetitive DNA family from Arabidopsis arenosa and relationships between Arabidopsis species. Plant Mol Biol 27:853–862Google Scholar
  27. Kenton A, Parokonny AS, Gleba YY, Bennett MD (1993) Characterization of the Nicotiana tabacum L. genome by molecular cytogenetics. Mol Gen Genet 240:159–169Google Scholar
  28. Leitch IJ, Heslop-Harrison JS (1992) Physical mapping of the 18 s-5.8 s-25 s rRNA genes in barley by in situ hybridization. Genome 35:1013–1018Google Scholar
  29. Leitch IJ, Heslop-Harrison JS (1993) Physical mapping of four sites of 5 s rDNA sequences and one site of the alpha-amylase-2 gene in barley (Hordeum vulgare). Genome 36:517–523Google Scholar
  30. Leitch AR, Schwarzacher T, Wang ML, Leitch IJ, Sulan-Momirovich G, Moore G, Heslop-Harrison JS (1993) Molecular cytogenetic analysis of repeated sequences in a long-term wheat suspension culture. Plant Cell Tissue Org Cult 33:287–296Google Scholar
  31. Maluszynska J, Heslop-Harrison JS (1993a) Physical mapping of rDNA in Brassica species. Genome 36:774–781Google Scholar
  32. Maluszynska J, Heslop-Harrison JS (1993b) Molecular cytogenetics of the genus Arabidopsis: in situ localization of rDNA sites, chromosome numbers and diversity in centromeric heterochromatin. Ann Bot 71:479–484Google Scholar
  33. Maréchal R, Mascherpa JM, Stainier F (1978) Etude taxonomique d'un groupe complese d'especes des genres Phaseolus et Vigna (Papilionaceae) sur la base de donnees morphologiques et polliniques, traitèes par l'analyse informatique. Boissiera 28:40–48Google Scholar
  34. Mukai Y, Endo TR, Gill BS (1991) Physical mapping of the 18 s rRNA multigene family in common wheat: identification of a new locus. Chromosoma 100:71–78Google Scholar
  35. Mukai Y, Friebe B, Hatchett JH, Yamamoto M, Gill BS (1993) Molecular cytogenetic analysis of radiation-induced wheat-rye terminal and intercalary chromosomal translocations and the detection of rye chromatin specifying resistance to Hessian fly. Chromosoma 102:88–95Google Scholar
  36. Ng NQ, Maréchal R (1985) Cowpea taxonomy, origin and germplasm. In Singh SR, Rachie KO (eds) Cowpea research, production and utilization, John Wiley and Sons Ltd. pp 11–21Google Scholar
  37. Nowak R (1994) Mining treasures from ‘Junk DNA’. Science 263:608–609Google Scholar
  38. Pardue ML, Gall JG (1970) Chromosomal localization of mouse satellite DNA. Science 168:1356–1358Google Scholar
  39. Quiros CF, Ochoa O, Kianian SF, Douches D (1986) Evolutionary trends in Brassica:gathering evidence from chromosome addition lines. Cruciferae Newslett 11:22–23Google Scholar
  40. Richards EJ, Ausubel FM (1988) Isolation of a higher eukaryotic telomere from Arabidopsis thaliana. Cell 53:127–136PubMedGoogle Scholar
  41. Scherthan H (1990) Localization of the repetitive telomeric sequence (TTAGGG)n in two muntjac species and implications for their karyotypic evolution. Cytogenet Cell Genet 53:115–117Google Scholar
  42. Schmidt T, Heslop-Harrison JS (1994) Variablity and evolution of highly repeated DNA sequences in the genus Beta. Genome 36:1074–1079Google Scholar
  43. Schubert I, Schriever-Schwemmer G, Werner T, Adler ID (1992) Telomeric signals in Robertsonian fusion and fission chromosomes: implications of the origin of pseudoaneuploidy. Cytogenet Cell Genet 59:6–9Google Scholar
  44. Schwarzacher T, Heslop-Harrison JS (1991) In situ hybridization to plant telomeres using synthetic oligomers. Genome 34:317–323Google Scholar
  45. Schwarzacher T, Letich AR, Bennett MD, Heslop-Harrison JS (1989) In situ localization of parental genomes in wide hybrids. Ann Bot 64:315–324Google Scholar
  46. Schwarzacher T, Anamthawat-Jónsson K, Harrison GE, Islam AKMR, Jia JZ, King IP, Leitch AR, Miller TE, Reader SM, Rogers WJ, Shi M, Heslop-Harrison JS (1992) Genomic in situ hybridization to identify alien chromosomes and chromosome segments in wheat. Theor Appl Genet 84:778–786Google Scholar
  47. Schweizer D (1976) Reverse fluorescent chromosome banding with chromomycin and DAPI. Chromosoma 58:307–324Google Scholar
  48. Schweizer D (1980) Fluorescent chromosome banding in plants: applications, mechanisms, and implications for chromosome structure. In: Davies DR, Hopwood DA (eds) Proc 4th John Innes Symp, The Plant Genome. Norwich, UK, pp 61–72Google Scholar
  49. Sen NK, Bhowal JG (1960) Cytotaxonomy studies on Vigna. Cytologia 25:195–207Google Scholar
  50. Tsuchiya T (1960) Cytogenetics studies of trisomies in barley. Jap J Bot 17:177–213Google Scholar
  51. UN (1935) Genome analysis in Brassica with special reference to the experimental formation of B. napus and its peculiar mode of fertilization. Jap J Bot 7:389–452Google Scholar
  52. Vaillancourt RE, Weeden NF (1993) Lack of isozyme similarity between Vigna unguiculata and other species of the subgenus Vigna (Leguminosae). Can J Bot 71:586–591Google Scholar
  53. Wu KS, Tanksley SD (1993) Genetic and physical mapping of telomeres and macrosatellites of rice. Plant Mol Biol 22:861–872Google Scholar
  54. Young ND (1992) Restriction fragment length polymorphisms (RFLPs) and crop improvement. Exp Agric 28:385–397Google Scholar
  55. Zheng JY, Nakata M, Irifune K, Tanaka R, Morikawa H (1993) Fluorescent banding-pattern analysis of eight taxa of Phaseolus and Vigna in relation to their phylogenetic relationships. Theor Appl Genet 87:38–43Google Scholar

Copyright information

© Springer-Verlag 1995

Authors and Affiliations

  • I. Galasso
    • 1
  • T. Schmidt
    • 2
  • D. Pignone
    • 1
  • J. S. Heslop-Harrison
    • 2
  1. 1.Istituto del Germoplasma, CNRBariItaly
  2. 2.Karyobiology Group, Department of Cell BiologyJohn Innes CentreNorwichUK

Personalised recommendations