Biologia Plantarum

, Volume 60, Issue 1, pp 25–36 | Cite as

Precise karyotyping of carrot mitotic chromosomes using multicolour-FISH with repetitive DNA

  • A. Nowicka
  • E. Grzebelus
  • D. Grzebelus
Original Papers


Carrot (Daucus carota L.) chromosomes are small and uniform in shape and length. Here, mitotic chromosomes were subjected to multicolour fluorescence in situ hybridization (mFISH) with probes derived from conserved plant repetitive DNA (18-25S and 5S rDNA, telomeres), a carrot-specific centromeric repeat (Cent-Dc), carrot-specific repetitive elements (DCREs), and miniature inverted-repeat transposable elements (MITEs). A set of major chromosomal landmarks comprising rDNA and telomeric and centromeric sequences in combination with chromosomal measurements enabled discrimination of carrot chromosomes. In addition, reproducible and unique FISH patterns generated by three carrot genome-specific repeats (DCRE22, DCRE16, and DCRE9) and two transposon families (DcSto and Krak) in combination with telomeric and centromeric reference probes allowed identification of chromosome pairs and construction of detailed carrot karyotypes. Hybridization patterns for DCREs were observed as pericentromeric and interstitial dotted tracks (DCRE22), signals in pericentromeric regions (DCRE16), or scattered signals (DCRE9) along chromosomes similar to those observed for both MITE families.

Additional key words

Daucus carota repetitive elements fluorescence in situ hybridization miniature inverted-repeat transposable elements 



bacterial artificial chromosome




Daucus carota repetitive element


fluorescence in situ hybridization


fluorescein isothiocyanate


multicolour fluorescence in situ hybridization


miniature inverted-repeat transposable element


nucleolar organizer region


saline-sodium citrate


transposable element


terminal inverted repeat


telomeric repeat sequence


target site duplications


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Supplementary material

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  1. Altinkut, A., Kotseruba, V., Kirzhner, V.M., Nevo, E., Raskina, O., Belyayev, A.: Ac-like transposons in populations of wild diploid Triticeae species: comparative analysis of chromosomal distribution. — Chromosome Res. 14: 307–317, 2006.CrossRefPubMedGoogle Scholar
  2. Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J., Zhang, Z., Miller, W., Lipman, D.J.: Gapped BLAST and PSIBLAST; a new generation of protein database search programs. — Nucl. Acids Res. 25: 3389–3402, 1997.PubMedCentralCrossRefPubMedGoogle Scholar
  3. Arscott, S.A., Tanumihardjo, S.A.: Carrots of many colors provide basic nutrition and bioavailable phytochemicals acting as a functional food. — Comp. Rev. Food Sci. Safety 9: 223–239, 2010.CrossRefGoogle Scholar
  4. Arumuganathan, K., Earle, E.D.: Nuclear DNA content of some important plant species. — Plant mol. biol. Rep. 9: 208–218, 1991.CrossRefGoogle Scholar
  5. Boyle, S., Rodesch, M.J., Halvensleben, H.A., Jeddeloh, J.A., Bickmore, W.A.: Fluorescence in situ hybridization with high-complexity repeat-free oligonucleotide probes generated by massively parallel synthesis. — Chromosome Res. 19: 901–909, 2011.PubMedCentralCrossRefPubMedGoogle Scholar
  6. Cavagnaro, P.F., Chung, S.M., Szklarczyk, M., Grzebelus, D., Senalik, D., Atkins, A.E., Simon, P.W.: Characterization of a deep-coverage carrot (Daucus carota L.) BAC library and initial analysis of BAC-end sequences. — Mol. Genet. Genomics 281: 273–288, 2009.CrossRefPubMedGoogle Scholar
  7. Cheng, Z., Dong, F., Langdon, T., Ouyang, S., Buell, R., Gu, M., Blattner, F.R., Jiang, J.: Functional rice centromeres are marked by a satellite repeat and a centromere-specific retrotransposon. — Plant Cell 14: 1691–1704, 2002.PubMedCentralCrossRefPubMedGoogle Scholar
  8. Chomczynski, P., Sacchi, N.: Single-step method of RNA isolation by acid guanidinium thiocyanate-phenolchloroform extraction. — Anal. Biochem. 162: 156–159, 1987.CrossRefPubMedGoogle Scholar
  9. Dechyeva, D., Gindullis, F., Schmidt, T.: Divergence of satellite DNA and interspersion of dispersed repeats in the genome of the wild beet Beta procumbens. — Chromosome Res. 11; 3–21, 2003.CrossRefPubMedGoogle Scholar
  10. Dydak, M., Kolano, B., Nowak, T., Siwinska, D,. Maluszynska, J.: Cytogenetic studies of three European species of Centaurea L. (Asteraceae). — Hereditas 146: 152–161, 2009.CrossRefPubMedGoogle Scholar
  11. Essad, S.: [Banding and biometry applied to karyotype analysis in Daucus carota L.] — Agronomie 5: 871–876, 1985. [In French]CrossRefGoogle Scholar
  12. Feschotte, C., Swamy, L., Wessler, S.R.: Genome-wide analysis of Mariner-like transposable elements in rice reveals complex relationships with Stowaway miniature inverted repeat transposable elements (MITEs). — Genetics 163: 747–758, 2003.PubMedCentralPubMedGoogle Scholar
  13. Findley, S.D., Cannon, S., Varala, K., Du, J., Ma, J., Hudson, M.E., Birchler, J.A., Stacey, G.: A fluorescence in situ hybridization system for karyotyping soybean. — Genetics 185: 727–744, 2010.PubMedCentralCrossRefPubMedGoogle Scholar
  14. Fuchs, J., Brandes, A., Schubert, I.: Telomere sequence localization and karyotype evolution in higher plants. — Plant Syst. Evol. 196: 227–241, 1995.CrossRefGoogle Scholar
  15. Gerlach, W.L., Bedbrook, J.R.: Cloning and characterization of ribosomal RNA genes from wheat and barley. — Nucl. Acids Res. 109: 1346–1352, 1979.Google Scholar
  16. Gerlach, W.L., Dyer, T.A.: Sequence organization of the repeating units in the nucleus of wheat which contain 5S rRNA genes. — Nucl. Acids Res. 8: 4851–4865, 1980.PubMedCentralCrossRefPubMedGoogle Scholar
  17. Grabowska-Joachimiak, A., Kula, A., Gernand-Kliefoth, D., Joachimiak, A.J.: Karyotype structure and chromosome fragility in the grass Phleum echinatum Host. — Protoplasma 252: 301–306, 2015.PubMedCentralCrossRefPubMedGoogle Scholar
  18. Grzebelus, D., Jagosz, B., Simon, P.W.: The DcMaster transposon display maps polymorphic insertion sites in the carrot (Daucus carota L.) genome. — Gene 390: 67–74, 2007.CrossRefPubMedGoogle Scholar
  19. Grzebelus, D., Simon, P.W.: Diversity of DcMaster-like elements of the PIF/Harbinger superfamily in the carrot genome. — Genetica 135: 347–353, 2009.CrossRefPubMedGoogle Scholar
  20. Grzebelus, D., Yau, Y.-Y., Simon, P.W.: Master: a novel family of PIF/Harbinger-like transposable elements identified in carrot (Daucus carota L.). Mol. Genet. Genomics 275: 450–459, 2006.CrossRefPubMedGoogle Scholar
  21. Hajdera, I., Siwinska, D,. Hasterok, R., Maluszynska, J.; Molecular cytogenetic analysis of genome structure in Lupinus angustifolius and Lupinus cosentinii. — Theor. appl. Genet. 107: 988–996, 2003.CrossRefPubMedGoogle Scholar
  22. Hasterok, R., Jenkins, G., Langdon, T., Jones, R.N., Maluszynska, J.: Ribosomal DNA is an effective marker of Brassica chromosomes. — Theor. appl. Genet. 103: 486–490, 2001.CrossRefGoogle Scholar
  23. Hasterok, R., Langdon, T., Taylor, S., Jenkins, G.; Combinatorial labelling of DNA probes enables multicolour fluorescence in situ hybridisation in plants. — Folia histochem. cytobiol. 40: 319–332, 2002.PubMedGoogle Scholar
  24. Heslop-Harrison, J.S.: Comparative genome organization in plants: from sequence and markers to chromatin and chromosomes. — Plant Cell 12: 617–635, 2000.PubMedCentralCrossRefPubMedGoogle Scholar
  25. Heslop-Harrison, J.S., Brandes, A., Taketa, S., Schmidt, T., Vershinin, A.V., Alkhimova, E.G., Kamm, A., Doudrick, R.L., Schwarzacher, T., Katsiotis, A., Kubis, S., Kumar, A., Pearce, S.R., Flavell, A.J., Harrison, G.E.: The chromosomal distributions of Ty1-copia group retrotransposable elements in higher plants and their implications for genome evolution. — Genetica 100: 197–204, 1997.CrossRefPubMedGoogle Scholar
  26. Hizume, M., Shibata, F., Matsusaki, Y., Garajova, Z.; Chromosome identification and comparative karyotypic analyses of four Pinus species. — Theor. appl. Genet. 105; 491–497, 2002.CrossRefPubMedGoogle Scholar
  27. Horakova, M., Fajkus, J.: TAS49 - a dispersed repetitive sequence isolated from subtelomeric regions of Nicotiana tomentosiformis chromosomes. — Genome 43: 273–284, 2000.PubMedGoogle Scholar
  28. Hoshi, Y., Yagi, K., Matsuda, M., Matoba, H., Tagashira, N., Plader, W., Malepszy, S., Nagano, K., Morikawa, A.: A comparative study of the three cucumber cultivars using fluorescent staining and fluorescence in situ hybridization. — Cytologia 76: 3–10, 2011.CrossRefGoogle Scholar
  29. Hueros, G., Loarce, Y., Ferrer, E.: A structural and evolutionary analysis of a dispersed repetitive sequence. — Plant mol. Biol. 22: 635–643, 1993.CrossRefPubMedGoogle Scholar
  30. Idziak, D., Hazuka, I., Poliwczak, B., Wiszynska, A., Wolny, E., Hasterok, R.: Insight into the karyotype evolution of Brachypodium species using comparative chromosome barcoding. — PLoS ONE 9: e93503, 2014.PubMedCentralCrossRefPubMedGoogle Scholar
  31. Iovene, M., Grzebelus, E., Carputo, D., Jiang, J., Simon, P.W.; Major cytogenetic landmarks and karyotype analysis in Daucus carota and other Apiaceae. — Amer. J. Bot. 95: 793–804, 2008.CrossRefGoogle Scholar
  32. Iovene, M., Cavagnaro, P.F., Senalik, D., Buell, C.R., Jiang, J., Simon, P.W.: Comparative FISH mapping of Daucus species (Apiaceae family). — Chromosome Res. 19: 493–506, 2011.CrossRefPubMedGoogle Scholar
  33. Itoh, Y., Hasebe, M., Davies, E., Takeda, J., Ozeki, Y.: Survival of Tdc transposable elements of the En/Spm superfamily in the carrot genome. — Mol. Genet. Genomics 269: 49–59, 2003.PubMedGoogle Scholar
  34. Jiang, N., Feschotte, C., Zhang, X., Wessler, S.R.: Using rice to understand the origin and amplification of miniature inverted repeat transposable elements (MITEs). — Curr. Opin. Plant Biol. 7: 115–119, 2004.CrossRefPubMedGoogle Scholar
  35. Jin, W., Melob, J.R., Nagaki, K., Talbert, P.B., Henikoff, S., Kelly, D.R., Jiang, J.: Maize centromeres: organization and functional adaptation in the genetic background of oat. —Plant Cell 16: 571–581, 2004.PubMedCentralCrossRefPubMedGoogle Scholar
  36. Jurka, J., Kapitonov, V.V., Pavlicek, A., Klonowski, P., Kohany, O., Walichiewicz, J.: Repbase update, a database of eukaryotic repetitive elements. — Cytogenet. Genome Res. 110: 462–467, 2005.CrossRefPubMedGoogle Scholar
  37. Kato, A., Lamb, J.C., Birchler, J.A.: Chromosome painting using repetitive DNA sequences as probes for somatic chromosome identification in maize. — Proc. nat. Acad. Sci. USA 101: 13554–13559, 2004.PubMedCentralCrossRefPubMedGoogle Scholar
  38. Kiefer-Meyer, M.C., Reddy, A.S., Delseny, M.: Complex arrangement of dispersed repeated DNA sequences in Oryza officinalis. — Genome 39: 183–190, 1996.CrossRefPubMedGoogle Scholar
  39. Kolano, B., Plucienniczak, A., Kwasniewski, M., Maluszynska, J.: Chromosomal localization of a novel repetitive sequence in the Chenopodium quinoa genome. — J. appl. Genet. 49; 313–320, 2008.CrossRefPubMedGoogle Scholar
  40. Kolano, B., Gardunia, B.W., Michalska, M., Bonifacio, A., Fairbanks, D., Maughan, P.J., Coleman, C.E., Stevens, M.R., Jellen, E.N., Maluszynska, J.: Chromosomal localization of two novel repetitive sequences isolated from the Chenopodium quinoa Willd. — Genome 54: 710–717, 2011.CrossRefPubMedGoogle Scholar
  41. Kubis, S., Schmidt, T., Seymour, J., Heslop-Harrison, J.S.; Repetitive DNA elements as a major component of plant genomes. — Ann. Bot. 82: 45–55, 1998.CrossRefGoogle Scholar
  42. Kulikova, O., Gualtieri, G., Guerts, R., Kim, D.J., Cook, D.; Integration of the FISH pachytene and genetic maps of Medicago truncatula. — Plant J. 27: 49–58, 2001.CrossRefPubMedGoogle Scholar
  43. Kulikova, O., Geurts, R., Lamine, M., Kim, D.J., Cook, D.R., Leunissen, J., De Jong, H., Roe, B.A., Bisseling, T.; Satellite repeats in the functional centromere and pericentromeric heterochromatin of Medicago truncatula. — Chromosoma 113: 276–283, 2004.CrossRefPubMedGoogle Scholar
  44. Kumar, P., Widholm, J.M.: Techniques for chromosome analysis of carrot culture cells. — Plant mol. biol. Rep. 2: 37–42, 1984.CrossRefGoogle Scholar
  45. Kumar, A., Bennetzen, J.L.: Plant retrotransposons. — Annu. Rev. Genet. 33: 479–532, 1999.CrossRefPubMedGoogle Scholar
  46. Levan, A., Fredga, K., Sandberg, A.A.: Nomenclature for centromeric position on chromosomes. — Hereditas 52: 201–220, 1964.CrossRefGoogle Scholar
  47. Macko-Podgorni, A., Nowicka, A., Grzebelus, E., Simon, P.W., Grzebelus, D.: DcSto: carrot Stowaway-like elements are abundant, diverse, and polymorphic. — Genetica 141: 255–267, 2013.PubMedCentralCrossRefPubMedGoogle Scholar
  48. Menzel, G., Dechyeva, D., Keller, H., Lange, C., Himmelbauer, H., Schmidt, T.: Mobilization and evolutionary history of miniature inverted-repeat transposable elements (MITEs) in Beta vulgaris L. — Chromosome Res. 14: 831–844, 2006.CrossRefPubMedGoogle Scholar
  49. Menzel, G., Dechyeva, D., Wenke, T., Holtgraewe, D., Weisshaar, B., Schmidt, T.: Diversity of a complex centromeric satellite and molecular characterization of dispersed sequence families in sugar beet (Beta vulgaris). — Ann. Bot. 102: 521–530, 2008.PubMedCentralCrossRefPubMedGoogle Scholar
  50. Navratilova, A., Neumann, P., Macas, J.: Karyotype analysis of four Vicia species using in situ hybridization with repetitive sequences. — Ann. Bot. 91: 921–926, 2003.PubMedCentralCrossRefPubMedGoogle Scholar
  51. Neumann, P., Nouzova, M., Macas, J.: Molecular and cytogenetic analysis of repetitive DNA in pea (Pisum sativum L.). — Genome 44: 716–728, 2001.CrossRefPubMedGoogle Scholar
  52. Nowicka, A., Grzebelus, E., Grzebelus, D.: Fluorescent in situ hybridization with arbitrarily amplified DNA fragments differentiates carrot (Daucus carota L.) chromosomes. —Genome 55: 205–213, 2012.CrossRefPubMedGoogle Scholar
  53. Ozeki, Y., Davies, E., Takeda, J.: Somatic variation during long term subculturing of plant cells caused by insertion of a transposable element in a phenylalanine ammonia-lyase (PAL) gene. — Mol. gen. Genet. 254: 407–416, 1997.CrossRefPubMedGoogle Scholar
  54. Paesold, S., Borchardt, D., Schmidt, T., Dechyeva, D.: A sugar beet (Beta vulgaris L.) reference FISH karyotype for chromosome and chromosome-arm identification, integration of genetic linkage groups and analysis of major repeat family distribution. — Plant J. 72: 600–611, 2012.CrossRefPubMedGoogle Scholar
  55. Rozen, S., Skaletsky, H.J.: Primer3 on the www for general users and for biologist programmers. — In: Krawetz, S., Misener, S. (ed.): Bioinformatics Methods and Protocols; Methods in Molecular Biology. Pp. 365–386. Humana Press, Totowa 2000.Google Scholar
  56. Schmidt, T., Kubis, S., Katsiotis, A., Jung, C., Heslop-Harrison, J.S.: Molecular and chromosomal organization of two repetitive DNA sequences with intercalary locations in sugar beet and other Beta species. — Theor. appl. Genet. 97; 696–704, 1998.CrossRefGoogle Scholar
  57. Schrader, O., Ahne, R., Fuchs, J.: Karyotype analysis of Daucus carota L. using Giemsa C-Banding and FISH of 5S and 18S/25S rRNA specific genes. — Caryologia 56: 149–154, 2003.CrossRefGoogle Scholar
  58. Schwarzacher, T.: DNA, chromosomes, and in situ hybridization. — Genome 46: 953–962, 2003.CrossRefPubMedGoogle Scholar
  59. Stebbins, G.L.: Chromosomal Evolution in Higher Plants. — Edward Arnold Publishers, London 1971.Google Scholar
  60. Szinay, D., Chang, S.B., Khrustaleva, L., Peters, S., Schijlen, E., Bai, Y., Stiekema, W.J., Van Ham, R., De Jong, H., Klein Lankhorst, R.: High-resolution chromosome mapping of BACs using multi-colour FISH and pooled-BAC FISH as a backbone for sequencing tomato chromosome 6. — Plant J. 56: 627–637, 2008.CrossRefPubMedGoogle Scholar
  61. Turcotte, K., Srinivasan, S., Bureau, T.E.: Survey of transposable elements from rice genomic sequences. — Plant J. 25: 169–179, 2001.CrossRefPubMedGoogle Scholar
  62. Weber, B., Wenke, T., Frommel, U., Schmidt, T., Heitkam, T.; The Ty1-copia families SALIRE and Cotzilla populating the Beta vulgaris genome show remarkable differences in abudance, chromosomal distribution, and age. — Chromosome Res. 18: 247–263, 2010.CrossRefPubMedGoogle Scholar
  63. Wicker, T., Sabot, F., Hua-Van, A., Bennetzen, J.L., Capy, P., Chalhoub, B., Flavell, A., Leroy, P., Morgante, M., Panaud, O., Paux, E., San Miguel, P., Schulman, A.H.: A unified classification system for eukaryotic transposable elements. — Nat. Rev. Genet. 8: 973–982, 2007.CrossRefPubMedGoogle Scholar
  64. Wolny, E., Fidyk, W., Hasterok, R.: Karyotyping of Brachypodium pinnatum (2n = 18) chromosomes using cross-species BAC-FISH. — Genome 56: 239–243, 2013.CrossRefPubMedGoogle Scholar
  65. Yu, W., Lamb, J.C., Han, F., Birchler, J.A.: Cytological visualization of DNA transposons and their transposition pattern in somatic cells of maize. — Genetics 175: 31–39, 2007.PubMedCentralCrossRefPubMedGoogle Scholar

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Authors and Affiliations

  1. 1.Department of Cell Biology, Franciszek Gorski Institute of Plant PhysiologyPolish Academy of SciencesKrakowPoland
  2. 2.Department of Genetics, Plant Breeding and Seed Science, Institute of Plant Biology and Biotechnology, Faculty of Biotechnology and HorticultureUniversity of Agriculture in KrakowKrakowPoland

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