Chromosome Research

, Volume 21, Issue 8, pp 739–751 | Cite as

Mapping nonrecombining regions in barley using multicolor FISH

  • M. Karafiátová
  • J. Bartoš
  • D. Kopecký
  • L. Ma
  • K. Sato
  • A. Houben
  • N. Stein
  • J. Doležel


Fluorescence in situ hybridization (FISH) is a widely used method to localize DNA sequences on chromosomes. Out of the many uses, FISH facilitates construction of physical maps by ordering contigs of large-insert DNA clones, typically bacterial artificial chromosome (BAC) and establishing their orientation. This is important in genomic regions with low recombination frequency where genetic maps suffer from poor resolution. While BAC clones can be mapped directly by FISH in plants with small genomes, excess of repetitive DNA hampers this application in species with large genomes. Mapping single-copy sequences such as complementary DNA (cDNA) is an attractive alternative. Unfortunately, localization of single-copy sequences shorter than 10 kb remains a challenging task in plants. Here, we present a highly efficient FISH technique that enables unambiguous localization of single copy genes. We demonstrated its utility by mapping 13 out of 15 full-length cDNAs of variable length (2,127–3,400 bp), which were genetically defined to centromeric and pericentromeric regions of barley chromosome 7H. We showed that a region of 1.2 cM (0.7 %) on genetic map represented more than 40 % of the physical length of the chromosome. Surprisingly, all cDNA probes occasionally revealed hybridization signals on other chromosomes, indicating the presence of partially homologous sequences. We confirmed the order of 10 cDNA clones and suggested a different position for three cDNAs as compared to published genetic order. These results underline the need for alternative approaches such as FISH, which can resolve the order of markers in genomic regions where genetic mapping fails.


cDNA multicolor FISH low-copy FISH nonrecombining regions physical mapping genetic mapping 



Bacterial artificial chromosome-fluorescence in situ hybridization

Cy3, Cy5

cyanine dyes


Deoxyuridine triphosphate




Full-length complementary DNA


Fluorescence in situ hybridization


Fluorescein isothiocyanate




Ribosomal DNA



We appreciate technical advice from our colleague Marie Kubaláková. This work was supported jointly by the Czech Academy of Sciences and the German Academic Exchange Service–DAAD (grant award no. CZ07-DE12/2013-2014) by Internal Grant Agency of Palacky University in Olomouc (grant award no. IGA PrF/2012/001) and by the Ministry of Education, Youth and Sports of the Czech Republic and the European Regional Development Fund (Operational Programme Research and Development for Innovations No. ED0007/01/01).

Supplementary material

10577_2013_9380_MOESM1_ESM.doc (328 kb)
ESM_1 Additional hybridization signals observed after FISH with probes for cDNA on complete metaphase spreads (DOC 327 kb)


  1. Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402PubMedCentralPubMedCrossRefGoogle Scholar
  2. Birnbiom HC (1983) A rapid alkaline extraction method for the isolation of plasmid DNA. Methods Enzymol 100:243–255Google Scholar
  3. Cai W, Ling J, Irvin B, Ohler L, Rose E et al (1998) High-resolution restriction maps of bacterial artificial chromosomes constructed by optical mapping. Proc Natl Acad Sci U S A 95:3390–3395PubMedCentralPubMedCrossRefGoogle Scholar
  4. Cao M, Sleper DA, Dong F, Jiang J (2000) Genomic in situ hybridization (GISH) reveals high chromosome pairing affinity between Lolium perenne and Festuca mairei. Genome 43:398–403PubMedGoogle Scholar
  5. Cheng Z, Presting GG, Buell CR, Wing RA, Jiang J (2001) High-resolution pachytene chromosome mapping of bacterial artificial chromosomes anchored by genetic markers reveals the centromere location and the distribution of genetic recombination along chromosomes 10 of rice. Genetics 157:1749–1757PubMedGoogle Scholar
  6. de Jong JH, Fransz P, Zabel P (1999) High resolution FISH in plants—techniques and applications. Trends Plant Sci 4:258–263CrossRefGoogle Scholar
  7. Devos KM, Gale MD (1993) Extended genetic maps of the homeologous group-3 chromosomes of wheat, rye and barley. Theor Appl Genet 85:649–652PubMedGoogle Scholar
  8. Doležel J, Greilhuber J, Lucretti S, Meister A, Lysak MA, Nardi L, Obermayer R (1998) Plant genome size estimation by flow-cytometry: inter-laboratory comparison. Ann Bot 82:17–26CrossRefGoogle Scholar
  9. Endo TR (1990) Gametocidal chromosomes and their induction of chromosome mutation in wheat. Jpn J Genet 65:135–152CrossRefGoogle Scholar
  10. Endo TR (2007) The gametocidal chromosomes as a tool for chromosome manipulation in wheat. Chromosome Res 15:67–75PubMedCrossRefGoogle Scholar
  11. Farré A, Cuadrado A, Lacasa-Benito I, Cistué L, Schubert I, Comadran J, Jansen J, Romagosa I (2012) Genetic characterization of a reciprocal translocation present in a widely grown barley variety. Mol Breed 30:1109–1119PubMedCentralPubMedCrossRefGoogle Scholar
  12. Fischer JA, Favreau MB (1991) Plasmid purification by phenol extraction from guanidinium thiocyanate solution: development of an automated protocol. Anal Biochem 194:309–315CrossRefGoogle Scholar
  13. Fuchs J, Schubert I (1995) Localization of seed protein genes on metaphase chromosomes of Vicia faba via fluorescence in situ hybridization. Chromosome Res 3:94–100PubMedCrossRefGoogle Scholar
  14. Fukui K, Kamisugi Y, Sakai F (1994) Physical mapping of 5S rDNA loci by direct-cloned biotinylated probes in barley chromosomes. Genome 37:105–117PubMedCrossRefGoogle Scholar
  15. Gamborg OL, Wetter LR (1975) Plant tissue culture methods. National Research Council of Canada, SaskatoonGoogle Scholar
  16. Giacalone J, Delobette V, Gibaja L, Ni L, Skidas Y et al (2000) Optical mapping of BAC clones from the human Y chromosome DAZ locus. Genome Res 10:1421–1429PubMedCrossRefGoogle Scholar
  17. Harwood WA, Bilham LJ, Travella S, Salvo-Garrido H, Snape JW (2005) Fluorescence in situ hybridization to localize transgenes in plant chromosomes. Methods Mol Biol 286:327–340PubMedGoogle Scholar
  18. Heng HH, Tsui LC (1998) High resolution free chromatin/DNA fiber fluorescent in situ hybridization. J Chromatogr 86:219–229CrossRefGoogle Scholar
  19. Janda J, Šafář J, Kubaláková M, Bartoš J, Kovářová P, Suchánková P, Pateyron S, Číhalíková J, Sourdille P, Šimková H, Faivre-Rampant P, Hřibová E, Bernard M, Lukaszewski A, Doležel J, Chalhoub B (2006) Advanced resources for plant genomics: BAC library specific for the short arm of wheat chromosome 1B. Plant J 47:977–986PubMedCrossRefGoogle Scholar
  20. Joshi GP, Nasuda S, Endo TR (2011) Dissection and cytological mapping of barley chromosome 2H in the genetic background of common wheat. Genes Genet Syst 86:231–248PubMedCrossRefGoogle Scholar
  21. Kato A (2011) High-density fluorescence in situ hybridization signal detection on barley (Hordeum vulgare L.) chromosomes with improved probe screening and reprobing procedures. Genome 54:151–159PubMedCrossRefGoogle Scholar
  22. Kato A, Vega JM, Han FP, Lamb JC, Birchler JA (2005) Advances in plant chromosome identification and cytogenetic techniques. Curr Opin Plant Biol 8:148–154PubMedCrossRefGoogle Scholar
  23. Kato A, Albert PS, Vega JM, Bichler JA (2006) Sensitive fluorescence in situ hybridization signal detection in maize using directly labelled probes produced by high concentration DNA polymerase nick translation. Biotech Histochem 81:71–78PubMedCrossRefGoogle Scholar
  24. Kocsis E, Trus BL, Steer CJ, Bisher ME, Steven AC (1991) Image averaging of flexible fibrous macromolecules: the clathrin triskelion has an elastic proximal segment. J Struct Biol 107:6–14PubMedCrossRefGoogle Scholar
  25. Kumar A, Simons K, Iqbal MJ, de Jiménez MM, Bassi FM, Ghavami F, Al-Azzam O, Drader T, Wang Y, Luo MC, Gu YQ, Denton A, Lazo GR, Xu SS, Dvorak J, Kianian PM, Kianian SF (2012) Physical mapping resources for large plant genomes: radiation hybrids for wheat d-genome progenitor Aegilops tauschii. BMC Genomics 13:597PubMedCentralPubMedCrossRefGoogle Scholar
  26. Künzel G, Korzun L, Meister A (2000) Cytologically integrated physical restriction fragment length polymorphism maps for the barley genome based on translocation breakpoints. Genetics 154:397–412PubMedGoogle Scholar
  27. Leicht IJ, Heslop-Harrison JS (1992) Physical mapping of 18S-5,8S-26S rRNA genes in barley by in situ hybridization. Genome 35:1013–1018CrossRefGoogle Scholar
  28. Leicht IJ, Heslop-Harrison JS (1993) Physical mapping of 4 sites of 5S rDNA sequences and one site of the aplha-amylase-2 gene in barley (Hordeum vulgare). Genome 36:517–523CrossRefGoogle Scholar
  29. Linde-Laursen IB (1978) Giemsa C-banding of barley chromosomes; II. Banding patterns of trisomics and telotrisomics. Hereditas 89:37–41CrossRefGoogle Scholar
  30. Lysák MA, Číhalíková J, Kubaláková M, Šimková H, Künzel G, Doležel J (1999) Flow karyotyping and sorting of mitotic chromosomes of barley (Hordeum vulgare L.). Chromosome Res 7:431–444PubMedCrossRefGoogle Scholar
  31. Ma L, Vu GTH, Schubert V, Watanabe K, Stein N, Houben A, Schubert I (2010) Synteny between Brachypodium distachyon and Hordeum vulgare as revealed by FISH. Chromosome Res 18:841–850PubMedCrossRefGoogle Scholar
  32. Masoudi-Nejad A, Nasuda S, McIntosh RA, Endo TR (2002) Transfer of rye chromosome segments to wheat by gametocidal system. Chromosome Res 10:349–357PubMedCrossRefGoogle Scholar
  33. Mayer KFX, Martis M, Hedley PE, Šimková H, Liu H, Morris JA, Steurnagel B, Taudien S, Roessner S, Gundlach H, Kubaláková M, Suchánková P, Murat F, Felder M, Nussbaumer T, Graner A, Slase J, Endo T, Sakai H, Tanaka T, Itoh T, Sato K, Platzer M, Matsumoto T, Scholz U, Doležel J, Waugh R, Stein N (2011) Unlocking the barley genome by chromosomal and comparative genomics. Plant Cell 23:1249–1263PubMedCentralPubMedCrossRefGoogle Scholar
  34. Meyers BC, Scalabrin S, Morgante M (2004) Mapping and sequencing complex genomes: let’s get physical! Nat Rev Genet 5:578–588PubMedCrossRefGoogle Scholar
  35. Michalak de Jimenez MK, Bassi FM, Ghavami F, Simons K, Dizon R, Seetan RI, Alnemer LM, Denton AM, Doğramacı M, Šimková H, Doležel J, Seth K, Luo MC, Dvorak J, Gu YQ, Kianian SF (2013) A radiation hybrid map of chromosome 1D reveals synteny conservation at a wheat speciation locus. Funct Integr Genomics 13:19–32PubMedCrossRefGoogle Scholar
  36. Paux E, Sourdille P, Salse J, Saintenac C, Choulet F, Leroy P, Korol A, Michalak M, Kianian S, Spielmeyer W, Lagudah E, Somers D, Kilian A, Alaux M, Vautrin S, Bergès H, Eversole K, Appels R, Safar J, Simkova H, Dolezel J, Bernard M, Feuillet C (2008) A physical map of the 1-gigabase bread wheat chromosome 3B. Science 322:101–104PubMedCrossRefGoogle Scholar
  37. Pérez R, de Bustos A, Cuadrado Á (2009) Localization of Rad50, a single-copy gene, on group 5 chromosomes of beat, using a FISH protocol employing tyramide for signal amplification (Tyr-FISH). Cytogenet Genome Res 125:321–328PubMedCrossRefGoogle Scholar
  38. Peterson DG, Lapitan NLV, Stack SM (1999) Localization of single- and low-copy sequences on tomato synaptonemal complex spreads using fluorescence in situ hybridization (FISH). Genetics 152:427–439PubMedGoogle Scholar
  39. Phillips D, Nibau C, Wnetrzak J, Jenkins G (2012) High resolution analysis of meiotic chromosome structure and behaviour in barley (Hordeum vulgare L.). PLoS One 7:e39539PubMedCentralPubMedCrossRefGoogle Scholar
  40. Riera-Lizarazu O, Vales MI, Kianian SF (2008) Radiation hybrid (PH) and happy mapping in plants. Cytogenet Genome Res 120:233–240PubMedCrossRefGoogle Scholar
  41. Sakai K, Nasuda S, Sato K, Endo TR (2009) Dissection of barley chromosome 3H in common wheat and comparison of 3H physical and genetic maps. Genes Genet Syst 84:25–34PubMedCrossRefGoogle Scholar
  42. Sato K, Shin-I T, Seki M, Shinozaki K, Yoshida H, Takeda K, Yamanaki Y, Conte M, Kohara Y (2009) Development of 5006 full-length CDNAs in barley: a tool for accessing cereal genomics resources. DNA Res 16:81–89PubMedCentralPubMedCrossRefGoogle Scholar
  43. Schnabel E, Kulikova O, Penmetsa RV, Bisseling T, Cook DR, Frugoli J (2003) An integrated physical, genetic and cytogenetic map around the sunn locus of Medicago truncatula. Genome 46:665–672PubMedCrossRefGoogle Scholar
  44. Schwartz DC, Li X, Hernandez LI, Ramnarain SP, Huff EJ, Wang YK (1993) Ordered restriction maps of Saccharomyces cerevisiae chromosomes constructed by optical mapping. Science 262:110–114PubMedCrossRefGoogle Scholar
  45. Singh RJ, Tsuchiya T (1982) An improved Giemsa N-banding technique for the identification of barley chromosomes. J Hered 73:227–229Google Scholar
  46. Tang X, Szinay D, Lang C, Ramanna MS, van der Vossen EAG, Datema E, Lankhorst RK, de Boer J, Peters SA, Bachem C, Stiekema W, VisserR GF, de Jong H, Bai Y (2008) Cross-species bacterial artificial chromosome-fluorescence in situ hybridization painting of the tomato and potato chromosome 6 reveals undescribed chromosomal rearrangements. Genetics 180:1319–1328PubMedCrossRefGoogle Scholar
  47. Tang X, de Boer JM, van Eck HJ, Bachem CH, Visser RGF, de Jong H (2009) Assignment of genetic linkage maps to diploid Solanum tuberosum pachytene chromosomes by BAC-FISH technology. Chromosome Res 17:399–915CrossRefGoogle Scholar
  48. The International Barley Genome Sequencing Consortium (2012) A physical, genetical and functional sequence assembly of the barley genome. Nature 491:711–716Google Scholar
  49. The International Brachypodium Initiative (2010) Genome sequencing and analysis of the model grass Brachypodium distachyon. Nature 463:763–768CrossRefGoogle Scholar
  50. Tjio JH, Hagberg A (1951) Cytological studies on some X-ray mutants of barley. Ann Aula Dei 2:149–167Google Scholar
  51. Tomato Genome Consortium (2012) The tomato genome sequence provides the insight into fleshy fruit evolution. Nature 485:635–641CrossRefGoogle Scholar
  52. Tsujimoto H, Mukai Y, Keisuke A, Nagaki K, Fujigaki J, Yamamoto M, Sasakuma T (1997) Identification of individual barley chromosomes based on repetitive sequences: conservative distribution of Afa-family repetitive sequences on the chromosome of barley and wheat. Genes Genet Syst 72:303–309PubMedCrossRefGoogle Scholar
  53. Valárik M, Bartoš J, Kovářová P, Kubaláková M, de Jong JH, Doležel J (2004) High-resolution FISH on super-stretched flow-sorted plant chromosomes. Plant J 37:940–950PubMedCrossRefGoogle Scholar
  54. Wang CH-JR, Harper L, Cande WZ (2006) High-resolution single-copy gene fluorescence in situ hybridization and its use in the construction of a cytogenetic map of maize chromosome 9. Plant Cell 18:529–544PubMedCentralPubMedCrossRefGoogle Scholar
  55. Wardrop J, Sape J, Powell W, Machray GC (2002) Constructing plant radiation hybrids panel. Plant J 31:223–228PubMedCrossRefGoogle Scholar
  56. Wicker T, Mayer KFX, Gundlach H, Martis M, Steuernagel B, Scholz U, Šimková H, Kubaláková M, Choulet F, Taudien S, Platzer M, Feuillet C, Fahima T, Budak H, Doležel J, Keller B, Stein N (2011) Frequent gene movement and pseudogene evolution is common to the large and complex genomes of wheat, barley, and their relatives. Plant Cell 23:1706–1718PubMedCentralPubMedCrossRefGoogle Scholar
  57. Yang K, Zhang H, Converse R, Wang Y, Rong X, Wu Z, Luo B, Yue L, Jian L, Zhu L, Wang X (2011) Fluorescence in situ hybridization on plant extended chromatin DNA fibres for single-copy and repetitive DNA sequences. Plant Cell Rep 30:1779–1786PubMedCrossRefGoogle Scholar
  58. Zhou S, Bechner MC, Place M, Churas CP, Pape L, Leong SA, Runnheim R, Forrest D, Goldstein S, Livny M, Schwartz DC (2007) Validation of rice genome sequence by optical mapping. BMC Genomics 8:278PubMedCentralPubMedCrossRefGoogle Scholar
  59. Zhou S, Wei F, Nguyen J, Bechner M, Potamousis K, Goldstain S, Pape L, Mehan MR, Churas C, Pasternak S, Forrest DK, Wise R, Ware D, Wing RA, Waterman MS, Livny M, Schwartz DC (2009) A single molecule scaffold for the maize genome. PLoS Genet 5:e1000711PubMedCentralPubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2013

Authors and Affiliations

  • M. Karafiátová
    • 1
  • J. Bartoš
    • 1
  • D. Kopecký
    • 1
  • L. Ma
    • 2
  • K. Sato
    • 3
  • A. Houben
    • 2
  • N. Stein
    • 2
  • J. Doležel
    • 1
    • 4
  1. 1.Centre of the Region Haná for Biotechnological and Agricultural ResearchInstitute of Experimental BotanyOlomouc-HoliceCzech Republic
  2. 2.Leibniz Institute of Plant Genetics and Crop Plant Research (IPK)SeelandGermany
  3. 3.Institute of Plant Science and ResourcesOkayama UniversityKurashikiJapan
  4. 4.Institute of Experimental BotanyOlomouc-HoliceCzech Republic

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