Plant Molecular Biology Reporter

, Volume 31, Issue 2, pp 289–295 | Cite as

Flow Karyotyping of Wheat Addition Line “T240” with a Haynaldia villosa 6VS Telosome

  • Dong-wei Guo
  • Dong-hong Min
  • Zhao-shi Xu
  • Ming Chen
  • Lian-cheng Li
  • Muhammad Ashraf
  • Abdul Ghafoor
  • You-zhi Ma
Original Paper


To construct a chromosome-specific DNA library of chromosome 6VS from Haynaldia villosa, a wheat alien chromosome addition line T240 with a 6VS chromosome arm and its parental common wheat cv. CA921 were used to optimize protocols for preparing chromosome suspension amenable to flow sorting of the 6VS chromosome. Our results showed that root tips incubated sequentially in Hogland’s solution containing 1.25 mM hydroxyurea (DNA synthesis inhibitor) for 18 h, a hydroxyurea-free period for 2 h, and 1 μM trifluralin for 4 h (metaphase blocking reagent) increased the metaphase index (MI) by up to 62 % . Many metaphase chromosomes were released to form a chromosome suspension when root tips fixed in 2 % paraformaldehyde were treated in a homogenizer at 9,500 rpm for 10–15 s. Most of the released chromosomes had intact morphology. The background solution of chromosome suspension was clear and relatively few of cell debris and chromosome clumps. Univariate flow karyotypes were established with chromosome suspension flow through FACS Vantage 2000 SE flow cytometer. The flow karyotype of CA921 consisted of four chromosome peaks, whereas that for T240 had a fifth peak. This fifth peak in T240 contained the telosome, which was further confirmed to be 6VS through fluorescence in situ hybridization.


Flow cytometry Chromosome sorting FISH 



We sincerely thank the Nature Science Foundation of Shaanxi province of China for support.


  1. Arumuganathan K, Martin GB, Telenius H, Tanksley SD, Earle ED (1994) Chromosome 2-specific DNA clones from-sorted chromosomes of tomato. Mol Gen Genet 242:51–558CrossRefGoogle Scholar
  2. Chester M, Leitch AR, Soltis PS, Soltis DE (2010) Review of the application of modern cytogenetic methods (FISH/GISH) to the study of reticulation (polyploidy/hybridization). Genes 1:166–192CrossRefGoogle Scholar
  3. Clain E, Brulfert A (1980) Hydroxyurea-induced mitotic synchronization in Allium sativum root meristems. Planta 150:26–31CrossRefGoogle Scholar
  4. Dolezel J, Lucretti S (1995) High resolution flow karyotyping and chromosome sorting in Vicia faba lines with standard and reconstructed karyotypes. Theor Appl Genet 90:797–802CrossRefGoogle Scholar
  5. Dolezel J, Safar J, Janda J, Bartos J, Kubalakova M, Cihalikova J, Simkova H, Sourdille, P, Bernard M, Chalhoub B (2003) Development of flow cytogenetics for wheat genome mapping. In: Pogna NE, Romano M, Pogna EA, Galterio G (eds) Proc 10th Int Wheat Genet Symp, vol 1. Instituto Sperimentale per la Cereal coltura, Roma, pp 65–68Google Scholar
  6. Dolezel J, Kubaláková M, Paux E, Bartos J, Feuillet C (2007) Chromosome-based genomics in the cereals. Chromosom Res 15:51–66CrossRefGoogle Scholar
  7. Doležel J, Šimková H, Kubaláková M, Šafář J, Suchánková P, Číhalíková J, Bartoš J, Valárik M (2009) Genetics and genomics of the triticeae. In: Feuillet C, Muehlbauer GJ (eds) Plant genetics and genomics: crops and models, vol 7, part 2. Springer, Berlin, pp 285–316Google Scholar
  8. Endo TR, Gill BS (1996) The deletion stocks of common wheat. Heredity 87:295–307CrossRefGoogle Scholar
  9. Fawcett JJ, Longmire JL, Martin JC, Deaven LL, Cram LS (1994) Large-scale chromosome sorting. Methods Cell Biol 42:319–330PubMedCrossRefGoogle Scholar
  10. Foote T (2004) Bacterial artificial chromosome (BAC) libraries and their applications in modern agriculture. Outlooks Pest Manag 15:80–82CrossRefGoogle Scholar
  11. Fukui K, Minezawa M, Kamisugi Y, Ishikawa M, Ohmido N, Yanagisawa T, Fujishita M, Sakai F (1992) Microdissection of plant chromosomes by argon-ion laser beam. Theor Appl Genet 84:787–791CrossRefGoogle Scholar
  12. Fukui K, Ohmido N, Khush GS (1994) Variability in rDNA loci in genus Oryza detected through fluorescence in situ hybridization. Theor Appl Genet 87:893–899CrossRefGoogle Scholar
  13. Gill KS, Arumuganathan K, Lee JH (1999) Isolating individual wheat (Triticum aestivum) chromosome arms by flow cytometric analysis of ditelosomic lines. Theor Appl Genet 98:1248–1252CrossRefGoogle Scholar
  14. Guo DW, Ma YZ, Li LC, Chen YF (2006) Flow sorting of wheat chromosome arms from the ditelosomic line 7BL. Plant Mol Biol Rep 24:23–31CrossRefGoogle Scholar
  15. Heslop-Harrison JS, Schwarzacher T (2011) Organisation of the plant genome in chromosomes. Plant J 66:18–33PubMedCrossRefGoogle Scholar
  16. Hultdin M, Gronlund E, Norrback K, Eriksson-Lindstrom E, Just T, Roos G (1998) Telomere analysis by fluorescence in situ hybridization and flow cytometry. Nucleic Acids Res 16:3651–3656CrossRefGoogle Scholar
  17. Ibrahim SF, van den Engh G (2004) High-speed chromosome sorting. Chromosom Res 12:5–14CrossRefGoogle Scholar
  18. Janda J, Bartos J, Safar J, Kubalakova M, Valarik M, Cihalikova J, Simkova H, Caboche M, Sourdille P, Benard M, Chalhoub B, Dolezel J (2004) Construction of a subgenomic BAC library specific for chromosomes 1D, 4D and 6D of hexaploid wheat. Theor Appl Genet 109:1337–1345PubMedCrossRefGoogle Scholar
  19. Janda J, Safar J, Kubalakova M, Bartos J, Kovarova P, Suchankova P, Pateyron S, Cihalikova J, Sourdille P, Simkova H, Faivre-Rampant P, Hribova E, Bernard M, Lukaszewski A, Dolezel J, Chalhoub B (2006) Advanced resources for plant genomics: a BAC library specific for the short arm of wheat chromosome 1B. Plant J 47:977–986PubMedCrossRefGoogle Scholar
  20. Kasprzak A, Safar J, Janda J, Dolezel J, Wolko B, Naganowska B (2006) The bacterial artificial chromosome (BAC) library of the narrow-leafed lupin (Lupinus angustifolius L.). Cell Mol Biol Lett 11:396–407PubMedCrossRefGoogle Scholar
  21. Kim S, Rayburn Lane A, Parrish A, Lee DK (2012) Cytogeographic distribution and genome size variation in Prairie Cordgrass (Spartina pectinata Bosc ex Link). Plant Mol Biol Rep. doi: 10.1007/s11105-012-0414-x
  22. Kubalakova M, Lysak MA, Vrana J, Simkova H, Cihalikova J, Dolezel J (2000) Rapid identification and determination of purity of flow-sorted plant chromosomes using C-PRINS. Cytometry 41:102–108PubMedCrossRefGoogle Scholar
  23. Kubalakova M, Bartos J, Kovarova P, Cihalikova J, Watanabe N, Kianian SF, Dolezel J (2003a) Chromosome analysis and sorting in durum wheat and physical mapping of repetitive DNA sequences. In: Pogna NE, Romano M, Pogna EA, Galterio G (eds) Proc 10th Intel Wheat Genet Symp, Vol 2. Instituto Sperimentale per la Cereal coltura, Roma, pp 601–603Google Scholar
  24. Kubalakova M, Bartos J, Kovarova P, Cihalikova J, Watanabe N, Kianian SF, Dolezel J (2003b) Analysis and sorting of rye (Secale cereale L.) chromosomes using flow cytometry. Genome 46:893–905PubMedCrossRefGoogle Scholar
  25. Lee JH, Arumuganathan K, Kaeppler SM, Kaeppler HF, Papa CM (1996) Cell synchronization and isolation of metaphase chromosomes from maize (Zea mays L.) root tips for flow cytometric analysis and sorting. Genome 39:697–703PubMedCrossRefGoogle Scholar
  26. Lee JH, Arumuganathan K, Yen Y, Kaeppler S, Kaeppler H, Baenziger PS (1997) Root tip cell cycle synchronization and metaphase chromosome isolation suitable for flow sorting in common wheat (Triticum aestivum L.). Genome 40:633–638PubMedCrossRefGoogle Scholar
  27. Lee JH, Arumuganathan K, Chung YH, Kim KY, Chung WB, Bae KS, Kim DH, Chung DS, Kwon OC (2000) Flow cytometric analysis and chromosome sorting of barley (Hordeum vulgare L.). Mol Cell 10:619–625Google Scholar
  28. Lee JH, Ma YZ, Wako T, Li LC, Kim KY, Park SW, Uchiyama S, Fukui K (2004) Flow karyotypes and chromosomal DNA contents of genus Triticum species and rye (Secale cereale). Chromosom Res 12:93–102CrossRefGoogle Scholar
  29. Li LJ, Arumugnathan K (2000) High recovery of large molecular weight DNA from sorted maize chromosomes. Plant Mol Biol Rep 18:41–45CrossRefGoogle Scholar
  30. Lukaszewski AJ (1993) Reconstruction in wheat of complete chromosomes 1B and 1R from the 1RS.1BL translocation of “Kavkaz” origin. Genome 36:821–824PubMedCrossRefGoogle Scholar
  31. Lysak MA, Cihalikova J, Kubalakova M, Simkova H, Kunzel G, Dolezel J (1999) Flow karyotyping and sorting of mitotic chromosomes of barley (Hordeum vulgare L.). Chromosom Res 7:431–444CrossRefGoogle Scholar
  32. Macas J, Navratilova A, Kubalakova M, Dolezel J (2006) PRINS on plant chromosomes. Methods Mol Biol 33:133–140Google Scholar
  33. Mccormick MKE, Campbell E, Deaven L, Moyzis R (1993) Low-frequency chimeric yeast artificial chromosome libraries from flow-sorted human chromosomes 16 and 21. Proc Nat Acad Sci USA 90:1063–1067PubMedCrossRefGoogle Scholar
  34. Menke M, Fuchs J, Schubert I (1998) A comparison of sequence resolution on plant chromosomes: PRINS versus FISH. Theor Appl Genet 97:1314–1320CrossRefGoogle Scholar
  35. Mirzaghaderi G (2010) A simple metaphase chromosome preparation from meristematic root tip cells of wheat for karyotyping or in situ hybridization. Afr J Biotechnol 9:314–318Google Scholar
  36. Naganowska B, Wolko B, Sliwinska E, Kaczmarek Z (2003) Nuclear DNA content variation and species relationships in the genus Lupinus (Fabaceae). Ann Bot 92:349–355PubMedCrossRefGoogle Scholar
  37. Nakamura M, Fukui K (1997) A chromosome-oriented approach to genome analysis in a woody plant—Sequoiadendron giganteum (Lindl.) Buchholz. In: Borzan Z, Schlarbaum SE (eds) Cytogenetic studies of forest trees and shrub species. Zagreb: Hrvatske Sume and Sumarski Fakultet Sveucilista, pp 89–102Google Scholar
  38. Neumann P, Pozarkova D, Vrana J, Dolezel J, Macas J (2002) Chromosome sorting and PCR-based physical mapping in pea (Pisum sativum L). Chromosom Res 10:63–71CrossRefGoogle Scholar
  39. Pan WH, Houben A, Schlegel R (1993) Highly effective cell synchronization in plant roots by hydroxyurea and amiprophos-methyl or colchicines. Genome 36:387–390PubMedCrossRefGoogle Scholar
  40. Peng R, Liu F, Hu X, Wang C, Li S, Zhang X, Wang Y, Wang K (2012) Microdissection and microcloning of chromosome 5 in gossypium arboretum. Plant Mol Biol Rep. doi: 10.1007/s11105-012-0438-2
  41. Roux N, Toloza A, Radecki Z, Zapata-Arias FJ, Dolezel J (2003) Rapid detection of aneuploidy in Musa using flow cytometry. Plant Cell Rep 21:483–490PubMedGoogle Scholar
  42. Safar J, Bartos J, Janda J, Bellec A, Kubalakova M, Valarik M, Pateyron S, Weiserova J, Tuskova R, Cihalikova J, Vrana J, Simkova H, Faivre-ramant P, Sourdille P, Caboche M, Benard M, Dolezel J, Chalhoub B (2004) Construction of a subgenomic BAC library specific for chromosomes 1D, 4D and 6D of hexaploid wheat. Theor Appl Genet 109:1337–1345PubMedCrossRefGoogle Scholar
  43. Simkova H, Cihalikova J, Vrana J, Lysak MA, Dolezel J (2003) Preparation of HWM DNA from plant nuclei and chromosomes isolated from root tips. Biol Plant 46:369–373CrossRefGoogle Scholar
  44. Suchankova P, Kubalakova M, Kovarova P, Bartos J, Cihalikova J, Molnar-Lang MR, Endo T, Dolezel J (2006) Dissection of the nuclear genome of barley by chromosome flow sorting. Theor Appl Genet 113:651–659PubMedCrossRefGoogle Scholar
  45. Uberall I, Vrana J, Bartos J, Smerda J, Dolezel J, Havel L (2004) Isolation of chromosomes from Picea abies and their analysis by flow cytometry. Biol Plant 48:199–203CrossRefGoogle Scholar
  46. Valarik M, Bartos J, Kovarova P, Kubalakova M, De Jong JH, Dolezel J (2004) High-resolution FISH on super-stretched flow-sorted plant chromosomes. Plant J 37:940–950PubMedCrossRefGoogle Scholar
  47. Vrana J, Kubalakova M, Simkova H, Cihalikova J, Lysak MA, Dolezel J (2000) Flow sorting of mitotic chromosomes in common wheat (Triticum aestivum L.). Genetics 156:2033–2041PubMedGoogle Scholar
  48. Wang ML, Leitch AR, Schwarzacher T, Heslop-Harrison JS, Moore G (1992) Construction of a chromosome-enriched Hpall library from flow-sorted wheat chromosomes. Nucleic Acids Res 20:1897–1901PubMedCrossRefGoogle Scholar
  49. Yuan WY, Tomita M, Sun SC, Yasumuro Y (1998) Genomic rearrangement analysis in derived lines from the trigeneric hybrids involving Triticum, Secale and Haynaldia using fluorescence in situ hybridization. In: Proceedings of the 9th International Wheat Genet Symposium, vol 2. University Extension Press, Univ. of Saskatchewan, Canada, pp 127–130Google Scholar
  50. Zhang HB, Zhao XP, Ding XL, Paterson AH, Wing RA (1995) Preparation of megabase-size DNA from plant nuclei. Plant J 7:175–184CrossRefGoogle Scholar
  51. Zhou YH, Dang BY, Wang HA, Hu ZM, Wang LL, Chen ZH (2001) Microdissection of a single chromosome and construction of the microclone library from soybean. Euphytica 121:129–135CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Dong-wei Guo
    • 2
  • Dong-hong Min
    • 2
  • Zhao-shi Xu
    • 1
  • Ming Chen
    • 1
  • Lian-cheng Li
    • 1
  • Muhammad Ashraf
    • 3
  • Abdul Ghafoor
    • 3
  • You-zhi Ma
    • 1
  1. 1.Key Laboratory of Crop Genetics and Breeding, Ministry of Agriculture, Institute of Crop SciencesChinese Academy of Agriculture SciencesBeijingChina
  2. 2.College of AgronomyNorthwest A & F UniversityYanglingChina
  3. 3.National Agriculture Research CenterIslamabadPakistan

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