Advertisement

Some Peculiarities in Application of Denaturating and Non-Denaturating In Situ Hybridization on Chromosomes of Cereals

  • V. M. Kuznetsova
  • O. V. Razumova
  • G. I. Karlov
  • T. X. Dang
  • P. Yu. Kroupin
  • M. G. DivashukEmail author
RESEARCH ARTICLE
  • 10 Downloads

Abstract

Non-denaturing fluorescent in situ hybridization (ND FISH) is a convenient method of cytogenetic research. Compared to the standard method, ND FISH is fast and easy to perform and requires less time, reagents, and tools. Thus, it is gaining increasing popularity among different groups of scientists and is used to accomplish various scientific tasks. However, when using this method to visualize the chromosomes of wheat and its wild relatives, we faced some peculiarities of its application when oligonucleotide probes are used. In this paper, we compare the three following methods: two different versions of denaturating and non-denaturating FISH. In the standard procedure and its modifications, chromosomes are treated with formamide at high temperature that results in the denaturation of supercoiled DNA of plant chromosomes. In the non-denaturing FISH, this step is omitted, which makes it possible to keep the native chromosome structure and, thus, is more time and cost effective. In our work, all methods demonstrated their efficiency. Non-denaturing FISH is characterized by ease and convenience but less reproducibility in a series of experiments. The standard protocol and its modifications are most stable and reliable, but negatively affect chromosome morphology. In successive hybridizations on the same slide (sequential FISH), we recommend a combination of these methods, with primary testing using a standard protocol and subsequent hybridization using the ND-FISH method.

Keywords:

FISH ND FISH nondenatured in situ hybridization molecular cytogenetics methods triticale. 

Notes

FUNDING

This work was performed with support from the Russian Foundation for Basic Research (project no. 16-16-00097).

CONFLICT OF INTEREST

The authors declare that they do not have any conflicts of interest.

COMPLIANCE WITH ETHICAL STANDARTS

The studies were performed without the use of animals and without the involvement of people as subjects.

REFERENCES

  1. 1.
    Rosato, M., Álvarez, I., Nieto Feliner, G., and Rosselló, J.A., High and uneven levels of 45S rDNA site-number variation across wild populations of a diploid plant genus (Anacyclus, Asteraceae), PLoS ONE, 2017, vol. 12, no. 10.Google Scholar
  2. 2.
    Han, F.P., Lamb, J.C., and Birchler, J.A., High frequency of centromere inactivation resulting in stable dicentric chromosomes of maize, Proc. Natl. Acad. Sci. U.S.A., 2006, vol. 103, no. 9, pp. 3238–3243.CrossRefGoogle Scholar
  3. 3.
    Tang, Z.X., Yang, Z.J., and Fu, S.L., Oligonucleotides replacing the roles of repetitive sequences pAs1, pSc119.2, pTa-535, pTa71, CCS1, and pAWRC.1 for FISH analysis, J. Appl. Genet., 2014, vol. 55, no. 3, pp. 313–318.CrossRefGoogle Scholar
  4. 4.
    Fu, S.L., Chen, L., Wang, Y.Y., Li, M., Yang, Z.J., Qiu, L., Yan, B.J., Ren, Z.L., and Tang, Z.X., Oligonucleotide probes for ND-D-FISH analysis to identify rye and wheat chromosomes, Sci. Rep., 2015, vol. 5, p. 10552.CrossRefGoogle Scholar
  5. 5.
    Xiao, Z., Tang, S., Qiu, L., Tang, Z., and Fu, S., Oligonucleotides and ND-FISH displaying different arrangements of tandem repeats and identification of Dasypyrum villosum chromosomes in wheat backgrounds, Molecules, 2017, vol. 22, p. 973.CrossRefGoogle Scholar
  6. 6.
    Badaeva, E.D., Amosova, A.V., Goncharov, N.P., Macas, J., Ruban, A.S., Grechishnikova, I.V., Zo-shchuk, S.A., and Houben, A., A set of cytogenetic markers allows the precise identification of all A-genome chromosomes in diploid and polyploidy wheat, Cytogenet. Genome Res., 2015, vol. 146, no. 1, pp. 71–79.CrossRefGoogle Scholar
  7. 7.
    Li, G.R., Gao, D., Zhang, H.G., Li, J.B., Wang, H.J., La, S.X., Ma, J.W., and Yang, Z.J., Molecular cytogenetic characterization of Dasypyrum breviaristatum chromosomes in wheat background revealing the genomic divergence between Dasypyrum species, Mol. Cytogenet., 2016, vol. 9, no. 1, p. 6.CrossRefGoogle Scholar
  8. 8.
    Du, P., Zhuang, L.F., Wang, Y.Z., Yuan, Q., Wang, D.R., Dawadondup, Tan, L.J., Shen, J., Xu, H.B., and Zhao, H., Development of oligonucleotides and multiplex probes for quick and accurate identification of wheat and Thinopyrum bessarabicum chromosomes, Genome, 2017, vol. 60, no. 2, pp. 93–103.CrossRefGoogle Scholar
  9. 9.
    Puterova, J., Razumova, O., Martinek, T., Alexandrov, O., Divashuk, M., Kubat, Z., Hobza, R., Karlov, G., and Kejnovsky, E., Satellite DNA and transposable elements in seabuckthorn (Hippophae rhamnoides), a dioecious plant with small Y and large X chromosomes, Genome Biol. Evol., 2017, vol. 9, no. 1, pp. 197–212.Google Scholar
  10. 10.
    Xin, H., Zhang, T., Han, Y., Wu, Y., Shi, J., Xi, M., and Jiang, J., Chromosome painting and comparative physical mapping of the sex chromosomes in Populus tomentosa and Populus deltoides, Chromosoma, 2018, vol. 127, no. 3, pp. 313–321.CrossRefGoogle Scholar
  11. 11.
    Tang, S., Tang, Z., Qiu, L., Yang, Z., Li, G., Lang, T., Zhu, W., Zhang, J., and Fu, S., Developing new Oligo probes to distinguish specific chromosomal segments and the A, B, D genomes of wheat (Triticum aestivum L.) using ND-FISH, Front. Plant Sci., 2018, vol. 9, p. 1104.CrossRefGoogle Scholar
  12. 12.
    Jiang, M., Xaio, Z.Q., Fu, S.L., and Tang, Z.X., FISH karyotype of 85 common wheat cultivars/lines displayed by ND-FISH using oligonucleotide probes, Cereal Res. Commun., 2017, vol. 45, no. 4, pp. 549–563.CrossRefGoogle Scholar
  13. 13.
    Kirov, I.V., Kiseleva, A.V., Van Laere, K., Van Roy, N., and Khrustaleva, L.I., Tandem repeats of Allium fistulosum associated with major chromosomal landmarks, Mol. Genet. Genomics, 2017, vol. 292, no. 2, pp. 453–464.CrossRefGoogle Scholar
  14. 14.
    Alexandrov, O.S. and Karlov, G.I., Molecular cytogenetic analysis and genomic organization of major DNA repeats in castor bean (Ricinus communis L.), Mol. Genet. Genomics, 2016, vol. 291, no. 2, pp. 775–787.CrossRefGoogle Scholar
  15. 15.
    Komuro, S., Endo, R., Shikata, K., and Kato, A., Genomic and chromosomal distribution patterns of various repeated DNA sequences in wheat revealed by a fluorescence in situ hybridization procedure, Genome, 2013, vol. 56, no. 3, pp. 131–137.CrossRefGoogle Scholar
  16. 16.
    Kato, A., High-density fluorescence in situ hybridization signal detection on barley (Hordeum vulgare L.) chromosomes with improved probe screening and reprobing procedures, Genome, 2011, vol. 54, no. 2, pp. 151–159.CrossRefGoogle Scholar
  17. 17.
    Iwata-Otsubo, A., Radke, B., Findley, S., Abernathy, B., Vallejos, C.E., and Jackson, S.A., Fluorescence in situ hybridization (FISH)-based karyotyping reveals rapid evolution of centromeric and subtelomeric repeats in common bean (Phaseolus vulgaris) and relatives, G3: Genes Genomes Genet., 2016, vol. 6, no. 4, pp. 1013–1022.Google Scholar
  18. 18.
    Badaeva, E.D. and Ruban, A.S., Evolution of the S-genomes in Triticum–Aegilops alliance: Evidences from chromosome analysis, Front. Plant Sci., 2018, vol. 9, p. 1756.CrossRefGoogle Scholar
  19. 19.
    Badaeva, E.D., Ruban, A.S., Aliyeva-Schnorr, L., Municio, C., Hesse, S., and Houben, A., In situ hybridization to plant chromosomes, in Fluorescence In situ Hybridization (FISH). Springer Protocols Handbooks, Liehr, T., Ed., Berlin, Heidelberg: Springer, 2017, pp. 477–494.Google Scholar
  20. 20.
    Lang, T., Li, G., Wang, H., Yu, Z., Chen, Q., Yang, E., Fu, S., Tang, Z., and Yang, Z., Physical location of tandem repeats in the wheat genome and application for chromosome identification, Planta, 2018, vol. 249, no. 3, pp. 663–675.CrossRefGoogle Scholar

Copyright information

© Allerton Press, Inc. 2019

Authors and Affiliations

  • V. M. Kuznetsova
    • 1
    • 2
  • O. V. Razumova
    • 1
    • 3
  • G. I. Karlov
    • 1
    • 2
  • T. X. Dang
    • 2
  • P. Yu. Kroupin
    • 1
    • 2
  • M. G. Divashuk
    • 1
    • 2
    Email author
  1. 1.Laboratory of Applied Genomics and Crop Breeding, All-Russia Research Institute of Agricultural BiotechnologyMoscowRussia
  2. 2.Center for Molecular Biotechnology, Russian State Agrarian University (MTAA)MoscowRussia
  3. 3.Laboratory of Molecular Systematics, Tsitsin Main Botanical Garden, Russian Academy of SciencesMoscowRussia

Personalised recommendations