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