Chromosomal DNA Probe Labeling and FISH

Spreading of mitotic chromosomes was carried out according to Hoang^{29 (link)}. 5S rDNA, 18S and 26S rDNA probes were generated from S. polyrhiza and from S. intermedia genomic DNA each by using designed primer pairs^{16 (link),21 (link),65 (link),66 (link)} as described^{24 (link)}. Ribosomal DNA, A. thaliana type telomere and S. polyrhiza BAC probes were labeled with Cy3-dUTP (GE Healthcare Life Science), Alexa Fluor 488-5-dUTP, Texas red-12-dUTP, biotin-dUTP or digoxigenin-dUTP (Life Technologies) and precipitated as described^{29 (link)}.
Denaturation of mitotic chromosomes and probes, hybridization, post-hybridization washing and signal detection were carried out according to Lysak et al.^{67 (link)}. Probe stripping and re-hybridization were done as described^{29 (link)}.

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Hoang P.T., Fiebig A., Novák P., Macas J., Cao H.X., Stepanenko A., Chen G., Borisjuk N., Scholz U, & Schubert I. (2020). Chromosome-scale genome assembly for the duckweed Spirodela intermedia, integrating cytogenetic maps, PacBio and Oxford Nanopore libraries. Scientific Reports, 10, 19230.

Publication 2020

Cy3-dUTP Alexa Fluor 488-5-dUTP Texas red-12-dUTP biotin-dUTP

Alexa fluor 488 Biotin Chromosomes Cy3 dutp Digoxigenin Dutp Genomic Hybridization Primer Ribosomal dna Signal detection Telomere

Corresponding Organization :

Other organizations : Leibniz Institute of Plant Genetics and Crop Plant Research, Institute of Plant Molecular Biology, Czech Academy of Sciences, Biology Centre, Czech Academy of Sciences, Huaiyin Normal University

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Variable analysis

independent variables

Spreading of mitotic chromosomes

dependent variables

Signal detection of labeled probes on mitotic chromosomes

control variables

Denaturation of mitotic chromosomes and probes
Hybridization
Post-hybridization washing
Probe stripping and re-hybridization

controls

Positive controls: Not explicitly mentioned
Negative controls: Not explicitly mentioned

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