In order to generate probes for FISH, genomic DNA (gDNA) from hexaploid C. gibelio was used as a template for amplification of the U1 and U2 snDNA regions, and H3 histone. DNA was extracted from adult fish tissues using the DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Primers used for amplification are listed in Table 2 . The annealing temperature was 54 °C and the elongation step 30 s for all polymerase chain reactions (PCR); other conditions for PCR amplification with PPP Master Mix (Top-Bio, Prague, Czech Republic) followed the manufacturer’s recommendations. PCR amplification of the U1, U2, and H3 genes consistently resulted in 112, 140, and 375 bp long fragments, respectively. The search using the blastn algorithm confirmed the locus- and species-specificity of each amplicon: 98.98% identity with the U1 DNA sequence of C. gibelio (accession number XR_008182931.1), 97.10% identity with the U2 DNA sequence of C. gibelio (accession number XR_008154662.1), and 97.53% identity with H3 DNA of C. gibelio (accession number XM_052561945.1). Labeling PCR was performed as described in Knytl and Fornaini (2021) (link). Digoxigenin-11-deoxyuridine triphosphate (dUTP) (Jena Bioscience, Jena, Germany) was used for U2 and H3 labeling, and biotin-16-dUTP (Jena Bioscience) was used for U1 labeling. Carassius gibelio U1 and U2 snDNA and H3 probes were then hybridized to chromosome spreads of C. carassius and C. auratus and tetraploid and hexaploid C. gibelio. The procedures for hybridization mixture preparation, denaturation, and subsequent overnight hybridization were previously described for rDNA FISH (Knytl et al. 2023 (link)). Post-hybridization stringency washing and blocking reactions were performed as described for painting FISH in Krylov et al. (2010) (link). Probe signal was visualized following Knytl et al. (2017) (link).
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