Sex chromosomes of H. punctata were selected to be used as probes since this species exhibits the closest 2n relative to the proposed ancestral state for the genus and, at the same time, it possesses a multiple sex chromosome system of the X1X1X2X2/X1X2Y type28 (link),32 (link),34 (link),35 (link),42 (link). Fifteen copies of the X1 and X2 chromosomes were isolated by glass-needle-based microdissection under an inverted microscope (Zeiss Axiovert 135). The collected DNA material was then amplified in a primary degenerated oligonucleotide-primed polymerase chain reaction (DOP-PCR)76 . The probes were then labeled in the secondary DOP-PCR reaction using 1 µL of the initial amplified product as a template DNA77 . The probe derived from the X1 chromosome (HPU-X1) was labeled with Spectrum Orange-dUTP (red), and the one derived from the X2 chromosome (HPU-X2) with Spectrum Green-dUTP (green) (Vysis, Downers Grove, United States).
The 5S and 18S rDNA fragments were obtained by PCR from the wolf fish Hoplias malabaricus genome using primers and thermal profiles described in previous studies78 (link)–80 . The labelling was done by nick translation using Atto550-dUTP (red) for the 5S rDNA and Alexa Fluor 488-dUTP (green) for the 18S rDNA (both Jena Biosciences, Jena, Germany), according to manufacturer's protocol.
The 5S and 18S rDNA fragments were obtained by PCR from the wolf fish Hoplias malabaricus genome using primers and thermal profiles described in previous studies78 (link)–80 . The labelling was done by nick translation using Atto550-dUTP (red) for the 5S rDNA and Alexa Fluor 488-dUTP (green) for the 18S rDNA (both Jena Biosciences, Jena, Germany), according to manufacturer's protocol.
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