Nucleospin kits for tissue

gDNA was extracted using NucleoSpin Kits for Tissue (Macherey-Nagel, Duren, Germany) as described above. A gdf6b fragment comprising the two exons, the intron and 2,260 bp of the proximal promoter (with a total size of 4,368 bp) was amplified by PCR in a total volume of 50 μL. The mixture contained 0.5 μM of each primer, a final concentration of 20 ng/μL gDNA, 1X of 10X AccuPrime PCR Buffer II, 1U/reaction of AccuPrime Taq DNA Polymerase, High Fidelity (Thermofisher), was adjusted to 50 μL with autoclaved and distilled water. Cycling conditions were as follows: 94°C for 45 s, then 35 cycles of (94° C for 15 s + 64°C for 30 s + 68° C for 5 min and 30 s), and 68°C for 5 min. The resulting PCR product was cloned into TOPO TA cloning Kit XL (Thermofisher) and after sequence verification it was purified using NucleoSpin plasmid DNA purification kit (Machery-Nagel, Düren, Germany) according to the supplier’s indications. This Pachón cave gdf6b cloned DNA fragment was labeled by nick translation with Cy3-dUTP using Cy3 NT Labeling Kit (Jena Bioscience, Jena, Germany). The optimal fragment size of the probe (approx. 200-500 bp) was achieved after 30 min of incubation at 15°C.

For fish genotyping, gDNA was extracted from fin clips stored in 90% ethanol, after lysis in 5% chelex^{80 (link)} and 10 mg Proteinase K at 55°C for 2 h, followed by 10 min at 99°C. Following extraction, samples were centrifuged and the supernatant containing the gDNA was transferred in clean tubes and stored at −20°C. For pool-sequencing and TaqMan assay, gDNA was extracted using NucleoSpin Kits for Tissue (Macherey-Nagel, Düren, Germany) according to the supplier’s recommendations. For long-read male genome sequencing, high molecular weight (HMW) gDNA was extracted from a mature testis grounded in liquid nitrogen and lysed in TNES-Urea buffer (TNES-Urea: 4M urea; 10 mM Tris-HCl, pH 7.5; 120 mM NaCl; 10 mM EDTA; 5% SDS) for two weeks at room temperature. For HMW gDNA extraction the TNES-Urea solution was supplemented with Proteinase K at a final concentration of 150 μg/mL and incubated at 37°C overnight. HMW gDNA was extracted with a modified phenol-chloroform protocol as previously described.^{43 (link)} The gDNA concentrations for both pool-seq and genome sequencing were quantified with Qubit3 fluorometer (Invitrogen, Carlsbad, CA) and HMW gDNA quality and purity were assessed using spectrophotometry, fluorometry, and capillary electrophoresis.