Sexing by genotype
Upon completion of the observer trials, all trout were euthanised with
an overdose of benzocaine solution. Genomic DNA was extracted from
adipose fin clips using a NucleoSpin™ Tissue kit (Macherey-Nagel GmbH &
Co. KG, Düren, Germany) following the manufacturer’s instructions;
quality controlled with spectrophotometry (NanoDrop, ThermoFisher
Scientific, Waltham, MA, USA); and quantified fluorometrically (Qubit
2.0, ThermoFisher Scientific, Waltham, MA, USA). Extracted DNA was
diluted to 20 ng μL-1.
To determine the sex of each trout, following a modification of
established protocols (Anglès d’Auriac et al., 2014; Lavender et al.,
2024), duplex PCR amplified the male-specific Y-chromosome gene,sdY (forward primer: CCC AGC ACT GTT TTC TTG TCT CA; reverse
primer: CTT AAA ACC ACT CCA CCC TCC AT), using the 18S gene as a
positive amplification control (forward primer: GTY CGA AGA CGA TCA GAT
ACC GT; reverse primer: CCG CAT AAC TAG TTA GCA TGC CG). PCR was
performed using 3 μL of DNA with 0.3 μL of each sdY primer,
0.075 μL of each 18S primer, 7.5 μL of Qiagen Multiplex PCR mix
containing 3 mM MgCl2, HotStarTaq DNA polymerase and
proprietary buffer (Qiagen N.V., Hilden, Germany), and 2.2 μL
nuclease-free H2O. Thermal cycling consisted of
initialisation for 15 minutes at 95 °C, followed by 35 amplification
cycles of 30 seconds at 94 °C, 90 seconds at 63 °C and 90 seconds at
72 °C, with a final extension for 10 minutes at 72 °C. The resulting PCR
products were visualised with 2 % agarose gel electrophoresis.