Dnadvance kit

Both the calcium phosphate precipitation method [29 ] and the lipofection method using the Lipofectamine 2,000 Reagent (Invitrogen, CA, USA) were used to transfer the Geneticin® resistant gene, pSV2neo, into the yellowtail fin cells. The treated cells were cultured in L-15 medium (Invitrogen, USA) containing Geneticin® (1 mg/mL, Invitrogen, USA) at 22°C to select the transformed cells. More than 300 independent Geneticin®-resistant clones were pooled for the fusion experiments. These cells were irradiated by exposure to 3,000, 4,000, 5,000, 6,000, 8,000, or 10,000 rads (MBR-1520R; Hitachi Power Solutions Co., Ltd., Ibaraki, Japan), and were fused with derivative mouse B78 cells at a 2:1 ratio in the presence of polyethylene glycol 1500 (Roche, Basel, Switzerland). The cells were cultivated with DMEM medium (Invitrogen, USA) containing Geneticin® for 3–4 weeks until hybrid clone colonies appeared. DNA was extracted from individual clones using a DNAdvance Kit (Beckman Coulter, CA, USA).

Genomic DNA was extracted using the Agencourt DNAdvance kit according to the manufacturer’s instructions (Beckmann Coulter, CA, USA). The extracted DNA was quantified and analyzed for its quality using the Spectrophotometer 190 (Molecular Devices, CA, USA) device. As quality criteria, a concentration higher than 40 ng/µl and an absorbance ratio 260/280 of 1.8–2 were used. The integrity of the DNA was also taken into account by electrophoretic running. Next, the Infinium HTS Automated Protocol (Illumina) [19 ] was followed with the objective of genotyping the samples. For this, the beadchip Global Screening Array-24 + V1.0/HTS CODIGO46_2017_01 was used. Genotypes described in Table 1 were determined using Genome Studio software.

DNA was extracted from saliva in Oragene collection vials (DNA Genotek Inc, Ontario, Canada) using the DNAdvance kit (Beckman Coulter Genomics, Danvers, MA). Approximately 2 ml of saliva was collected from each participant. Our previous research comparing methylation in brain, blood and saliva found that saliva produced methylation data comparable to brain tissue^{26 (link)}. Genomic DNA was bisulfite converted using the EZ-96 DNA Methylation Kit (Zymo Research), and DNA methylation levels were interrogated for >480,000 CpG sites using the Illumina HumanMethylation450 BeadChip. Hybridization and processing was performed according to the manufacturer’s instructions as previously described^{27 (link)}. Beta values were generated with BeadStudio and were set to missing (no call) if detection p-values exceeded 0.001. All samples had probe detection call rates <95% and an average intensity value of either <50% of the experiment-wide sample mean or <2,000 arbitrary units (AU). CpG sites with missing data for >10% of samples were excluded from analysis. Normalization of probe distribution and background differences between Type I and type II probes was conducted using Beta Mixture Quantile Normalization (BMIQ)^{28 (link)}, and ComBat was used to account for sources of technical variations including batch and positional effects^{29 (link)}.

DNA from eggs was isolated as described by Mateos‐Rivera et al. (2020 ), while cod tissue was suspended in lysis buffer from the DNAdvance kit (Beckman Coulter, CA, USA) and processed using a Biomek i5 Automated Workstation (Beckman Coulter), following the manufacturer's instructions.
All samples (eggs and tissues) were genotyped with 21 microsatellite loci. Microsatellites were chosen as the marker of choice as extensive experience in genetic identification of farmed salmonids and gadoids (Glover et al., 2008 , 2010 (link), 2011 ) has illustrated that they are effective at detecting founder effects and drift that are rife in aquaculture and that they effectively resolve parent‐offspring and sibship relations (Duval et al., 2021 (link); Quintela et al., 2016 ). Detailed genotyping conditions are summarized in Table S1. PCR products were diluted 1:20 and analysed on an ABI3730 sequencer (Applied Biosystems, MA, USA). Microsatellite alleles were scored using GeneMapper v6.0 (Applied Biosystems).