Sampling and DNA isolation
A total of 200 European sea bass individuals that were reared and managed under the same environmental conditions (natural photoperiod in net cages) in a commercial fish farm were collected randomly from the male individuals in the reproduction period at the age of 24 months from Processing Factory in İzmir (location 38°11'1.54"N 26°27'22.70"E). The fishes were categorized as a commercial grading in 4 different groups of weights (1st group, 210–300 g; 2nd group, 300–450 g; 3rd group, 450–600 g; 4th group, 600–750 g). The body depth (BD, cm), standard length (SL, cm), head length (HL, cm), body length (BL, cm), pre-anal length (PAL, cm), abdominal length (AL, cm), post-anal length (POSTAL, cm), head depth (HD, cm), total weight (TW, g), fillet weight (FL, g) of each sample were measured and muscle tissue samples were taken and stored in 96% ethanol at -20°C until DNA extraction. Genomic DNA was extracted by using GeneMATRIX Tissue & Bacterial DNA Purification Kit (EURx Ltd, Gdansk, Poland) according to the manufacturer's protocols. The purity and concentration of DNA samples were checked by using 1% agarose gel electrophoresis and spectrophotometer (MN-913 MaestroNano Micro-Volume Spectrophotometer, Maestrogen, Taiwan).
Primer design and PCR amplification of GH gene
Primer sequences of the GH gene were designed based on the European sea bass sequence retrieved from GenBank (Accession number GQ918491) using the Primer-BLAST algorithm (https:// www.ncbi.nlm.nih.gov/tools/primer-blast/). Primer sequences of GH gene are F: 5’- GTGATCAGTCGGGTTCAGGT-3’ and R: 5’-CGTTGTGTCTCGTGCTTGTC-3’. For amplification reactions, the 50 µL PCR volume contained: 100 ng genomic DNA, 0.5 µM of each primer and 2X MyTaq™ Mix (Meridian Bioscience, USA). The PCR temperature cycling conditions were as follows: initial denaturation at 95°C for 3 min; 35 cycles of denaturation at 95°C for 30 s, annealing at 60°C for 30 s, and elongation at 72°C for 60 s. The final cycle was followed by an extension at 72°C for 10 min. Afterward, the PCR products were checked on 1.5% agarose gel using horizontal electrophoresis and the gels were stained using RedSafe™ (iNtRON Biotechnology, Korea)
Sequencing
The 576 bp of the GH gene region was sequenced on an Applied Biosystems 3500XL Genetic Analyzer System (Applied Biosystems, USA). The sequences were checked by ChromasPro Version 2.1.8 (Technelysium Pty. Ltd. Australia). Expasy resource portal from the Swiss Institute of Bioinformatics (SIB) was used to translate the nucleotide sequence of the GH gene region to amino acid sequence (https://web.expasy.org/translate/). The 3D tertiary structure of the proteins based on the studied GH gene region was predicted using the I-TASSER server (Yang et al., 2015).
Statistical Analysis of GH gene
Genotypes of SNPs were tested for the Hardy–Weinberg equilibrium (HWE) with “HardyWeinberg” package in R software (R version R-3.4.3) (R Core Team, 2013). The associations between the genotypes of GH locus and the measured traits were analyzed via SPSS Ver. 21.0 (IBM Corporation, NY, USA) according to a general linear model and an alpha value of 0.05 was chosen as the significance level.
Linear Model I = Yijk = µ + Bi + Gj + eijk
Where Yijk represents the traits; µ represents the intercept; Bi represents the group effect, Gj represents the effect of GH genotype and eijk is the random error.
The significance of differences between groups was determined using one-way analysis of variance (ANOVA) and Tukey’s multiple range test. The thresholds for significant and highly significant differences were P < 0.05 and P < 0.01, respectively.