Sampling and DNA isolation
A total of 200 harvest size (24 months old) European sea bass male individuals were randomly sampled at TÜMAY SEAFOOD Corp. Cage Farm. The best sampling time to determine the growth performance of fish in commercial fish farms is usually 24 months of age. It was observed that all individuals were male at the time of sampling (24 months) in the studied commercial fish farm (Vandeputte et al., 2012).
Same age fish larvae came from two commercial hatcheries that were reared and managed under the similar conditions. Fish larvae were produced in single day by mass spawning from a total of 2 broodstock tanks in which 15 males and 15 females were stocked of each hatchery. The stocking density of larvae was 75 larvae/liter, the larval survival rate was 45%, survival rate during the survey and adaptation period was 80% in the hatcheries. After 150 days of care in the hatcheries, the fish reaching fingerling size (2 g) were stocked in two 16 m diameter cages in Çeşme-İzmir (GPS location: 38°11'3.87"N 26°27'25.19"E). The fish were fed with commercial diets (protein ratio 35–45%, fish oil ratio: 20–30%) depending on water temperature and body weight of stock in the cage environment. The sampling for the study was carried out during harvest stage at the age of 24 months. As the fish were grouped according to their size during harvest, while sampling 100 fish from each hatchery 4 size groups were selected. That means; random 25 fish from each size group were sampled adding up 100 samples from each of the two hatcheries. 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
A region of 576 bp covering 1st partial intron, 2nd exon, 2nd intron and 3rd partial exon regions 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). The haplotypes were performed by the “Haploview4.1” (Barrett et al., 2005). 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
SNPs genotypes 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 genotypes, haplotypes and growth traits were analysed (via SPSS Inc. V. 18.0, IBM, Chicago, IL, 2009) using the general linear model (GLM) and an alpha value of 0.05 was considered significant.
Linear Model I = Yjk = µ + Gj + ejk
Where Yijk represents the traits; µ represents the intercept; Gj represents the fixed effect of GH genotype or haplotypes (j = 1, 2 or 3 for each SNPs or haplotypes) and eijk is the random error.
The significance of differences between genotypes of each locus was determined using Bonferroni multiple range test. The thresholds for significant and highly significant differences were P < 0.05 and P < 0.01, respectively.