Background
Lipid deposition is an important factor of production efficiency, product quality and health in animal production. The liver is a major lipid storage site in European seabass (Dias et al., 2005). However, excess lipid accumulation in the liver can negatively affect production efficiency , liver function and health, with onset of fatty liver disorder. The lipid content of muscle tissue affects organoleptic and processing qualities of fish fillets, and the omega-3 fatty acid content is important for the nutritional quality. As a significant component of the Mediterranean diet, seabass is an important nutritional source of the health-promoting essential omega-3 fatty acids EPA and DHA.
Substantial genetic variation has been reported for muscle lipid content in seabass ( Besson et al., 2019; Saillant et al., 2009). Studies in other fish species have revealed a genetic component to the omega-3 levels of fillets (Horn et al. 2018; Wang et al. 2019). The genetic parameters of fatty acid composition traits and liver fat have not yet been explored in seabass. The aim of this study was to estimate genetic parameters of lipid and omega-3 fatty acid content in muscle and liver of European seabass . For the first time in seabass, a GWAS on lipid and fatty acid traits was performed.
Materials and methods
European seabass originating from ABSA-Culmarex were used in this experiment. Fish were fed commercial feed and kept in commercial sea cages in Murcia, Spain. Samples of liver (n = 87) and muscle (n = 313) were homogenized and total lipids were extracted using the Folch extraction method. The fatty acid composition of the total lipids was determined using the Mason & Waller method by means of gas chromatography.
The fish were genotyped using the 60K MedFish array (Peñaloza et al. 2020). Genotypic data was filtered using the Plink software. Approximately 21K SNPs passed filters and quality control. A genomic relationship matrix between the animals was generated with the “-grm ” function implemented in GCTA software, and used for the estimation of the genetic parameters . The genomic relationship matrix was computed according to VanRaden (2008) as where is the allele frequency of the second allele and is the total number of SNP markers.
(Co)Variance components and the corresponding heritability were estimated from bivariate and univariate restricted maximum likelihood (GREML) analyses, including effect of sex and batch as fixed effects. GWAS was performed using a linear mixed animal model implemented in GCTA program with the “–mlma-loco” function. With fixed effects including (phenotypic) sex, batch, and 5 principal components as covariates (only sex and batch for liver traits were included due to the small dataset). The following traits were analysed for both muscle and liver tissue: Total fat (%), DHA (%), EPA (%), DHA/ALA ratio, omega-3/omega-6 ratio.
Results and discussion
On average, the fish weighed 346 g and had a muscle fat content of 9 %, ranging from 2 to 21 %. The phenotypic and genetic correlation between muscle fat and liver fat was weak, suggesting independent regulation of the two lipid deposits. This was supported by the high heritability of muscle fat (0.59) , which was within the range of previous estimates (e.g. Besson et al. 2019; Saillant et al. 2009) , and the lower heritability of liver fat (0.17) .
We found high heritability for omega-3 related traits in both tissues, but for different specific traits, o ne exception being DHA, which had high heritability in both liver (0.45) and muscle (0.51). In liver, EPA and DHA/ALA ratio had high heritability (0.43 and 0.42, respectively) , while in muscle omega-3/omega-6 ratio had a high heritability (0.41) . The high heritability of DHA and omega-3/omega-6 ratio in muscle indicates substantial potential to improve the fatty acid profile of fish fillets through selective breeding, by increasing the ratio of anti-inflammatory omega-3 to pro-inflammatory omega-6 fatty acids.
The proportional content of DHA was 6.4 % and 7.3 % in muscle and liver, respectively, which was higher than the proportion supplied in the feed (3.9 %). There was a positive phenotypic correlation between muscle DHA and liver DHA/ALA ratio (0.6), and both traits had a high heritability (>0.4) . As the DHA/ALA ratio can be seen as a marker of omega-3 bioconversion of the shorter-chain omega-3 ALA to DHA, these results may imply that European seabass has omega-3 bioconversion activity in liver that has a strong genetic component to it, and that could impact the DHA content of muscle.
The GWAS showed a clear signal on LG X with chromosome-wide significant peaks for the four traits muscle and liver omega-3/omega-6 ratio, muscle DHA and liver EPA. Although these traits did not have the same top significant SNPs, some were in the same region; 5,6-5,8Mb and 7-8Mb. One strong candidate gene was identified here: Peroxisome proliferator-activated receptor alpha (PPARα) , a nuclear transcription factor central in regulation of genes involved in lipid metabolism, including mitochondrial beta-oxidation. Another signal was found for the SNP AX-172297229 on LG 11 for the two traits DHA and DHA/ALA ratio in liver. Three candidate genes involved in lipid and/or omega-3 metabolism were found ± 300Kb region of this SNP : ALOX5, PLCB3 and SPTLC3. ALOX5 is especially interesting as it can mediate the lipoxygenation of DHA.
Conclusions
D ifferences in genetic parameters between liver and muscle tissues reflect differences in lipid metabolism. There is a substantial potential to increase DHA content of seabass fillets through selective breeding. The r esults also suggest that there is omega-3 bioconversion activity in liver of seabass that has a strong genetic component linked to the DHA content of fillets. GWAS signals on LG X for omega-3 traits across tissues implicate the candidate gene PPARα.
Acknowledgements
This study was made possible by the EU project MedAID (H2020 grant agreement No 727315).
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