Selective breeding and genetic improvement for robustness, disease and stress tolerance in the European sea bass using NGS technologies

The project aims at developing biotechnological tools to improve our understanding of the genomic basis for growth and robustness (resistance/tolerance to diseases e.g. VNN, vibriosis, pasteurellosis and stress) in European sea bass (Dicentrarchus labrax), one of the most intensively farmed teleost species in the Mediterranean. The species shows high susceptibility to stress and displays high basal cortisol (i.e. primary stress hormone) concentrations. Since stress is being considered as an important co-factor for reproductive dysfunctions and disease outbreaks in this species which in turn hampers production. Genetic improvement of European sea bass through advanced selective breeding and tools is expected to provide long term sustainable production and minimize production vulnerabilities/risks. Selection of individuals for increased survival against pathogens and response to acute stress conditions is expected to produce robust future generations in the European sea bass populations. STRESS INDICATORS: The stress test experiment was completed using 20 tagged fish from 20 families (10 stronger, and 10 susceptible). All fish were sampled without exposure to acute stress of handling, and 20 days later after exposure to acute stress. The stress sampling was repeated 3 times in total, with 20-days intervals between samplings. The measurements of weight, cortisol, glucose, lactate and lysozyme were recorded in all the above-mentioned four samplings. All physiological parameters were shown to be replicable, with significant inter-individual (stronger vs. susceptible) differences i.e. cortisol = 0.441, glucose = 0.307, lactate = 0.360 and lysozyme = 0.545. Therefore, cortisol responsiveness could become a suitable trait for use in selective breeding programs to improve resistance against stress. GENETICS OF STRESS INDICATORS: The stress and disease experiments were performed where European sea bass individuals (n=960) were recorded for stress markers (e.g. cortisol), immune parameters (e.g. lysozyme) and body weight. The sub-group of 960 individuals (n=420) were tested for mortality after vibrio injection. The genotyping of 620 individuals (out of 960) was performed using 35 DNA markers (microsatellite). The objective of these experiments and trait recordings were to unravel genetics of traits, detect genes affecting traits, and genetic correlations among the traits. The genetic analyses showed the high effect of gene variations on trait variations (heritability=0.36, 0.55, and 0.38 for cortisol, lysozyme levels, and mortality after vibriosis, respectively). A favorable genetic correlation (-0.43) was observed between cortisol level and the body weight which means selection for growth would also improve tolerance to stress due to correlated response. The quantitative trait loci (QTL) detection analysis revealed detection of loci/genes at linkage groups 3 and 14 for the cortisol levels which also showed effects on stress indicators. EPIGENOMIC LANDSCAPES IN RESPONSE TO STRESS: The genomes of 74 pre- and post-stressed individuals were scanned using epiGBS (epiGenotyping by sequencing) approach. The analysis revealed a strong family effect with 57 distinct differentially methylated cytosines (DMCs) found between pre- and post-stress individuals, indicating the effect of stress on genome. The methylated genome changes were found inside or in the vicinity of 51 distinct genes which have previously shown association with stress. GENETICS OF DISEASE RESISTANCE: The challenge tests were performed for three diseases viral nervous necrosis (VNN), vibriosis, pasteurellosis and for the stress. The tests for vibriosis and pasteurellosis failed to produce informative results/estimates (98.49% and non-significant mortalities, respectively) while tests for VNN and stress were accomplished successfully. The successful VNN challenge test was performed on 92 families produced by Nireus aquaculture using 85 males and 35 females. Additionally, another related year class comprising 89 families which were previously challenged with VNN was also included in the analysis. All the recorded individuals for survival (dead/alive) trait were tissue sampled. The subset of individuals was selected (n~1700) from both year classes using statistical method which were genotyped using Axiom DlabCHIP (~57K SNPs). The data was analyzed using pedigree (PBLUP), genomic (GBLUP), and combined hybrid information (HBLUP). The genetic analyses showed low to moderate effect of gene variations on survival with heritability estimates of 0.18±0.03, 0.26±0.04, and 0.22±0.03 using PBLUP, GBLUP and HBLUP respectively. The gene/loci detection analysis resulted in a strong signal at linkage group 12. Finally, the evaluations using genomic information produced three times improvement in accuracy to select parents for next generation compared to classical pedigree information.

-The successful conduction of challenge test against VNN virus, and the computation of genetic variation (0.18±0.03) for resistance against virus, indicating strong potential for inclusion into breeding programs. -Identification of quantitative trait loci (QTL) for resistance against VNN, detected at LG12. The results may help in improving health status of the fish, and the efficient genetic improvement of the stocks. -The cortisol level could become a suitable trait for use in selective breeding programs to improve stress tolerance. The results generated on epigenomic landscapes in response to acute stress and the detected loci associated with cortisol level may ultimately assist in improving fish welfare. -The genomic selection for resistance against VNN showed ~30% increase in accuracy of prediction compared to classical pedigree-based model representing strong potential for efficient development of stocks.

The project aims at developing biotechnological tools to improve our understanding of the genomic basis for growth and robustness in European sea bass, one of the most intensively farmed teleost species in the Mediterranean. Particular concern will be granted to increase species resistance to diseases, namely vibriosis and Viral Nervous Necrosis infection. A family based breeding program, running disease challenge tests on siblings to breeding candidates, can improve resistance by selecting broodstock from genetically best families. Exposing breeding candidates directly to disease to identify superior individuals is not indicated, but genomic analysis can be applied to efficiently discriminate among breeding candidates from the same family that otherwise will be ranked equally if only using family values. Therefore, genetic improvement of European sea bass through advanced selective breeding and molecular tools is expected to reduce production cost in the long term while minimize production vulnerabilities and risk. In addition, the species shows high susceptibility to stress and displays high basal cortisol concentrations. Preliminary results showed that cortisol responsiveness was a repeated trait and fish with constantly Low or High resting and post-stress cortisol concentrations were identified. Interestingly, some quantitative trait loci (QTL) that influence cortisol concentrations seem to be in the same genomic regions with QTL affecting weight. Since stress is being considered as an important factor for reproductive dysfunctions and disease outbreaks in this species, we aim to investigate more deeply how stress response measured by cortisol level and weight/growth interfere to develop a promising selection index in future breeding programs. Selection based on rigorous phenotype and genotype measurements for increased survival against pathogens and response to acute stress conditions is expected to lead to more robust future generations of the species.