Development of high-throughput technologies for species of veterinary relevance. Investigation of the genetic basis of mandibular prognathism in the European seabass (Dicentrarchus labrax)

In the last 20 years, the production of farmed marine fish species has increased rapidly, mainly as a consequence of improved breeding methods and technologies. Skeletal malformations and others severe production bottlenecks such as high larval mortality or susceptibility to stress and disease, remain to be solved. Skeletal anomalies in farmed fish are a relevant issue affecting animal welfare and health and cause significant economic losses. The constant progress of genomic technologies promises to rapidly increase our knowledge on molecular mechanisms underlying productive traits of economic relevance in farmed species. The main objective of this PhD research was to investigate the molecular basis underlying the mandibular prognathism (MP), a skeletal malformation with a moderate incidence in intensively reared European seabass (Dicentrarchus labrax). To this aim, we applied a 2bRAD (type IIB endonucleases restriction-site associated DNA) pipeline, a widely used NGS (Next Generation Sequencing) technique for genome-wide genotyping. The reliability and reproducibility of the 2bRAD protocol has been preliminary tested on two organisms of veterinary relevance: the yellowfin tuna (Thunnus albacares), a fish with great biological and economic importance at global scale and the Listeria monocytogenes, a pathogenic bacterium that causes the infection listeriosis. In total, 6772 high-quality genome-wide SNPs (Single Nucleotide Polymorphism) were identified for the yellowfin tuna, in across population samples collected from the Atlantic, Indian and Pacific oceans and covering the entire distribution area. Discriminant Analysis of Principal Components (DAPC) endorsed the presence of genetically discrete yellowfin tuna populations among three oceanic pools. These results showed the efficiency of this genotyping technique in assessing genetic divergence in a marine fish with high dispersal potential. A total of 1279 unique SNPs loci were identified for Listeria monocytogenes. This research represents a first example of application of RAD-like technologies in microbial genetics. Results obtained for L. monocytogenes suggest that 2b-RAD is an effective tool for molecular epidemiology and public health, as well as proved in other areas such as phylogenetics, taxonomy, population genetics and biosafety. Once the 2bRAD technique was optimized as described above, a high-density genetic map of European seabass for QTL mapping of jaw deformity was constructed and a genome-wide association study (GWAS) was carried out on a total of 298 juveniles, 148 of which belonged to four full-sib families. A total of 7362 SNP markers was genotyped in more than 80% of the experimental population. Three significant QTLs were detected as significantly associated to MP by applying a half-sib regression analysis. The first QTL was located on linkage group LG18, the second one on LG20 and the third on LG22, each of them explaining 11-13% of the phenotypic variation. Two SNPs associated with MP were identified with GWAS analysis. Candidate markers were located on chromosome ChrX and chromosome Chr17, both in close proximity with the peaks of the two most significant QTLs. Notably, the SNP marker on Chr17 was positioned within the Sobp (Sine Oculis Binding Protein) gene coding region, which plays a pivotal role in craniofacial development. The analysis of differentially expressed genes in jaw-deformed animals highlighted the “nervous system development” as a crucial pathway in MP. In particular, Zic2, a key gene for craniofacial morphogenesis in model species, was significantly down-regulated in MP-affected animals. By integrating transcriptomic and GWA methods, the analysis developed during this PhD study, provides evidence for putative mechanisms underlying seabass jaw deformity