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

An integrated genomic approach for the study of mandibular prognathism in the European seabass (Dicentrarchus labrax)

Skeletal anomalies in farmed fish are a relevant issue affecting animal welfare and health and causing significant economic losses. Here, 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. Out of 298 fish, 107 were affected by mandibular prognathism (MP). Three significant QTLs and two candidate SNPs associated with MP were identified. The two GWAS candidate markers were located on ChrX and 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 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. Gene expression data revealed also a significant down-regulation of Sobp in deformed larvae. Our analyses, integrating transcriptomic and GWA methods, provide evidence for putative mechanisms underlying seabass jaw deformity

fshr, a fish sex-determining locus shows variable incomplete penetrance across flathead grey mullet populations

Whole-genome sequence data were produced from a single flathead grey mullet female and assembled into a draft genome sequence, whereas publicly available sequence data were used to obtain a male draft sequence. Two pools, each consisting of 60 unrelated individuals respectively of male and female fish were analysed using Pool-Sequencing. Mapping and analysis of Pool-Seq data against the draft genome(s) revealed >30 loci potentially associated with sex, the most promising locus of which, encoding the follicle stimulating hormone receptor (fshr) and harbouring two missense variants, was genotyped on 245 fish from four Mediterranean populations. Genotype data showed that fshr represents a previously unknown sex-determining locus, though the incomplete association pattern between fshr genotype and sex-phenotype, the variability of such pattern across different populations, and the presence of other candidate loci, reveal that a greater complexity underlies sex determination in the flathead grey mullet

Multi-tissue RNA-Seq Analysis and Long-read-based Genome Assembly Reveal Complex Sex-specific Gene Regulation and Molecular Evolution in the Manila Clam

The molecular factors and gene regulation involved in sex determination and gonad differentiation in bivalve molluscs are unknown. It has been suggested that doubly uniparental inheritance (DUI) of mitochondria may be involved in these processes in species such as the ubiquitous and commercially relevant Manila clam, Ruditapes philippinarum. We present the first long-read-based de novo genome assembly of a Manila clam, and a RNA-Seq multi-tissue analysis of 15 females and 15 males. The highly contiguous genome assembly was used as reference to investigate gene expression, alternative splicing, sequence evolution, tissue-specific co-expression networks, and sexual contrasting SNPs. Differential expression (DE) and differential splicing (DS) analyses revealed sex-specific transcriptional regulation in gonads, but not in somatic tissues. Co-expression networks revealed complex gene regulation in gonads, and genes in gonad-associated modules showed high tissue specificity. However, male gonad-associated modules showed contrasting patterns of sequence evolution and tissue specificity. One gene set was related to the structural organization of male gametes and presented slow sequence evolution but high pleiotropy, whereas another gene set was enriched in reproduction-related processes and characterized by fast sequence evolution and tissue specificity. Sexual contrasting SNPs were found in genes overrepresented in mitochondrial-related functions, providing new candidates for investigating the relationship between mitochondria and sex in DUI species. Together, these results increase our understanding of the role of DE, DS, and sequence evolution of sex-specific genes in an understudied taxon. We also provide resourceful genomic data for studies regarding sex diagnosis and breeding in bivalves

SNP discovery using next generation transcriptomic sequencing in Atlantic herring (Clupea harengus)

The introduction of Next Generation Sequencing (NGS) has revolutionised population genetics, providing studies of non-model species with unprecedented genomic coverage, allowing evolutionary biologists to address questions previously far beyond the reach of available resources. Furthermore, the simple mutation model of Single Nucleotide Polymorphisms (SNPs) permits cost-effective high-throughput genotyping in thousands of individuals simultaneously. Genomic resources are scarce for the Atlantic herring (Clupea harengus), a small pelagic species that sustains high revenue fisheries. This paper details the development of 578 SNPs using a combined NGS and high-throughput genotyping approach. Eight individuals covering the species distribution in the eastern Atlantic were bar-coded and multiplexed into a single cDNA library and sequenced using the 454 GS FLX platform. SNP discovery was performed by de novo sequence clustering and contig assembly, followed by the mapping of reads against consensus contig sequences. Selection of candidate SNPs for genotyping was conducted using an in silico approach. SNP validation and genotyping were performed simultaneously using an Illumina 1,536 GoldenGate assay. Although the conversion rate of candidate SNPs in the genotyping assay cannot be predicted in advance, this approach has the potential to maximise cost and time efficiencies by avoiding expensive and time-consuming laboratory stages of SNP validation. Additionally, the in silico approach leads to lower ascertainment bias in the resulting SNP panel as marker selection is based only on the ability to design primers and the predicted presence of intron-exon boundaries. Consequently SNPs with a wider spectrum of minor allele frequencies (MAFs) will be genotyped in the final panel. The genomic resources presented here represent a valuable multi-purpose resource for developing informative marker panels for population discrimination, microarray development and for population genomic studies in the wild

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.Negli ultimi 20 anni, l’allevamento di specie ittiche marine ha avuto un rapido incremento, grazie principalmente al continuo sviluppo e miglioramento delle tecniche di produzione. Rimangono comunque ancora da risolvere diversi problemi nei processi di produzione, come ad esempio la presenza di malformazioni scheletriche, l'elevata mortalità delle larve o la suscettibilità allo stress e le malattie. Le anomalie scheletriche nei pesci d'allevamento incidono in maniera rilevante anche sul benessere e la salute degli animali, causando notevoli perdite economiche per gli allevatori. Tuttavia, il costante progresso delle tecniche di biologia molecolare e di genetica hanno permesso di ampliare notevolmente le nostre conoscenze sui meccanismi molecolari alla base di molti caratteri produttivi di rilevanza economica nelle specie allevate. L'obiettivo principale di questo studio è stato quello di indagare le basi genetiche del prognatismo mandibolare (MP), una malformazione scheletrica presente, con una moderata incidenza, negli allevamenti intensivi di branzino (Dicentrarchus labrax). A questo scopo, abbiamo applicato un particolare protocollo di analisi, il 2bRAD (type IIB endonucleases restriction-site associated DNA), ampiamente utilizzato per lo sviluppo di marcatori genetici affiancato a tecniche di sequenziamento massivo NGS (Next Generation Sequencies). L'affidabilità e la riproducibilità del protocollo 2bRAD è stata prima testata su due organismi di una certa rilevanza veterinaria: il tonno pinna gialla (Thunnus albacares), un pesce importante sia dal punto di vista biologico che economico, e la Listeria monocytogenes, un batterio patogeno che può causare la listeriosi. Nel tonno pinna gialla sono stati identificati 6772 marcatori SNP (Single Nucleotide Polymorphism) in popolazioni campionate su tutto l’areale di distribuzione negli Oceani Atlantico, Indiano e Pacifico. L’analisi discriminante delle componenti principali (DAPC) ha permesso di rilevare la presenza di popolazioni distinte di tonno pinna gialla nei tre Oceani. Questi risultati hanno dimostrato quindi l'efficacia del 2bRAD nello studio della divergenza genetica in un pesce marino ad alto potenziale di dispersione. Un totale di 1279 loci SNP sono stati identificati per Listeria monocytogenes, e questo lavoro rappresenta un primo esempio di applicazione di tecnologie “RAD-like” nel campo della genetica microbica. I risultati ottenuti suggeriscono che il 2bRAD potrebbe rivelarsi uno strumento estremamente utile per l'epidemiologia molecolare, così come in altri settori di studio come la filogenesi, la tassonomia, la genetica di popolazione e la salute pubblica. Nel lavoro sul prognatismo mandibolare del branzino, è stata inizialmente sviluppata una mappa di linkage ad alta densità utilizzata per la mappatura dei QTL potenzialmente associati alla patologia; successivamente è stato fatto uno studio di associazione (GWAS) scansionando l’intero genoma di branzino per trovare marcatori SNP putativamente associati al prognatismo. Sono stati utilizzati 298 esemplari giovanili in totale, di cui 148 appartenenti a quattro famiglie full-sib. Un totale di 7362 marcatori SNP sono stati genotipizzati in più del 80% della popolazione sperimentale. Tre QTL significativi sono stati rilevati per il prognatismo utilizzando un’analisi di regressione half-sib. Il primo è stato mappato sul gruppo di linkage LG18, il secondo su LG20 e il terzo sul gruppo di linkage LG22; ogni QTL spiegava circa l’11-13% della variazione fenotipica. Due SNP associati al MP sono stati identificati con l'analisi GWAS. I marcatori candidati sono stati individuati sul cromosoma ChrX e sul cromosoma Chr17, in stretta vicinanza ai due QTL più significativi. In particolare, il marcatore SNP su Chr17 è posizionato all'interno della regione codificante del gene Sobp, che svolge un ruolo fondamentale nello sviluppo craniofacciale. Inoltre, l’analisi dei geni differenzialmente espressi nei branzini prognati ha evidenziato lo “sviluppo del sistema nervoso" come un pathway cruciale nella formazione del MP. In particolare, Zic2, un gene chiave per la morfogenesi craniofacciale nelle specie modello, risulta significativamente sotto-espresso negli animali affetti da prognatismo. Il lavoro svolto in questo studio quindi, integrando la trascrittomica e l’analisi di marcatori molecolari sviluppati sull’intero genoma, fornisce validi risultati per comprendere meglio i meccanismi molecolari che sono alla base dell’insorgenza del prognatismo mandibolare nei branzini di allevamento

The mitochondrial genome of the ascalaphid owlfly Libelloides macaronius and comparative evolutionary mitochondriomics of neuropterid insects

BACKGROUND: The insect order Neuroptera encompasses more than 5,700 described species. To date, only three neuropteran mitochondrial genomes have been fully and one partly sequenced. Current knowledge on neuropteran mitochondrial genomes is limited, and new data are strongly required. In the present work, the mitochondrial genome of the ascalaphid owlfly Libelloides macaronius is described and compared with the known neuropterid mitochondrial genomes: Megaloptera, Neuroptera and Raphidioptera. These analyses are further extended to other endopterygotan orders. RESULTS: The mitochondrial genome of L. macaronius is a circular molecule 15,890 bp long. It includes the entire set of 37 genes usually present in animal mitochondrial genomes. The gene order of this newly sequenced genome is unique among Neuroptera and differs from the ancestral type of insects in the translocation of trnC. The L. macaronius genome shows the lowest A+T content (74.50%) among known neuropterid genomes. Protein-coding genes possess the typical mitochondrial start codons, except for cox1, which has an unusual ACG. Comparisons among endopterygotan mitochondrial genomes showed that A+T content and AT/GC-skews exhibit a broad range of variation among 84 analyzed taxa. Comparative analyses showed that neuropterid mitochondrial protein-coding genes experienced complex evolutionary histories, involving features ranging from codon usage to rate of substitution, that make them potential markers for population genetics/phylogenetics studies at different taxonomic ranks. The 22 tRNAs show variable substitution patterns in Neuropterida, with higher sequence conservation in genes located on the α strand. Inferred secondary structures for neuropterid rrnS and rrnL genes largely agree with those known for other insects. For the first time, a model is provided for domain I of an insect rrnL. The control region in Neuropterida, as in other insects, is fast-evolving genomic region, characterized by AT-rich motifs. CONCLUSIONS: The new genome shares many features with known neuropteran genomes but differs in its low A+T content. Comparative analysis of neuropterid mitochondrial genes showed that they experienced distinct evolutionary patterns. Both tRNA families and ribosomal RNAs show composite substitution pathways. The neuropterid mitochondrial genome is characterized by a complex evolutionary history

Is It an Ant or a Butterfly? Convergent Evolution in the Mitochondrial Gene Order of Hymenoptera and Lepidoptera

Insect mitochondrial genomes (mtDNA) are usually double helical and circular molecules containing 37 genes that are encoded on both strands.The arrangementof thegenes isnot constant for all species, andproducesdistinctgeneorders (GOs) thathave proven to be diagnostic in defining clades at different taxonomic levels. In general, it is believed that distinct taxa have a very low chance of sharing identically arranged GOs. However, examples of identical, homoplastic local rearrangements occurring in distinct taxa do exist. In this study, we sequenced the complete mtDNAs of the ants Formica fusca and Myrmica scabrinodis (Formicidae, Hymenoptera) and compared their GOs with those of other Insecta. The GO of F. fusca was found to be identical to the GO of Dytrisia (the largest clade of Lepidoptera). This finding is the first documented case of an identical GO shared by distinct groups of Insecta, andit is the oldestknownevent ofGOconvergent evolutioninanimals. BothHymenoptera andLepidoptera acquired thisGO early in their evolution. Using a phylogenetic approach combined with new bioinformatic tools, the chronological order of the evolutionary events that produced the diversity of the hymenopteran GOs was determined. Additionally, new local homoplastic rearrangementssharedbydistinctgroupsof insectswere identified.Our study showedthat localandglobalhomoplasies affecting the insect GOs are more widespread than previously thought. Homoplastic GOs can still be useful for characterizing the various clades, provided that they are appropriately considered in a phylogenetic and taxonomic context

Macrostructural Evolution of the Mitogenome of Butterflies (Lepidoptera, Papilionoidea)

The mitogenome of the species belonging to the Papilionodea (Lepidoptera) is a double stranded circular molecule containing the 37 genes shared by Metazoa. Eight mitochondrial gene orders are known in the Papilionoidea. MIQGO is the plesiomorphic gene order for this superfamily, while other mitochondrial arrangements have a very limited distribution. 2S1GO gene order is an exception and is present in several Lycaenidae and one species of Hesperiidae. We studied the macrostructural changes generating the gene orders of butterflies by analysing a large data set (611 taxa) containing 5 new mitochondrial sequences/assemblies and 87 de novo annotated mitogenomes. Our analysis supports a possible origin of the intergenic spacer trnQ-nad2, characterising MIQGO, from trnM. We showed that the homoplasious gene order IMQGO, shared by butterflies, species of ants, beetles and aphids, evolved through different transformational pathways. We identify a complicated evolutionary scenario for 2S1GO in Lycaenidae, characterised by multiple events of duplication/loss and change in anticodon of trnS1. We show that the gene orders ES1GO and S1NGO originated through a tandem duplication random loss mechanism. We describe two novel gene orders. Ampittia subvittatus (Hesperiidae) exhibits the gene order 2FFGO, characterised by two copies of trnF, one located in the canonical position and a second placed in the opposite strand between trnR and trnN. Bhutanitis thaidina (Papilionidae) exhibits the gene order 4QGO, characterised by the quadruplication of trnQ

Combining Culture-Dependent and Culture-Independent Methods: New Methodology Insight on the Vibrio Community of Ruditapes philippinarum

Vibrios represent a natural contaminant of seafood products. V. alginolyticus, V. cholerae, V. parahaemolyticus and V. vulnificus are the most hazardous species to human health. Given the worldwide consumption of mollusc products, reliable detection of Vibrio spe-cies is recommended to prevent human vibriosis. In this study, culture-dependent and -independent methods were compared and integrated to implement knowledge of the Manila clam Vibrio community composition. Here, 16S and recA-pyrH metabarcoding were applied to compare the microbial communities of homogenate clam samples (cul-ture-independent method) and their culture-derived samples plated on three different media (culture-dependent method). In addition, a subset of plated clam samples was in-vestigated using shotgun metagenomics. Homogenate metabarcoding characterized the most abundant taxa (16S) and Vibrio species (recA-pyrH). Culture-dependent metabar-coding detected the cultivable taxa, including rare species. Moreover, marine agar me-dium was found to be a useful substrate for the recovery of several Vibrio species, in-cluding the main human pathogenic ones. The culture-dependent shotgun metagenomics detected all the main human pathogenic Vibrio species and a higher number of vibrios with respect to the recA-pyrH metabarcoding. The study revealed that integration of cul-ture-dependent and culture-independent methods might be a valid approach for the characterization of Vibrio biodiversity