Microsatellite genetic analysis of European sea bass (Dicentrarchus labrax) samples from an area subject to a restocking program in the Venice Lagoon

Lo studio presentato in questa tesi consiste nell’analisi, basata su 9 marcatori microsatellite, di campioni di branzino (Dicentrarchus labrax) provenienti dalla Laguna di Venezia. L’area di campionamento è stata soggetta, alcuni mesi prima dei campionamenti stessi, al rilascio di un gruppo di 30,000 branzini da parte dell’ ‘Associazione Pescatori della Laguna di Venezia’, che lamentava la povertà della zona in animali di questa specie. I giovanili utilizzati per il ripopolamento provenivano dall’allevamento ‘Ca’ Zuliani’ di Pila di Porto Tolle. Due dei campioni analizzati provenivano dall’area della bocca di porto di Malamocco interessata dal ripopolamento, mentre il terzo, commissionato ai membri dell’associazione, è costituito da branzini provenienti da un’area più vasta della laguna e non meglio definita. Per le analisi sono stati utilizzati anche i dati di alcuni campioni della popolazione selvatica della Laguna di Venezia, dei riproduttori utilizzati per generare gli animali liberati e, infine, dati della popolazione selvatica di provenienza di questi riproduttori (area del Delta del Po). Le analisi effettuate comprendono la ricerca di similarità tra i gruppi attraverso l’analisi con il software Structure; un’analisi di parentela tra gli individui dei gruppi campionati e i riproduttori dell allevamento Ca’ Zuliani (per verificare la presenza di individui rilasciati tra i campionati); l’assegnazione, basata sui 9 loci microsatellite, dei campioni alle possibili poplazioni d’origine (Laguna, nel caso di animali selvatici o Delta del Po, nel caso di animali rilasciati).ope

Development of tools for tracebility and for assessing the genetic impact of aquaculture

Overexploitation of natural food resources, among which marine resources, put in serious risk the survival of many species and its availability as human food. Reduction of harvest, restocking with farmed fish and farming as alternative source of product, are commonly used to grant high quality food in a sustainable way. Nevertheless, the last two solutions have potential side effects, among which the genetic impact on natural populations that are involved in restocking actions or escapees from fattening cages and farms. Study the genetic structure of the species in the wild and farming environment is a key aspect to understand the real risks related to aquaculture. At the same time, genetic tools developed in the process can be used to trace wild and farmed origin of fish product, which is an aspect that is gaining great interest among consumers. In the study presented in the thesis, genetic analysis based on RAD genotyping allowed the study of more than 1000 wild and farmed samples with 1216 SNP. The results obtained suggest a subdivision of natural samples in four genetically distinct groups: Atlantic, West Mediterranean, Ionian Sea and Aegean Sea. The analysis carried out on many European broodstocks revealed a higher genetic differentiation compared to wild groups, probably due to founder effects and genetic drift; broodstocks are characterized by lower genetic variability, that in some cases fell below the minimum threshold to avoid inbreeding; finally, some of the broodstocks showed genetic traits that could make offspring unfit to the natural environment they would find in case of restocking of escapees. Comparing wild and farmed groups stimulated a discussion on the potential impact of aquaculture on natural populations, considering the reduction in fitness and the loss of inter/intra groups genetic variability, that cause a loss in long-term adaptation potential. The analytical techniques used and the results obtained are important for the development of gilthead sea bream aquaculture in Europe and for the correct management and protection of natural populations from the areas involved in production

Low impact of different SNP panels from two building-loci pipelines on RAD-Seq population genomic metrics: case study on five diverse aquatic species

The irruption of Next-generation sequencing (NGS) and restriction site-associated DNA sequencing(RAD-seq) in the last decade has led to the identification of thousands of molecular markers and their genotyping for refined genomic screening. This approach has been especially useful for non-model organisms with limited genomic resources. Many building-loci pipelines have been developed to obtain robust single nucleotide polymorphism (SNPs) genotyping datasets using a de novo RAD-seq approach, i.e. without reference genomes.Here, the performances of two building-loci pipelines, STACKS 2 and Meyer’s 2b-RAD v2.1 pipeline, were compared using a diverse set of aquatic species representing different genomic and/or population structure scenarios. Two bivalve species (Manila clam and common edible cockle) and three fish species (brown trout, silver catfish and small-spotted catshark) were studied. Four SNP panels were evaluated in each species to test both different building-loci pipelines and criteria for SNP selection. Furthermore, for Manila clam and brown trout, a reference genome approach was used as control

Genomic survey of edible cockle (Cerastoderma edule) in the Northeast Atlantic: a baseline for sustainable management of its wild resources.

Knowledge on correlations between environmental factors and genome divergence between populations of marine species is crucial for sustainable management of fisheries and wild populations. The edible cockle (Cerastoderma edule) is a marine bivalve distributed along the Northeast Atlantic coast of Europe and is an important resource from both commercial and ecological perspectives. We performed a population genomics screening using 2b‐RAD genotyping on 9309 SNPs localized in the cockle's genome on a sample of 536 specimens pertaining to 14 beds in the Northeast Atlantic Ocean to analyse the genetic structure with regard to environmental variables. Larval dispersal modelling considering species behaviour and interannual/interseasonal variation in ocean conditions was carried out as an essential background to which compare genetic information. Cockle populations in the Northeast Atlantic displayed low but significant geographical differentiation between populations (F (ST) = 0.0240; p < 0.001), albeit not across generations. We identified 742 and 36 outlier SNPs related to divergent and balancing selection in all the geographical scenarios inspected, and sea temperature and salinity were the main environmental correlates suggested. Highly significant linkage disequilibrium was detected at specific genomic regions against the very low values observed across the whole genome. Two main genetic groups were identified, northwards and southwards of French Brittany. Larval dispersal modelling suggested a barrier for larval dispersal linked to the Ushant front that could explain these two genetic clusters. Further genetic subdivision was observed using outlier loci and considering larval advection. The northern group was divided into the Irish/Celtic Seas and the English Channel/North Sea, while the southern group was divided into three subgroups. This information represents the baseline for the management of cockles, designing conservation strategies, founding broodstock for depleted beds and producing suitable seed for aquaculture production

Low impact of different SNP panels from two building-loci pipelines on RAD-Seq population genomic metrics: case study on five diverse aquatic species

Información complementaria: https://doi.org/10.1186/s12864-021-07465-w.Background: The irruption of Next-generation sequencing (NGS) and restriction site-associated DNA sequencing (RAD-seq) in the last decade has led to the identification of thousands of molecular markers and their genotyping for refined genomic screening. This approach has been especially useful for non-model organisms with limited genomic resources. Many building-loci pipelines have been developed to obtain robust single nucleotide polymorphism (SNPs) genotyping datasets using a de novo RAD-seq approach, i.e. without reference genomes. Here, the performances of two building-loci pipelines, STACKS 2 and Meyer’s 2b-RAD v2.1 pipeline, were compared using a diverse set of aquatic species representing different genomic and/or population structure scenarios. Two bivalve species (Manila clam and common edible cockle) and three fish species (brown trout, silver catfish and small-spotted catshark) were studied. Four SNP panels were evaluated in each species to test both different building-loci pipelines and criteria for SNP selection. Furthermore, for Manila clam and brown trout, a reference genome approach was used as control. Results: Despite different outcomes were observed between pipelines and species with the diverse SNP calling and filtering steps tested, no remarkable differences were found on genetic diversity and differentiation within species with the SNP panels obtained with a de novo approach. The main differences were found in brown trout between the de novo and reference genome approaches. Genotyped vs missing data mismatches were the main genotyping difference detected between the two building-loci pipelines or between the de novo and reference genome comparisons. Conclusions: Tested building-loci pipelines for selection of SNP panels seem to have low influence on population genetics inference across the diverse case-study scenarios here studied. However, preliminary trials with different bioinformatic pipelines are suggested to evaluate their influence on population parameters according with the specific goals of each study

Heterogeneous microgeographic genetic structure of the common cockle (Cerastoderma edule) in the Northeast Atlantic Ocean: biogeographic barriers and environmental factors

Knowledge of genetic structure at the finest level is essential for the conservation of genetic resources. Despite no visible barriers limiting gene flow, significant genetic structure has been shown in marine species. The common cockle (Cerastoderma edule) is a bivalve of great commercial and ecological value inhabiting the Northeast Atlantic Ocean. Previous population genomics studies demonstrated significant structure both across the Northeast Atlantic, but also within small geographic areas, highlighting the need to investigate fine-scale structuring. Here, we analysed two geographic areas that could represent opposite models of structure for the species: (1) the SW British Isles region, highly fragmented due to biogeographic barriers, and (2) Galicia (NW Spain), a putative homogeneous region. A total of 9250 SNPs genotyped by 2b-RAD on 599 individuals from 22 natural beds were used for the analysis. The entire SNP dataset mostly confirmed previous observations related to genetic diversity and differentiation; however, neutral and divergent SNP outlier datasets enabled disentangling physical barriers from abiotic environmental factors structuring both regions. While Galicia showed a homogeneous structure, the SW British Isles region was split into four reliable genetic regions related to oceanographic features and abiotic factors, such as sea surface salinity and temperature. The information gathered supports specific management policies of cockle resources in SW British and Galician regions also considering their particular socio-economic characteristics; further, these new data will be added to those recently reported in the Northeast Atlantic to define sustainable management actions across the whole distribution range of the species

Low impact of different SNP panels from two building-loci pipelines on RAD-Seq population genomic metrics: case study on five diverse aquatic species

Background: The irruption of Next-generation sequencing (NGS) and restriction site-associated DNA sequencing (RAD-seq) in the last decade has led to the identification of thousands of molecular markers and their genotyping for refined genomic screening. This approach has been especially useful for non-model organisms with limited genomic resources. Many building-loci pipelines have been developed to obtain robust single nucleotide polymorphism (SNPs) genotyping datasets using a de novo RAD-seq approach, i.e. without reference genomes. Here, the performances of two building-loci pipelines, STACKS 2 and Meyer’s 2b-RAD v2.1 pipeline, were compared using a diverse set of aquatic species representing different genomic and/or population structure scenarios. Two bivalve species (Manila clam and common edible cockle) and three fish species (brown trout, silver catfish and small-spotted catshark) were studied. Four SNP panels were evaluated in each species to test both different building-loci pipelines and criteria for SNP selection. Furthermore, for Manila clam and brown trout, a reference genome approach was used as control. Results: Despite different outcomes were observed between pipelines and species with the diverse SNP calling and filtering steps tested, no remarkable differences were found on genetic diversity and differentiation within species with the SNP panels obtained with a de novo approach. The main differences were found in brown trout between the de novo and reference genome approaches. Genotyped vs missing data mismatches were the main genotyping difference detected between the two building-loci pipelines or between the de novo and reference genome comparisons. Conclusions: Tested building-loci pipelines for selection of SNP panels seem to have low influence on population genetics inference across the diverse case-study scenarios here studied. However, preliminary trials with different bioinformatic pipelines are suggested to evaluate their influence on population parameters according with the specific goals of each studyThe work undertaken in this project was funded by Xunta de Galicia Autonomous Government (GRC2014/010), Interreg Atlantic Area (Cockles project, EAPA_458/2016) and Girona University (MPCUdG2016/060) projects. Adrián Casanova was a Xunta de Galicia fellowship (ED481A-2017/091)S

Disentangling genetic variation for resistance and endurance to scuticociliatosis in turbot using pedigree and genomic information

Selective breeding for improving host responses to infectious pathogens is a promising option for disease control. In fact, disease resilience, the ability of a host to survive or cope with infectious challenge, has become a highly desirable breeding goal. However, resilience is a complex trait composed of two different host defence mechanisms, namely resistance (the ability of a host to avoid becoming infected or diseased) and endurance (the ability of an infected host to survive the infection). While both could be targeted for genetic improvement, it is currently unknown how they contribute to survival, as reliable estimates of genetic parameters for both traits obtained simultaneously are scarce. A difficulty lies in obtaining endurance phenotypes for genetic analyses. In this study, we present the results from an innovative challenge test carried out in turbot whose design allowed disentangling the genetic basis of resistance and endurance to Philasterides dicentrarchi, a parasite causing scuticociliatosis that leads to substantial economic losses in the aquaculture industry. A noticeable characteristic of the parasite is that it causes visual signs that can be used for disentangling resistance and endurance. Our results showed the existence of genetic variation for both traits (heritability = 0.26 and 0.12 for resistance and endurance, respectively) and for the composite trait resilience (heritability = 0.15). The genetic correlation between resistance and resilience was very high (0.90) indicating that both are at a large extent the same trait, but no significant genetic correlation was found between resistance and endurance. A total of 18,125 SNPs obtained from 2b-RAD sequencing enabled genome-wide association analyses for detecting QTLs controlling the three traits. A candidate QTL region on linkage group 19 that explains 33% of the additive genetic variance was identified for resilience. The region contains relevant genes related to immune response and defence mechanisms. Although no significant associations were found for resistance, the pattern of association was the same as for resilience. For endurance, one significant association was found on linkage group 2. The accuracy of genomic breeding values was also explored for resilience, showing that it increased by 12% when compared with the accuracy of pedigree-based breeding values. To our knowledge, this is the first study in turbot disentangling the genetic basis of resistance and endurance to scuticociliatosis.This work was supported by the European Union’s Seventh Framework Programme (KBBE.2013.1.2-659 10) under Grant Agreement No. 613611, Ministerio de Ciencia, Innovación y Universidades, Spain (Grant CGL2016-75904-C2-2-P), and Fondos FEDER.S

Genome-wide analysis clarifies the population genetic structure of wild gilthead sea bream (Sparus aurata)

Gilthead sea bream is an important target for both recreational and commercial fishing in Europe, where it is also one of the most important cultured fish. Its distribution ranges from the Mediterranean to the African and European coasts of the North-East Atlantic. Until now, the population genetic structure of this species in the wild has largely been studied using microsatellite DNA markers, with minimal genetic differentiation being detected. In this geographically widespread study, 958 wild gilthead sea bream from 23 locations within the Mediterranean Sea and Atlantic Ocean were genotyped at 1159 genome-wide SNP markers by RAD sequencing. Outlier analyses identified 18 loci potentially under selection. Neutral marker analyses identified weak subdivision into three genetic clusters: Atlantic, West, and East Mediterranean. The latter group could be further subdivided into an Ionian/Adriatic and an Aegean group using the outlier markers alone. Seascape analysis suggested that this differentiation was mainly due to difference in salinity, this being also supported by preliminary genomic functional analysis. These results are of fundamental importance for the development of proper management of this species in the wild and are a first step toward the study of the potential genetic impact of the sea bream aquaculture industry

Development of tools for tracebility and for assessing the genetic impact of aquaculture

Overexploitation of natural food resources, among which marine resources, put in serious risk the survival of many species and its availability as human food. Reduction of harvest, restocking with farmed fish and farming as alternative source of product, are commonly used to grant high quality food in a sustainable way. Nevertheless, the last two solutions have potential side effects, among which the genetic impact on natural populations that are involved in restocking actions or escapees from fattening cages and farms. Study the genetic structure of the species in the wild and farming environment is a key aspect to understand the real risks related to aquaculture. At the same time, genetic tools developed in the process can be used to trace wild and farmed origin of fish product, which is an aspect that is gaining great interest among consumers. In the study presented in the thesis, genetic analysis based on RAD genotyping allowed the study of more than 1000 wild and farmed samples with 1216 SNP. The results obtained suggest a subdivision of natural samples in four genetically distinct groups: Atlantic, West Mediterranean, Ionian Sea and Aegean Sea. The analysis carried out on many European broodstocks revealed a higher genetic differentiation compared to wild groups, probably due to founder effects and genetic drift; broodstocks are characterized by lower genetic variability, that in some cases fell below the minimum threshold to avoid inbreeding; finally, some of the broodstocks showed genetic traits that could make offspring unfit to the natural environment they would find in case of restocking of escapees. Comparing wild and farmed groups stimulated a discussion on the potential impact of aquaculture on natural populations, considering the reduction in fitness and the loss of inter/intra groups genetic variability, that cause a loss in long-term adaptation potential. The analytical techniques used and the results obtained are important for the development of gilthead sea bream aquaculture in Europe and for the correct management and protection of natural populations from the areas involved in production.Il sovrasfruttamento delle risorse naturali di cibo, tra cui quelle marine, ha messo in serio pericolo la sopravvivenza di molte specie e la loro disponibilità per il consumo da parte dell’uomo. La riduzione del prelievo, il ripopolamento degli stock naturali con pesci allevati e l’allevamento stesso come fonte alternativa di prodotto sono mezzi comunemente utilizzati per risolvere il duplice problema di garantire cibo di qualità e preservare l’ambiente naturale. Tuttavia, le ultime due misure presentano potenziali effetti collaterali, tra i quali un impatto sulla diversità genetica delle popolazioni naturali soggette a ripopolamenti o fughe dagli allevamenti. Per comprendere i rischi legati a questi due eventi, è fondamentale studiare le caratteristiche genetiche delle popolazioni selvatiche e dei riproduttori usati in allevamento. Allo stesso tempo, le tecniche di analisi sviluppate possono essere sfruttate per la tracciabilità del prodotto allevato e selvatico, aspetto che sta guadagnando sempre maggiore importanza tra i consumatori. L’analisi basata su tecniche di caratterizzazione genetica di tipo RAD ha permesso lo studio di più di 1000 campioni di orata con 1240 marcatori SNPs. I risultati suggeriscono una suddivisione dei campioni naturali in quattro gruppi geneticamente distinti: Atlantico, Mediterraneo Ovest, Ionio e Egeo. L’analisi dei broodstocks dei maggiori allevamenti europei ha rivelato una differenziazione genetica tra i gruppi più elevata di quella osservata tra i selvatici, probabilmente dovuta all’uso di un ridotto numero di riproduttori e alla deriva genetica; è stata rilevata anche una minore variabilità genetica all’interno dei gruppi allevati, talvolta al di sotto dei limiti considerati sicuri per evitare l’inbreeding; infine, alcuni riproduttori portano tratti genetici che potrebbero rendere la prole non adatta all’ambiente naturale che incontrerebbe in caso di fuga o rilascio. Il confronto delle caratteristiche genetiche dei gruppi allevati e selvatici ha permesso di discutere il potenziale impatto dell'acquacoltura sulla fitness e sul potenziale adattativo delle popolazioni selvatiche. Le tecniche messe a punto e i risultati ottenuti sono di grande importanza per lo sviluppo del settore dell'acquacoltura di orata e per la corretta gestione e salvaguardia delle popolazioni naturali delle zone coinvolte nella produzione