Analysing sex determination in farmed fish using Next Generation DNA sequencing

The aim of the current thesis was the analysis of the genetics of sex determination of farmed fish with sexual dimorphism, using Next Generation Sequencing. Three different species of farmed fish with sex-determining systems of varying complexity were studied. Both full-sibs and more distantly related specimens of Atlantic halibut (Hippoglossus hippoglossus), Nile tilapia (Oreochromis niloticus) and European sea bass (Dicentrarchus labrax) were used for this study. Application of Restriction-site Associated DNA sequencing (RAD-seq) and double digest Restriction-site Associated DNA sequencing (ddRAD-seq), two related techniques based on next generation sequencing, allowed the identification of thousands of Single Nucleotide Polymorphisms (SNPs; > 3,000) for each of the above species. The first SNP-based genetic maps for the above species were constructed during the current study. The first evidence concerning the location of the sex-determining region of Atlantic halibut is provided in this study. In the case of Nile tilapia both novel sex-determining regions and fine mapping of the major sex-determining region are presented. In the study of European sea bass evidence concerning the absence of a major sex-determining gene was provided. Indications of putative sex-determining regions in this species are also provided. The results of the current thesis help to broaden current knowledge concerning sex determination in three important farmed fish. In addition the results of the current thesis have practical applications as well, towards the production of mono-sex stocks of those species for the aquaculture industry

Genotyping Strategies Using ddRAD Sequencing in Farmed Arctic Charr (Salvelinus alpinus)

Simple SummaryAnimal breeding in recent years has benefited greatly from the availability of large-scale genetic information. The most widely applied genomic tools in selective breeding are specialized arrays that use DNA hybridization. However, the high financial investments accompanying this practice impair the profitability of emerging aquaculture species, including Arctic charr. The aim of the current study was to assess and compare the potential of two cost-efficient genotyping strategies applicable in a variety of breeding-related tasks, such as pedigree verification, genetic diversity screening and detection of genomic regions that are associated with phenotypes of economic importance. Both strategies are based on reduced representation sequencing but differ in sequencing coverage (low and high). The low coverage strategy offers a higher density of DNA markers, but also presents a greater proportion of missing data in the marker set and is characterized by more uncertainty in determining heterozygosity compared to high coverage. Our results show that while high coverage genotyping performs better in genetic diversity and kinship analyses, a low coverage strategy is more successful in identifying genomic regions associated with phenotypic traits, leading to the conclusion that both strategies could be of value into selection schemes.Incorporation of genomic technologies into fish breeding programs is a modern reality, promising substantial advances regarding the accuracy of selection, monitoring the genetic diversity and pedigree record verification. Single nucleotide polymorphism (SNP) arrays are the most commonly used genomic tool, but the investments required make them unsustainable for emerging species, such as Arctic charr (Salvelinus alpinus), where production volume is low. The requirement to genotype a large number of animals for breeding practices necessitates cost effective genotyping approaches. In the current study, we used double digest restriction site-associated DNA (ddRAD) sequencing of either high or low coverage to genotype Arctic charr from the Swedish national breeding program and performed analytical procedures to assess their utility in a range of tasks. SNPs were identified and used for deciphering the genetic structure of the studied population, estimating genomic relationships and implementing an association study for growth-related traits. Missing information and underestimation of heterozygosity in the low coverage set were limiting factors in genetic diversity and genomic relationship analyses, where high coverage performed notably better. On the other hand, the high coverage dataset proved to be valuable when it comes to identifying loci that are associated with phenotypic traits of interest. In general, both genotyping strategies offer sustainable alternatives to hybridization-based genotyping platforms and show potential for applications in aquaculture selective breeding

Microbiome structure of milt and ovarian fluid in farmed Arctic charr (Salvelinus alpinus)

Limited knowledge exists about the residing microbiome in gamete-related samples in fish. A potential effect between the seminal microbiome composition and sperm quality traits has been previously suggested in humans and livestock. Using a metabarcoding approach, we aimed to gain insights into the structure of the residing prokaryotes and microbial eukaryotes in ovarian fluid (n = 10) and milt (n = 84) from farmed Arctic charr -a species with highly variable reproductive success in captivity. In addition, sperm quality traits were recorded on the sampled males to investigate potential associations with the residing seminal microbiome. Higher microbial diversity was found in the ovarian fluid compared to the milt habitat. Even though the residing microbiome showed distinct differences between the two habitats, substantial overlap was observed, with >70% of the milt core microbiome being found in the ovarian fluid habitat. Statistically significant associations were found be-tween the Shannon diversity index and sperm motility-related traits. Additionally, a fungal operational taxo-nomic unit (OTU) potentially belonging to the Leotiomycetes class was associated with sperm concentration and motility. Overall, our study documents the microbiome structure of gamete-related samples from Arctic charr. Even though some associations were obtained between sperm quality parameters and either microbiome di-versity or with a fungal OTU, follow-up studies on a larger scale with more tank replicates are needed to confirm the robustness and causality of these relationships

Evaluating the potential of improving sperm quality traits in farmed Arctic charr (Salvelinus alpinus) using selective breeding

Arctic charr (Salvelinus alpinus) is a high-value species for the Nordic aquaculture. The highly variable reproductive performance that is commonly observed in commercial farms is hindering the expansion of the Arctic charr industry in Sweden. Traits related to sperm motility (total motility; curvilinear velocity; average path velocity; straight-line velocity) and concentration can play a pivotal role in male fertility. Selective breeding practices could offer solutions and contribute to improving male fertility. The current study aimed to investigate the magnitude of genetic variance for sperm quality traits in a selectively bred population of Arctic charr from Sweden and evaluate the possibility of their improvement through selection. Sperm motility and concentration were recorded using a computer-assisted semen analysis (CASA) system and a NucleoCounter, respectively, in over 400 males from year-class 2017. Double digest restriction-site associated DNA sequencing (ddRAD-seq) was applied in a subset of the recorded animals (n = 329), resulting in the detection of over 5000 single nucleotide polymorphisms (SNPs). Moderate heritability estimates were obtained for the recorded semen traits using both pedigree (0.21-0.32; SE 0.09) and genomic (0.23-0.26; SE 0.09) relationship matrices. A genome-wide association study (GWAS) detected a single SNP significantly associated (P < 1e-05) with total sperm motility on chromosome LG7 in relatively close proximity (500 Kb) to PTPN11 a gene previously associated with sperm quality traits in mammals. Moreover, weighted single-step genomic best linear unbiased prediction (WssGBLUP) pinpointed genomic regions explaining more than 3 % of the additive genetic variance for both the motility traits and the sperm concentration. Finally, the efficiency of genomic prediction was tested using a 3-fold cross-validation scheme. Higher prediction accuracy for total motility and velocities (both curvilinear and average path) was obtained using genomic information (0.26-0.29, SE 0.03-0.06) compared to pedigree (0.20-0.28, SE 0.04-0.07), while for sperm concentration a pedigree-based model (0.22 SE 0.03) was more efficient than the genomic model (0.14 SE 0.04). Overall, our results indicate that the recorded sperm quality traits are heritable, and could be improved through selective breeding practices

Genetic parameters and genomic prediction of resistance to koi herpesvirus disease using a low-density SNP panel on two Amur mirror carp populations

Koi herpesvirus disease (KHVD), caused by Cyprinid herpesvirus-3 (CyHV-3), is one of the most serious threats to carp farming. In the present study, we investigated the efficiency of a low-density (LD) SNP panel for estimating genetic parameters and breeding values to KHVD resistance in the Amur mirror carp (AMC). Two populations (Pop 1 and Pop 2) of AMC generated from unrelated parents were created using a partial factorial design. One-year old fish (Pop 1 = 1500 individuals.; Pop 2 = 1200 individuals) were challenged with CyHV-3 and phe-notyped to KHVD resistance. 218 SNPs originating from a medium genotyping platform previously applied to Pop 1 (15615 SNPs; denoted as MD panel) with the highest association to KHVD resistance were used as a LD panel to genotype individuals of Pop 2. Genetic parameters and estimated pedigree-based BLUP (EBV) and genomic-based GBLUP (GEBV_MD and GEBV_LD) breeding values were calculated and obtained for Pop 1 using either pedigree, MD or LD panel and for Pop 2 using either pedigree or the LD panel. The heritability estimates of KHVD resistance were very high for both populations ranging from 0.42 to 0.96. Selection for KHVD resistance in Pop 2 using the LD panel would have led to a relative increase of similar to 7% in prediction accuracy compared to the pedigree-based selection. Pearson correlation coefficients between pedigree-based and genomic-based estimated breeding values (EBV vs. GEBV_MD; EBV vs. GEBV_LD; GEBV_MD vs. GEBV_LD) showed a strong association for both populations (0.79 - 0.91). In addition, the concordance rate of individuals selected by pedigree-based (EBV) and genomic-based breeding values (GEBV_MD and GEBV_LD) within selection pressures of 5%, 10% and 20% were not statistically different in most cases. In conclusion, the low-density SNP panel could be useful for a selection program focused on the genetic improvement of KHVD resistance

Whole-genome re-sequencing provides key genomic insights in farmed Arctic charr (Salvelinus alpinus) populations of anadromous and landlocked origin from Scandinavia

Arctic charr (Salvelinus alpinus) is a niche-market high-value species for Nordic aquaculture. Similar to other salmonids, both anadromous and landlocked populations are encountered. Whole-genome re-sequencing (22X coverage) was performed on two farmed populations of anadromous (Sigerfjord; n = 24) and landlocked (Arctic Superior; n = 24) origin from Norway and Sweden respectively. More than 5 million SNPs were used to study their genetic diversity and to scan for selection signatures. The two populations were clearly distinguished through principal component analysis, with the mean fixation index being similar to 0.12. Furthermore, the levels of genomic inbreeding estimated from runs of homozygosity were 6.23% and 8.66% for the Norwegian and the Swedish population respectively. Biological processes that could be linked to selection pressure associated primarily with the anadromous background and/or secondarily with domestication were suggested. Overall, our study provided insights regarding the genetic composition of two main strains of farmed Arctic charr from Scandinavia. At the same time, ample genomic resources were produced in the magnitude of millions of SNPs that could assist the transition of Nordic Arctic charr farming in the genomics era

High resolution mapping of the recombination landscape of the phytopathogen Fusarium graminearum suggests two-speed genome evolution

International audienceRecombination is a major evolutionary force, increasing genetic diversity and permitting efficient coevolution of fungal pathogen(s) with their host(s). The ascomycete Fusarium graminearum is a devastating pathogen of cereal crops, and can contaminate food and feed with harmful mycotoxins. Previous studies have suggested a high adaptive potential of this pathogen, illustrated by an increase in pathogenicity and resistance to fungicides. In this study, we provide the first detailed picture of the crossover events occurring during meiosis and discuss the role of recombination in pathogen evolution. An experimental recombinant population (n = 88) was created and genotyped using 1306 polymorphic markers obtained from restriction site-associated DNA sequencing (RAD-seq) and aligned to the reference genome. The construction of a high-density linkage map, anchoring 99% of the total length of the reference genome, allowed the identification of 1451 putative crossovers, positioned at a median resolution of 24 kb. The majority of crossovers (87.2%) occurred in a relatively small portion of the genome (30%). All chromosomes demonstrated recombination-active sections, which had a near 15-fold higher crossover rate than non-active recombinant sections. The recombination rate showed a strong positive correlation with nucleotide diversity, and recombination-active regions were enriched for genes with a putative role in host-pathogen interaction, as well as putative diversifying genes. Our results confirm the preliminary analysis observed in other F. graminearum strains and suggest a conserved 'two-speed' recombination landscape. The consequences with regard to the evolutionary potential of this major fungal pathogen are also discussed

Mapping and Validation of the Major Sex-Determining Region in Nile Tilapia (Oreochromis niloticus L.) Using RAD Sequencing

Sex in Oreochromis niloticus (Nile tilapia) is principally determined by an XX/XY locus but other genetic and environmental factors also influence sex ratio. Restriction Associated DNA (RAD) sequencing was used in two families derived from crossing XY males with females from an isogenic clonal line, in order to identify Single Nucleotide Polymorphisms (SNPs) and map the sex-determining region(s). We constructed a linkage map with 3,802 SNPs, which corresponded to 3,280 informative markers, and identified a major sex-determining region on linkage group 1, explaining nearly 96% of the phenotypic variance. This sex-determining region was mapped in a 2 cM interval, corresponding to approximately 1.2 Mb in the O. niloticus draft genome. In order to validate this, a diverse family (4 families; 96 individuals in total) and population (40 broodstock individuals) test panel were genotyped for five of the SNPs showing the highest association with phenotypic sex. From the expanded data set, SNPs Oni23063 and Oni28137 showed the highest association, which persisted both in the case of family and population data. Across the entire dataset all females were found to be homozygous for these two SNPs. Males were heterozygous, with the exception of five individuals in the population and two in the family dataset. These fish possessed the homozygous genotype expected of females. Progeny sex ratios (over 95% females) from two of the males with the "female" genotype indicated that they were neomales (XX males). Sex reversal induced by elevated temperature during sexual differentiation also resulted in phenotypic males with the "female" genotype. This study narrows down the region containing the main sex-determining locus, and provides genetic markers tightly linked to this locus, with an association that persisted across the population. These markers will be of use in refining the production of genetically male O. niloticus for aquaculture

A new SNP-based vision of the genetics of sex determination in European sea bass (Dicentrarchus labrax)

Background: European sea bass (Dicentrarchus labrax) is one of the most important farmed species in Mediterranean aquaculture. The observed sexual growth and maturity dimorphism in favour of females adds value towards deciphering the sex determination system of this species. Current knowledge indicates the existence of a polygenic sex determining determination system that interacts with temperature. This was explored by restriction-site associated DNA (RAD) marker analysis in a test panel of 175 offspring that originated from a factorial cross between two dams and four sires from a single full-sib family. Results: The first high-density single nucleotide polymorphism (SNP) based linkage map for sea bass was constructed, consisting of 6706 SNPs on 24 linkage groups. Indications for putative sex-determining QTL (quantitative trait loci) that were significant at the genome-wide threshold were detected on linkage groups 6, 11 and 18 to 21, although a genome-wide association study (GWAS) did not identify individual significant SNPs at a genome-wide threshold. A preliminary genomic prediction approach that tested the efficiency of SNP-based selection for female sea bass showed a slight advantage compared to traditional pedigree-based selection. However, when the same models were tested on the same animals for selection for greater length, a clear advantage of the SNP-based selection was observed. Conclusions: Overall, the results of this study provide additional support to the polygenic sex determination hypothesis in sea bass. In addition, identification of sex-ratio QTL may provide new opportunities for sex-ratio control in sea bass