Genome-Wide Association and Genomic Selection for Resistance to Amoebic Gill Disease in Atlantic Salmon

Abstract Amoebic gill disease (AGD) is one of the largest threats to salmon aquaculture, causing serious economic and animal welfare burden. Treatments can be expensive and environmentally damaging, hence the need for alternative strategies. Breeding for disease resistance can contribute to prevention and control of AGD, providing long-term cumulative benefits in selected stocks. The use of genomic selection can expedite selection for disease resistance due to improved accuracy compared to pedigree-based approaches. The aim of this work was to quantify and characterize genetic variation in AGD resistance in salmon, the genetic architecture of the trait, and the potential of genomic selection to contribute to disease control. An AGD challenge was performed in ∼1,500 Atlantic salmon, using gill damage and amoebic load as indicator traits for host resistance. Both traits are heritable (h2 ∼0.25-0.30) and show high positive correlation, indicating they may be good measurements of host resistance to AGD. While the genetic architecture of resistance appeared to be largely polygenic in nature, two regions on chromosome 18 showed suggestive association with both AGD resistance traits. Using a cross-validation approach, genomic prediction accuracy was up to 18% higher than that obtained using pedigree, and a reduction in marker density to ∼2,000 SNPs was sufficient to obtain accuracies similar to those obtained using the whole dataset. This study indicates that resistance to AGD is a suitable trait for genomic selection, and the addition of this trait to Atlantic salmon breeding programs can lead to more resistant stocks.</jats:p

Optimizing Low-Cost Genotyping and Imputation Strategies for Genomic Selection in Atlantic Salmon

Genomic selection enables cumulative genetic gains in key production traits such as disease resistance, playing an important role in the economic and environmental sustainability of aquaculture production. However, it requires genome-wide genetic marker data on large populations, which can be prohibitively expensive. Genotype imputation is a cost-effective method for obtaining high-density genotypes, but its value in aquaculture breeding programs which are characterised by large full-sibling families has yet to be fully assessed. The aim of this study was to optimise the use of low-density genotypes and evaluate genotype imputation strategies for cost-effective genomic prediction. Phenotypes and genotypes (78,362 SNPs) were obtained for 610 individuals from a Scottish Atlantic salmon breeding program population (Landcatch, UK) challenged with sea lice, Lepeophtheirus salmonis. The genomic prediction accuracy of genomic selection was calculated using GBLUP approaches and compared across SNP panels of varying densities and composition, with and without imputation. Imputation was tested when parents were genotyped for the optimal SNP panel, and offspring were genotyped for a range of lower density imputation panels. Reducing SNP density had little impact on prediction accuracy until 5,000 SNPs, below which the accuracy dropped. Imputation accuracy increased with increasing imputation panel density. Genomic prediction accuracy when offspring were genotyped for just 200 SNPs, and parents for 5,000 SNPs, was 0.53. This accuracy was similar to the full high density and optimal density dataset, and markedly higher than using 200 SNPs without imputation. These results suggest that imputation from very low to medium density can be a cost-effective tool for genomic selection in Atlantic salmon breeding programs

Gene Expression Response to Sea Lice in Atlantic Salmon Skin: RNA Sequencing Comparison Between Resistant and Susceptible Animals

Sea lice are parasitic copepods that cause large economic losses to salmon aquaculture worldwide. Frequent chemotherapeutic treatments are typically required to control this parasite, and alternative measures such as breeding for improved host resistance are desirable. Insight into the host–parasite interaction and mechanisms of host resistance can lead to improvements in selective breeding, and potentially novel treatment targets. In this study, RNA sequencing was used to study the skin transcriptome of Atlantic salmon (Salmo salar) parasitized with sea lice (Caligus rogercresseyi). The overall aims were to compare the transcriptomic profile of skin at louse attachment sites and “healthy” skin, and to assess differences in gene expression response between animals with varying levels of resistance to the parasite. Atlantic salmon pre-smolts were challenged with C. rogercresseyi, growth and lice count measurements were taken for each fish. 21 animals were selected and RNA-Seq was performed on skin from a louse attachment site, and skin distal to attachment sites for each animal. These animals were classified into family-balanced groups according to the traits of resistance (high vs. low lice count), and growth during infestation. Overall comparison of skin from louse attachment sites vs. healthy skin showed that 4,355 genes were differentially expressed, indicating local up-regulation of several immune pathways and activation of tissue repair mechanisms. Comparison between resistant and susceptible animals highlighted expression differences in several immune response and pattern recognition genes, and also myogenic and iron availability factors. Components of the pathways involved in differential response to sea lice may be targets for studies aimed at improved or novel treatment strategies, or to prioritize candidate functional polymorphisms to enhance genomic selection for host resistance in commercial salmon breeding programs

Potential of low-density genotype imputation for cost-efficient genomic selection for resistance to Flavobacterium columnare in rainbow trout (Oncorhynchus mykiss)

Background Flavobacterium columnare is the pathogen agent of columnaris disease, a major emerging disease that afects rainbow trout aquaculture. Selective breeding using genomic selection has potential to achieve cumulative improvement of the host resistance. However, genomic selection is expensive partly because of the cost of genotyping large numbers of animals using high-density single nucleotide polymorphism (SNP) arrays. The objective of this study was to assess the efciency of genomic selection for resistance to F. columnare using in silico low-density (LD) panels combined with imputation. After a natural outbreak of columnaris disease, 2874 challenged fsh and 469 fsh from the parental generation (n=81 parents) were genotyped with 27,907 SNPs. The efciency of genomic prediction using LD panels was assessed for 10 panels of diferent densities, which were created in silico using two sampling methods, random and equally spaced. All LD panels were also imputed to the full 28K HD panel using the parental generation as the reference population, and genomic predictions were re-evaluated. The potential of prioritizing SNPs that are associated with resistance to F. columnare was also tested for the six lower-density panels. Results The accuracies of both imputation and genomic predictions were similar with random and equally-spaced sampling of SNPs. Using LD panels of at least 3000 SNPs or lower-density panels (as low as 300 SNPs) combined with imputation resulted in accuracies that were comparable to those of the 28K HD panel and were 11% higher than the pedigree-based predictions. Conclusions Compared to using the commercial HD panel, LD panels combined with imputation may provide a more afordable approach to genomic prediction of breeding values, which supports a more widespread adoption of genomic selection in aquaculture breeding programme

Analysis of qPCR reference genes stability determination methods and a practical approach for efficiency calculation on a turbot (Scphthalmus maximus) gonad dataset

Gene expression analysis by reverse transcription quantitative PCR (qPCR) is the most widely used method for analyzing the expression of a moderate number of genes and also for the validation of microarray results. Several issues are crucial for a successful qPCR study, particularly the selection of internal reference genes for normalization and efficiency determination. There is no agreement on which method is the best to detect the most stable genes neither on how to perform efficiency determination. In this study we offer a comprehensive evaluation of the characteristics of reference gene selection methods and how to decide which one is more reliable when they show discordant outcomes. Also, we analyze the current efficiency calculation controversy. Our dataset is composed by gonad samples of turbot at different development times reared at different temperatures. Turbot (Scophthalmus maximus) is a relevant marine aquaculture European species with increasing production in the incoming years. Since females largely outgrow males, identification of genes related to sex determination, gonad development and reproductive behavior, and analysis of their expression profiles are of primary importance for turbot industryVersión del edito

Discovery and Functional Annotation of Quantitative Trait Loci Affecting Resistance to Sea Lice in Atlantic Salmon

<p>Sea lice (Caligus rogercresseyi) are ectoparasitic copepods which have a large negative economic and welfare impact in Atlantic salmon (Salmo salar) aquaculture, particularly in Chile. A multi-faceted prevention and control strategy is required to tackle lice, and selective breeding contributes via cumulative improvement of host resistance to the parasite. While host resistance has been shown to be heritable, little is yet known about the individual loci that contribute to this resistance, the potential underlying genes, and their mechanisms of action. In this study we took a multifaceted approach to identify and characterize quantitative trait loci (QTL) affecting host resistance in a population of 2,688 Caligus-challenged Atlantic salmon post-smolts from a commercial breeding program. We used low and medium density genotyping with imputation to collect genome-wide SNP marker data for all animals. Moderate heritability estimates of 0.28 and 0.24 were obtained for lice density (as a measure of host resistance) and growth during infestation, respectively. Three QTL explaining between 7 and 13% of the genetic variation in resistance to sea lice (as represented by the traits of lice density) were detected on chromosomes 3, 18, and 21. Characterisation of these QTL regions was undertaken using RNA sequencing and pooled whole genome sequencing data. This resulted in the identification of a shortlist of potential underlying causative genes, and candidate functional mutations for further study. For example, candidates within the chromosome 3 QTL include a putative premature stop mutation in TOB1 (an anti-proliferative transcription factor involved in T cell regulation) and an uncharacterized protein which showed significant differential allelic expression (implying the existence of a cis-acting regulatory mutation). While host resistance to sea lice is polygenic in nature, the results of this study highlight significant QTL regions together explaining between 7 and 13 % of the heritability of the trait. Future investigation of these QTL may enable improved knowledge of the functional mechanisms of host resistance to sea lice, and incorporation of functional variants to improve genomic selection accuracy.</p

The nedd-8 activating enzyme gene underlies genetic resistance to infectious pancreatic necrosis virus in Atlantic salmon

Genetic resistance to infectious pancreatic necrosis virus (IPNV) in Atlantic salmon is a rare example of a trait where a single locus (QTL) explains almost all of the genetic variation. Genetic marker tests based on this QTL on salmon chromosome 26 have been widely applied in selective breeding to markedly reduce the incidence of the disease. In the current study, whole genome sequencing and functional annotation approaches were applied to characterise genes and variants in the QTL region. This was complemented by an analysis of differential expression between salmon fry of homozygous resistant and homozygous susceptible genotypes challenged with IPNV. These analyses pointed to the NEDD-8 activating enzyme 1 (nae1) gene as a putative functional candidate underlying the QTL effect. The role of nae1 in IPN resistance was further assessed via CRISPR-Cas9 knockout of the nae1 gene and chemical inhibition of the nae1 protein activity in Atlantic salmon cell lines, both of which resulted in highly significant reduction in productive IPNV replication. In contrast, CRISPR-Cas9 knockout of a candidate gene previously purported to be a cellular receptor for the virus (cdh1) did not have a major impact on productive IPNV replication. These results suggest that nae1 is the causative gene underlying the major QTL affecting resistance to IPNV in salmon, provide further evidence for the critical role of neddylation in host-pathogen interactions, and highlight the value in combining high-throughput genomics approaches with targeted genome editing to understand the genetic basis of disease resistance

The structural variation landscape in 492 Atlantic salmon genomes

Structural variants (SVs) are a major source of genetic and phenotypic variation, but remain challenging to accurately type and are hence poorly characterized in most species. We present an approach for reliable SV discovery in non-model species using whole genome sequencing and report 15,483 high-confidence SVs in 492 Atlantic salmon (Salmo salar L.) sampled from a broad phylogeographic distribution. These SVs recover population genetic structure with high resolution, include an active DNA transposon, widely affect functional features, and overlap more duplicated genes retained from an ancestral salmonid autotetraploidization event than expected. Changes in SV allele frequency between wild and farmed fish indicate polygenic selection on behavioural traits during domestication, targeting brain-expressed synaptic networks linked to neurological disorders in humans. This study offers novel insights into the role of SVs in genome evolution and the genetic architecture of domestication traits, along with resources supporting reliable SV discovery in non-model species.Peer reviewe

Resultados preliminares de la influencia de la temperatura de cultivo sobre la proporción de sexos en el rodaballo (Scophthalmus maximus L.)

Las larvas de tres familias de rodaballos se cultivaron a temperaturas de 15'C, 18'C y 22'C desde el día dos hasta día 90 de vida y a temperatura ambiente hasta el dia 210. En las familias 1 y 2 las proporciones sexuales, determinadas por el fenotipo, oscilaron en los tres grupos entre el 40%- 60%,y no se observaron diferencias siqníñcaffvas entre los diferentes grupos de temperatura (p>0.05). En la familia 3, el porcentaje de hembras fue mayor que el de machos en los tres grupos de temperatura, y además se observó diferencia significativa (p<0.05) entre el grupo de peces cultivados a temperatura fría con respecto a los cultivados en agua ambiente. En tas familias 1 y 2 el sexo genético coincidió en gran medida con~ sexo fenolípico, siendo la discrepancia menor del 10%. Sin embargo, en la familia 3 se observó que el 36,5% de los machos genéticos eran hembras a 15'C, el 29% a 18ºC y el 18% a 23'C. Los resultados sugieren la interacción temperatura-familia en la determinación sexual del rodaballo, que debe ser confirmada en un mayor número de familias.Larvae from three families of turbot were cultured at 15'C, 18'C and 22'C from 2 to 90 days old, and then at ambient temperature until210 daysold. Regarding families 1 and 2, \he sexual proportions determined by \he phenotype varied between 40%·60%, regardless of \he cunure temperature (p>O.05). Gontrary, in family 3, \he percentage of females was higher than for males in all three cunure temperatures. Furthermore, \here were differences (¡¡<O.05) between families cultured at 15'C and 18'C. For families 1 and 2, sex determined by genotype was similarto \hat detennined by the phenotype, wi\h differences <1 0%. Gontrary, for family 3,\he \he percentage of males determined by \he genotype \hat were phenotipycatt¡ females was 36, 29 and 18% for animals cunured at 15'C, 18'C and 22'C, respectively. Results suggest an interaction temperature-family in \he turbot sex determination which should be checked on a higher numberoffamilies

Applying genetic technologies to combat infectious diseases in aquaculture

Disease and parasitism cause major welfare, environmental and economic concerns for global aquaculture. In this review, we examine the status and potential of technologies that exploit genetic variation in host resistance to tackle this problem. We argue that there is an urgent need to improve understanding of the genetic mechanisms involved, leading to the development of tools that can be applied to boost host resistance and reduce the disease burden. We draw on two pressing global disease problems as case studies—sea lice infestations in salmonids and white spot syndrome in shrimp. We review how the latest genetic technologies can be capitalised upon to determine the mechanisms underlying inter- and intra-species variation in pathogen/ parasite resistance, and how the derived knowledge could be applied to boost disease resistance using selective breeding, gene editing and/or with targeted feed treatments and vaccines. Gene editing brings novel opportunities, but also implementation and dissemination challenges, and necessitates new protocols to integrate the technology into aquaculture breeding programmes. There is also an ongoing need to minimise risks of disease agents evolving to overcome genetic improvements to host resistance, and insights from epidemiological and evolutionary models of pathogen infestation in wild and cultured host populations are explored. Ethical issues around the different approaches for achieving genetic resistance are discussed. Application of genetic technologies and approaches has potential to improve fundamental knowledge of mechanisms affecting genetic resistance and provide effective pathways for implementation that could lead to more resistant aquaculture stocks, transforming global aquaculture.publishedVersio