AquaLeap: Innovation in Genetics and Breeding to Advance UK Aquaculture Production

Lead Research Organisation: University of Edinburgh
Department Name: The Roslin Institute

Abstract

Sustainable and profitable aquaculture in the UK relies on high quality stock. In contrast to terrestrial agriculture, the sources of stock for aquaculture species range from use of wild stock for several species, to pedigree-based breeding programmes incorporating genomic tools in salmon. Well managed programmes of domestication and breeding have huge potential for cumulative gains in production, including by preventing infectious disease outbreaks. Barriers to applying such approaches in commercial aquaculture include knowledge gaps in the genetic basis of economically important traits, and a lack of genetic tools and expertise applied to aquaculture. 'AquaLeap' establishes a leading interdisciplinary hub focused on innovation in aquaculture genetics to enable each sector to take a 'step' or 'leap' forward in stock enhancement.

We will target advances for four species of economic importance or potential for UK aquaculture; European lobster (Homarus gammarus), European flat oyster (Ostrea edulis), lumpfish (Cyclopterus lumpus) and Atlantic salmon (Salmo salar). For each of these species, we will develop genomic tools and methods which will then be used to tackle industry-defined barriers to progress in stock enhancement.

The genomic tools include high quality reference genome sequences using cutting-edge sequencing technology for the species for which they are currently lacking (lobster, oyster, lumpfish). These genome sequences will be used to exploit standard (e.g. single nucleotide polymorphism, SNP) and novel [e.g. copy number variation (CNV) and epigenetic modifications] sources of variation. Gene editing techniques will be developed, as this technology is likely to lead to breakthroughs in addressing aquaculture problems in the near future.

Lobsters are a high value species with potential for diversifying UK aquaculture. Building on previous studies into the on-growing of hatchery-reared lobsters in aquaculture systems, and using the aforementioned genomic tools, we will assess the contribution of genetic and epigenetic variation to growth and survival traits. These results will inform selective breeding, hatchery conditions and choice of juveniles for on-growing, and has potential to improve the performance of lobsters at sea.

Native oysters have declined dramatically in recent years, and there is significant interest in restocking from both an aquaculture and ecological perspective. A major barrier to hatchery-based restocking and production is the parasitic disease Bonamia. We will build on previous genomic tool development to identify SNP markers that can be used to predict breeding animals with innate resistance to Bonamia, informing selection of native oysters for stocking and tackling a major production issue.

Lumpfish are used extensively as cleaner fish for biological control of sea lice in salmon farming. Hatchery reproduction is now possible, and the next step is selective breeding for traits to enhance their robustness and performance. To help facilitate this, we will assess wild stock diversity to inform base populations for breeding, to estimate genetic parameters for production traits, and develop SNP marker panels for stock management.

Breeding of salmon is advanced, and uses genomic tools to enhance trait improvement and inbreeding control via genomic selection (GS). We will apply innovative approaches to improve the cost-efficiency of GS, and test these approaches for the emerging aquaculture species. We will assess the role of potential novel sources of genetic variation (CNVs) in gill health traits. Finally, we will use gene editing to modify a specific gene causing resistance to a viral disease in salmon, with a view to future editing of salmon genes to improve resistance to infectious diseases.

The scientific programme is complemented by a series of training, dissemination and public engagement activities, including addressing skills gaps identified by the ARCH-UK network.

Technical Summary

The primary goal of AquaLeap is to tackle industry-defined barriers to advances in selective breeding and domestication of aquaculture species. To achieve this goal across diverse aquaculture sectors, we will develop appropriate underpinning genomic tools and techniques, and then apply those to specific end-user-defined problems in three 'emerging' species (European lobster, European flat oyster, Lumpfish) and one 'established' species (Atlantic salmon.)

The reference genome assemblies for the emerging species will be created using 10X genomics and PacBio sequencing approaches. These will underpin several downstream tasks, including choice of SNP marker panels for stock management and breeding value calculations, genotype imputation and study of epigenetic marks using bisulphite sequencing.

In lobster, we will estimate heritability for growth and survival traits using mixed model approaches, and will assess the relative contribution of genetics and epigenetics to these traits. In oyster, we will use a recently developed SNP array to study resistance to Bonamia, harnessing data from a large-scale laboratory disease challenge. In lumpfish, we will assess stock diversity using RAD-Sequencing to inform choice of animals for base populations, and will assess heritability of production traits. In salmon, we will assess the contribution of copy number variants to genetic variation in disease resistance, with a focus on gill health traits.

Across all species, we will develop SNP marker panels for parentage and stock management, and also use the genomic resources to test imputation approaches to improve the affordability of genomic selection via combined parentage - imputation panels.

Gene editing has transformative potential for aquaculture and we will improve CRISPR-Cas9 editing techniques in salmon, and use editing to target putative causative variants underlying a major disease resistance QTL in salmon cell lines and embryos.

Planned Impact

AquaLeap is based on close cooperation and interdependency between the academic and non-academic partners, providing clear routes for immediate translation of research results. This is augmented by exchange of personnel and skills between partners, across sectors, including industrial placements for PDRAs. The broader impacts will arise from the creation of a hub of expertise in breeding and genetics, including animal and plant breeding experts, with associated training and capacity building. The following groups can expect positive impact from the proposal:

(i) UK and global aquaculture production: The immediate impacts will be via project partners. For the National Lobster Hatchery, the outputs include tools and knowledge to inform selection of lobsters for breeding to improve performance and robustness at sea. For Tethys oysters, the outputs will include methods to inform selection of stock with increased resistance to Bonamia in the field, with downstream benefits for survival and robustness of stocks. For Otterferry Sea Farms, the outputs will include validated tools to inform lumpfish selective breeding for improved stock to tackle sea lice in the salmon industry. For Hendrix Genetics, the primary output will be improvement of gene editing methods and methods for use of new breeding technologies to improve disease resistance in salmon. The longer term impacts include improved performance and reliability of stock in UK aquaculture, making step advances in the various sectors. In turn, this offers potential for cumulative gains in production, including disease resistance. This will help tackle existing and emerging disease threats in an environmentally friendly and sustainable manner, helping to address animal welfare concerns. The focus on several emerging species will also assist with UK aquaculture diversification, which is an important component of maximising sustainable production and minimising risk.

(ii) Genetic services industry: There are several companies in the UK and globally whose core business is to support aquaculture breeding and production by offering genetic services, including management of breeding programmes. For project partner Xelect, the outputs will include potential new products which can be offered to new and existing customers, all of which may be useful to other partners in the project (e.g. marker panels for parentage and cost-effective estimation of breeding values, CNV assays for marker-assisted selection).

(iii) UK economy: This project has potential for long term impact for the UK economy via improved sustainable production of various high quality food products with reduced environmental impact. There will be direct contribution to the UK treasury via improved competitiveness and market share for project partners, and also downstream positive impacts on fish farming companies, and the communities that depend on these industries.

(iv) UK science capacity. This project will enable capacity and expertise for use of genetic and genomic tools to answer fundamental biological questions via research programs in academia and industry. This includes the development of universal genomic resources such as reference genome assemblies and SNP panels. This should help cement the position of the UK as a leading country in aquaculture bioscience.

(v) Political and regulatory bodies. Aquaculture is assuming increasing political importance, and solutions to production and environmental issues are key to its expansion. The outputs of this project may influence ethical and regulatory frameworks to encourage exploitation of new breeding technologies such as gene editing.

(vi) General public and society. This project has potential to influence societal attitudes to aquaculture, including use of selective breeding and gene editing. In the longer term, there will be direct benefits to society via improved economic stability and reduced environmental impact of the aquaculture industry.

Publications

10 25 50
 
Title Additional file 1 of Efficient CRISPR/Cas9 genome editing in a salmonid fish cell line using a lentivirus delivery system 
Description Additional file 1 Figure S1. Puromycin can be used to select for resistant cells. CHSE-EC cells were treated for 7 days with different concentrations of Puromycin and the survival was calculated by CellTiter-Glo. A concentration of 0.25 µg/mL of puromycin was found to be the minimal concentration to efficiently kill all non-antibiotic-resistant cells. 
Type Of Art Film/Video/Animation 
Year Produced 2020 
URL https://springernature.figshare.com/articles/Additional_file_1_of_Efficient_CRISPR_Cas9_genome_editi...
 
Title Additional file 1 of Efficient CRISPR/Cas9 genome editing in a salmonid fish cell line using a lentivirus delivery system 
Description Additional file 1 Figure S1. Puromycin can be used to select for resistant cells. CHSE-EC cells were treated for 7 days with different concentrations of Puromycin and the survival was calculated by CellTiter-Glo. A concentration of 0.25 µg/mL of puromycin was found to be the minimal concentration to efficiently kill all non-antibiotic-resistant cells. 
Type Of Art Film/Video/Animation 
Year Produced 2020 
URL https://springernature.figshare.com/articles/Additional_file_1_of_Efficient_CRISPR_Cas9_genome_editi...
 
Title Additional file 2 of Efficient CRISPR/Cas9 genome editing in a salmonid fish cell line using a lentivirus delivery system 
Description Additional file 2 Figure S2. Editing efficiency estimation, The analysis of the editing of pooled cell population samples using ICE online software. a. The chromatograms (.ab1 file) from the control (non-edited) and edited samples, along with the gRNA sequence are uploaded on ice.sythego.com. b. The platform verifies that the cut site corresponds to the start of the mixed population chromatogram and deconvolutes the picks to original sequences + or - a few bases. c. The results are presented as the percentage of each edited sample present in the pooled population contributing to the mixed chromatogram. 
Type Of Art Film/Video/Animation 
Year Produced 2020 
URL https://springernature.figshare.com/articles/Additional_file_2_of_Efficient_CRISPR_Cas9_genome_editi...
 
Title Additional file 2 of Efficient CRISPR/Cas9 genome editing in a salmonid fish cell line using a lentivirus delivery system 
Description Additional file 2 Figure S2. Editing efficiency estimation, The analysis of the editing of pooled cell population samples using ICE online software. a. The chromatograms (.ab1 file) from the control (non-edited) and edited samples, along with the gRNA sequence are uploaded on ice.sythego.com. b. The platform verifies that the cut site corresponds to the start of the mixed population chromatogram and deconvolutes the picks to original sequences + or - a few bases. c. The results are presented as the percentage of each edited sample present in the pooled population contributing to the mixed chromatogram. 
Type Of Art Film/Video/Animation 
Year Produced 2020 
URL https://springernature.figshare.com/articles/Additional_file_2_of_Efficient_CRISPR_Cas9_genome_editi...
 
Title Additional file 3 of Efficient CRISPR/Cas9 genome editing in a salmonid fish cell line using a lentivirus delivery system 
Description Additional file 3 Figure S3. Off-target editing. Off-target evaluation of RIG-I editing. a: The sequence of the top 2 off-target sites are represented along the gRNA sequence used for targeting RIG-I (red box in the middle). Blue letters indicate differences with the original sequence. An additional nucleotide was sequenced in the CHSE-EC cell line, not present in the published sequence (Otsh_v1.0, green box). b: Diagram representing the editing efficiency in the off-target regions (OffT1: ch7:73101728-73,102,398 and OffT2: ch14:42341999-42,342,859). No off-target was detected by Sanger sequencing in either sample (Puro- and Puro+; all sequences, including CHSE-EC (wt) were compared to CHSEwt). c: Representative chromatogram from the sequencing of off-target region 1 (OffT1) in CHSEwt (Control sample, bottom track) and CHSE-EC-RIG-I Puro + (Edited Sample, top track). 
Type Of Art Film/Video/Animation 
Year Produced 2020 
URL https://springernature.figshare.com/articles/Additional_file_3_of_Efficient_CRISPR_Cas9_genome_editi...
 
Title Additional file 3 of Efficient CRISPR/Cas9 genome editing in a salmonid fish cell line using a lentivirus delivery system 
Description Additional file 3 Figure S3. Off-target editing. Off-target evaluation of RIG-I editing. a: The sequence of the top 2 off-target sites are represented along the gRNA sequence used for targeting RIG-I (red box in the middle). Blue letters indicate differences with the original sequence. An additional nucleotide was sequenced in the CHSE-EC cell line, not present in the published sequence (Otsh_v1.0, green box). b: Diagram representing the editing efficiency in the off-target regions (OffT1: ch7:73101728-73,102,398 and OffT2: ch14:42341999-42,342,859). No off-target was detected by Sanger sequencing in either sample (Puro- and Puro+; all sequences, including CHSE-EC (wt) were compared to CHSEwt). c: Representative chromatogram from the sequencing of off-target region 1 (OffT1) in CHSEwt (Control sample, bottom track) and CHSE-EC-RIG-I Puro + (Edited Sample, top track). 
Type Of Art Film/Video/Animation 
Year Produced 2020 
URL https://springernature.figshare.com/articles/Additional_file_3_of_Efficient_CRISPR_Cas9_genome_editi...
 
Description There are several key findings from this award, which are now being translated to publications and impact. The first is that ribonucleoprotein complexes are highly efficient at genome editing of salmon cell lines, and this is reflected in a recent publication in the journal Marine Biotechnology. The second is that this technique can be effectively transferred from cell line to animals and provide a basis for studying gene function in vivo. The third is the development of an effective disease challenge for the bonamia parasite in flat oysters, led by Centre for Environment, Aquaculture and Fisheries Science. The scientists involved in the project also led the writing and publication of a seminal review on aquaculture genetics which was published in the top journal in the field, Nature Reviews Genetics. There have also been significant training and engagement activities, such as a workshop discussing the future of genetic improvement in aquaculture with industry and academic and funding representatives.
Exploitation Route The developments of the techniques and results described above are useful for both academic and industry researchers to take forward research in genome editing and disease resistance in salmonids and oysters.
Sectors Agriculture

Food and Drink

 
Description Scientists involved in the project have been contributing to dialogue on the regulatory processes for genome editing in aquaculture and agriculture, including USDA, CGIAR, FSA, and Defra discussions. They have also contributed to policy documents relating to the restoration of native oyster species in Europe, and the ICES report on aquatic genetic resources.
First Year Of Impact 2020
Sector Agriculture, Food and Drink,Environment
Impact Types Societal

Policy & public services

 
Description BBSRC Responsive Mode
Amount £1,640,000 (GBP)
Funding ID BB/V009818/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 08/2021 
End 08/2025
 
Description Development and validation of a multi-species SNP-array to support genetic improvement in Qatar aquaculture
Amount $493,805 (USD)
Funding ID MME02-0908-200012 
Organisation Qatar Foundation 
Department Qatar National Research Fund
Sector Charity/Non Profit
Country Qatar
Start 11/2021 
End 11/2024
 
Description Genome editing for resistance to viral disease in rainbow trout
Amount £161,786 (GBP)
Organisation Hendrix Genetics 
Sector Private
Country Netherlands
Start 11/2020 
End 02/2022
 
Description Seafood Innovation Fund
Amount £189,968 (GBP)
Funding ID RD135 
Organisation Centre For Environment, Fisheries And Aquaculture Science 
Sector Public
Country United Kingdom
Start 03/2022 
End 03/2023
 
Title Additional file 4 of Efficient CRISPR/Cas9 genome editing in a salmonid fish cell line using a lentivirus delivery system 
Description Additional file 4 Table S1. Predicted Off-target sites. List of all predicted off-targets from CRISPOR website for 354 bp in RIG-I exon 2 (Tab1). The selected gRNA is labeled 224 forw and the list of off-targets can be found lines 2127-2240. Tab2 summarises the predicted off-targets for gRNA 224 forw. The sum of the two off-target scores (MIT and CDF) were calculated to rank the results. The 2 targets selected are highlighted. 
Type Of Material Database/Collection of data 
Year Produced 2020 
Provided To Others? Yes  
URL https://springernature.figshare.com/articles/Additional_file_4_of_Efficient_CRISPR_Cas9_genome_editi...
 
Title Additional file 4 of Efficient CRISPR/Cas9 genome editing in a salmonid fish cell line using a lentivirus delivery system 
Description Additional file 4 Table S1. Predicted Off-target sites. List of all predicted off-targets from CRISPOR website for 354 bp in RIG-I exon 2 (Tab1). The selected gRNA is labeled 224 forw and the list of off-targets can be found lines 2127-2240. Tab2 summarises the predicted off-targets for gRNA 224 forw. The sum of the two off-target scores (MIT and CDF) were calculated to rank the results. The 2 targets selected are highlighted. 
Type Of Material Database/Collection of data 
Year Produced 2020 
Provided To Others? Yes  
URL https://springernature.figshare.com/articles/Additional_file_4_of_Efficient_CRISPR_Cas9_genome_editi...
 
Title Historical translocations and stocking alter the genetic structure of a Mediterranean lobster fishery 
Description Stocking is often used to supplement wild populations that are overexploited or have collapsed, yet it is unclear how this affects the genetic diversity of marine invertebrate populations. During the 1970s, a lobster stock enhancement programme was carried out around the island of Corsica in the Mediterranean using individuals translocated from the Atlantic coast of France. This included the release of thousands of hatchery-reared post-larval lobsters and several adult individuals, but no monitoring plan was established to assess whether these animals survived and recruited to the population. In this study, we sampled European lobster (Homarus gammarus) individuals caught around Corsica and tested whether they showed Atlantic ancestry. Due to a natural marked phylogeographic break between Atlantic and Mediterranean lobsters, we hypothesised that lobsters with dominant (>0.50) Atlantic ancestry were descended from historical stocking releases. Twenty Corsican lobsters were genotyped at 79 single nucleotide polymorphisms and assignment analysis showed that the majority (13) had dominant Atlantic ancestry. This suggests that the hatchery stocking programme carried out in Corsica during the 1970s, using individuals translocated from the Atlantic coast of France, has likely augmented local recruitment but at a cost of altering the genetic structure of the Corsican lobster population. 
Type Of Material Database/Collection of data 
Year Produced 2020 
Provided To Others? Yes  
URL https://datadryad.org/stash/dataset/doi:10.5061/dryad.cvdncjt1d
 
Title Supporting data for "A chromosome-level genome assembly for the Pacific oyster (Crassostrea gigas)" 
Description The Pacific oyster (Crassostrea gigas) is a bivalve mollusc species with vital roles in coastal ecosystems and aquaculture globally. While extensive genomic tools are available for C. gigas, highly contiguous reference genomes are required to support both fundamental and applied research. Herein we report the creation and annotation of a chromosome-level assembly for the Pacific oyster.In the current study, high coverage long and short read sequence data generated on Pacific Biosciences and Illumina platforms from a single female individual specimen was used to generate an initial assembly, which was then scaffolded into 10 pseudo chromosomes using both Hi-C sequencing and a high density SNP linkage map. The final assembly has a scaffold N50 of 58.4 Mb and a contig N50 of 1.8 Mb, representing a step advance on the previously published C. gigas assembly. The new assembly was annotated using Pacific Biosciences Iso-Seq and Illumina RNA-Seq data, identifying ~30K putative protein coding genes, with an average of 3.1 transcripts per gene. Annotation of putative repeat elements highlighted an inverse relationship with gene density and identified putative centromeres of the metacentric chromosomes. An enrichment of Helitron rolling circle transposable elements was observed, suggesting their potential role in shaping the evolution of the C. gigas genome. This new chromosome-level assembly will be an enabling resource for genetics and genomics studies to support fundamental insight into bivalve biology, as well as for genetic improvement of C. gigas in aquaculture breeding programmes. 
Type Of Material Database/Collection of data 
Year Produced 2021 
Provided To Others? Yes  
URL http://gigadb.org/dataset/100875
 
Description Strategic research partnership with WorldFish 
Organisation Worldfish
Country Malaysia 
Sector Charity/Non Profit 
PI Contribution Roslin and Worldfish have a strategic research partnership since 2018 which focuses on selective breeding of Nile tilapia, one of the world's most important farmed fish species. Roslin scientists are developing genomic tools, and investigating how those genomic tools can be used to improve disease resistance of tilapia via breeding.
Collaborator Contribution The partners WorldFish run a tilapia breeding programme which provides samples and data to support the research undertaken at Roslin. This breeding programme also provides the route to impact by which the improved strains can be disseminated to benefit low and middle income country fish farmers.
Impact This has led to a peer reviewed publication https://www.sciencedirect.com/science/article/pii/S0044848619331837 describing genetic resistance to tilapia lake virus, which is one of the most problematic pathogens for global aquaculture. The story was presented in news form via the University of Edinburgh https://www.ed.ac.uk/edinburgh-infectious-diseases/news/news/genetic-resistance-to-lethal-virus-in-farmed-fish and also Science magazine https://www.sciencemag.org/news/2020/03/emerging-virus-killing-farmed-fish-breeders-can-help-them-fight-back
Start Year 2017
 
Description New Scientist Future of Food Webinar 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Ross Houston took part in the New Scientist Future of Food and Agriculture workshop by taking part in a panel discussion on genome editing.
Year(s) Of Engagement Activity 2020
URL https://www.newscientist.com/science-events/future-food-agriculture/