The diversity of ocean life on Earth is under extreme threat and in need of immediate intervention. Genetic rescue offers promising approaches to preserving and restoring the biodiversity of vulnerable species and even ecosystems. The term genetic rescue ranges from the use of genomic insight to uncover new ways to better manage natural resources, to potential gene editing techniques that could introduce genetic resilience to a changing environment. Though genetic rescue is expansive in scope, as a foundation, all applications require two things: (1) A high coverage reference genome ideally based upon a collection of genome sequences of individuals from various populations of the species, and (2) a source of viable cells or tissue (a voucher) from that species. These essential resources for genetic rescue efforts are desperately lacking for a wide range of marine fish, wildlife and ecosystems.

During the research on this Horizon Scan, the interviewed experts often highlighted a widespread lack of genetic resources available to researchers.  These researchers cited several sources of this inertia: the pace of change in genomics; a lack of understanding of the potential for molecular biology to provide new perspectives on their taxon of study; an academic publishing focus that only views sequencing as a means to an end; and, a lack of funding to prepare and pursue a reference-level genome.

The fact that we are losing marine species diversity – before science has even had a chance to bank or sequence it – is the most compelling reason to rapidly increase the pace and scale of banking and sequencing. The urgency of the situation requires that the number of reference genomes and preserved tissue samples of marine species be drastically increased, especially for those species that are known to occupy ecologically significant niches in their ecosystems. This genetic knowledge would support conventional conservation, as well as targeted genetic manipulation.

To-date, most efforts to collect genetic sequence information or cryopreserved samples from wild marine species have not prioritized the application of the resulting information to advance conservation. A fund dedicated to coordinating reference-level sequencing and biobanking efforts with priority conservation projects could be transformative in marine conservation.

The importance of sequencing for conservation

The use of sequencing data to understand the biological activity and responses of an organism to its environment can collectively be referred to as functional “-omics.” Researchers can now compile a reference genome from individuals of a given species and then re-sequence individual genomes of that same species to uncover individual variations. In conservation applications, these functional “-omics” can uncover the breadth of adaptive variation within a species as differences in allele sequences, epigenetic patterns, and gene expression patterns. In marine settings, these insights into the genetic sources of fitness can provide new perspectives on the population ecology of priority species to inform and guide conservation measures. From a restoration perspective, genomic insights could guide precision breeding and potentially even genetic engineering programs directed at facilitating adaptation to stressors (e.g. disease or climate).

The story of salmon regulation exemplifies the power of having high quality genetic insights to the ecology of a species. Genetic insights have enabled rigorous protection and management strategies for salmon. These insights may also enable planned adaptation in restoration efforts as a changing climate alters conditions for these important members of North Pacific marine and terrestrial ecosystems.

To date, very few conservation-relevant marine species have been sequenced, preventing opportunities for genetic rescue. Furthermore, the limited genomic resources that have been gathered are not well suited to inform conservation efforts because they are either (1) derived from a single individual, making it impossible to identify the gene regions relevant to adaptive genetic variation, or (2) lack meaningful associated metadata (on sample origin) or vouchered samples.

The collection of genomic sequences in large databanks has been enabled by the dramatic reduction in sequencing costs that is now fairly routine for humans and commercially important animals. Technical challenges still remain for conservation-relevant organisms, but these tend to be specific to organism type. For example, sequence analysis of corals is complicated by the fact that they are holobionts (assemblages of up to 1000 different species). The sequences of the symbiotic algae and bacteria that are so critical to coral health must also be studied. Likewise, kelp genomics could provide crucial insight for breeding strains that are resilient to climate change; however, like other plants, kelp species have enormous genomes that will be difficult to annotate.

There is an emerging consensus that a broader mix of disciplines will be needed to transform conservation genetics. Analytic methods like computational statistics and data management need to be adapted or invented for use in conservation genomics. A thoughtful framing of these data heavy questions could help to predict ecological interactions across long time scales and changing climates. This kind of increased attention to the computer sciences and bioinformatics will help to transform conservation practice by unlocking the first step in the functional genomics pipeline, and driving it towards actionable insights.

The importance of cryopreservation for conservation

Cryopreservation of living tissue samples will enable the development of experimental platforms by providing researchers with long-term access to tissues and reducing reliance on lengthy field sampling. Research can then be done to determine the molecular mechanisms responsible for adaptation and resilience such as tolerance to warming ocean waters, or can serve as a test bed for hypothetical interventions (like gene edits) to infectious diseases. One could potentially identify lost adaptive capacity in a bottlenecked population. In extreme cases, frozen tissue samples can be used as sources of genetic material in reproductive cloning projects. Though the ability to bring extinct individuals back through reproductive technology is still experimental, this field is moving forward rapidly.

Having a preserved source of living tissue will enable cryopreserved species to take advantage of future technologies. Existing cryopreservation initiatives tend to add to their collections opportunistically rather than strategically; therefore, a focused attention on priority taxa is needed. Methods need to be tailored to specific species, but this is experimentally challenging and requires skills and resources. Standardization followed by automation of cryopreservation methods could be very beneficial for lowering costs and increasing scale, as shown by the pharmaceutical and agricultural industries.

Revive & Restore proposes to establish a Marine Banking & Sequencing Fund that would accelerate the banking and sequencing of marine species of high conservation significance.

Potential projects would be evaluated according to: timeliness and urgency (Is the species at risk?), the ecological role of the targeted species (Is the species a keystone species?), the species’ potential role in providing ecosystem services, and the intended use of the data (How will the scientists apply the information to affect a positive conservation outcome?).

The Marine Sequencing Fund would have two main objectives:

1. Identify potential marine conservation solutions that are currently hampered by a lack of genomic insight
2. Fund the creation of a high-quality reference genome from cryopreserved samples

The steps of the funding process will be:

1. A request for proposals for the development of reference-level genomes will be announced.
2. An Advisory Board would help to determine the level of need for the proposed work and the likely impact on ocean conservation efforts.
3. Approved projects would be funded under stipulation that all sequencing and bioinformatic data and results be shared through open source publications, and that all sequencing and cryopreserved samples be stored in approved banking facilities, and be made freely available for conservation efforts.
4. All data will be entered into GenBank (or other open source site), rather than languishing on the researcher’s hard drive.

This targeted but comprehensive approach will accelerate the pace of genetic rescue for marine environments and allow research data to be fully leveraged for the benefit of all life on Earth.

Sequencing Objectives

The fund will prioritize sequencing initiatives focused on specific conservation-relevant questions. The fund may also focus on sequencing initiatives of model species that could provide a novel capability for all genetic rescue efforts. Such models could unlock high-impact conservation applications for multiple species.

Funding priorities to expand conservation focused sequencing may include the following areas:

• Population-based genomic sequencing approaches, especially whole genome analysis, or genome wide association studies, which enable the identification of adaptive genotypes and mutations.
• Research to uncover gene expression patterns connected to stressful environments, especially those that make use of standardized stress response assays, and measure gene expression responses to simulated environmental stressors.

Potential priority projects aimed at improving sequencing capabilities in marine conservation include:

• Developing universal standards to ensure that data is portable between different sequencing and bioinformatics platforms and groups. This may include the development of discrete standards for sample collection and publication, or collecting and logging high-resolution metadata associated with samples and marine environments.
• Organizing collaborations between conservation groups and sequencing companies so that conservation groups can bring conservation problems to commercial sequencing sponsors.
• Establishing visiting fellowships to fund trained bioinformaticians from other fields to work in conservation laboratories.

Cryopreservation Objectives

The proposed fund will select cryopreservation projects that will bank rare or critically endangered marine species, rapidly increase the number of marine specimens preserved in biobanks, or develop standardized cryopreservation methods for marine species. The fund will prioritize the cryopreservation of cells and tissues that could yield viable organisms (i.e. germplasm and embryos or potentially stem cells or tissue fragments from clonal organisms) to ensure these resources can be leveraged for future genetic rescue work. The cryopreservation of species that occupy important functional roles that drive or maintain ecosystem integrity is especially important to fulfilling the objective of this fund. Furthermore, cryopreservation projects must be well-designed with sound metadata standards and redundant sample collections, as well as samples from multiple locations to bank a diverse set of genotypes adapted to different environments.

Funding priorities to expand cryopreservation operations may include the following the areas.

• Establishing cryopreservation technical capabilities at marine conservation centers. Many conservation groups have access to priority species but can’t preserve and ship samples. Basic training and equipment (freezers, reagents, shippers) are simple investments that would accelerate the bio-banking of priority species.
• Develop cryopreservation protocols for new species. Significant experimentation is needed to a develop cryopreservation protocol for a particular marine species.
• Develop incentive-based business models to draw new entrants to cryopreservation, including citizen scientists, fishermen, and commercial businesses.

Priorities for developing and standardizing cryopreservation protocols and advancing technologies include:

• Cryoprotectant development and improvement: Recombinant protein engineering and computational protein design have enabled novel cryoprotectants that could be used to preserve as-yet-unsampled species or cell types for which existing chemical-based approaches have failed.
• Cryopreservation automation and standardization: Funds will be awarded to innovative projects that seek to standardize methods for adapting a cryopreservation protocol to a new species, which may include the development of predictive models based on easily measured cell properties. Other projects may involve high-throughput sample collection systems, and high throughput freeze-thaw platforms.

Increasing cryopreservation expertise: Visiting fellowships will support experienced cryopreservation technicians and pioneers from the medical life sciences to take positions in conservation laboratories. Ideal candidates will be able to lead conservation-focused cryopreservation initiatives and train new practitioners.

Project Manager – Revive & Restore 

Revive & Restore, with a mission to enhance biodiversity through the new techniques of genetic rescue for endangered and extinct species, is uniquely positioned to administer the Marine Sequencing Fund. The organization sits at the intersection of biotechnology and conservation science, employing experts in both areas. Last year, the organization launched the Catalyst Science Fund, dedicated to identifying and supporting transformative research projects with potential to impact genetic rescue efforts. The Catalyst Science team has established a pipeline for soliciting and reviewing proposals, and for managing funded efforts to enable maximal chances of success. The program manager of the Catalyst Science Fund has a technical background in biomedical science and many years’ experience managing R&D efforts at DARPA. The Marine Sequencing Fund would fit naturally within the existing structure of the Catalyst Fund at Revive & Restore. Furthermore, our organization has a history of convening interdisciplinary groups in order to inspire immediate action for specific genetic rescue missions.

Partners

As the administering body of the Marine Sequencing Fund, Revive & Restore will foster its relationships with the following organizations, which will be essential to the success of the fund. Each of these groups has a global conservation focus and a well-established reach to all relevant cryopreservation and sequencing scientists.

• Ocean Genome Legacy is a nonprofit, marine research organization and biological specimen repository dedicated to exploring and preserving the wealth of information contained in the DNA of marine organisms. OGL works to acquire, authenticate, study, preserve, develop, and distribute genetic materials, biological specimens, information, technology, and standards needed to advance basic and applied non-commercial research.
• Smithsonian Institute Cryo-Initiative is a partnership between the Smithsonian, the National Museum of Natural History, Smithsonian Tropical Research Institute, and the Smithsonian Environmental Research Center, with a mission to optimize management, care, and accessibility of Smithsonian frozen scientific collections.
• San Diego Frozen Zoo holds the largest and most diverse collection of cell cultures, sperm, oocytes and embryos in the world, with over 1,000 taxa preserved.
• Frozen Ark conducts research on how to best preserve samples of all species, especially endangered taxa; however, it currently operates on a small budget.
• Earth Biogenome project aims to sequence, catalog and characterize the genomes of all of Earth’s eukaryotic biodiversity over a period of ten years.
• Vertebrate Genome Project is a project of the G10K Consortium that aims to generate near error-free reference genome assemblies of all 66,000 extant vertebrate species.