Development and validation of a 66K SNP array for the hard clam (Mercenaria mercenaria).

BACKGROUND: The hard clam (Mercenaria mercenaria), a marine bivalve distributed along the U.S. eastern seaboard, supports a significant shellfish industry. Overharvest in the 1970s and 1980s led to a reduction in landings. While the transition of industry from wild harvest to aquaculture since that time has enhanced production, it has also exacerbated challenges such as disease outbreaks. In this study, we developed and validated a 66K SNP array designed to advance genetic studies and improve breeding programs in the hard clam, focusing particularly on the development of markers that could be useful in understanding disease resistance and environmental adaptability. RESULTS: Whole-genome resequencing of 84 individual clam samples and 277 pooled clam libraries yielded over 305 million SNPs, which were filtered down to a set of 370,456 SNPs that were used as input for the design of a 66K SNP array. This medium-density array features 66,543 probes targeting coding and non-coding regions, including 70 mitochondrial SNPs, to capture the extensive genetic diversity within the species. The SNPs were distributed evenly throughout the clam genome, with an average interval of 25,641 bp between SNPs. The array incorporates markers for detecting the clam pathogen Mucochytrium quahogii (formerly QPX), enhancing its utility in disease management. Performance evaluation on 1,904 samples demonstrated a 72.7% pass rate with stringent quality control. Concordance testing affirmed the array's repeatability, with an average agreement of allele calls of 99.64% across multiple tissue types, highlighting its reliability. The tissue-specific analysis demonstrated that some tissue types yield better genotyping results than others. Importantly, the array, including its embedded mitochondrial markers, effectively elucidated complex genetic relationships across different clam groups, both wild populations and aquacultured stocks, showcasing its utility for detailed population genetics studies. CONCLUSIONS: The 66K SNP array is a powerful and robust genotyping tool that offers unprecedented insights into the species' genomic architecture and population dynamics and that can greatly facilitate hard clam selective breeding. It represents an important resource that has the potential to transform clam aquaculture, thereby promoting industry sustainability and ecological and economic resilience.

Comparative analysis of the Mercenaria mercenaria genome provides insights into the diversity of transposable elements and immune molecules in bivalve mollusks.

BACKGROUND: The hard clam Mercenaria mercenaria is a major marine resource along the Atlantic coasts of North America and has been introduced to other continents for resource restoration or aquaculture activities. Significant mortality events have been reported in the species throughout its native range as a result of diseases (microbial infections, leukemia) and acute environmental stress. In this context, the characterization of the hard clam genome can provide highly needed resources to enable basic (e.g., oncogenesis and cancer transmission, adaptation biology) and applied (clam stock enhancement, genomic selection) sciences. RESULTS: Using a combination of long and short-read sequencing technologies, a 1.86 Gb chromosome-level assembly of the clam genome was generated. The assembly was scaffolded into 19 chromosomes, with an N50 of 83 Mb. Genome annotation yielded 34,728 predicted protein-coding genes, markedly more than the few other members of the Venerida sequenced so far, with coding regions representing only 2% of the assembly. Indeed, more than half of the genome is composed of repeated elements, including transposable elements. Major chromosome rearrangements were detected between this assembly and another recent assembly derived from a genetically segregated clam stock. Comparative analysis of the clam genome allowed the identification of a marked diversification in immune-related proteins, particularly extensive tandem duplications and expansions in tumor necrosis factors (TNFs) and C1q domain-containing proteins, some of which were previously shown to play a role in clam interactions with infectious microbes. The study also generated a comparative repertoire highlighting the diversity and, in some instances, the specificity of LTR-retrotransposons elements, particularly Steamer elements in bivalves. CONCLUSIONS: The diversity of immune molecules in M. mercenaria may allow this species to cope with varying and complex microbial and environmental landscapes. The repertoire of transposable elements identified in this study, particularly Steamer elements, should be a prime target for the investigation of cancer cell development and transmission among bivalve mollusks.

Identification of variants associated with hard clam, Mercenaria mercenaria, resistance to Quahog Parasite Unknown disease.

Severe losses in aquacultured and wild hard clam (Mercenaria mercenaria) stocks have been previously reported in the northeastern United States due to a protistan parasite called QPX (Quahog Parasite Unknown). Previous work demonstrated that clam resistance to QPX is under genetic control. This study identifies single nucleotide polymorphism (SNP) associated with clam survivorship from two geographically segregated populations, both deployed in an enzootic site. The analysis contrasted samples collected before and after undergoing QPX-related mortalities and relied on a robust draft clam genome assembly. ~200 genes displayed significant variant enrichment at each sampling point in both populations, including 18 genes shared between both populations. Markers from both populations were identified in genes related to apoptosis pathways, protein-protein interaction, receptors, and signaling. This research begins to identify genetic markers associated with clam resistance to QPX disease, leading the way for the development of resistant clam stocks through marker-assisted selection.

Global host molecular perturbations upon in situ loss of bacterial endosymbionts in the deep-sea mussel Bathymodiolus azoricus assessed using proteomics and transcriptomics.

BACKGROUND: Colonization of deep-sea hydrothermal vents by most invertebrates was made efficient through their adaptation to a symbiotic lifestyle with chemosynthetic bacteria, the primary producers in these ecosystems. Anatomical adaptations such as the establishment of specialized cells or organs have been evidenced in numerous deep-sea invertebrates. However, very few studies detailed global inter-dependencies between host and symbionts in these ecosystems. In this study, we proposed to describe, using a proteo-transcriptomic approach, the effects of symbionts loss on the deep-sea mussel Bathymodiolus azoricus' molecular biology. We induced an in situ depletion of symbionts and compared the proteo-transcriptome of the gills of mussels in three conditions: symbiotic mussels (natural population), symbiont-depleted mussels and aposymbiotic mussels. RESULTS: Global proteomic and transcriptomic results evidenced a global disruption of host machinery in aposymbiotic organisms. We observed that the total number of proteins identified decreased from 1118 in symbiotic mussels to 790 in partially depleted mussels and 761 in aposymbiotic mussels. Using microarrays we identified 4300 transcripts differentially expressed between symbiont-depleted and symbiotic mussels. Among these transcripts, 799 were found differentially expressed in aposymbiotic mussels and almost twice as many in symbiont-depleted mussels as compared to symbiotic mussels. Regarding apoptotic and immune system processes - known to be largely involved in symbiotic interactions - an overall up-regulation of associated proteins and transcripts was observed in symbiont-depleted mussels. CONCLUSION: Overall, our study showed a global impairment of host machinery and an activation of both the immune and apoptotic system following symbiont-depletion. One of the main assumptions is the involvement of symbiotic bacteria in the inhibition and regulation of immune and apoptotic systems. As such, symbiotic bacteria may increase their lifespan in gill cells while managing the defense of the holobiont against putative pathogens.

Differential Gene Expression in Five Isolates of the Clam Pathogen, Quahog Parasite Unknown (QPX).

Quahog parasite unknown (QPX) is a thraustochytrid protist that infects the hard clam, Mercenaria mercenaria, causing significant economic losses along the northeastern coast of North America. Previous investigations noted differences in growth dynamics and virulence in QPX cells from different geographic locations. In order to probe the molecular determinants for these variations, we investigated the transcriptomic profiles of five geographically distinct QPX isolates using custom 15k 60-mer oligonucleotide arrays. A total of 1,263 transcripts were differentially expressed (DE) among the five QPX isolates. The hierarchical clustering of gene expression profiles showed that the QPX isolates from Raritan Bay (RB, NY) and from Provincetown Harbor (MA) were more similar to each other and diverged from QPX isolates from Peconic Bay (PB, NY) and Old Plantation Creek (VA), which had more similar gene expression profiles. The most prominent difference was based on 78 transcripts coding for heat shock proteins DE between the five QPX isolates. The study generated contrasting transcriptomic profiles for QPX isolated from northern (MA) and deeper (RB, NY) locations as compared to southern (VA) and shallower (PB, NY) areas, suggesting the adaptation of the parasite to local environmental, in particular temperature, conditions.

Alterations of the immune transcriptome in resistant and susceptible hard clams (Mercenaria mercenaria) in response to Quahog Parasite Unknown (QPX) and temperature.

Quahog Parasite Unknown (QPX) is a fatal protistan parasite that causes severe losses in the hard clam (Mercenaria mercenaria) fisheries along the northeastern coast of the US. Field and laboratory studies of QPX disease have demonstrated a major role for water temperature and M. mercenaria genetic origin in disease development. Infections are more likely to occur at cold temperatures, with clam stocks originating from southern states being more susceptible than clams from northern origin where disease is enzootic. Even though the influence of temperature on QPX infection have been examined in susceptible and resistant M. mercenaria at physiological and cellular scales, the underlying molecular mechanisms associated with host-pathogen interactions remain largely unknown. This study was carried out to explore the molecular changes in M. mercenaria in response to temperature and QPX infection on the transcriptomic level, and also to compare molecular responses between susceptible and resistant clam stocks. A M. mercenaria oligoarray (15 K Agilent) platform was produced based on our previously generated transcriptomic data and was used to compare gene expression profiles in naive and QPX-infected susceptible (Florida stock) and resistant (Massachusetts) clams maintained at temperatures favoring disease development (13 °C) or clam healing (21 °C). In addition, transcriptomic changes reflecting focal (the site of infection, mantle) and systemic (circulating hemocytes) responses were also assessed using the oligoarray platform. Results revealed significant regulation of multiple biological pathways by temperature and QPX infection, mainly associated with immune recognition, microbial killing, protein synthesis, oxidative protection and metabolism. Alterations were widely systemic with most changes in gene expression revealed in hemocytes, highlighting the role of circulating hemocytes as the first line of defense against pathogenic stress. A large number of complement-related recognition molecules with fibrinogen or C1q domains were shown to be specially induced following QPX challenge, and the expression of these molecules was significantly higher in resistant clams as compared to susceptible ones. These highly variable immune proteins may be potent candidate molecular markers for future study of M. mercenaria resistance against QPX. Beyond the specific case of clam response to QPX, this study also provides insights into the primitive complement-like system in the hard clam.

The use of -omic tools in the study of disease processes in marine bivalve mollusks.

Our understanding of disease processes and host-pathogen interactions in model species has benefited greatly from the application of medium and high-throughput genomic, metagenomic, epigenomic, transcriptomic, and proteomic analyses. The rate at which new, low-cost, high-throughput -omic technologies are being developed has also led to an expansion in the number of studies aimed at gaining a better understanding of disease processes in bivalves. This review provides a catalogue of the genetic and -omic tools available for bivalve species and examples of how -omics has contributed to the advancement of marine bivalve disease research, with a special focus in the areas of immunity, bivalve-pathogen interactions, mechanisms of disease resistance and pathogen virulence, and disease diagnosis. The analysis of bivalve genomes and transcriptomes has revealed that many immune and stress-related gene families are expanded in the bivalve taxa examined thus far. In addition, the analysis of proteomes confirms that responses to infection are influenced by epigenetic, post-transcriptional, and post-translational modifications. The few studies performed in bivalves show that epigenetic modifications are non-random, suggesting a role for epigenetics in regulating the interactions between bivalves and their environments. Despite the progress -omic tools have enabled in the field of marine bivalve disease processes, there is much more work to be done. To date, only three bivalve genomes have been sequenced completely, with assembly status at different levels of completion. Transcriptome datasets are relatively easy and inexpensive to generate, but their interpretation will benefit greatly from high quality genome assemblies and improved data analysis pipelines. Finally, metagenomic, epigenomic, proteomic, and metabolomic studies focused on bivalve disease processes are currently limited but their expansion should be facilitated as more transcriptome datasets and complete genome sequences become available for marine bivalve species.

Characterization of the transcriptome and temperature-induced differential gene expression in QPX, the thraustochytrid parasite of hard clams.

BACKGROUND: The hard clam or northern quahog, Mercenaria mercenaria, is one of the most valuable seafood products in the United States representing the first marine resource in some Northeastern states. Severe episodes of hard clam mortality have been consistently associated with infections caused by a thraustochytrid parasite called Quahog Parasite Unknown (QPX). QPX is considered as a cold/temperate water organism since the disease occurs only in the coastal waters of the northwestern Atlantic Ocean from Maritime Canada to Virginia. High disease development at cold temperatures was also confirmed in laboratory studies and is thought to be caused predominantly by immunosuppression of the clam host even though the effect of temperature on QPX virulence has not been fully investigated. In this study, the QPX transcriptome was sequenced using Roche 454 technology to better characterize this microbe and initiate research on the molecular basis of QPX virulence towards hard clams. RESULTS: Close to 18,000 transcriptomic sequences were generated and functionally annotated. Results revealed a wide array of QPX putative virulence factors including a variety of peptidases, antioxidant enzymes, and proteins involved in extracellular mucus production and other secretory proteins potentially involved in interactions with the clam host. Furthermore, a 15 K oligonucleotide array was constructed and used to investigate the effect of temperature on QPX fitness and virulence factors. Results identified a set of QPX molecular chaperones that could explain its adaptation to cold temperatures. Finally, several virulence-related factors were up-regulated at low temperature providing molecular targets for further investigations of increased QPX pathogenicity in cold water conditions. CONCLUSIONS: This is one of the first studies to characterize the transcriptome of a parasitic labyrinthulid, offering new insights into the molecular bases of the pathogenicity of members of this group. Results from the oligoarray study demonstrated the ability of QPX to cope with a wide range of environmental temperatures, including those considered to be suboptimal for clam immunity (low temperature) providing a mechanistic scenario for disease distribution in the field and for high disease prevalence and intensity at low temperature. These results will serve as basis for studies aimed at a better characterization of specific putative virulence factors.

Transcriptional changes in Manila clam (Ruditapes philippinarum) in response to Brown Ring Disease.

Brown Ring Disease (BRD) is a bacterial infection affecting the economically-important clam Ruditapes philippinarum. The disease is caused by a bacterium, Vibrio tapetis, that colonizes the edge of the mantle, altering the biomineralization process and normal shell growth. Altered organic shell matrices accumulate on the inner face of the shell leading to the formation of the typical brown ring in the extrapallial space (between the mantle and the shell). Even though structural and functional changes have been described in solid (mantle) and fluid (hemolymph and extrapallial fluids) tissues from infected clams, the underlying molecular alterations and responses remain largely unknown. This study was designed to gather information on clam molecular responses to the disease and to compare focal responses at the site of the infection (mantle and extrapallial fluid) with systemic (hemolymph) responses. To do so, we designed and produced a Manila clam expression oligoarray (15K Agilent) using transcriptomic data available in public databases and used this platform to comparatively assess transcriptomic changes in mantle, hemolymph and extrapallial fluid of infected clams. Results showed significant regulation in diseased clams of molecules involved in pathogen recognition (e.g. lectins, C1q domain-containing proteins) and killing (defensin), apoptosis regulation (death-associated protein, bcl-2) and in biomineralization (shell matrix proteins, perlucin, galaxin, chitin- and calcium-binding proteins). While most changes in response to the disease were tissue-specific, systemic alterations included co-regulation in all 3 tested tissues of molecules involved in microbe recognition and killing (complement-related factors, defensin). These results provide a first glance at molecular alterations and responses caused by BRD and identify targets for future functional investigations.

Chromosome-level genome assembly of the bay scallop Argopecten irradians.

The bay scallop, Argopecten irradians, is a species of major commercial, cultural, and ecological importance. It is endemic to the eastern coast of the United States, but has also been introduced to China, where it supports a significant aquaculture industry. Here, we provide an annotated chromosome-level reference genome assembly for the bay scallop, assembled using PacBio and Hi-C data. The total genome size is 845.9 Mb, distributed over 1,503 scaffolds with a scaffold N50 of 44.3 Mb. The majority (92.9%) of the assembled genome is contained within the 16 largest scaffolds, corresponding to the 16 chromosomes confirmed by Hi-C analysis. The assembly also includes the complete mitochondrial genome. Approximately 36.2% of the genome consists of repetitive elements. The BUSCO analysis showed a completeness of 96.2%. We identified 33,772 protein-coding genes. This genome assembly will be a valuable resource for future research on evolutionary dynamics, adaptive mechanisms, and will support genome-assisted breeding, contributing to the conservation and management of this iconic species in the face of environmental and pathogenic challenges.

Cadmium Highlights Common and Specific Responses of Two Freshwater Sentinel Species, Dreissena polymorpha and Dreissena rostriformis bugensis.

Zebra mussel (ZM), Dreissena polymorpha, commonly used as a sentinel species in freshwater biomonitoring, is now in competition for habitat with quagga mussel (QM), Dreissena rostriformis bugensis. This raises the question of the quagga mussel's use in environmental survey. To better characterise QM response to stress compared with ZM, both species were exposed to cadmium (100 µg·L-1), a classic pollutant, for 7 days under controlled conditions. The gill proteomes were analysed using two-dimensional electrophoresis coupled with mass spectrometry. For ZM, 81 out of 88 proteoforms of variable abundance were identified using mass spectrometry, and for QM, 105 out of 134. Interestingly, the proteomic response amplitude varied drastically, with 5.6% of proteoforms of variable abundance (DAPs) in ZM versus 9.4% in QM. QM also exhibited greater cadmium accumulation. Only 12 common DAPs were observed. Several short proteoforms were detected, suggesting proteolysis. Functional analysis is consistent with the pleiotropic effects of the toxic metal ion cadmium, with alterations in sulphur and glutathione metabolisms, cellular calcium signalling, cytoskeletal dynamics, energy production, chaperone activation, and membrane events with numerous proteins involved in trafficking and endocytosis/exocytosis processes. Beyond common responses, the sister species display distinct reactions, with cellular response to stress being the main category involved in ZM as opposed to calcium and cytoskeleton alterations in QM. Moreover, QM exhibited greater evidence of proteolysis and cell death. Overall, these results suggest that QM has a weaker stress response capacity than ZM.

Detection and characterisation of mutations responsible for allele-specific protein thermostabilities at the Mn-superoxide dismutase gene in the deep-sea hydrothermal vent polychaete Alvinella pompejana.

Alvinella pompejana (Polychaeta, Alvinellidae) is one of the most thermotolerant marine eukaryotes known to date. It inhabits chimney walls of deep-sea hydrothermal vents along the East Pacific Rise (EPR) and is exposed to various challenging conditions (e.g. high temperature, hypoxia and the presence of sulphides, heavy metals and radiations), which increase the production of dangerous reactive oxygen species (ROS). Two different allelic forms of a manganese-superoxide dismutase involved in ROS detoxification, ApMnSOD1 and ApMnSOD2, and differing only by two substitutions (M110L and A138G) were identified in an A. pompejana cDNA library. RFLP screening of 60 individuals from different localities along the EPR showed that ApMnSOD2 was rare (2 %) and only found in the heterozygous state. Dynamic light scattering measurements and residual enzymatic activity experiments showed that the most frequent form (ApMnSOD1) was the most resistant to temperature. Their half-lives were similarly long at 65 °C (>110 min) but exhibited a twofold difference at 80 °C (20.8 vs 9.8 min). Those properties are likely to be explained by the occurrence of an additional sulphur-containing hydrogen bond involving the M110 residue and the effect of the A138 residue on the backbone entropy. Our results confirm the thermophily of A. pompejana and suggest that this locus is a good model to study how the extreme thermal heterogeneity of the vent conditions may help to maintain old rare variants in those populations.

Transcriptomic and cellular response to bacterial challenge (pathogenic Vibrio parahaemolyticus) in farmed juvenile Haliotis rufescens fed with or without probiotic diet.

The abalone production in Chile has increased considerably in recent years with no sign of tapering off. Open and semi-closed circuits in the marine water zones in the north and south of Chile are the preferred areas of culture. Coastal ecosystems are subjected to a wide variety of contaminants that generate stress that affects populations via their impacts to individuals at both physiological and genetic levels. This work investigated the genomic and cellular response of post-weaning juvenile Haliotis rufescens abalone under hatchery conditions, fed with probiotic diets, and subsequently challenged with Vibrio parahaemolyticus. The expression patterns of 16 selected genes associated with different metabolic pathways were analyzed using Real-Time PCR. Gene expression was then compared to immunological response parameters in the abalone and quantification of V. parahaemolyticus during the experimental period. Both transcriptomic and immunological analyses indicated significant alteration of physiological processes in H. rufescens correlated to exposure to the pathogenic bacteria, as well as to probiotic nutrition.

Molecular Features Associated with Resilience to Ocean Acidification in the Northern Quahog, Mercenaria mercenaria.

The increasing concentration of CO2 in the atmosphere and resulting flux into the oceans will further exacerbate acidification already threatening coastal marine ecosystems. The subsequent alterations in carbonate chemistry can have deleterious impacts on many economically and ecologically important species including the northern quahog (Mercenaria mercenaria). The accelerated pace of these changes requires an understanding of how or if species and populations will be able to acclimate or adapt to such swift environmental alterations. Thus far, studies have primarily focused on the physiological effects of ocean acidification (OA) on M. mercenaria, including reductions in growth and survival. However, the molecular mechanisms of resilience to OA in this species remains unclear. Clam gametes were fertilized under normal pCO2 and reared under acidified (pH ~ 7.5, pCO2 ~ 1200 ppm) or control (pH ~ 7.9, pCO2 ~ 600 ppm) conditions before sampled at 2 days (larvae), 32 days (postsets), 5 and 10 months (juveniles) and submitted to RNA and DNA sequencing to evaluate alterations in gene expression and genetic variations. Results showed significant shift in gene expression profiles among clams reared in acidified conditions as compared to their respective controls. At 10 months of exposure, significant shifts in allele frequency of single nucleotide polymorphisms (SNPs) were identified. Both approaches highlighted genes coding for proteins related to shell formation, bicarbonate transport, cytoskeleton, immunity/stress, and metabolism, illustrating the role these pathways play in resilience to OA.

Combination of RNAseq and RADseq to Identify Physiological and Adaptive Responses to Acidification in the Eastern Oyster (Crassostrea virginica).

Ocean acidification (OA) is a major stressor threatening marine calcifiers, including the eastern oyster (Crassostrea virginica). In this paper, we provide insight into the molecular mechanisms associated with resilience to OA, with the dual intentions of probing both acclimation and adaptation potential in this species. C. virginica were spawned, and larvae were reared in control or acidified conditions immediately after fertilization. RNA samples were collected from larvae and juveniles, and DNA samples were collected from juveniles after undergoing OA-induced mortality and used to contrast gene expression (RNAseq) and SNP (ddRADseq) profiles from animals reared under both conditions. Results showed convergence of evidence from both approaches, particularly in genes involved in biomineralization that displayed significant changes in variant frequencies and gene expression levels among juveniles that survived acidification as compared to controls. Downregulated genes were related to immune processes, supporting previous studies demonstrating a reduction in immunity from exposure to OA. Acclimation to OA via regulation of gene expression might confer short-term resilience to immediate threats; however, the costs may not be sustainable, underscoring the importance of selection of resilient genotypes. Here, we identified SNPs associated with survival under OA conditions, suggesting that this commercially and ecologically important species might have the genetic variation needed for adaptation to future acidification. The identification of genetic features associated with OA resilience is a highly-needed step for the development of marker-assisted selection of oyster stocks for aquaculture and restoration activities.

Characterisation and genetic polymorphism of metallothionein gene CgMT4 in experimental families of Pacific oyster Crassostrea gigas displaying summer mortality.

Summer mortality events have been observed in Pacific oyster Crassostrea gigas for several decades. This paper examines the selective pressure exerted by summer mortality on the polymorphism of a newly identified oyster metallothionein gene. CgMT4 cDNA and genomic sequences were obtained. CgMT4 was studied in two generations of oysters reared in three sites on the French Atlantic coast, using single strand conformation polymorphism analysis. Four alleles were detected. Individuals carrying genotype MT4-CD seem to have higher susceptibility to summer risk conditions. The MT4 gene could be a potential new genetic marker for susceptibility; further validation studies are recommended.

Does the environmental history of mussels have an effect on the physiological response to additional stress under experimental conditions?

Expected effects on marine biota of the ongoing elevation of water temperature and high latitudes is of major concern when considering the reliability of coastal ecosystem production. To compare the capacity of coastal organisms to cope with a temperature increase depending on their environmental history, responses of adult blue mussels (Mytilus spp.) taken from two sites differentially exposed to chemical pollution were investigated during an experimental exposure to a thermal stress. Immune parameters were notably altered by extreme warming and transcriptional changes for a broad selection of genes were associated to the temperature increase following a two-step response pattern. Site-specific responses suggested an influence of environmental history and support the possibility of a genetic basis in the physiological response. However no meaningful difference was detected between the response of hybrids and M galloprovincialis. This study brings new information about the capacity of mussels to cope with the ongoing elevation of water temperature in these coastal ecosystems.

Two parallel chromosome-level reference genomes to support restoration and aquaculture of European flat oyster Ostrea edulis.

This volume of Evolutionary Applications sees the publication of two genomes for the European native flat oyster Ostrea edulis, a species of significant evolutionary, ecological and commercial value. Each is a highly contiguous chromosome-level assembly from individuals of different genetic backgrounds, which have been benchmarked against one another. This situation has resulted from the serendipitous discovery that two independent research groups were both deep into the process of building, annotating and investigating separately produced assemblies. Due to constraints with funder requirements and the need to recognize early career researchers for their work, alongside the technical challenge of integrating assemblies from two very different genomes, there was limited capacity to merge the sequences into one publication at the stage of discovery. This issue is likely to become very common over the next few years until the technologies for working with multiple genomes at once, for example, graph genomes, become commonplace in nonmodel species. Consequently, both of our teams have decided to collaborate rather than compete, recognizing the benefit to copublishing two separate genome resources for the research community, each with distinct scientific investigations, and working collaboratively to benchmark the assemblies.

Chromosome-level reference genome for European flat oyster (Ostrea edulis L.).

The European flat oyster (Ostrea edulis L.) is a bivalve naturally distributed across Europe, which was an integral part of human diets for centuries, until anthropogenic activities and disease outbreaks severely reduced wild populations. Despite a growing interest in genetic applications to support population management and aquaculture, a reference genome for this species is lacking to date. Here, we report a chromosome-level assembly and annotation for the European Flat oyster genome, generated using Oxford Nanopore, Illumina, Dovetail OmniC™ proximity ligation and RNA sequencing. A contig assembly (N50: 2.38 Mb) was scaffolded into the expected karyotype of 10 pseudochromosomes. The final assembly is 935.13 Mb, with a scaffold-N50 of 95.56 Mb, with a predicted repeat landscape dominated by unclassified elements specific to O. edulis. The assembly was verified for accuracy and completeness using multiple approaches, including a novel linkage map built with ddRAD-Seq technology, comprising 4016 SNPs from four full-sib families (eight parents and 163 F1 offspring). Annotation of the genome integrating multitissue transcriptome data, comparative protein evidence and ab-initio gene prediction identified 35,699 protein-coding genes. Chromosome-level synteny was demonstrated against multiple high-quality bivalve genome assemblies, including an O. edulis genome generated independently for a French O. edulis individual. Comparative genomics was used to characterize gene family expansions during Ostrea evolution that potentially facilitated adaptation. This new reference genome for European flat oyster will enable high-resolution genomics in support of conservation and aquaculture initiatives, and improves our understanding of bivalve genome evolution.

Chromosomal assembly of the flat oyster (Ostrea edulis L.) genome as a new genetic resource for aquaculture.

The European flat oyster (Ostrea edulis L.) is a native bivalve of the European coasts. Harvest of this species has declined during the last decades because of the appearance of two parasites that have led to the collapse of the stocks and the loss of the natural oyster beds. O. edulis has been the subject of numerous studies in population genetics and on the detection of the parasites Bonamia ostreae and Marteilia refringens. These studies investigated immune responses to these parasites at the molecular and cellular levels. Several genetic improvement programs have been initiated especially for parasite resistance. Within the framework of a European project (PERLE 2) that aims to produce genetic lines of O. edulis with hardiness traits (growth, survival, resistance) for the purpose of repopulating natural oyster beds in Brittany and reviving the culture of this species in the foreshore, obtaining a reference genome becomes essential as done recently in many bivalve species of aquaculture interest. Here, we present a chromosome-level genome assembly and annotation for the European flat oyster, generated by combining PacBio, Illumina, 10X linked, and Hi-C sequencing. The finished assembly is 887.2 Mb with a scaffold-N50 of 97.1 Mb scaffolded on the expected 10 pseudochromosomes. Annotation of the genome revealed the presence of 35,962 protein-coding genes. We analyzed in detail the transposable element (TE) diversity in the flat oyster genome, highlighted some specificities in tRNA and miRNA composition, and provided the first insight into the molecular response of O. edulis to M. refringens. This genome provides a reference for genomic studies on O. edulis to better understand its basic physiology and as a useful resource for genetic breeding in support of aquaculture and natural reef restoration.