The oyster genome reveals stress adaptation and complexity of shell formation.

The Pacific oyster Crassostrea gigas belongs to one of the most species-rich but genomically poorly explored phyla, the Mollusca. Here we report the sequencing and assembly of the oyster genome using short reads and a fosmid-pooling strategy, along with transcriptomes of development and stress response and the proteome of the shell. The oyster genome is highly polymorphic and rich in repetitive sequences, with some transposable elements still actively shaping variation. Transcriptome studies reveal an extensive set of genes responding to environmental stress. The expansion of genes coding for heat shock protein 70 and inhibitors of apoptosis is probably central to the oyster's adaptation to sessile life in the highly stressful intertidal zone. Our analyses also show that shell formation in molluscs is more complex than currently understood and involves extensive participation of cells and their exosomes. The oyster genome sequence fills a void in our understanding of the Lophotrochozoa.

Aquaculture genomics, genetics and breeding in the United States: current status, challenges, and priorities for future research.

Advancing the production efficiency and profitability of aquaculture is dependent upon the ability to utilize a diverse array of genetic resources. The ultimate goals of aquaculture genomics, genetics and breeding research are to enhance aquaculture production efficiency, sustainability, product quality, and profitability in support of the commercial sector and for the benefit of consumers. In order to achieve these goals, it is important to understand the genomic structure and organization of aquaculture species, and their genomic and phenomic variations, as well as the genetic basis of traits and their interrelationships. In addition, it is also important to understand the mechanisms of regulation and evolutionary conservation at the levels of genome, transcriptome, proteome, epigenome, and systems biology. With genomic information and information between the genomes and phenomes, technologies for marker/causal mutation-assisted selection, genome selection, and genome editing can be developed for applications in aquaculture. A set of genomic tools and resources must be made available including reference genome sequences and their annotations (including coding and non-coding regulatory elements), genome-wide polymorphic markers, efficient genotyping platforms, high-density and high-resolution linkage maps, and transcriptome resources including non-coding transcripts. Genomic and genetic control of important performance and production traits, such as disease resistance, feed conversion efficiency, growth rate, processing yield, behaviour, reproductive characteristics, and tolerance to environmental stressors like low dissolved oxygen, high or low water temperature and salinity, must be understood. QTL need to be identified, validated across strains, lines and populations, and their mechanisms of control understood. Causal gene(s) need to be identified. Genetic and epigenetic regulation of important aquaculture traits need to be determined, and technologies for marker-assisted selection, causal gene/mutation-assisted selection, genome selection, and genome editing using CRISPR and other technologies must be developed, demonstrated with applicability, and application to aquaculture industries.Major progress has been made in aquaculture genomics for dozens of fish and shellfish species including the development of genetic linkage maps, physical maps, microarrays, single nucleotide polymorphism (SNP) arrays, transcriptome databases and various stages of genome reference sequences. This paper provides a general review of the current status, challenges and future research needs of aquaculture genomics, genetics, and breeding, with a focus on major aquaculture species in the United States: catfish, rainbow trout, Atlantic salmon, tilapia, striped bass, oysters, and shrimp. While the overall research priorities and the practical goals are similar across various aquaculture species, the current status in each species should dictate the next priority areas within the species. This paper is an output of the USDA Workshop for Aquaculture Genomics, Genetics, and Breeding held in late March 2016 in Auburn, Alabama, with participants from all parts of the United States.

De Novo assembly of the Manila clam Ruditapes philippinarum transcriptome provides new insights into expression bias, mitochondrial doubly uniparental inheritance and sex determination.

Males and females share the same genome, thus, phenotypic divergence requires differential gene expression and sex-specific regulation. Accordingly, the analysis of expression patterns is pivotal to the understanding of sex determination mechanisms. Many bivalves are stable gonochoric species, but the mechanism of gonad sexualization and the genes involved are still unknown. Moreover, during the period of sexual rest, a gonad is not present and sex cannot be determined. A mechanism associated with germ line differentiation in some bivalves, including the Manila clam Ruditapes philippinarum, is the doubly uniparental inheritance (DUI) of mitochondria, a variation of strict maternal inheritance. Two mitochondrial lineages are present, one transmitted through eggs and the other through sperm, as well as a mother-dependent sex bias of the progeny. We produced a de novo annotation of 17,186 transcripts from R. philippinarum and compared the transcriptomes of males and females and identified 1,575 genes with strong sex-specific expression and 166 sex-specific single nucleotide polymorphisms, obtaining preliminary information about genes that could be involved in sex determination. Then we compared the transcriptomes between a family producing predominantly females and a family producing predominantly males to identify candidate genes involved in regulation of sex-specific aspects of DUI system, finding a relationship between sex bias and differential expression of several ubiquitination genes. In mammalian embryos, sperm mitochondria are degraded by ubiquitination. A modification of this mechanism is hypothesized to be responsible for the retention of sperm mitochondria in male embryos of DUI species. Ubiquitination can additionally regulate gene expression, playing a role in sex determination of several animals. These data enable us to develop a model that incorporates both the DUI literature and our new findings.

Phenotypic effects of cattle mitochondrial DNA in American bison.

Hybridization between endangered species and more common species is a significant problem in conservation biology because it may result in extinction or loss of adaptation. The historical reduction in abundance and geographic distribution of the American plains bison (Bison bison bison) and their recovery over the last 125 years is well documented. However, introgression from domestic cattle (Bos taurus) into the few remaining bison populations that existed in the late 1800s has now been identified in many modern bison herds. We examined the phenotypic effect of this ancestry by comparing weight and height of bison with cattle or bison mitochondrial DNA (mtDNA) from Santa Catalina Island, California (U.S.A.), a nutritionally stressful environment for bison, and of a group of age-matched feedlot bison males in Montana, a nutritionally rich environment. The environmental and nutritional differences between these 2 bison populations were very different and demonstrated the phenotypic effect of domestic cattle mtDNA in bison over a broad range of conditions. For example, the average weight of feedlot males that were 2 years of age was 2.54 times greater than that of males from Santa Catalina Island. In both environments, bison with cattle mtDNA had lower weight compared with bison with bison mtDNA, and on Santa Catalina Island, the height of bison with cattle mtDNA was lower than the height of bison with bison mtDNA. These data support the hypothesis that body size is smaller and height is lower in bison with domestic cattle mtDNA and that genomic integrity is important for the conservation of the American plains bison.

Overt and concealed genetic loads revealed by QTL mapping of genotype-dependent viability in the Pacific oyster Crassostrea gigas.

Understanding the genetic bases of inbreeding depression, heterosis, and genetic load is integral to understanding how genetic diversity is maintained in natural populations. The Pacific oyster Crassostrea gigas, like many long-lived plants, has high fecundity and high early mortality (type-III survivorship), manifesting a large, overt, genetic load; the oyster harbors an even greater concealed genetic load revealed by inbreeding. Here, we map viability QTL (vQTL) in six interrelated F2 oyster families, using high-density linkage maps of single nucleotide polymorphisms generated by genotyping-by-sequencing (GBS) methods. Altogether, we detect 70 vQTL and provisionally infer 89 causal mutations, 11 to 20 per family. Genetic mortality caused by independent (unlinked) vQTL ranges from 94.2% to 97.8% across families, consistent with previous reports. High-density maps provide better resolution of genetic mechanisms, however. Models of one causal mutation present in both identical-by-descent (IBD) homozygotes and heterozygotes fit genotype frequencies at 37 vQTL; consistent with the mutation-selection balance theory of genetic load, 20 are highly deleterious, completely recessive mutations and 17 are less deleterious, partially dominant mutations. Another 22 vQTL require pairs of recessive or partially dominant causal mutations, half showing selection against recessive mutations linked in repulsion, producing pseudo-overdominance. Only eight vQTL appear to support the overdominance theory of genetic load, with deficiencies of both IBD homozygotes, but at least four of these are likely caused by pseudo-overdominance. Evidence for epistasis is absent. A high mutation rate, random genetic drift, and pseudo-overdominance may explain both the oyster's extremely high genetic diversity and a high genetic load maintained primarily by mutation-selection balance.

Sex determination: genetic models for oysters.

In oysters, sex is determined partly by environment, but previous studies employing controlled crosses suggest that genetic factors are also important. Sex ratios in both full- and half-sib families of the Pacific oyster show paternal control of sex ratio and suggest that a single major gene with 2 genotypes controls sex in the Pacific oyster, with FM oysters being male and FF oysters maturing as male or female. Here, we show that such a model does indeed produce a stable polymorphism for either single or multiple age-class populations, though under limited ranges of f, the probability that an FF individual matures as a female. However, this 2-genotype model cannot explain observed heterogeneity of sex ratios among progeny from different dams within half-sib families. We propose an alternative 3-genotype model that also produces a stable polymorphism, for either single or multiple age-class populations, but over all values of f between zero and one. This model accounts for sex ratio heterogeneity among male half-sib families because it features 2 types of females, a protandric FM and a fixed female FF. Furthermore, the 3-genotype model, accounts for the frequencies of mating types inferred from the observed sex ratios of families more closely than the 2-genotype model. Although the mechanism of sex determination may ultimately prove more complex, simple genetic mechanisms can account for the broad features of sexual maturation in oyster families and the stability of sex ratios in populations.

High-Density Linkage Maps Based on Genotyping-by-Sequencing (GBS) Confirm a Chromosome-Level Genome Assembly and Reveal Variation in Recombination Rate for the Pacific Oyster Crassostrea gigas.

Studies of linkage and linkage mapping have advanced genetic and biological knowledge for over 100 years. In addition to their growing role, today, in mapping phenotypes to genotypes, dense linkage maps can help to validate genome assemblies. Previously, we showed that 40% of scaffolds in the first genome assembly for the Pacific oyster Crassostrea gigas were chimeric, containing single nucleotide polymorphisms (SNPs) mapping to different linkage groups. Here, we merge 14 linkage maps constructed of SNPs generated from genotyping-by-sequencing (GBS) methods with five, previously constructed linkage maps, to create a compendium of nearly 69 thousand SNPs mapped with high confidence. We use this compendium to assess a recently available, chromosome-level assembly of the C. gigas genome, mapping SNPs in 275 of 301 contigs and comparing the ordering of these contigs, by linkage, to their assembly by Hi-C sequencing methods. We find that, while 26% of contigs contain chimeric blocks of SNPs, i.e., adjacent SNPs mapping to different linkage groups than the majority of SNPs in their contig, these apparent misassemblies amount to only 0.08% of the genome sequence. Furthermore, nearly 90% of 275 contigs mapped by linkage and sequencing are assembled identically; inconsistencies between the two assemblies for the remaining 10% of contigs appear to result from insufficient linkage information. Thus, our compilation of linkage maps strongly supports this chromosome-level assembly of the oyster genome. Finally, we use this assembly to estimate, for the first time in a Lophotrochozoan, genome-wide recombination rates and causes of variation in this fundamental process.

Estimation of preferential pairing rates in second-generation autotetraploid pacific oysters (Crassostrea gigas).

Although previously disregarded, polyploidy, and in particular autopolyploidy, is now believed to have played a prominent role in the evolution of plants and animals. We estimated the rate of preferential pairing in second-generation autotetraploid Pacific oysters from gametic frequencies. We found significant levels of preferential pairing in these recently generated autopolyploids, suggesting that genetic variation in standing populations may play a role in meiotic mechanisms of polyploids derived from these populations.

Linkage maps of microsatellite DNA markers for the Pacific oyster Crassostrea gigas.

We constructed male and female consensus linkage maps for the Pacific oyster Crassostrea gigas, using a total of 102 microsatellite DNA markers typed in 11-day-old larvae from three families. We identified 11 and 12 linkage groups in the male and female consensus maps, respectively. Alignment of these separate maps, however, suggests 10 linkage groups, which agrees with the haploid chromosome number. The male linkage map comprises 88 loci and spans 616.1 cM, while the female map comprises 86 loci and spans 770.5 cM. The male and the female maps share 74 loci; 2 markers remain unlinked. The estimated coverages for the consensus linkage maps are 79% for the male and 70-75% for the female, on the basis of two estimates of genome length. Ninety-five percent of the genome is expected to lie within 16 and 21 cM of markers on the male and female maps, respectively, while 95% of simulated minimum distances to the male and female maps are within 10.1 and 13.6 cM, respectively. Females have significantly more recombination than males, across 118 pairs of linked markers in common to the parents of the three families. Significant differences in recombination and orders of markers are also evident among same-sex parents of different families as well as sibling parents of opposite sex. These observations suggest that polymorphism for chromosomal rearrangements may exist in natural populations, which could have profound implications for interpreting the evolutionary genetics of the oyster. These are the first linkage maps for a bivalve mollusc that use microsatellite DNA markers, which should enable them to be transferred to other families and to be useful for further genetic analyses such as QTL mapping.

Second-Generation Linkage Maps for the Pacific Oyster Crassostrea gigas Reveal Errors in Assembly of Genome Scaffolds.

The Pacific oyster Crassostrea gigas, a widely cultivated marine bivalve mollusc, is becoming a genetically and genomically enabled model for highly fecund marine metazoans with complex life-histories. A genome sequence is available for the Pacific oyster, as are first-generation, low-density, linkage and gene-centromere maps mostly constructed from microsatellite DNA makers. Here, higher density, second-generation, linkage maps are constructed from more than 1100 coding (exonic) single-nucleotide polymorphisms (SNPs), as well as 66 previously mapped microsatellite DNA markers, all typed in five families of Pacific oysters (nearly 172,000 genotypes). The map comprises 10 linkage groups, as expected, has an average total length of 588 cM, an average marker-spacing of 1.0 cM, and covers 86% of a genome estimated to be 616 cM. All but seven of the mapped SNPs map to 618 genome scaffolds; 260 scaffolds contain two or more mapped SNPs, but for 100 of these scaffolds (38.5%), the contained SNPs map to different linkage groups, suggesting widespread errors in scaffold assemblies. The 100 misassembled scaffolds are significantly longer than those that map to a single linkage group. On the genetic maps, marker orders and intermarker distances vary across families and mapping methods, owing to an abundance of markers segregating from only one parent, to widespread distortions of segregation ratios caused by early mortality, as previously observed for oysters, and to genotyping errors. Maps made from framework markers provide stronger support for marker orders and reasonable map lengths and are used to produce a consensus high-density linkage map containing 656 markers.

Separating the nature and nurture of the allocation of energy in response to global change.

Understanding and predicting biological stability and change in the face of rapid anthropogenic modifications of ecosystems and geosystems are grand challenges facing environmental and life scientists. Physiologically, organisms withstand environmental stress through changes in biochemical regulation that maintain homeostasis, which necessarily demands tradeoffs in the use of metabolic energy. Evolutionarily, in response to environmentally forced energetic tradeoffs, populations adapt based on standing genetic variation in the ability of individual organisms to reallocate metabolic energy. Combined study of physiology and genetics, separating "Nature and Nurture," is, thus, the key to understanding the potential for evolutionary adaptation to future global change. To understand biological responses to global change, we need experimentally tractable model species that have the well-developed physiological, genetic, and genomic resources necessary for partitioning variance in the allocation of metabolic energy into its causal components. Model species allow for discovery and for experimental manipulation of relevant phenotypic contrasts and enable a systems-biology approach that integrates multiple levels of analyses to map genotypic-to-phenotypic variation. Here, we illustrate how combined physiological and genetic studies that focus on energy metabolism in developmental stages of a model marine organism contribute to an understanding of the potential to adapt to environmental change. This integrative research program provides insights that can be readily incorporated into individual-based ecological models of population persistence under global change.

Quantitative trait locus analysis of stage-specific inbreeding depression in the Pacific oyster Crassostrea gigas.

Inbreeding depression and genetic load have been widely observed, but their genetic basis and effects on fitness during the life cycle remain poorly understood, especially for marine animals with high fecundity and high, early mortality (type-III survivorship). A high load of recessive mutations was previously inferred for the Pacific oyster Crassostrea gigas, from massive distortions of zygotic, marker segregation ratios in F(2) families. However, the number, genomic location, and stage-specific onset of mutations affecting viability have not been thoroughly investigated. Here, we again report massive distortions of microsatellite-marker segregation ratios in two F(2) hybrid families, but we now locate the causative deleterious mutations, using a quantitative trait locus (QTL) interval-mapping model, and we characterize their mode of gene action. We find 14-15 viability QTL (vQTL) in the two families. Genotypic frequencies at vQTL generally suggest selection against recessive or partially recessive alleles, supporting the dominance theory of inbreeding depression. No epistasis was detected among vQTL, so unlinked vQTL presumably have independent effects on survival. For the first time, we track segregation ratios of vQTL-linked markers through the life cycle, to determine their stage-specific expression. Almost all vQTL are absent in the earliest life stages examined, confirming zygotic viability selection; vQTL are predominantly expressed before the juvenile stage (90%), mostly at metamorphosis (50%). We estimate that, altogether, selection on vQTL caused 96% mortality in these families, accounting for nearly all of the actual mortality. Thus, genetic load causes substantial mortality in inbred Pacific oysters, particularly during metamorphosis, a critical developmental transition warranting further investigation.

Unequal and genotype-dependent expression of mitochondrial genes in larvae of the pacific oyster Crassostrea gigas.

Mitochondria are essential for regulation of energy metabolism, but little is known about patterns of mitochondrial genome expression in invertebrates. To explore the association of mitochondrial expression with differential growth of Crassostrea gigas, the Pacific oyster, we crossed two inbred lines to produce inbred and hybrid larvae, which grew at different rates under the same environmental conditions. Using high-throughput cloning and sequencing methods, we identified 1.1 million expressed sequence tags from the mitochondrial genome, 96.7% of which were perfect matches to genes targeted by the method. Expression varied significantly among genes, ranging over nearly four orders of magnitude, from mt:lRNA, which constituted 21% of all transcripts, to mt:CoII, which constituted less than 0.02% of all transcripts. Variable expression of genes coding for subunits of macromolecular complexes (e.g., mt:CoI and mt:CoII) implies that stoichiometry in these complexes must be regulated post-transcriptionally. Surprisingly, the mitochondrial transcriptome contained non-coding transcripts, which may play a role in the regulation of mitochondrial function. Finally, mitochondrial expression depended strongly on maternal factors and nuclear-cytoplasmic interactions, which may explain previously observed growth differences between reciprocal hybrids. Differences in mitochondrial gene expression could provide a biochemical index for the metabolic basis of genetically determined differences in larval growth.

Transcriptomic analysis of growth heterosis in larval Pacific oysters (Crassostrea gigas).

Compared with understanding of biological shape and form, knowledge is sparse regarding what regulates growth and body size of a species. For example, the genetic and physiological causes of heterosis (hybrid vigor) have remained elusive for nearly a century. Here, we investigate gene-expression patterns underlying growth heterosis in the Pacific oyster (Crassostrea gigas) in two partially inbred (f = 0.375) and two hybrid larval populations produced by a reciprocal cross between the two inbred families. We cloned cDNA and generated 4.5 M sequence tags with massively parallel signature sequencing. The sequences contain 23,274 distinct signatures that are expressed at statistically nonzero levels and show a highly positively skewed distribution with median and modal counts of 9.25 million and 3 transcripts per million, respectively. For nearly half of these signatures, expression level depends on genotype and is predominantly nonadditive (hybrids deviate from the inbred average). Statistical contrasts suggest approximately 350 candidate genes for growth heterosis that exhibit concordant nonadditive expression in reciprocal hybrids; this represents only approximately 1.5% of the >20,000 transcripts. Patterns of gene expression, which include dominance for low expression and even underdominance of expression, are more complex than predicted from classical dominant or overdominant explanations of heterosis. Preliminary identification of ribosomal proteins among candidate genes supports the suggestion from previous studies that efficiency of protein metabolism plays a role in growth heterosis.

Evolution of a perfect simple sequence repeat locus in the context of its flanking sequence.

Microsatellites, which have rapidly become the preferred markers in population genetics, reliably assign individual chinook salmon to the winter, fall, late-fall, or spring chinook runs in the Sacramento River in California's Central Valley (Banks et al. 2000. Can. J. Fish. Aquat. Sci. 57:915-927). A substantial proportion of this discriminatory power comes from Ots-2, a simple CA repeat, which is expected to evolve rapidly under the stepwise mutation model. We have sequenced a 300-bp region around this locus and typed 668 microsatellite-flanking sequence haplotypes to explore further the basis of this microsatellite divergence. Three sites of nucleotide polymorphism in the Ots-2 flanking sequence define five haplotypes that are shared by the Californian and Canadian populations. The Ots-2 microsatellite alleles are nonrandomly distributed among these five haplotypes in a pattern of gametic disequilibrium that is also shared among populations. Divergence between the winter run and other Central Valley stocks appears to be caused by a combination of surprisingly static evolution at Ots-2 within a context of more rapidly changing haplotype frequencies.

GENETIC DETERMINANTS OF PROTANDRIC SEX IN THE PACIFIC OYSTER, CRASSOSTREA GIGAS THUNBERG.

A unique feature of sex in Crassostrea oysters is the coexistence of protandric sex change, dioecy, and hermaphroditism. To determine whether such a system is genetically controlled, we analyzed sex ratios in 86 pair-mated families of the Pacific oyster, Crassostrea gigas Thunberg. The overall female ratios of one-, two-, and three-year-old oysters were 37%, 55%, and 75%, respectively, suggesting that a significant proportion of oysters matured first as males and changed to females in later years. Detailed analysis of sex ratios in factorial and nested crosses revealed significant paternal effects, which corresponded to two types of sires. No major maternal effects on sex were observed. Major genetic control of sex was further indicated by the distribution of family sex ratios in two to four apparently discreet groups. These and other data from the literature are compatible with a single-locus model of primary sex determination with a dominant male allele (M) and a protandric female allele (F), so that MF are true males and FF are protandric females that are capable of sex change. The rate of sex change of FF individuals may be influenced by secondary genes and/or environmental factors. Strong maternal and weak paternal effects on sexual maturation or time of spawning were also suggested.