Development and Evaluation of High-Density SNP Arrays for the Eastern Oyster Crassostrea virginica.

The eastern oyster Crassostrea virginica is a major aquaculture species for the USA. The sustainable development of eastern oyster aquaculture depends upon the continued improvement of cultured stocks through advanced breeding technologies. The Eastern Oyster Breeding Consortium (EOBC) was formed to advance the genetics and breeding of the eastern oyster. To facilitate efficient genotyping needed for genomic studies and selection, the consortium developed two single-nucleotide polymorphism (SNP) arrays for the eastern oyster: one screening array with 566K SNPs and one breeders' array with 66K SNPs. The 566K screening array was developed based on whole-genome resequencing data from 292 oysters from Atlantic and Gulf of Mexico populations; it contains 566,262 SNPs including 47K from protein-coding genes with a marker conversion rate of 48.34%. The 66K array was developed using best-performing SNPs from the screening array, which contained 65,893 oyster SNPs including 22,984 genic markers with a calling rate of 99.34%, a concordance rate of 99.81%, and a much-improved marker conversion rate of 92.04%. Null alleles attributable to large indels were found in 13.1% of the SNPs, suggesting that copy number variation is pervasive. Both arrays provided easy identification and separation of selected stocks from wild progenitor populations. The arrays contain 31 mitochondrial SNPs that allowed unambiguous identification of Gulf mitochondrial genotypes in some Atlantic populations. The arrays also contain 756 probes from 13 oyster and human pathogens for possible detection. Our results show that marker conversion rate is low in high polymorphism species and that the two-step process of array development can greatly improve array performance. The two arrays will advance genomic research and accelerate genetic improvement of the eastern oyster by delineating genetic architecture of production traits and enabling genomic selection. The arrays also may be used to monitor pedigree and inbreeding, identify selected stocks and their introgression into wild populations, and assess the success of oyster restoration.

Genome-wide analysis of acute low salinity tolerance in the eastern oyster Crassostrea virginica and potential of genomic selection for trait improvement.

As the global demand for seafood increases, research into the genetic basis of traits that can increase aquaculture production is critical. The eastern oyster (Crassostrea virginica) is an important aquaculture species along the Atlantic and Gulf Coasts of the United States, but increases in heavy rainfall events expose oysters to acute low salinity conditions, which negatively impact production. Low salinity survival is known to be a moderately heritable trait, but the genetic architecture underlying this trait is still poorly understood. In this study, we used ddRAD sequencing to generate genome-wide single-nucleotide polymorphism (SNP) data for four F2 families to investigate the genomic regions associated with survival in extreme low salinity (<3). SNP data were also used to assess the feasibility of genomic selection (GS) for improving this trait. Quantitative trait locus (QTL) mapping and combined linkage disequilibrium analysis revealed significant QTL on eastern oyster chromosomes 1 and 7 underlying both survival and day to death in a 36-day experimental challenge. Significant QTL were located in genes related to DNA/RNA function and repair, ion binding and membrane transport, and general response to stress. GS was investigated using Bayesian linear regression models and prediction accuracies ranged from 0.48 to 0.57. Genomic prediction accuracies were largest using the BayesB prior and prediction accuracies did not substantially decrease when SNPs located within the QTL region on Chr1 were removed, suggesting that this trait is controlled by many genes of small effect. Our results suggest that GS will likely be a viable option for improvement of survival in extreme low salinity.

Mitotic instability in triploid and tetraploid one-year-old eastern oyster, Crassostrea virginica, assessed by cytogenetic and flow cytometry techniques.

For commercial oyster aquaculture, triploidy has significant advantages. To produce triploids, the principal technology uses diploid × tetraploid crosses. The development of tetraploid brood stock for this purpose has been successful, but as more is understood about tetraploids, it seems clear that chromosome instability is a principal feature in oysters. This paper is a continuation of work to investigate chromosome instability in polyploid Crassostrea virginica. We established families between tetraploids-apparently stable (non-mosaic) and unstable (mosaic)-and normal reference diploids, creating triploid groups, as well as tetraploids between mosaic and non-mosaic tetraploids. Chromosome loss was about the same for triploid juveniles produced from either mosaic or non-mosaic tetraploids or from either male or female tetraploids. However, there was a statistically significant difference in chromosome loss in tetraploid juveniles produced from mosaic versus non-mosaic parents, with mosaics producing more unstable progeny. These results confirm that chromosome instability, as manifested in mosaic tetraploids, is of little concern for producing triploids, but it is clearly problematic for tetraploid breeding. Concordance between the results from cytogenetics and flow cytometry was also tested for the first time in oysters, by assessing the ploidy of individuals using both techniques. Results between the two were non-concordant.

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.

Aneuploid progeny of the American oyster, Crassostrea virginica, produced by tetraploid × diploid crosses: another example of chromosome instability in polyploid oysters.

The commercial production of triploids, and the creation of tetraploid broodstock to support it, has become an important technique in aquaculture of the eastern oyster, Crassostrea virginica. Tetraploids are produced by cytogenetic manipulation of embryos and have been shown to undergo chromosome loss (to become a mosaic) with unknown consequences for breeding. Our objective was to determine the extent of aneuploidy in triploid progeny produced from both mosaic and non-mosaic tetraploids. Six families of triploids were produced using a single diploid female and crossed with three mosaic and non-mosaic tetraploid male oysters. A second set of crosses was performed with the reciprocals. Chromosome counts of the resultant embryos were tallied at 2-4 cell stage and as 6-hour(h)-old embryos. A significant level of aneuploidy was observed in 6-h-old embryos. For crosses using tetraploid males, aneuploidy ranged from 53% to 77% of observed metaphases, compared to 36% in the diploid control. For crosses using tetraploid females, 51%-71% of metaphases were aneuploidy versus 53% in the diploid control. We conclude that somatic chromosome loss may be a regular feature of early development in triploids, and perhaps polyploid oysters in general. Other aspects of chromosome loss in polyploid oysters are also discussed.

Genetic improvement for disease resistance in oysters: A review.

Oyster species suffer from numerous disease outbreaks, often causing high mortality. Because the environment cannot be controlled, genetic improvement for disease resistance to pathogens is an attractive option to reduce their impact on oyster production. We review the literature on selective breeding programs for disease resistance in oyster species, and the impact of triploidy on such resistance. Significant response to selection to improve disease resistance was observed in all studies after two to four generations of selection for Haplosporidium nelsoni and Roseovarius crassostrea in Crassostrea virginica, OsHV-1 in Crassostrea gigas, and Martelia sydneyi in Saccostrea glomerata. Clearly, resistance in these cases was heritable, but most of the studies failed to provide estimates for heritability or genetic correlations with other traits, e.g., between resistance to one disease and another. Generally, it seems breeding for higher resistance to one disease does not confer higher resistance or susceptibility to another disease. For disease resistance in triploid oysters, several studies showed that triploidy confers neither advantage nor disadvantage in survival, e.g., OsHV-1 resistance in C. gigas. Other studies showed higher disease resistance of triploids over diploid as observed in C. virginica and S. glomerata. One indirect mechanism for triploids to avoid disease was to grow faster, thus limiting the span of time when oysters might be exposed to disease.

Systematic factor optimization for sperm cryopreservation of tetraploid Pacific oysters, Crassostrea gigas.

The availability of tetraploid Pacific oysters provides a unique opportunity for comparative studies of sperm cryopreservation between diploids and tetraploids. In parallel to studies with sperm from diploid oysters, this study reports systematic factor optimization for sperm cryopreservation of tetraploid oysters. Specifically, this study evaluated the effects of cooling rate, single or combined cryoprotectants at various concentrations, equilibration time (exposure to cryoprotectant), and straw size. Similar to sperm from diploids, the optimal cooling rate was 5 degrees C/min to -30 degrees C, followed by cooling at 45 degrees C/min to -80 degrees C before plunging into liquid nitrogen. Screening of single or combined cryoprotectants at various concentrations showed that a combination of the cryoprotectants 6% polyethylene glycol/4% propylene glycol and 6% polyethylene glycol/4% dimethyl sulfoxide yielded consistently high post-thaw motility. A long equilibration (60 min) yielded higher percent fertilization, and confirmed that extended equilibration could be beneficial when low concentrations of cryoprotectant are used. There was no significant difference in post-thaw motility between straw sizes of 0.25 and 0.5 mL. Despite low post-thaw fertilization (<10%) in general for sperm from tetraploids, optimized protocols in the present study effectively retained post-thaw motility for sperm from tetraploid oysters. This study confirmed that sperm from tetraploid Pacific oysters were more negatively affected by cryopreservation than were those of diploids. One possible explanation is that sperm from these two ploidies are different in their plasma membrane properties (e.g., structure, permeability, and elasticity), and the plasma membrane of sperm from tetraploids is more sensitive to cryopreservation effects. The fact that combinations of non-permeating and permeating cryoprotectants improved post-thaw motility in sperm from tetraploids provided presumptive evidence for this interpretation.

Genetic variation in wild and hatchery stocks of Suminoe Oyster (Crassostrea ariakensis) assessed by PCR-RFLP and microsatellite markers.

Genetic variation in wild Asian populations and U.S. hatchery stocks of Crassostrea ariakensis was examined using polymerase chain reactions with restriction fragment length polymorphism (PCR-RFLP) analysis of both the mitochondrial COI gene and the nuclear internal transcribed spacer (ITS) 1 region and using 3 microsatellite markers. Hierarchical analysis of molecular variance and pairwise comparisons revealed significant differentiation (P < 0.05) between samples from the northern region, represented by collections from China and Japan, and 2 of 3 samples from southern China. PCR-RFLP patterns were identified that were diagnostic for the northern (N-type) and southern (S-type) groups. Microsatellite marker profiles were used to assign each oyster to one of the two northern or two southern populations. Results for more than 97% of the oysters were consistent with the PCR-RFLP patterns observed for each individual in that oysters with N-type patterns were assigned to one of the northern populations and those with S-type patterns to one of the southern populations. At one site of the Beihai (B) region in southern China a mix of individuals with either the N-type or S-type PCR-RFLP genotypes was found. No heterozygotes at the nuclear ITS-1 locus were found in the sample, possibly indicating reproductive isolation in sympatry. Microsatellite assignment test results of the B individuals were also consistent with identifications as either the N-type or S-type based on PCR-RFLP patterns. The parental population for one hatchery stock was this B sample, which initially was composed of almost equal numbers of northern and southern genetic types. After hatchery spawns, however, more than 97% of the progeny fell into the northern genetic group by PCR-RFLP and microsatellite assignment test analyses, indicating that the individuals with the southern genotype contributed little to the spawn, owing to gametic incompatibility, differential larval survival, or a difference in timing of sexual maturity. Overall, results suggested that oysters collected as C. ariakensis in this study, and likely in other studies as well, include two different sympatric species with some degree of reproductive isolation.

Commercial-scale sperm cryopreservation of diploid and tetraploid Pacific oysters, Crassostrea gigas.

Cryopreservation of sperm from tetraploid organisms (the possession of four chromosome sets) is essentially unexplored. This is the first cryopreservation study to address sperm from tetraploid Pacific oysters, Crassostrea gigas, and addresses the commercial production of triploid oysters (three chromosome sets). Initial motility, refrigerated storage of undiluted sperm, osmolality of extender solutions, sperm concentrations, equilibration time, and cryoprotectants of propylene glycol and dimethyl sulfoxide were evaluated with sperm from diploid and tetraploid oysters. Unlike most teleost fishes, in which the duration of active motility is typically brief, the motility of sperm from oysters lasts for hours. The present study showed that responses to treatment effects by sperm from tetraploids were different from diploids. The majority of tetraploid experiments resulted in less than 10% motility after thawing and less than 5% fertilization. The highest fertilization obtained for thawed sperm was 96% for sperm from diploid oysters and 28% for sperm from tetraploid oysters. Differential responses to treatments by sperm from tetraploid and diploid oysters may be due to differences in gonadal development. However, the use of cryopreserved sperm from tetraploid Pacific oysters produced 100% triploid offspring by fertilization of eggs from diploid females as determined by flow cytometry of larvae. This study demonstrates that sperm from tetraploid oysters can be collected, frozen, and stored for production of triploid offspring.

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.