Comparison of Myosepta Development and Transcriptome Profiling between Blunt Snout Bream with and Tilapia without Intermuscular Bones.

Intermuscular bones (IBs) are small spicule-like bones located in the myosepta of basal teleosts, which negatively affect the edibleness and economic value of fish. Blunt snout bream (Megalobrama amblycephala, with epineural and epipleural IBs) and tilapia (Oreochromis niloticus, without epineural and epipleural IBs) are two major aquaculture species and ideal models for studying the formation mechanisms of fish IBs. Here, we compared myosepta development between M. amblycephala and O. niloticus, based on histological analysis, transcriptome profiling, and expression analysis of bone-related genes. The histological results showed that dye condensation began to appear in the myosepta 20 days post hatching (dph) in M. amblycephala, and IBs could be clearly observed 50 dph in the myosepta, based on different staining methods. However, in O. niloticus, dye condensation was not observed in the myosepta from 10 to 60 dph. Differentially expressed genes (DEGs) at different developmental stages were screened by comparing the transcriptomes of M. amblycephala and O. niloticus, and KEGG analysis demonstrated that these DEGs were enriched in many bone-related pathways, such as focal adhesion, calcium, and Wnt signaling pathways. Quantitative PCR was performed to further compare the expression levels of some bone-related genes (scxa, scxb, runx2a, runx2b, bgp, sp7, col1a2, entpd5a, entpd5b, phex, alpl, and fgf23). All the tested genes (except for alpl) exhibited higher expression levels in M. amblycephala than in O. niloticus. A comparison of the dorsal and abdominal muscle tissues between the two species also revealed significant expression differences for most of the tested genes. The results suggest that scxa, scxb, runx2a, runx2b, entpd5a, col1a2, and bgp may play important roles in IB development. Our findings provide some insights into the molecular mechanisms of IB formation, as well as clues for further functional analysis of the identified genes to better understand the development of IBs.

Identification of Distant Regulatory Elements Using Expression Quantitative Trait Loci Mapping for Heat-Responsive Genes in Oysters.

Many marine ectotherms, especially those inhabiting highly variable intertidal zones, develop high phenotypic plasticity in response to rapid climate change by modulating gene expression levels. Herein, we examined the regulatory architecture of heat-responsive gene expression plasticity in oysters using expression quantitative trait loci (eQTL) analysis. Using a backcross family of Crassostrea gigas and its sister species Crassostrea angulata under acute stress, 56 distant regulatory regions accounting for 6-26.6% of the gene expression variation were identified for 19 heat-responsive genes. In total, 831 genes and 164 single nucleotide polymorphisms (SNPs) that could potentially regulate expression of the target genes were screened in the eQTL region. The association between three SNPs and the corresponding target genes was verified in an independent family. Specifically, Marker13973 was identified for heat shock protein (HSP) family A member 9 (HspA9). Ribosomal protein L10a (RPL10A) was detected approximately 2 kb downstream of the distant regulatory SNP. Further, Marker14346-48 and Marker14346-85 were in complete linkage disequilibrium and identified for autophagy-related gene 7 (ATG7). Nuclear respiratory factor 1 (NRF1) was detected approximately 3 kb upstream of the two SNPs. These results suggested regulatory relationships between RPL10A and HSPA9 and between NRF1 and ATG7. Our findings indicate that distant regulatory mutations play an important role in the regulation of gene expression plasticity by altering upstream regulatory factors in response to heat stress. The identified eQTLs provide candidate biomarkers for predicting the persistence of oysters under future climate change scenarios.

Identification of SNPs involved in Zn and Cu accumulation in the Pacific oyster (Crassostrea gigas) by genome-wide association analysis.

Oysters accumulate high concentrations of zinc (Zn) and copper (Cu), which can be transferred to human due to sea food consumption. Breeding new oyster varieties with low Zn and Cu accumulations is one important way to improve food safety. However, the genetic basis for metal accumulation in mollusks is not well understood. To address this issue, oysters collected in the field were used for genome-wide association study (GWAS) and then the identified genes were used for mRNA expressions analysis in laboratory. First, GWAS were conducted for Zn and Cu accumulation in 288 wild Pacific oysters (Crassostrea gigas) farmed in the same ocean environment. The oysters did not show obvious population structure or kinship but exhibited 8.43- and 10.0- fold changes of Zn and Cu contents respectively. GWAS have identified 11 and 12 single nucleotide polymorphisms (SNPs) associated with Zn and Cu, respectively, as well as 16 genes, which were Zn-containing proteins or participated in caveolae-dependent endocytosis. Second, the mRNA expressions of these 16 genes were observed under Zn and Cu exposure. After 9 days of Zn exposure, Zn contents increased 3.1-fold, while the mRNA expression of cell number regulator 3 increased 1.65-fold. Under 9 days of Cu exposure, Cu contents increased 1.97-fold, while the mRNA expression of caveolin-1 decreased 0.61-fold. These provide the evidence for their roles in regulating physiological levels of these two metals. The findings advance our understanding of the genetic basis of Zn and Cu accumulation in mollusks, which can be useful for breeding new, less toxic varieties of oysters.

Genome-wide association analysis of nutrient traits in the oyster Crassostrea gigas: genetic effect and interaction network.

BACKGROUND: Oyster is rich in glycogen and free amino acids and is called "the milk of sea". To understand the main genetic effects of these traits and the genetic networks underlying their correlation, we have conducted the whole genome resequencing with 427 oysters collected from the world-wide scale. RESULTS: After association analysis, 168 clustered significant single nucleotide polymorphism (SNP) loci were identified for glycogen content and 17 SNPs were verified with 288 oyster individuals in another wide populations. These were the most important candidate loci for oyster breeding. Among 24 genes in the 100-kb regions of the leading SNP loci, cytochrome P450 17A1 (CYP17A1) contained a non-synonymous SNP and displayed higher expressions in high glycogen content individuals. This might enhance the gluconeogenesis process by the transcriptionally regulating the expression of phosphoenolpyruvate carboxykinase (PEPCK) and glucose 6-phosphatase (G6Pase). Also, for amino acids content, 417 clustered significant SNPs were identified. After genetic network analysis, three node SNP regions were identified to be associated with glycogen, protein, and Asp content, which might explain their significant correlation. CONCLUSION: Overall, this study provides insights into the genetic correlation among complex traits, which will facilitate future oyster functional studies and breeding through molecular design.

Divergence and plasticity shape adaptive potential of the Pacific oyster.

The interplay between divergence and phenotypic plasticity is critical to our understanding of a species' adaptive potential under rapid climate changes. We investigated divergence and plasticity in natural populations of the Pacific oyster Crassostrea gigas with a congeneric oyster Crassostrea angulata from southern China used as an outgroup. Genome re-sequencing of 371 oysters revealed unexpected genetic divergence in a small area that coincided with phenotypic divergence in growth, physiology, heat tolerance and gene expression across environmental gradients. These findings suggest that selection and local adaptation are pervasive and, together with limited gene flow, influence population structure. Genes showing sequence differentiation between populations also diverged in transcriptional response to heat stress. Plasticity in gene expression is positively correlated with evolved divergence, indicating that plasticity is adaptive and favoured by organisms under dynamic environments. Divergence in heat tolerance-partly through acetylation-mediated energy depression-implies differentiation in adaptive potential. Trade-offs between growth and survival may play an important role in local adaptation of oysters and other marine invertebrates.