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BACKGROUND:Shell color polymorphisms of Mollusca have contributed to development of evolutionary biology and population genetics, while the genetic bases and molecular mechanisms underlying shell pigmentation are poorly understood. The Pacific oyster (Crassostrea gigas) is one of the most important farmed oysters worldwide. Through successive family selection, four shell color variants (white, golden, black and partially pigmented) of C. gigas have been developed. To elucidate the genetic mechanisms of shell coloration in C. gigas and facilitate the selection of elite oyster lines with desired coloration patterns, differentially expressed genes (DEGs) were identified among the four shell color variants by RNA-seq. RESULTS:Digital gene expression generated over fifteen million reads per sample, producing expression data for 28,027 genes. A total number of 2,645 DEGs were identified from pair-wise comparisons, of which 432, 91, 43 and 39 genes specially were up-regulated in white, black, golden and partially pigmented shell of C. gigas, respectively. Three genes of Abca1, Abca3 and Abcb1 which belong to the ATP-binding cassette (ABC) transporters super-families were significantly associated with white shell formation. A tyrosinase transcript (CGI_10008737) represented consistent up-regulated pattern with golden coloration. We proposed that white shell variant of C. gigas could employ "endocytosis" to down-regulate notch level and to prevent shell pigmentation. CONCLUSION:This study discovered some potential shell coloration genes and related molecular mechanisms by the RNA-seq, which would provide foundational information to further study on shell coloration and assist in selective breeding in C. gigas.

Environmental salinity creates a key barrier to limit the distribution of most aquatic organisms. Adaptation to osmotic fluctuation is believed to be a factor facilitating species diversification. Adaptive evolution often involves beneficial mutations at more than one locus. Bivalves hold great interest, with numerous species living in waters, as osmoconformers, who maintain the osmotic pressure balance mostly by free amino acids. In this study, 107,076,589 reads from two groups of Crassostrea hongkongensis were produced and the assembled into 130,629 contigs. Transcripts putatively involved in stress-response, innate immunity and cell processes were identified according to Gene ontology and KEGG pathway analyses. Comparing with the transcriptome of C. gigas to characterize the diversity of transcripts between species with osmotic divergence, we identified 182,806 high-quality single nucleotide polymorphisms (SNPs) for C. hongkongensis, and 196,779 SNPs for C. gigas. Comparison of 11,602 pairs of putative orthologs allowed for identification of 14 protein-coding genes that experienced strong positive selection (Ka/Ks>1). In addition, 45 genes that may show signs of moderate positive selection (1 ≥ Ka/Ks>0.5) were also identified. Based on Ks ratios and divergence time between the two species published previously, we estimated a neutral transcriptome-wide substitution mutation rate of 1.39 × 10(-9) per site per year. Several genes were differentially expressed across the control and treated groups of each species. This is the first time to sequence the transcriptome of C. hongkongensis and provide the most comprehensive transcriptomic resource available for it. The increasing amount of transcriptome data on Crassostrea provides an excellent resource for phylogenetic analysis. A large number of SNPs identified in this work are expected to provide valuable resources for future marker and genotyping assay development. The analysis of natural selection provides an innovative view on the adaptation within species and sets the basis for future genetic and evolutionary studies.

BACKGROUND: Low salinity is one of the main factors limiting the distribution and survival of marine species. As a euryhaline species, the Pacific oyster Crassostrea gigas is considered to be tolerant to relative low salinity. The genes that regulate C. gigas responses to osmotic stress were monitored using the next-generation sequencing of whole transcriptome with samples taken from gills. By RNAseq technology, transcript catalogs of up- and down-regulated genes were generated from the oysters exposed to low and optimal salinity seawater. METHODOLOGY/PRINCIPAL FINDINGS: Through Illumina sequencing, we reported 1665 up-regulated transcripts and 1815 down-regulated transcripts. A total of 45771 protein-coding contigs were identified from two groups based on sequence similarities with known proteins. As determined by GO annotation and KEGG pathway mapping, functional annotation of the genes recovered diverse biological functions and processes. The genes that changed expression significantly were highly represented in cellular process and regulation of biological process, intracellular and cell, binding and protein binding according to GO annotation. The results highlighted genes related to osmoregulation, signaling and interactions of osmotic stress response, anti-apoptotic reactions as well as immune response, cell adhesion and communication, cytoskeleton and cell cycle. CONCLUSIONS/SIGNIFICANCE: Through more than 1.5 million sequence reads and the expression data of the two libraries, the study provided some useful insights into signal transduction pathways in oysters and offered a number of candidate genes as potential markers of tolerance to hypoosmotic stress for oysters. In addition, the characterization of C. gigas transcriptome will not only provide a better understanding of the molecular mechanisms about the response to osmotic stress of the oysters, but also facilitate research into biological processes to find underlying physiological adaptations to hypoosmotic shock for marine invertebrates.