The shell color polymorphism widely exists in economic shellfish, which not only results in a better visual perception but also shows great value as an economic trait for breeding. Small numbers of reddish-orange shell Yesso scallops, Patinopecten yessoensis, were found in cultured populations compared to the brown majority. In this study, a genome-wide association study was conducted to understand the genetic basis of shell color. Sixty-six 2b-RAD libraries with equal numbers of reddish-orange and brown shell individuals were constructed and sequenced using the Illumina HiSeq 2000 platform. A total of 322,332,684 high-quality reads were obtained, and the average sequencing depth was 18.4×. One genomic region on chromosome 11 that included 239 single-nucleotide polymorphisms (SNPs) was identified as significantly associated with shell color. After verification by high-resolution melting in another population, two SNPs were selected as specific loci for reddish-orange shell color. These two SNPs could be used to improve the selective breeding progress of true-breeding strains with complete reddish-orange scallops. In addition, within the significantly associated genomic region, candidate genes were identified using marker sequences to search the draft genome of Yesso scallop. Three genes (LDLR, FRIS, and FRIY) with known functions in carotenoid metabolism were identified. Further study using high-performance liquid chromatography proved that the relative level of carotenoids in the reddish-orange shells was 40 times higher than that in the brown shells. These results suggested that the accumulation of carotenoids contributes to the formation of reddish-orange shells.

Long non-coding RNA (lncRNA) structurally resembles mRNA but cannot be translated into protein. Although the systematic identification and characterization of lncRNAs have been increasingly reported in model species, information concerning non-model species is still lacking. Here, we report the first systematic identification and characterization of lncRNAs in two sea cucumber species: (1) Apostichopus japonicus during lipopolysaccharide (LPS) challenge and in heathy tissues and (2) Holothuria glaberrima during radial organ complex regeneration, using RNA-seq datasets and bioinformatics analysis. We identified A. japonicus and H. glaberrima lncRNAs that were differentially expressed during LPS challenge and radial organ complex regeneration, respectively. Notably, the predicted lncRNA-microRNA-gene trinities revealed that, in addition to targeting protein-coding transcripts, miRNAs might also target lncRNAs, thereby participating in a potential novel layer of regulatory interactions among non-coding RNA classes in echinoderms. Furthermore, the constructed coding-non-coding network implied the potential involvement of lncRNA-gene interactions during the regulation of several important genes (e.g., Toll-like receptor 1 [TLR1] and transglutaminase-1 [TGM1]) in response to LPS challenge and radial organ complex regeneration in sea cucumbers. Overall, this pioneer systematic identification, annotation, and characterization of lncRNAs in echinoderm pave the way for similar studies and future genetic, genomic, and evolutionary research in non-model species.

Genetic prediction of quantitative traits is a critical task in plant and animal breeding. Genomic selection is an accurate and efficient method of estimating genetic merits by using high-density genome-wide single nucleotide polymorphisms (SNP). In the framework of linear mixed models, we extended genomic best linear unbiased prediction (GBLUP) by including additional quantitative trait locus (QTL) information that was extracted from high-throughput SNPs by using least absolute shrinkage selection operator (LASSO). GBLUP was combined with three LASSO methods—standard LASSO (SLGBLUP), adaptive LASSO (ALGBLUP), and elastic net (ENGBLUP)—that were used for detecting QTLs, and these QTLs were fitted as fixed effects; the remaining SNPs were fitted using a realized genetic relationship matrix. Simulations performed under distinct scenarios revealed that (1) the prediction accuracy of SLGBLUP was the lowest; (2) the prediction accuracies of ALGBLUP and ENGBLUP were equivalent to or higher than that of GBLUP, except under scenarios in which the number of QTLs was large; and (3) the persistence of prediction accuracy over generations was strongest in the case of ENGBLUP. Building on the favorable computational characteristics of GBLUP, ENGBLUP enables robust modeling and efficient computation to be performed for genomic selection.

Attachment is an essential physiological process in life histories of many marine organisms. Using a combination of transcriptomic and proteomic approach, scallop byssal proteins (Sbps) and their associated regulatory network genes were investigated for the first time. We built the first scallop foot transcriptome library, and 75 foot-specific genes were identified. Through integration of transcriptomic-proteomic approach, seven unique Sbps were identified. Of them, three showed significant amino acid sequence homology to known proteins. In contrast, the rest did not show significant protein matches, indicating they are possibly novel proteins. Our transcriptomic and proteomic analyses also suggest that post-translational modification may be one of the significant features for Sbps as well. Taken together, our study provides the first multidimensional collection of a core set of genes that may be potentially involved in scallop byssal attachment.

•A proteomic approach was used to study carotenoid accumulation in Yesso scallop.•PPARs may play a vital role in carotenoid metabolism in Yesso scallop.•It will help understanding the mechanism of carotenoid accumulation in mollusks.Carotenoids are powerful antioxidants that affect many physiological functions. As an important source of natural carotenoids, marine mollusks contain various types of carotenoids and are receiving increasing research attention. To better understand the molecular mechanism underlying carotenoid accumulation in marine mollusks, a new variety of carotenoid-enriched Yesso scallop (Patinopecten yessoensis), named “Haida golden scallop”, was used in this study. A proteomic approach was applied to explore the differences between the new variety and common individuals, resulting in seven differentially expressed proteins. Real-time PCR showed that four of the corresponding genes were also significantly up-regulated at the mRNA level in the new variety. Genes involved in various biological processes, such as lipid and glucose metabolism, protein-folding and degradation, were altered. Peroxisome proliferator-activated receptors (PPARs) may play a vital role in these changes. This study represents the first step towards future work on the genetic basis of carotenoid accumulation in marine mollusks.

The Yesso scallop (Patinopecten yessoensis) is one of the most important aquacultural scallops in the north of China. We identified a rare colour variant that occurs in about 0.2% of the natural population. Orange variants have not been reported before in shellfish, including scallops. Identification of the molecular basis of this colour variation will provide guidance when developing a breeding plan for these scallops. The main pigment was isolated from the muscle tissues of the variant Yesso scallop by silica gel column chromatography, and was characterised by mass spectrometry and 1H and 13C nuclear magnetic resonance methods. Its structure was identified as that of pectenolone (3,3′-dihydroxy-β,β-caroten-4–one). This is the first report of pectenolone, a type of carotenoid, in the muscle of the Yesso scallop.

Rotifer growth performance and microbial community changes associated with rotifer cultures were monitored while different feed types (Nannochloropsis oculata paste and the commercial yeast based feed CS-3000), different regimes (daily changes, changes per batch and no changes) and mixtures of three probionts (Phenylobacterium sp.; Gluconobacter sp. and Paracoccus denitrificans) were provided. It was shown that the dominant bacterial species in the cultures receiving either N. oculata or CS-3000 were different. However, in cultures receiving both feeds (either switching between feeds on a daily basis or on a batch basis), a high similarity in microbial community fingerprint was found. The presence of probionts was detected by the end of four batch culture cycles in spite of strong shifts of the bacterial community. By group discriminant analysis, it was found that Phenylobacterium sp. and Paracoccus sp. contributed positively to the CS-3000-fed group, while Gluconobacter sp. contributed positively to the N. oculata-fed group, although they did not appear as very dominant species.

Marine scallops, with extended planktonic larval stages which can potentially disperse over large distances when advected by marine currents, are expected to possess low geographical differentiation. However, the sessile lifestyle as adult tends to form discrete “sea beds” with unique population dynamics and structure. The narrow distribution of Zhikong scallop (Chlamys farreri), its long planktonic larval stage, and the extremely hydrographic complexity in its distribution range provide an interesting case to elucidate the impact of marine currents on geographical differentiation for marine bivalves at a fine geographical scale. In this study, we analyzed genetic variation at nine microsatellite DNA loci in six locations throughout the distribution of Zhikong scallop in the Northern China. Very high genetic diversity was present in all six populations. Two populations sampled from the same marine gyre had no detectable genetic differentiation (FST = 0.0013); however, the remaining four populations collected from different marine gyres or separated by strong marine currents showed low but significant genetic differentiation (FST range 0.0184–0.0602). Genetic differentiation was further analyzed using the Monmonier algorithm to identify genetic barriers and using the assignment test conducted by software GeneClass2 to ascertain population membership of individuals. The genetic barriers fitting the orientation of marine gyres/currents were clearly identified, and the individual assignment analysis indicated that 95.6% of specimens were correctly allocated to one of the six populations sampled. The results support the hypothesis that significant population structure is present in Zhikong scallop at a fine geographical scale, and marine currents can be responsible for the genetic differentiation.

Scallop adductor muscles are very popular delicious seafood distributed in the international markets. Identification of commercial adductor muscles to the species level is an important issue to assure the correct labeling for their distribution. In the present study, we established the species-specific microsatellite markers for four main economic scallop species to identify commercial adductor muscles. A total of 140 adductors derived from three different food processes, the frozen, the dried and the canned, were collected from markets and analyzed using the species-specific microsatellite markers. Although the DNA extracted from the commercial samples was highly degraded, all samples were well identified using the method established in the present study. This method well demonstrated high precision and high sensitivity, and more competence in the identification of mixed commercial samples. Moreover, the results in this study gave the experimental evidences for the potential use of species-specific microsatellite markers for species identification in the market management.

Two novel elements (CFG1 and PYG1) of Mag lineage of Ty3/Gypsy retrotransposons were cloned from Zhikong scallop (Chlamys farreri) and Japanese scallop (Patinopecten yessoensis). The total length of the CFG1 element is 4826 bp, including 5′-LTR (192 bp), the entire ORF (4047 bp) and 3′-LTR (189 bp). The entire ORFs of both CFG1 and PYG1 elements are composed of 1348 aa and do not have any frameshifts. Their closest relative is Jule element from the poeciliid fish (Xiphophorus maculatus). On average, the diploid genome of C. farreri contains approximately 84 copies of CFG1 elements. We summarize the major features of CFG1, PYG1 and other elements of Mag lineage of the Ty3/Gypsy group. mRNA expression of CFG1 element in larvae increases gradually before the gastrulae stage and decreases gradually afterward, whereas in adductor such expression in adductor muscle and digestive gland are lower than those in other tissues. Overall, mRNA expression of CFG1 element in the early larvae is significantly higher than that in adult tissues. In muscle tissue, while the promoter and partial GAG domain of CFG1 element are unmethylated, the partial RT domain is highly methylated. These results suggest that CFG1 expression may be controlled by a post-transcriptional gene silencing mechanism that is associated with coding-region (RT domain) methylation.

Chromosomal structural rearrangement in four scallops, Chlamys farreri (n = 19), Patinopecten yessoensis (n = 19), Chlamys nobilis (n = 16) and Argopecten irradians (n = 16), was studied by fluorescence in situ hybridization using histone H3 gene probes. The results show that histone H3 gene sites differ strikingly with regard to number, location, and intensity among, or even within these species. For example, two histone H3 gene loci were detected on the metaphase chromosomes of P. yessoensis, while one locus was found in the others. In P. yessoensis, differing intensities of hybridization signals were detected between homologues 5 and 11, and within homologue 11. These data suggest that the histone H3 gene is a qualified chromosome marker for the preliminary understanding of the historical chromosomal reconstructing of the Pectinidae family. The variable distribution patterns of the histone H3 gene suggest that gene duplication/diminution as well as chromosome rearrangements by inversion and translocation may have played important roles in the genomic evolution of Pectinidae. We also compiled our present results with former published data regarding the chromosome mapping of rDNAs in species of the Pectinidae family. Such comparative chromosomal mapping should improve our understanding of historical chromosomal reconstructions of modern-day scallops.

It is at present difficult to accurately position gaps in sequence alignment and to determine substructural homology in structure alignment when reconstructing phylogenies based on highly divergent sequences. Therefore, we have developed a new strategy for inferring phylogenies based on highly divergent sequences. In this new strategy, the whole secondary structure presented as a string in bracket notation is used as phylogenetic characters to infer phylogenetic relationships. It is no longer necessary to decompose the secondary structure into homologous substructural components. In this study, reliable phylogenetic relationships of eight species in Pectinidae were inferred from the structure alignment, but not from sequence alignment, even with the aid of structural information. The results suggest that this new strategy should be useful for inferring phylogenetic relationships based on highly divergent sequences. Moreover, the structural evolution of ITS1 in Pectinidae was also investigated. The whole ITS1 structure could be divided into four structural domains. Compensatory changes were found in all four structural domains. Structural motifs in these domains were identified further. These motifs, especially those in D2 and D3, may have important functions in the maturation of rRNAs.

Zhikong scallop (Chlamys farreri Jones et Preston, 1904) is one of the most commercially important bivalves in China, but research on its genome is underdeveloped. In this study, we constructed the first Zhikong scallop fosmid library, and analyzed the fosmid end sequences to provide a preliminary assessment of the genome. The library consists of 133,851 clones with an average insert size of about 40 kb, amounting to 4.3 genome equivalents. Fosmid stability assays indicate that Zhikong scallop DNA was stable during propagation in the fosmid system. Library screening with two genes and seven microsatellite markers yielded between two and eight positive clones, and none of those tested was absent from the library. End-sequencing of 480 individual clones generated 828 sequences after trimming, with an average sequence length of 624 bp. BLASTN searches of the nr and EST databases of GenBank and BLASTX searches of the nr database resulted in 213 (25.72%) and 44 (5.31%) significant hits (E < e−5), respectively. Repetitive sequences analysis resulted in 375 repeats, accounting for 15.84% of total length, which were composed of interspersed repetitive sequences, tandem repeats, and low-complexity sequences. The fosmid library, in conjunction with the fosmid end sequences, will serve as a useful resource for physical mapping and positional cloning, and provide a better understanding of the Zhikong scallop genome.

•A full set of 61 PyHsp70 genes was characterized in the P. yessoensis genome.•Extensive expansion of Hsp70 genes occurred independently in the bivalve lineages.•PyHsp70s up-regulated after A. catenella exposure were all from the bivalve-specific expanded Hspa12 sub-family.•Responses to toxic stress in scallop may have arisen through adaptive evolution of tandem duplication of PyHsp70 genes.Heat shock protein 70 (Hsp70s) family members are present in virtually all living organisms and perform a fundamental role against different types of environmental stressors and pathogenic organisms. Marine bivalves live in highly dynamic environments and may accumulate paralytic shellfish toxins (PSTs), a class of well-known neurotoxins closely associated with harmful algal blooms (HABs). Here, we provide a systematic analysis of Hsp70 genes (PyHsp70s) in the genome of Yesso scallop (Patinopecten yessoensis), an important aquaculture species in China, through in silico analysis using transcriptome and genome databases. Phylogenetic analyses indicated extensive expansion of Hsp70 genes from the Hspa12 sub-family in the Yesso scallop and also the bivalve lineages, with gene duplication events before or after the split between the Yesso scallop and the Pacific oyster. In addition, we determined the expression patterns of PyHsp70s after exposure to Alexandrium catenella, the dinoflagellate producing PSTs. Our results confirmed the inducible expression patterns of PyHsp70s under PSTs stress, and the responses to the toxic stress may have arisen through the adaptive recruitment of tandem duplication of Hsp70 genes. These findings provide a thorough overview of the evolution and modification of the Hsp70 family, which will gain insights into the functional characteristics of scallop Hsp70 genes in response to different stresses.

The present study aimed to generate genome-wide single nucleotide polymorphisms (SNPs) for the deep-sea mussel Bathymodiolus platifrons via a combination of genome survey sequencing and the type IIB endonuclease restriction-site associated DNA (2b-RAD) sequencing, assess the potential use of SNPs in detecting fine-sale population genetic structure and signatures of divergent selection, as well as their cross-species application in other bathymodioline mussels. Genome survey sequencing was conducted for one individual of B. platifrons. De novo assembly resulted in 781,720 sequences with a scaffold N50 of 2.9 kb. Using these sequences as a reference, 9307 genome-wide SNPs were identified by 2b-RAD for 28 B. platifrons individuals collected from a seep and a vent population. Among these SNPs, nine outliers showed significant evidence for divergent selection, and their positions in the genes or scaffolds were identified. The FST estimated based on the putative neutral SNPs was low (0.0126) indicating the two B. platifrons populations having a high genetic connectivity. However, the permutation test detected significant differences (P<0.00001), indicating the two populations having clearly detectable genetic differentiation. The Bayesian clustering analyses and principle component analyses (PCA) performed based on either the putative neutral or outlier SNPs also showed that these two populations were genetically differentiated. In addition, 2b-RAD was also conducted to detect 10,199, 6429, and 3811 single nucleotide variants (SNVs) respectively in the bathymodioline mussels Bathymodiolus japonicus, Bathymodiolus aduloides and Idas sp. with different phylogenetic distances from B. platifrons. Overall, our study has demonstrated the feasibility and effectiveness of combining genome survey sequencing and 2b-RAD to rapidly generate genomic resources for use in fine-scale population genetic studies, and various cross-species applications.

The pearl oyster, Pinctada fucata martensii, is an important aquaculture species  in the production of free marine pearls in China and Japan; likewise, it is an ideal animal model for the study of biomineralization. A high-density genetic linkage map was constructed for P. fucata martensii with 3,117 SNP markers. The map spanned 990.74 cM whereas the genome coverage was 99.1% with an average interval of 0.39 cM, a resolution that was the highest in pearl oyster and even higher than that of any other mollusc. QTL mapping and association analysis suggested that six growth-related QTLs were identified, including two QTLs related with total weight and one QTL related with shell length, hinge line length, inner non-nacre covered shell length and shell nacre thickness. One marker, f394, was located at scaffold21710.1|size9744 and 117 bp away from the highly homologous region with intron 2 of N16, a matrix protein gene involved in nacreous layer formation. Taken together, our study generates, for the first time, a high-density genetic linkage map for P. fucata martensii, which enables us to fine-map a gene potentially crucial for pearl formation, thus providing a target gene for selective breeding of P. fucata martensii to improve pearl production.