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Genome editing using CRISPR/Cas9 methodology holds much potential to accelerate the genetic improvement in aquaculture, and has been extensively tested and applied to various aquaculture species. However, screening and identifying successfully edited individuals based on sensitive, low-cost, and rapid methods remains challenging. Here, we developed and optimized a high-resolution melting analysis (HRM) assay to screen CRISPR/Cas9-edited larval oysters. The target amplification size range was optimized to 100–200 bp. Compared with fluorescent dyes SYBR Green and SYTO-9, saturated dye EvaGreen showed high stability and repeatability when used in PCR master mixes. The HRM assay was sensitive enough to detect a temperature shift when a mixture of 10% mutant colonies and 90% WT colonies were present. Sanger sequencing analysis confirmed that oysters with indel mutations could be successfully identified by the HRM method. Together, these data indicate that the HRM assay developed in this study is a reproducible, sensitive, and efficient method for rapidly screening and identifying gene-edited oysters. • The HRM is sensitive enough to detect a mixture of 10% mutant colonies and 90% WT colonies were present. • Amplification size is between 100 and 200 bp, and the saturated dye EvaGreen Mix demonstrated repeatability and stability. • The use of HRM to screen CRISPR/Cas9 oysters has not been reported previously. [ABSTRACT FROM AUTHOR]

The Pacific oyster (Crassostrea gigas), as the most productive cultured shellfish in the world, has considerable economic importance and value. Yet research on its sex determination remains lacking. Here, a novel HMG-box-containing homolog of the sox gene was uncovered in C. gigas (named CgSox-like). Alignment with the HMG-box of sox genes from other animals revealed that CgSox-like had a high similarity to the Sox gene of scallops. Among different adult tissues, CgSox-like was mainly expressed in the gonads. qPCR analysis revealed that CgSox-like and CgSox8 , a sox gene family gene mainly expressed in male gonads, shared similar expression patterns, with elevated expression as the male gonads developed. RNA interference and dual luciferase reporter assays demonstrated that CgSox-like was located upstream of CgSox8 and promoted the expression of CgSox8. In addition, analysis of CgSox-like and CgSox8 methylation patterns indicated that both genes were in hypomethylation state during the gonadal development. Finally, demethylase inhibitors resulted in elevated expression levels of CgSox-like and CgSox8 genes, suggesting that epigenetics was engaged in modulating the expression of sex-related genes in the Pacific oyster. Taken together, the novel gene CgSox-like was implicated in the sex determination or gonadal development process in male oysters. These results will provide valuable information for the investigation of sex determination mechanism and sex control breeding. • CgSox-like is involved in sex determination or gonadal development in oysters. • CgSox-like was located upstream of CgSox8 and promoted the expression of CgSox8. • DNA methylation participates in the regulation of sex determination in Crassostrea gigas. [ABSTRACT FROM AUTHOR]

Abstract: Rapid, sensitive, and specific detection of pathogens is important for effective disease control in aquaculture. However, the use of conventional microbiological methods for pathogen detection is often limited by the time required to complete the assays. The aim of this study was to develop a DNA microarray assay to detect 8 species of pathogenic bacteria, i.e., Vibrio harveyi, V. alginolyticus, V. parahaemolyticus, V. anguillarum, V. cholerae, Nocardia seriolae, Aeromonas hydrophila, and Streptococcus iniae, in cultured aquatic animals. Probes were designed according to a variable region of the bacterial 16S rRNA gene and gryB sequences, and 23 probes were finally selected by hybridization with 25 bacterial type cultures. The microarray sensitivity for V. anguillarum detection in healthy fish blood was ≥100CFU/mL. Pathogenic bacteria in blood samples from 21 diseased aquacultural samples were analyzed in a blinded fashion by using the microarray, conventional culture, and 16S rRNA gene sequencing methods; the results of the microarray assay agreed well with those of the conventional culture and 16S rRNA gene sequencing methods. The results of this study show that the microarray method described is suitable for preliminary diagnoses or for confirmation of the main potential pathogens in aquatic animals. [Copyright &y& Elsevier]

Gain and loss-of-function analyses are powerful genetic approaches to uncover gene functions in basic and applied research. However, the genetic analysis in bivalve molluscs has been challenged by the lack of effective gene promoters for driving gene overexpression. The cosmopolitan Pacific oyster (Crassostrea gigas) is an economically important marine bivalve and a representative model species for ecological, evolutionary, and developmental studies. Here, we isolated the elongation factor1-α (EF-1α) gene promoter from the Pacific oyster and compared the promoter activity with the commonly used cytomegalovirus (CMV) and Xenopus EF-1α promoters in oyster embryos. We found that the 3465 bp DNA sequence prior to the ATG start codon in oyster EF-1α was able to drive enhanced green fluorescent protein (EGFP) expression in oyster embryos. In contrast, CMV and Xenopus EF-1α promoters failed to direct EGFP expression in oyster embryos. It appeared that the first intron sequence in oyster EF-1α gene was required for the promoter activity. DNA construct without the first intron located upstream of the ATG start codon failed to drive EGFP expression in oyster embryos. Collectively, these studies indicate that the endogenous EF-1α promoter is preferred promoter for driving gene expression in bivalves. • The first endogenous ubiquitous promoter that induces gene expression in bivalves. • CMV and Xenopus EF-1α promoters failed to direct EGFP expression in oyster embryos. • The first intron sequence in oyster EF -1α gene was required for the promoter activity. [ABSTRACT FROM AUTHOR]

Shell coloration in many molluscs is highly variable. A shell color segregation study with progenies from a full factorial cross generated among Pacific oysters exhibiting distinct shell colors (golden, white and black) was conducted to investigate the inheritance of the golden shell color and its correlation with dark pigmentation. Random samples from twenty-three full-sib families were obtained and the shell coloration of offspring within each family was recorded. Results revealed that golden coloration was inherited in a different pattern from dark pigmentation, indicating its different genetic basis. Dark pigmentation was identified as a foreground color while golden or white color were background ones. The locus controlling background colors has two alleles with the allele for golden background being dominant to the allele for white background. In addition, the overlying foreground pigmentation of shells with a golden background was significantly lighter than that of shells with a white background, which suggested an epistatic effect of background color on shell foreground pigmentation. All these findings will facilitate the selection of elite oyster lines with desired shell coloration for aquaculture. [ABSTRACT FROM AUTHOR]

The Pacific oyster ( Crassostrea gigas ) is one of the most important farmed oysters worldwide. To improve breeding progress, research was done to identify molecular markers linked to shell pigmentation, which has been viewed as a key factor for product value. An F 1 -segregating population exhibiting a bimodal distribution of shell pigmentation intensity was obtained by crossing two wild oysters with opposite shell pigmentation. Genomic DNA from nine individuals with lightest shell pigmentation and nine individuals with darkest shell pigmentation were equally pooled for amplified fragment length polymorphism (AFLP) screening. In bulked segregant analysis, six out of 225 selective primer pair combinations produced seven polymorphic fragments tightly associated with shell pigmentation across the segregating population. The seven AFLP markers were mapped onto a single linkage group, and 80% of phenotypic variance could be explained by this locus. In conversion of the seven fragments into sequence-characterized amplified region (SCAR) markers, only one was successfully converted into a co-dominant SCAR marker, named SP-170. The SCAR marker was integrated into a previously constructed linkage map. The SCAR marker obtained in this study will be useful for marker-assisted selection of the Pacific oyster. [ABSTRACT FROM AUTHOR]

The Pacific oyster (Crassostrea gigas) is a widely cultured shellfish species with great economic value; however, estimation of heritabilities for meat composition traits meat composition have not been studied with molecular pedigree. In this study, the heritabilities of six meat composition traits were studied using near-infrared reflectance spectroscopy (NIRS) in the golden shell strain of Pacific oyster. All 30 families used in this study were produced by nested mating. In total, progeny from crosses among 10 sires and 30 dams were communally reared and pedigree was reconstructed with six microsatellite markers. A total of 608 offspring were harvested at 16 months of age and unambiguously assigned to their respective parents. We estimated the heritability at 0.41 ± 0.19 for glycogen content, 0.51 ± 0.09 for total protein content, 0.89 ± 0.18 for total fat content, 0.40 ± 0.11 for zinc content, 0.87 ± 0.06 for selenium content and 0.65 ± 0.13 for ash content. Remarkably, there were moderate negative genetic correlations between protein and both glycogen (−0.65 ± 0.11) and fat content (−0.40 ± 0.09) as well as between shell height and glycogen content (−0.68 ± 0.11). Overall, this study demonstrated that in meat composition traits could be improved through selective breeding under commercial conditions. The correlations among composition traits and growth traits should be taken into consideration. • Genetic parameters for meat composition traits in the golden shell color strain of C. gigas was estimated. • The meat composition traits were determined with near-infrared reflectance spectroscopy (NIRS) technology. • The families of oysters were fostered communally and reconstructed with six microsatellite makers. [ABSTRACT FROM AUTHOR]

The Pacific oyster (Crassostrea gigas) is one of the most important species which has the largest production among all cultured shellfish. Recently, several shell color strains of C. gigas have been developed to improve the commercial values, however the inheritance of shell pigmentation is still unclear. A total of 29 families cross-mated between different pigmented oysters were produced to investigate the inheritance of the shell pigmentation and purple pigmentation. Random samples from each family were obtained and their shell pigmentation were recorded. Results revealed that shell pigmentation was controlled by two genetic locus, among which one is responsible for secretion of pigmentation and the other is responsible for distribution mode of pigmentation. The locus controlling the secretion of pigmentation has two alleles with the allele for the presence of pigmentation being dominant to the allele for the absence of pigmentation. Similarly, another locus controlling the distribution mode of pigmentation has two alleles with the allele for striped distribution being dominant to the allele for solid distribution. In addition, one independent locus with two alleles was suggested to control the purple-striped pigmentation, which one allele for shell devoid of purple pigmentation is dominant to another allele for shell with purple pigmentation. The findings will provide valuable information for the efficient selective breeding of shell color strains in the Pacific oyster. • A two-locus model for inheritance of shell pigmentation were demonstrated. • The allele for secretion of pigmentation is dominant to the other allele for absence of pigmentation. • The allele for striped distribution of pigmentation is dominant to the allele for solid distribution of pigmentation. • An indenpendent loci was found to control the inheritance of purple striped pigmentation. [ABSTRACT FROM AUTHOR]

Crassostrea nippnoa is a potential aquaculture species because of its unique flavor, delicious taste and edibility during the summer, while C. gigas is a major global aquaculture species due to the fast growth rate, high disease resistance and strong environmental adaptability. This study firstly described the reciprocal hybridization between the two oyster species, and four crosses were produced: C. gigas ♀ × C. gigas ♂ (GG), C. gigas ♀ × C. nippona ♂ (GN), C. nippona ♀ × C. gigas ♂ (NG) and C. nippona ♀ × C. nippona ♂ (NN). The embryonic development of reciprocal crosses was similar to that of the pure crosses. However, fertilization rates were obviously different between the reciprocal hybrids and parental species, where the fertilization rates of reciprocal hybrids were significantly lower than those of parental species. At larval stages, the survival rates of the hybrid cohorts were significantly lower than those of inbred cohorts, and the critical stage of reciprocal hybrids where highest mortality occurred was the umbo period. The growth rate of GN progeny was not different from the inbred cohorts, while the growth rate of the NG progeny was higher than that of inbred cohorts. Furthermore, both hybrid cohorts had normal metamorphosis and produced viable spats. During the spat stage, the survival rates of reciprocal hybrids were not different from that of GG spat but were much higher than that of the NN spat. The growth rates of reciprocal hybrids were comparable to that of GG spat but significantly higher than that of NN spat. The shell height of the reciprocal crosses GN and NG were increased by 46.66% and 60.75% compared with the NN cross at day 360, respectively. The reciprocal hybrids had significant survival advantage and growth heterosis compared to NN cohort. The results demonstrated that the cross NG was deemed to be the better hybridization combination for genetic improvement, which could have a potential utilization for oyster aquaculture. • C. nippona was successfully hybridized with Pacific oyster. • Reciprocal hybrids exhibited larger growth heterosis and survival advantage at spat stage. • The cross NG was deemed to be the better hybridization combination in oyster genetic improvement. [ABSTRACT FROM AUTHOR]

The pacific oyster ( Crassostrea gigas ) is one of the most widely farmed aquatic animal species and of great economic importance. Mantle edge color in C . gigas is highly variable and considered as a new potential trait for a better commercial value. In this study, heritability for mantle edge pigmentation and its correlation with shell pigmentation were explored in the white-shell strain of C . gigas with mixed-family method. A total of 460 offspring raised under communal conditions were successfully assigned to their parents using four multiplex PCR protocols based on 11 microsatellite loci. Mantle edge pigmentation was measured through assay of melanin content using spectrophotometric analysis. Animal model heritability estimate were 0.215 ± 0.092 for mantle edge pigmentation. The genetic and phenotypic correlations between two pigment traits were 0.980 ± 0.094 and 0.423 ± 0.042, respectively. Pearson analyses for average mantle edge and shell pigmentation of family also showed strong and significant correlation ( r = 0.729, P = 0.000, n = 29). Chi-square analyses showed that mantle edge pigmentation of family 7 segregated into “lighter” and “darker” groups in a 3:1 ratio ( P = 0.833), suggesting that a major locus might control mantle edge pigment trait. The results obtained in this study will benefit selective breeding of C . gigas with desired mantle and shell pigmentation. [ABSTRACT FROM AUTHOR]

The Pacific oyster ( Crassostrea gigas ) is an important aquatic animal species with enormous production and economic benefits, but heritability estimation for shell color applying mixed-family approach and computer vision system has not been performed. In this study, heritability for shell color-related traits was estimated in the golden shell strain of Pacific oyster by fostering a single cohort of 30 families in a nested mating design consisting of 10 sires and 30 dams. After parentage assignment inference based on six microsatellite markers, 690 offspring were included in the subsequent analyses. Using an animal model, at 16 months of age, heritability estimates were 0.10 ± 0.12 for L ⁎ , 0.41 ± 0.16 for a ⁎ , 0.23 ± 0.15 for b ⁎ and 0.33 ± 0.19 for Δ E . Genetic correlation between shell color-related traits and growth-related traits were generally inconspicuous ranging from − 0.02 to 0.11. Conclusions can be obtained from our study that favourable improvement in aquatic selective breeding would be predictable for the genetic potential of the golden shell color in this strain of Pacific oyster, whereas independent selection conducted at the same time rather than correlative selection would be feasible in the improvement of both types of traits. [ABSTRACT FROM AUTHOR]

Molluscan shellfish have a diverse form of shell coloration and sex determination, understanding the genetic mechanism under these two traits is crucial to their germplasm exploitation and conservation. In our breeding program, a novel orange-shell variant of C. gigas was obtained, and an F2 family was constructed by crossing an orange-shell male with a black-shell female. This F2 family provided an ideal material for QTL mapping of orange shell color and sex traits of C. gigas simultaneously. Here, a high-density genetic linkage map containing 1799 SNP markers was constructed using this F2 family by genotyping-by-sequencing. These markers were distributed on 10 linkage groups and the total map size was 828.31 cM, with an average marker interval of 0.58 cM. Based on this map, six chromosome-level significant QTLs associated with orange shell color were detected, with the phenotypic variance explained ranging from 9.10% to 12.30%. Within the QTL regions, four candidate genes, 3-ketoacyl-CoA thiolase A, fibroblast growth factor receptor 4, poly [ADP-ribose] polymerase tankyrase-1 and tripartite motif-containing protein 45-like, related to the organic matrix of mollusk shells or collagen were identified. Additionally, only one genome-wide significant sex-related QTL was detected, which explained 19.30% of phenotypic variance. Two sex-related genes, proteasome subunit beta type-3 and G-protein coupled estrogen receptor 1 were identified within the QTL region. The QTLs and candidate genes identified in this study provide valuable resources for further investigations on the molecular mechanisms of shell color and sex in C. gigas. • A high-density genetic linkage map containing 1799 SNP markers was constructed for C. gigas using an F2 family. • Six chromosome-level significant QTLs associate with orange shell color were detected in C. gigas. • Only one genome-wide significant sex-related QTL was detected in C. gigas. • Different description methods of phenotype have a great impact on the result of QTL mapping. [ABSTRACT FROM AUTHOR]