Genetic diversity and structure of domesticated strains of red sea bream,Pagrus major,inferred from microsatellite DNA markers

The genetic diversity and structure of nine domesticated strains of red sea bream used in a private hatchery were studied and compared to a wild population. A total of 313 individuals were genotyped at eight microsatellite loci. Average number of alleles per locus ranged from 5.5 to 9.4 in domesticated strains, but that of the wild population was 28.4. Heterozygosity of domesticated strains (ranged 0.697–0.804) was also lower compared to the wild population (0.952). Estimated Ne also decreased in all domesticated strains (ranged 10.3–126.0) compared to the wild population (1422.5). The UPGMA tree and 3‐D FCA showed that there were two main clusters containing domesticated strains, and the wild population was at the middle of both of the domesticated clusters. The STRUCTURE analysis also supported the phylogenetic analysis, and revealed three sub‐clusters in the domesticated strains. Pairwise F_ST revealed that all domesticated strains were statistically different from the wild population, and also the differentiation between domesticated strains was all statistically significant. Information on genetic diversity and structure of domesticated strains of red sea bream obtained in this study will be useful for future broodstock management and selective breeding programmes.     

Effects of replacing fishmeal with fermented and non‐fermented rapeseed meal on the growth,immune and antioxidant responses of red sea bream (Pagrus major)

The effect of rapeseed meal (RM) and Aspergillus oryzae fermented rapeseed meal (RM‐Koji) on red sea bream (Pagrus major) was examined. Three groups of fish (initial weight, 4.5 ± 0.02 g) were fed a basal diet (RM0) and two test diets where half of fishmeal was replaced by RM (RM50) and RM‐Koji (FRM50) for 56 days. The obtained results showed that fish fed RM0 and FRM50 exerted significantly higher growth performance, feed utilization and haemoglobin level but lower triglyceride and cholesterol than RM50 group (p < 0.05). Interestingly, except of antiprotease activity, all the immune parameters including lysozyme, respiratory burst (NBT) and bactericidal activities were significantly increased in fish fed RM0 and FRM50 diets compared to RM50 diet (p < 0.05). In addition, malondialdehyde and reactive oxygen metabolites were significantly reduced in RM0 and FRM50 groups over RM50 group (p < 0.05). The present results suggest that fermented RM induced better growth performance and immune responses than feeding red sea bream with non‐fermented RM and both RM and RM‐Koji improved the antioxidative status of fish, making RM‐Koji an interesting candidate as a functional feed for aquatic animals.     

A natural infection by the red sea bream iridovirus‐type Megalocytivirus in the golden mandarin fish Siniperca scherzeri

An outbreak of a Megalocytivirus infection was found in the golden mandarin fish Siniperca scherzeri during September and October 2016, in Korea. Phylogeny and genetic diversity based on the major capsid protein (MCP) and adenosine triphosphatase (ATPase) genes showed a new strain. Designated as GMIV, this strain derived from the golden mandarin fish was suggested to belong to the red sea bream iridovirus (RSIV)‐subgroup I. Additionally, this train clustered with the ehime‐1 strain from red sea bream Pagrus major in Japan and was distinguished from circulating isolates (RSIV‐type subgroup II and turbot reddish body iridovirus [TRBIV] type) in Korea. The infection level, evaluated by qPCR, ranged from 8.18 × 10² to 7.95 × 10⁶ copies/mg of tissue individually, suggesting that the infected fish were in the disease‐transmitting stage. The diseased fish showed degenerative changes associated with cytomegaly in the spleen as general sign of Megalocytivirus infection. The results confirm that the RSIV‐type Megalocytivirus might have crossed the environmental and species barriers to cause widespread infection in freshwater fish.     

Comparative genome sequencing to assess the genetic diversity and virulence attributes of 15 Vibrio anguillarum isolates

Vibrio anguillarum is the causative agent of vibriosis, a deadly haemorrhagic septicaemic disease affecting various marine and fresh/brackish water fish, bivalves and crustaceans. However, the diversity and virulence mechanisms of this pathogen are still insufficiently known. In this study, we aimed to increase our understanding of V. anguillarum diversity and virulence through comparative genome analysis of 15 V. anguillarum strains, obtained from different hosts or non‐host niches and geographical regions, among which 10 and 5 strains were found to be virulent and avirulent, respectively, against sea bass larvae. First, the 15 draft genomes were annotated and screened for putative virulence factors, including genes encoding iron uptake systems, transport systems and non‐ribosomal peptide synthetases. Second, comparative genome analysis was performed, focusing on single nucleotide polymorphisms (SNPs) and small insertions and deletions (InDels). Five V. anguillarum strains showed a remarkably high nucleotide identity. However, these strains comprise both virulent and avirulent strains towards sea bass larvae, suggesting that differences in virulence may be caused by subtle nucleotide variations. Clearly, the draft genome sequence of these 15 strains represents a starting point for further genetic research of this economically important fish pathogen.     

Detection and molecular characterization of infectious spleen and kidney necrosis virus from major ornamental fish breeding states in Peninsular Malaysia

‘Gold standard’ OIE reference PCR assay was utilized to detect the presence of infectious spleen and kidney necrosis virus (ISKNV) in freshwater ornamental fish from Malaysia. From total of 210 ornamental fish samples representing 14 species, ISKNV was detected in 36 samples representing 5 fish species. All positive cases did not show any clinical signs of ISKNV. Three restriction enzymes analyses showed that the fish were infected by identical strains of the same virus species within Megalocytivirus genus. Major capsid protein (MCP) genes of 10 ISKNV strains were sequenced and compared with 9 other reference nucleotide sequences acquired from GenBank. Sequence analysis of MCP gene showed that all strains detected in this study were closely related to the reference ISKNV with nucleotide sequence identity that was ranging from 99.8% to 100%. In addition, phylogenetic analysis of MCP gene revealed that viruses from genus Megalocytivirus can be divided into three genotypes: genotype 1 include reference ISKNV and all other strains that were detected in this study, genotype 2 include viruses closely related to red sea bream iridovirus (RSIV), and genotype 3 include viruses closely related turbot reddish body iridovirus (TRBIV).     

Effects of dietary docosahexaenoic acid on growth performance,fatty acid profile and lipogenesis of blunt snout bream (Megalobrama amblycephala)

Six diets were designed to investigate the effects of dietary docosahexaenoic acid (22:6n‐3; DHA) levels (0.5, 1.3, 2.3, 4.2, 8.1 and 15.9 g/kg diets) on growth performance, fatty acid profile and expression of some lipogenesis‐related genes of blunt snout bream (Megalobrama amblycephala). Fish (average weight: 26.40 ± 0.11 g) were randomly fed one of six diets for 8 weeks. Results indicated that the final body weight (FBW) and specific growth rate (SGR) of fish fed 1.3 g/kg DHA were significantly higher than other groups except for the 2.3 g/kg DHA (p < .05). Compared with other groups, the number of lipid droplet clusters of the liver stained with oil red O in the 2.3 g/kg DHA group was the highest, which was consistent with the lipid contents of whole body and liver. The DHA proportion in liver and muscle significantly increased with the increasing dietary DHA levels (p < .05), which reflected fatty acid profiles of diets. The highest mRNA expressions of acetyl‐CoA carboxylase α (ACCα), fatty acid synthase (FAS) and sterol regulatory element‐binding protein‐1 (SREBP‐1) occurred in the 1.3 g/kg DHA group, followed by 2.3 g/kg DHA. In summary, the supplementation of 1.3–2.3 g/kg DHA could improve growth performance and lipogenesis, and the dietary DHA could improve DHA and PUFA proportion in liver and muscle.     

Polymorphisms and haplotypes of the myostatin gene associated with growth in juvenile red sea bream Pagrus major

Polymorphisms of the myostatin gene (MSTN) have been studied in vertebrates including several aquaculture species, revealing their role in growth. We attempted to identify polymorphisms in MSTN associated with growth traits of juvenile farmed red sea bream Pagrus major, an important cultured fish species in Japan. Polymorphisms in the coding region of MSTN were screened, and six polymorphisms were found among three exons: a deletion in exon 1, a single nucleotide polymorphism (SNP) in exon 2 and four SNPs in exon 3. The deletion in exon 1 eliminated a codon (glutamine) from the region comprising the polyglutamine structure, but all SNPs were synonymous. We analysed the polymorphisms for association with growth phenotype in large and small P. major specimens obtained from commercial production at 50 days post hatching. Two SNPs located in exon 3 (c.+846T>C and c.+1140T>C) were significantly more frequent in the large group, while no SNPs were significantly associated with the small phenotype. Haplotypes were reconstructed using genotype information of the two phenotype groups, and nine haplotypes (Hap_1 to Hap_9) were successfully reconstructed. Hap_6 and Hap_7 were significantly more often observed in small and large groups respectively. Analysis of diplotypes (haplotype combinations) of individual specimens revealed 19 diplotypes. The Hap_7/Hap_7 diplotype was found in 46.7% of large phenotype specimens, significantly more frequently than in the small group (13.5%). These results will be useful for marker‐assisted selection of red sea bream to improve production with respect to growth.     

Correlation of changes in seasonal distribution and catch of red sea bream Pagrus major with winter temperature in the eastern Seto Inland Sea,Japan (1972–2010)

Red sea bream Pagrus major is a commercially important fish in Japan. In eastern Seto Inland Sea (SIS), the catch has increased from 297 tons in 1972 to 2,039 tons in 2010. We examined the relationship, 1972–2010, between increase in catch and winter temperature, based on the catch in February and March and the lowest water temperature at 10 m depth. In 1972–1986, the lowest water temperatures in the inner SIS areas (Osaka Bay, Harima‐nada, and Bisan‐seto) were <8°C, which is physiologically unfavorable for red sea bream. However, in 1987–2010 temperatures were generally ≥8°C. In the inner areas, the catch during winter had been minimal until the early 1980s, presumably because most red sea breams moved to the Kuroshio‐influenced (warmer) Kii Channel area. However, the winter catch in the inner areas of SIS increased from the late 1980s with warm winters. In addition, the catch between April and June, the spawning season, increased in the inner areas from the 1990s, and the catch rate of the inner areas was more than twice higher in the 2000s than in the 1980s. The results suggest that expansion of the distribution area during winter due to warm winter and increase in egg production in the inner areas greatly contributes to the increasing in catch in the eastern SIS.     

Microsatellite multiplex panels for population genetic analysis of red sea bream <Emphasis Type="Italic">Pagrus major</Emphasis>

In this study, multiplex PCR panels were developed to amplify 20 microsatellite markers for red sea bream, Pagrus major, with the aim of reducing labor and material costs associated with genetic analysis of this species. The usefulness of these panels was validated in 200 fish collected at five sampling locations. The occurrence of null alleles was suggested at five markers, which were dropped from further analysis. The remaining 15 microsatellite loci showed high levels of heterozygosity (H _E range 0.34–0.97, H _O range 0.32–1) and a wide range in the number of alleles per locus (A; range 5–46). Increasing the number of microsatellite loci from three to ten and 15 improved the performance of the panels for population differentiation (F _ST) and estimation of Nei’s (D _S) genetic distance. The results of this study confirm the usefulness of genotyping a large number of microsatellite DNA markers to expand our knowledge of red sea bream population genetics.     

Effects of dietary taurine levels on epidermal thickness and scale loss in red sea bream,Pagrus major

Taurine is often added to artificial fish diets to compensate for a reduction in fish meal (FM). However, the taurine content of FM‐based diets is typically lower than in diets consisting of raw fish, even in diets where FM is the only protein source. We evaluated the effects of dietary taurine in FM‐based diets on epidermal thickness and scale detachability in red sea bream Pagrus major. We compared the effect of diets containing 0% (control), 0.3% (Tau‐0.3%), 0.6% (Tau‐0.6%) and 1.0% (Tau‐1.0%) taurine. Red sea bream (average body weight, 39 g) were fed these diets for 7 weeks. Taurine supplementation had no effect on growth, feed intake, feeding efficiency, or survival. However, the epidermal thickness was higher in fish in the Tau‐0.6% and Tau‐1.0% groups than in the control and Tau‐0.3% groups. Similarly, scale loss was significantly higher in the control group than in the Tau‐0.6% and Tau‐1.0% groups. Our results suggest that supplementation with >0.6% taurine (1.0% in diet) improves skin condition.     

Spawning season,spawning grounds,and egg production of red sea bream in Hiuchi-nada,Seto Inland Sea

The spawning season and grounds of red sea bream in Hiuchi-nada, the central part of the Seto Inland Sea, were described using a new method based on monoclonal antibodies for identifying Pagrus major eggs, and the daily egg production (standardized by the incubation time and survival rate) was estimated. At the peak of spawning (May), the ranges of sea temperature, salinity, and chlorophyll a where red sea bream eggs occurred were 14.8–17.4°C, 32.0–33.0, 0.5–4.4 μg/L, respectively. The main spawning grounds of the red sea bream were confirmed as being the areas near the Geiyo Islands, Misaki Peninsula, Saijyo, Niihama. The spatial spread of red sea bream eggs increased with the egg developmental stage. The ranges of daily egg production in 2005 and 2006 were 0.3–19.3 and 0.2–6.7 × 10⁹ eggs/day, respectively. In Hiuchi-nada, aquaculture farms are located close to the spawning grounds, and the potential spawning population from the aquaculture farms equaled or exceeded that of the estimated spawning population obtained by the egg production method. Red sea bream eggs in Hiuchi-nada might be produced by both wild and aquaculture-based spawning populations.     

Effect of phytase supplementation of a plant‐based diet on phosphorus and nitrogen bioavailability in sea bream Sparus aurata

To assess to what extent addition of phytase to a plant‐based diet results in spatio‐temporal changes of phytate, available P, soluble protein, total amino acids and the activity of the main digestive proteases in gilthead sea bream, fish were fed two plant‐based diets with or without phytase. Stomach, proximal intestine and distal intestine contents were monitored for these parameters at 0, 1, 2, 4 and 6 h after feeding. A reduction (P < 0.0001) of the soluble P–IP6 in the stomach when phytase was added to the diet was observed. Within stomach, most of the total P–IP6 was precipitated (86%), possibly due to the low acidification capacity of the sea bream (pH > 4), but 57% of the dietary P–IP6 was dephosphorylated, suggesting that phytase could have the capacity to dephosphorylate insoluble IP6 at such pH. An increment (60%) (P < 0.01) in total gastric protease activity was observed by phytase addition, this being the first demonstration of the in vivo effect of IP6 on the pepsin activity in fish stomach. Gastric pH and residence time of the digesta inside the stomach are critical factors for an efficient phytase action and improve P and N bioavailability in plant‐based diets used in fish aquaculture.     

Germplasm differences among three species of genus Erythroculter based on morphological characters and gene sequences of mitochondrial CO I and Cyt b

Fish of genus Erythroculter (Cypriniformes, Cyprinidae, Abramidinae) are important commercial freshwater species in China. In this work, morphological characters and gene sequences of mitochondrial CO I and Cyt b were analysed to investigate the germplasm differences among E. mongolicus, E. dabryi and E. ilishaeformis. Results demonstrated that about countable trait, there were six traits (scale in/ above/ below lateral line, gill‐raker in first left gill‐arch and branched fin‐ray in anal/ pectoral fin) showed significant differences. Gene fragments of CO I and Cyt b was 905 bps and 1,142 bps respectively. In CO I, there were 54.3% base content of A + T, 14 haplotypes and 84 nucleotide variable sites. As to Cyt b, there were 56.3% base content of A + T, 20 haplotypes and 142 nucleotide variable sites. In both CO I and Cyt b, the highest and lowest nucleotide diversity was detected in E. ilishaeformis and E. mongolicus respectively. According to morphological distance, genetic distance, genetic differentiation coefficient (Fst) and gene flow, E. dabryi was grouped together with E. ilishaeformis firstly, and then clustered with E. mongolicus. These results might be useful in the conservation and management of fishery resources of these three species.     

Effects of dietary macroalgae meal and lipid source on growth performance and body wall fatty acid composition of sea cucumber Apostichopus japonicus

A 70‐day experiment was conducted to examine the effects of different macroalgal meals and lipid sources on growth, body wall composition and fatty acid (FA) profile of sea cucumber Apostichopus japonicus. Two macroalgal meals including Sargassum muticum (SM) and Gracilaria lemaneiformis (GL) and two lipid sources including fish oil (FO) and vegetable oil (VO) were formulated into four diets, i.e., S. muticum and fish oil (SF), S. muticum and vegetable oil (SV), G. lemaneiformis and fish oil (GF) and G. lemaneiformis and vegetable oil (GV). The results showed that the specific growth rates (SGR) of A. japonicus fed diets containing SM were significantly higher than those fed diets containing GL. No significant differences in SGR between the FO‐based and VO‐based groups were observed. Similar results were observed in the body wall lipid content. Most body wall FAs changed to resemble the dietary FA proportions because of the dietary effect. Concentrations of 20:4n‐6 of the SF and GF groups were significantly lower than the SV and GV groups, while levels of 20:5n‐3 and 22:6n‐3 were significantly higher than the SV and GV groups. The n‐3/n‐6 polyunsaturated fatty acids (PUFA) ratios of the SF and GF groups were significantly higher than the SV and GV groups. Moreover, the SF group had significantly higher 20:5n‐3 and 22:6n‐3 contents and n‐3/n‐6 PUFAs ratio than the GF group. These findings reveal that the SF diet can show beneficial effects on both growth performance and body wall n‐3 PUFAs content of A. japonicus.     

Bromodeoxyuridine DNA labelling reveals host and parasite proliferation in a fish‐myxozoan model

Enteromyxum leei is a myxozoan parasite responsible for enteritis in gilthead sea bream (Sparus aurata). The parasite proliferates in the paracellular space of the intestinal epithelium and induces an inflammatory reaction. To assess intestinal cell turnover and parasite proliferation, fish were infected with the parasite by anal intubation; after 17 and 64 days, the cell proliferative marker bromodeoxyuridine (BrdU) was administered; and after 24 hr, tissue samples were taken for immunohistochemical detection. Parasite exposure induced increased epithelial and immune cell proliferation in all intestinal segments at all time points, even before parasite establishment. This increased turnover was triggered early after intubation and mainly at a local level, as shown by an increased proliferating cell nuclear antigen (pcna) gene expression only at the posterior intestine after 17 days (not found in lymphohaematopoietic organs). Incorporation of BrdU in parasite secondary and tertiary daughter cells indicated that parasite endogeny is not by schizogonial division, which uses de novo synthesis pathway of pyrimidines. Altogether, BrdU immunolabelling and pcna gene expression showed the rapid proliferative response of the fish intestines upon a myxozoan infection and how this response is effectively triggered even before the parasite reaches or establishes in the site.     

Cloning and expression analysis of innate immune genes from red sea bream to assess different susceptibility to megalocytivirus infection

As suggested by the Office International des Epizooties (OIE), fishes belonging to the genus Oplegnathus are more sensitive to megalocytivirus infection than other fish species including red sea bream (Pagrus major). To assess the roles of the innate immune response to these different susceptibilities, we cloned the genes encoding inflammatory factors including IL‐8 and COX‐2, and the antiviral factor like Mx from red sea bream for the first time and performed phylogenetic and structural analysis. Analysed expression levels of IL‐1β, IL‐8 and COX‐2 and the antiviral factor like Mx genes performed with in vivo challenge experiment showed no difference in inflammatory gene expression or respiratory burst activity between red sea bream and rock bream (Oplegnathus fasciatus). However, the Mx gene expression levels in red sea bream were markedly higher than those in rock bream, suggesting the importance of type I interferon (IFN)‐induced proteins, particularly Mx, during megalocytivirus infection, rather than inflammation‐related genes. The in vitro challenge experiments using embryonic primary cultures derived from both fish species showed no difference in cytopathic effects (CPE), viral replication profiles, and inflammatory and Mx gene expression pattern between the two fish species.     

The use of preserved copepods in sea bream small‐scale culture: biometric,biochemical and molecular implications

Considering the well‐known problems arising from the use of rotifers and Artemia as live prey in larval rearing in terms of fatty acid deficiencies, the aim of this study was to evaluate a partial or complete replacement of traditional live prey with preserved copepods during the larviculture of gilthead sea bream (Sparus aurata). Sea bream larvae were randomly divided into 4 experimental groups in triplicates: group A larvae (control) fed rotifers followed by Artemia nauplii; group B fed a combined diet (50%) of rotifers–Artemia and preserved copepods; group C fed rotifers followed by preserved copepods; and group D fed preserved copepods solely. Survival and biometric data were analysed together with major molecular biomarkers involved in growth, lipid metabolism and appetite. Moreover, fatty acid content of prey and larvae was also analysed. At the end of 40 days treatment, a stress test, on the remaining larvae, was performed to evaluate the effects of different diets on stress response. Data obtained evidenced a positive effect of cofeeding preserved copepods during sea bream larviculture. Higher survival and growth were achieved in group B (fed combined diet) larvae respect to control. In addition, preserved copepods cofeeding was able to positively modulate genes involved in fish growth, lipid metabolism, stress response and appetite regulation.     

Effects of commonly used disinfectants on bacterial load,hatchability and survival of Bluefin Sea bream (Sparidentex hasta) eggs

The efficacy of four chemical reagents, iodophor, formalin, hydrogen peroxide and bronopol as fish egg surface disinfectants were evaluated in bluefin sea bream (Sparidentex hasta). Fertilized eggs were counted and subjected to a static bath dip treatment in different concentrations of the above chemicals for 4 min before being incubated at 20 ± 0.5°C for 40 h. Treatment efficacy of the different disinfectants was evaluated by assessing the bactericidal activity, egg hatch percentage and survival of larvae up to 3 days post hatch. Results showed that iodophor at medium concentrations (75 and 100 ppm) was the best of all tested disinfectants in bacterial killing ability (12% reduction in the bacterial counts), egg hatching per cent (99.8% and 99.6% respectively) and larval survival up to 3 days post hatch (50.8% and 54.8% respectively). Formalin was the second best disinfectant at levels of 100 and 150 ppm. Hydrogen peroxide gave good results compared with the control while, bronopol showed discouraging results. In conclusion, iodophor appeared to be suitable for bluefin sea bream eggs disinfection with a 4 min exposure to 75–100 ppm when applied 14–16 h after egg fertilization.     

Incidence and prevalence of Diplectanum aequans and its influence on the fitness of juvenile sea bass (Dicentrarchus labrax) in the Black Sea

High rates of unexplained mortalities (up to 70%) are anticipated in the cultured juvenile sea bass (Dicentrarchus labrax) in farms in the eastern Black Sea at least for the last 8 years. Diplectanum aequans, often blamed for the observed mortalities, is the only prevalent parasite impacting the health of sea bass in the brackish water of the Black Sea. To investigate the seasonal fluctuation of D. aequans prevalence, mean intensities and the potential effects of D. aequans on the fitness of sea bass, eight cages from one farm (Farm A) and four cages at another distinct site (Farm B) were surveyed monthly from May to October of 2008. Six hundred and sixty‐one juvenile sea bass from Farm A and 236 from Farm B were individually examined for the presence and intensity of the parasite. The prevalence of D. aequans in cultured sea bass (96.3 ± 11.11; Mean ± SD) ranged from 66.7% to 100% in June, the beginning of 6‐month growing season. The mean intensity was 6.08 ± 2.19, significantly higher than that (2.74 ± 0.87; P < 0.05) in December (P < 0.05), the end of the growing season. Fish with low fitness had significantly higher number of parasite than the fish with higher fitness (P < 0.005). Although D. aequans negatively influenced host's condition factor, the impact was not enough to lead mortalities in the cultured sea bass in the Black Sea.     

Rapid detection of SNPs in candidate genes regulating the growth of orange‐spotted grouper,Epinephelus coioides (Hamilton, 1822), using semiconductor sequencing

Orange‐spotted grouper (Epinephelus coioides) is one of the most important marine food species in Southeast Asia and China and has been cultured for decades. In this study, we fully utilized the limited capacity of semiconductor sequencing, the high efficiency of long‐range PCR for target enrichment and a non‐indexed pooling strategy to screen single‐nucleotide polymorphisms (SNPs) in a breeding population of orange‐spotted grouper. Forty‐one genomic DNA fragments, with a total length of approximately 180 kb, including 22 candidate genes that control growth, and from a DNA pool of 20 heaviest and 22 lightest individuals of the sampled population were successfully sequenced using an Ion Torrent Personal Genome Machine. 3 503 466 clear reads were produced with a length of 192 ± 56 bp, 86.8% of which were mapped to the reference with an average coverage depth of 2567‐fold and physical coverage of 98.8%. Finally, 1623 high‐quality SNPs were adopted. Compared with Sanger sequencing of three random common regions, the sensitivity and specificity of our approach were 39.4% and 100.0% respectively. A mutation located at the third position of the previously labelled start codon of growth hormone receptor type 1 invalidated the start codon. Furthermore, comparison of the frequencies of genotypes and alleles of this site between the two extreme groups, prediction of signal peptide and identification of conservative mRNA sequences suggested that the functional start codon is likely located at the position of another downstream in‐frame ATG in the mutant. These detected SNP markers will provide important tools for the selective breeding of orange‐spotted grouper.