Japanese Flounder Paralichthys Research Articles

The miR-200 Family Targeting amh Affects the Gonadal Development of Japanese Flounder

Four members of the miR-200 family in Japanese flounder (Paralichthys olivaceus) have sex-biased expression patterns, but their target genes and how they work in the development of the gonads are rarely known. Anti-Müllerian hormone (AMH) can inhibit the development of Muller’s duct in female mammals and regulate the formation of gametes after sexual maturity. There is no Muller’s duct in teleosts, but the amh gene still exists. Knockout of amh results in sex reversal from male to female. Therefore, it is essential to explore the relationship between the miR-200 family and amh to clarify what role miR-200 plays in the development of the gonads. In Japanese flounder, the two binding sites for the miR-200 family in the 3′UTR of amh were found through bioinformatic prediction. Double luciferase and green fluorescent protein reporter experiments demonstrated amh to be directly targeted by miR-200a and miR-200b. Moreover, miR-200a and miR-200b reduced the expression of amh through site 1 rather than site 2. To explore the regulatory role of miR-200a in gonadal development, we further overexpressed miR-200a in the primary Sertoli cells of the testis. With the overexpression of miR-200a, the expression of amh decreased, while the expression of the other two male sex-related genes, dmrt1 (doublesex and mab-3 related transcription factor 1) and gsdf (diagonal soma driven factor), increased significantly. This result indicates that the miR-200 family regulates the gonadal differentiation and development by targeting amh in Japanese flounder.

Brown seaweed ( Sargassum fulvellum ) inclusion in diets with fishmeal partially replaced with soy protein concentrate for Japanese flounder ( Paralichthys olivaceus ) juveniles

Brown seaweed ( <i>Sargassum fulvellum</i> ) inclusion in diets with fishmeal partially replaced with soy protein concentrate for Japanese flounder ( <i>Paralichthys olivaceus</i> ) juveniles

Two CD9 tetraspanin family members of Japanese flounder (Paralichthys olivaceus): characterization and comparative analysis of the anti-infectious immune function

CD9 is a glycoprotein of the transmembrane 4 superfamily that is involved in various cellular processes. Studies related to the immune functions and activities of CD9 in teleost fish are limited. In this study, we characterized two CD9 homologs, PoCD9.1 and PoCD9.3, from Japanese flounder (Paralichthys olivaceus). Sequence analysis showed that PoCD9.1 and PoCD9.3 possess characteristic transmembrane 4 superfamily (TM4SF) structures. PoCD9.1 shares 70.61% sequence identity with PoCD9.3. The expression of PoCD9.1 and PoCD9.3 in the three main immune tissues was significantly induced in a time-dependent manner by extracellular and intracellular pathogen infection, which indicates that the two CD9 homologs play an important role in the response to pathogenic infection. Following infection with the extracellular pathogen Vibrio anguillarum, the expression profiles of both PoCD9.1 and PoCD9.3 were similar. After infection with the intracellular pathogen Edwardsiella piscicida, the expression levels of PoCD9.1 and PoCD9.3 were different at different stages of infection, especially in the spleen. The spleen was the most important tissue for the PoCD9.1 and PoCD9.3 responses to pathogen infection among the three examined immune tissues. Knockdown of PoCD9.1 and PoCD9.3 attenuated the ability of host cells to eliminate pathogenic bacteria, and PoCD9.1 knockdown was more lethal than PoCD9.3 knockdown for host cells with E. piscicida infection. Overexpression of PoCD9.1 and PoCD9.3 promoted host or host cell defence against E. piscicida infection. These findings suggest that PoCD9.1 and PoCD9.3 serve as immune-related factors, play an important role in the immune defence system of Japanese flounder, and display different functions in response to different pathogens at different stages of infection.

Identification of candidate piRNAs in the gonads of Paralichthys olivaceus (Japanese flounder).

Piwi-interacting RNA (piRNA) plays an important role in the gonadal development and maintenance of Teleostei. In this study, piRNA libraries derived from the adult gonads of Japanese flounder (Paralichthys olivaceus) were generated using next-generation sequencing technology. Using zebrafish piRNAs as a reference, 5 865 unique candidate piRNAs were identified; 289 candidate piRNA clusters (PRCs) were generated from the above piRNAs. Among the isolated candidate PRCs, a total of 38 ovary-specific, 45 ovary-bias, 24 testis-specific, and 131 testis-bias PRCs were found. The relative expression levels of seven PRCs were validated through quantitative reverse transcription-polymerase chain reaction. The results of this study will help facilitate exploration of the development and maintenance of the phenotypic sex mechanism in P. olivaceus.

Quantitative trait loci detection of Edwardsiella tarda resistance in Japanese flounder Paralichthys olivaceus using bulked segregant analysis

In recent years, Edwardsiella tarda has become one of the most deadly pathogens of Japanese flounder (Paralichthys olivaceus), causing serious annual losses in commercial production. In contrast to the rapid advances in the aquaculture of P. olivaceus, the study of E. tarda resistance-related markers has lagged behind, hindering the development of a disease-resistant strain. Thus, a marker-trait association analysis was initiated, combining bulked segregant analysis (BSA) and quantitative trait loci (QTL) mapping. Based on 180 microsatellite loci across all chromosomes, 106 individuals from the F1333 (♀: F0768 ×♂: F0915) (Nomenclature rule: F+year+family number) were used to detect simple sequence repeats (SSRs) and QTLs associated with E. tarda resistance. After a genomic scan, three markers (Scaffold 404-21589, Scaffold 404-21594 and Scaffold 270-13812) from the same linkage group (LG)-1 exhibited a significant difference between DNA, pooled/bulked from the resistant and susceptible groups (P <0.001). Therefore, 106 individuals were genotyped using all the SSR markers in LG1 by single marker analysis. Two different analytical models were then employed to detect SSR markers with different levels of significance in LG1, where 17 and 18 SSR markers were identified, respectively. Each model found three resistance-related QTLs by composite interval mapping (CIM). These six QTLs, designated qE1–6, explained 16.0%–89.5% of the phenotypic variance. Two of the QTLs, qE-2 and qE-4, were located at the 66.7 cM region, which was considered a major candidate region for E. tarda resistance. This study will provide valuable data for further investigations of E. tarda resistance genes and facilitate the selective breeding of disease-resistant Japanese flounder in the future.

Locus Number Estimation of MHC Class II B in Stone Flounder and Japanese Flounder

Members of major histocompatibility complex (MHC) family are important in immune systems. Great efforts have been made to reveal their complicated gene structures. But many existing studies focus on partial sequences of MHC genes. In this study, by gene cloning and sequencing, we identified cDNA sequences and DNA sequences of the MHC class II B in two flatfishes, stone flounder (Kareius bicoloratus) and homozygous diploid Japanese flounder (Paralichthys olivaceus). Eleven cDNA sequences were acquired from eight stone flounder individuals, and most of the polymorphic sites distributed in exons 2 and 3. Twenty-eight alleles were identified from the DNA fragments in these eight individuals. It could be deduced from their Bayesian inference phylogenetic tree that at least four loci of MHC class II B exist in stone flounder. The detailed whole-length DNA sequences in one individual were analyzed, revealing that the intron length varied among different loci. Four different cDNA sequences were identified from one homozygous diploid Japanese flounder individual, implying the existence of at least four loci. Comparison of the cDNA sequences to the DNA sequence confirmed that six exons existed in this gene of Japanese flounder, which was a common feature shared by Pleuronectiformes fishes. Our results proved the multi-locus feature of MHC class II B. The sequences we obtained would provide detailed and systematic data for further research.

Comparative analysis of disease resistance among three successive generations of Japanese flounder (&lt;I&gt;Paralichthys olivaceus&lt;/I&gt;) fam-ilies

Japanese flounder(Paralichthys olivaceus) is a widely cultured marine fish species in China and is highly valued because of its fast growth and good flavor. However, frequent occurrence of disease and subsequent losses limit profitability and aquaculture development. Breeding a new flounder strain with enhanced disease resistance is an important task in marine fish aquaculture. In the present study, new Japanese flounder families were established based on Japanese stock(JS), Korean stock(KS), and stock with high resistance to Vibrio anguillarum(RS) by nested hybrid, cross, and gynogenesis. Forty-three and sixty-five Japanese flounder families were established in 2009 and 2012, respectively. Disease resistance was investigated using 33 families in 2009 and 43 in2012 through intraperitoneal injection challenged with V. anguillarum. The results demonstrate that different families exhibited variable survival rates and 13 families with high disease resistance were obtained. All 13 families exhibited significantly higher survival rates compared with the control, however, some were more significant than others(P0.01 in 3 families; P0.05 in 10 families). Of these 13 families, three were derived from the F2family, one from the F3 family, and two belonged to the first and second gynogenesis generations(G1 and G2). In these six families, disease resistance was high in the parents, with the exception of the F2 family; their survival rate variation coefficients were 10%. Analysis of disease resistance in the three successive generations revealed that, 13 of the 16 disease-resistance families which included 13 families mentioned above and 3 resistant families sereened in 2007 were derived from RS. In conclusion, some F2, F3, G1, and G2 families exhibited enhanced disease resistance inherited from their parents, indicating that the establishment of a disease-resistant strain based on these families is possible.

Complete Genome Sequence of Vibrio anguillarum M3, a Serotype O1 Strain Isolated from Japanese Flounder in China

Vibrio anguillarum is an important bacterial pathogen that causes vibriosis in marine fish. We present the complete genome sequence of V. anguillarum M3, a serotype O1 clinical strain isolated from Japanese flounder (Paralichthys olivaceus) in Shandong, China.

Data Mining for Simple Sequence Repeats in Expressed Sequence Tags from Japanese Flounder (Paralichthys olivaceus)

Based on ESTs of Japanese flounder (Paralichthys olivaceus) in the public database, EST-SSR makers were developed after mining and evaluating SSRs in them by bioinformatics methods. 5 927 non-redundant ESTs of Japanese flounder were screened and 390 SSRs were mined out. The frequency of these EST-SSRs was 7.95% and the average distance of distribution was 7.9 kb in non-redundant ESTs. The dinucleotide repest motif was dominant type (59.02%) with repeat motif AC being the most common (16.91%). The distribution of trinucleotide, tetranucleotide and hexanucleotide repeats were dispersive. 30 primer pairs for EST-SSRs were designed, 27 primer pairs showed the amplification, and 17 primer pairs showed polymorphisms, the rates of polymorphic EST-SSRs were 62.96% with the alleles per locus ranging from 2 to 6 (mean 3.5). The observed (HO) and expected (HE) heterozygosities of these EST-SSRs were 0.280.92 and 0.31550.8033, respectively. Two EST-SSR loci significantly deviated from the HardyWeinberg equilibrium (HWE) expectation, and theremaining 15 loci were in HWE. These new EST-SSR markers would provide sufficient polymorphism for population genetic studies and genome mapping of Japanese flounder.

Construction of genetic linkage map in Japannese flounder (Paralichthys olivaceus)

Using the microsatellite sequence got from genome sequencing,we constructed genetic linkage map with SSR markers for Japanese flounder(Paralichthys olivaceus),in which 681383B is the male parent and 6812E36 is the female parent.The male linkage map included 418 markers,which were distributed in 24 linkage groups,with 88.7% coverage.The length reached 1 418.1 cM,and the average marker interval was 3.62 cM.The female linkage map distributed in 24 linkage groups included 437 markers,with 89.1% coverage.spanning.The length reached 1 298.1 cM,and the average marker interval was 3.16 cM,.The medium density genetic linkage maps laid the groundwork for QTL analyses and marker-assisted selection breeding programs,and effectively promote the genetic improvement of Paralichthys,make flounder aquaculture sustainable development.

Analysis of homozygosity and genetic similarity between two succes-sive generations in a meiogynogenetic Japanese flounder family

The artificial induction of gynogenesis is a form of chromosome manipulation that has several applica-tions,including the rapid establishment of inbred lines or strains.The technique offers a high degree of homozy-gosity,sex-control,and accelerated elimination of recessive deleterious genes from a cultured population.We produced two meiotic gynogenetic Japanese flounder(Paralichthys olivaceus) family lines: meio-G1(the first generation) and meio-G2(the second generation).We evaluated the efficacy of artificial meiotic gynogenesis for producing pure inbred lines by quantifying the homozygosity and genetic similarity of meio-G1 and meio-G2 us-ing microsatellite markers The average homozygosity among 15 loci was 0.124 7 for meio-G1 and 0.221 5 for meio-G2,which was higher than that in the natural mating family(0.091 7).The average similarity indices among individuals within meio-G1 and meio-G2 were 0.891 7 and 0.923 8,which were higher than within the natural mating family(0.5603).The average similarity in meio-G2 was slightly higher than in meio-G1.The observed heterozygosity in meio-G1 and meio-G2 was 1 at the Poli9-8tuf,Poli18tuf,Poli107tuf loci,all of which were lo-cated at some distance from the centromere(i.e.,had high recombination frequencies).The loci(Poli33tuf,Po-li24MHFS) closest to the centromere(i.e.,with a low recombination frequency) were homozygous in meio-G1 and meio-G2.Our results suggest that artificially induced meiotic gynogenesis is an efficient method for inbreeding to purify the genome,increase genetic similarity,and fix maternal genetic traits in the Japanese flounder.The mei-ogynogenetic families cultured in our study may be used in further selective breeding research.

Analysis of genetic diversity in Bohai natural population ofParalichthys olivaceus

To analyze the genetic diversity of natural population and select the specific microsatellite markers for identifying effectively genetic characteristics of population in Japanese flounder( Paralichthys olivaceus)in this study,seventy-four w ild individuals captured from the Bohai Sea w ere used to form the experimental population. A set of 72 microsatellite markers located on different regions in linkage group w ere chosen to carry out the genetic analysis. In these markers,17 w ere located in the centromeric region,36 in the distant region from the centromere and 19 in the intermediate region of linkage groups. Analysis show ed that the number of alleles( A) ranged from 6. 400 to 7. 389,number of effective alleles( N e) ranged from 4. 469 to5. 129 and Shannon's information index( I) ranged from 1. 565 to 1. 683 in different regions of linkage group. The observed heterozygosity( H o),unbiased expected heterozygosity( H e) and polymorphism information content( PIC) w as from 0. 568 to 0. 593,from 0. 738 to 0. 753 and from 0. 707 to 0. 746,respectively. The Hardy-Weinberg departure value( d) w as low er than- 0. 200,w hile,genetic distance( G D)among individuals in population w as above than 0. 620. Each genetic parameter demonstrated that there w as richer genetic diversity in experimental population and relatively farther genetic distance among individuals.This population w as suitable for conducting selective breeding as founder population. Significant difference did not exist in the genetic parameters obtained by microsatellite markers located on different regions in linkage group,w hich verified that there w as no necessary correlation betw een the positions of marker located and the degree of genetic diversity of markers. How ever,these polymorphic markers could be used as marker candidates to analyze the genetic structure in population of Japanese flounder.

Effects of dietary soy isoflavones on feed intake, growth performance and digestibility in juvenile Japanese flounder (Paralichthys olivaceus)

An 8-week feeding trial was conducted to investigate the effects of dietary soy isoflavones on feeding intake, growth performance, and digestion of juvenile Japanese flounder (Paralichthys olivaceus). Four isonitrogenous (49% crude protein) and isoenergetic (20.1 MJ kg−1) diets were formulated to contain four graded levels of soy isoflavones, namely, 0, 1, 4 and 8 g soy isoflavones in 1 kg of diet. Each diet was randomly fed to triplicate tanks of fish (Initial average weight: 2.58 g ± 0.01 g), and each tank was stocked with 35 fish. No significant difference was observed among diets with levels of 0, 1 and 4 g kg−1 soy isoflavones in feed intake, weight gain, feed efficiency ratio (FER), proximate composition of fish whole body and apparent digestibility coefficients (ADC) of nutrients and energy (P>0.05). However, high dietary soy isoflavones level (8 g kg−1) significantly depressed weight gain, FER, whole-body crude lipid content of fish and ADC of nutrients (P<0.05). These results indicate that high level of dietary soy isoflavones (above 4 g kg−1) significantly depresses growth responses and FER of Japanese flounder. However, as the content of soy isoflavones in soybean meal is around 1 to 3 g kg−1, the adverse effects might be neglected when soybean products are used as a fish feed ingredient.

Cloning, characterization and expression of estrogen receptor beta in the male half-smooth tongue sole, Cynoglossus semilaevis

A full-length sequence encoding the estrogen receptor beta was isolated from half-smooth tongue sole, Cynoglossus semilaevis (hstsERβ) using reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends procedures. The hstsERβ cDNA clone was found to contain 1,791 nucleotides including an open reading frame that encodes 578 amino acids. The deduced hstsERβ protein consisted of six nuclear receptor-characteristic domains. Based on a phylogenetic analysis, the hstsERβ C and E domains are highly conserved compared to other fishes. The potential phosphorylation sites for PKC, CK-2 and PTK are also found in this protein. Highest amino acid identities were found for hstsERβ with common carp (Cyprinus carpio) ERβ (76%) and Japanese flounder (Paralichthys olivaceus) ERβ (76%). Tissue expression analysis confirmed that the hstsERβ was widely distributed and predominantly expressed in testis, brain and liver. Seasonal changes in the testis, brain and liver expression profiles of hstsERβ were examined by RT-PCR; the present results suggest that level of hstsERβ in brain increased to the highest then decreases with gonadal growth; whereas in the testis and liver, the hstsERβ mRNA level dropped to lowest then slightly increased.

Identification and expression analysis of IGFBP-1 gene from Japanese flounder (Paralichthys olivaceus)

Insulin-like growth factor binding protein-1 (IGFBP-1) plays an important role in IGF regulating vertebrate growth and development. In this study, we cloned IGFBP-1 cDNA from Japanese flounder (Paralichthys olivaceus) liver. The full-length cDNA is 1070 bp, including a 5'-untranslated region (UTR) of 69 bp, a 3'-UTR of 272 bp, and an open reading frame (ORF) of 729 bp encoding a polypeptide of 242 amino acids. Real-time quantitative PCR revealed that IGFBP-1 mRNA is mainly expressed in the liver, and a small amount of mRNAs was also found in other adult tissues. There are maternal transcripts of IGFBP-1 gene, and relatively low mRNA levels were observed in different embryonic stages. A higher level of IGFBP-1 mRNA was detected at 3 days post hatching (dph), and it got to the highest level at 29 dph (metamorphic climax), and finally brought back to a lower level at the end of metamorphosis. The expression of IGFBP-1 mRNA was greatly up-regulated in thyroid hormone (TH)-treated larvae, and declined after thiourea (TU) treatment. These results provide basic information for further studies on the role of IGF system in the P. olivaceus development and metamorphosis.

Mutations in exons of the CYP17-II gene affect sex steroid concentration in male Japanese flounder (Paralichthys olivaceus)

As a specific gene of fish, cytochrome P450c17-II (CYP17-II) gene plays a key role in the growth, development an reproduction level of fish. In this study, the single-stranded conformational polymorphism (SSCP) technique was used to characterize polymorphisms within the coding region of CYP17-II gene in a population of 75 male Japanese flounder (Paralichthys olivaceus). Three single nucleotide polymorphisms (SNPs) were identified in CYP17-II gene of Japanese flounder. They were c.G594A (p.G188R), c.G939A and c.G1502A (p.G490D). SNP1 (c.G594A), located in exon 4 of CYP17-II gene, was significantly associated with gonadosomatic index (GSI). Individuals with genotype GG of SNP1 had significantly lower GSI (P < 0.05) than those with genotype AA or AG. SNP2 (c.G939A) located at the CpG island of CYP17-II gene. The mutation changed the methylation of exon 6. Individuals with genotype AA of SNP2 had significantly lower serum testosterone (T) level and hepatosomatic index (HSI) compared to those with genotype GG. The results suggested that SNP2 could influence the reproductive endocrine of male Japanese flounder. However, the SNP3 (c.G1502A) located in exon 9 did not affect the four measured reproductive traits. This study showed that CYP17-II gene could be a potentially useful candidate gene for the research of genetic breeding and physiological aspects of Japanese flounder.

Proteomic aspects of infection by lymphocystis disease virus in Japanese flounder (Paralichthys olivaceus)

To reveal the key factor in self-healing from LCDV (lymphocystis disease virus)-infected Japanese flounder (Paralichthys olivaceus), serum proteins from self-healing and sick Japanese flounder were separated by two-dimensional electrophoresis to screen differentially expressed proteins. Protein spots demonstrating changes greater than two-fold in the expression level were digested and further identified in capillary liquid chromatography tandem mass spectrometry (LC-MS/MS). Two immunity-relevant proteins were thus identified as transferrin and the complement component C3 of Japanese flounder. These findings suggest that the two proteins may play important roles in the self-healing of lymphocystis in Japanese flounder. This is an important theoretical foundation to promote self-healing in LCDV-infected Japanese flounder by improving their innate immunity.

Development of polymorphic microsatellite markers in barfin flounder (Verasper moseri) and spotted halibut (Verasper variegatus) by the cross-species amplification

Barfin flounder (Verasper moseri) and spotted halibut (Verasper variegatus) are two commercially important flatfish species in the Northeast Asia. In the present study, we reported polymorphic microsatellite markers in V. moseri and V. variegatus by the cross-species amplification of microsatellite primers developed previously in two other related marine fish species. A total of 244 polymorphic microsatellite markers were selected for cross-species amplification in V. moseri and V. variegatus, of which 182 markers deriving from Atlantic halibut (Hippoglossus hippoglossus) and 62 markers deriving from Japanese flounder (Paralichthys olivaceus). A sample of 10 individuals were detected. As a result, a total of 67 loci showed polymorphisms in V. moseri, and 62 loci showed polymorphisms in V. variegatus, with the observed number of alleles per locus ranging from two to five in V. moseri, and from two to seven in V. variegatus, respectively. This paper provided more candidate microsatellite markers which could be useful for construction of genetic linkage maps, evaluation of population genetic structure and stock management of V. moseri and V. variegatus.

Cloning of &lt;I&gt;CYP19a&lt;/I&gt; gene and expression analysis during the reproductive cycle in males of &lt;I&gt;Verasper moseri&lt;/I&gt;

Estrogens are key players in sexual differentiation and development and their production is essential for normal sexual development in both females and males. Cytochrome P450 aromatase is the terminal enzyme in steroidogenic pathway that converts androgens into estrogens. In most of vertebrates,P450arom is the product of the CYP19 gene,which exists as a single copy per haploid genome. In contrast,there are two isoforms of aromatase in teleosts,which are encoded by two different genes,CYP19a and CYP19b,as well as have different distribution. This work describes the molecular cloning of the cDNA encoding the barfin flounder(Verasper moseri) gonad cytochrome P450 aromatase(CYP19a) by means of reverse transcriptase polymerase chain reaction(RT-PCR),degenerate primers PCR amplification and 5'-and 3'-rapid amplification of cDNA ends(RACE) analyses. We cloned the complete cDNA sequence of CYP19a gene and the length is 2 167 bp,encoding a protein of 523 amino acids. Via Blastn,the CYP19a cDNA displayed high identity with Atlantic halibut(Hippoglossus hippoglossus),Japanese flounder(Paralichthys olivaceus) and southern flounder(Paralichthys lethostigma). Alignment analysis using barfin flounder CYP19a gene deduced animo acid sequence and animo acid sequences of other species showed that there were transmembrane helix region,I-helix region,Ozol's peptide region,aromatase-specific conserved region and heme-binding region in the deduced animo acid sequence. Phylogenetic analysis suggested the occurrence of a gene duplication for cytochrome P450 aromatase in the teleost lineage after its divergence from the tetrapods. Furthermore,the higher identity between barfin flounder P450aromA and P450aromAs of other teleosts suggested that they have the same origin. A semiquantitative PCR was developed to measure mRNA expression levels of CYP19a gene of barfin flounder. Expression analysis in different tissues demonstrated that P450aromA transcripts were highly abundant in brain,ovary and testis,less abundant in intestine,liver and kidney,showing that not only gonads can produce steroids but also other tissues have steroidogenic capability. And we analyzed the relative mRNA expression level of CYP19a in testes at different stages of reproductive cycle,there were the highest level at stageⅡ and the lowest level at stageⅤ. These changes in expression profiles may imply differences in the functionality of the enzyme between different stages,suggesting an important role for P450aromA in barfin flounder testes development.

Single nucleotide polymorphism of FSHβ gene associated with reproductive traits in Japanese flounder (Paralichthys olivaceus)

Follicle stimulating hormone β (FSHβ) of Japanese flounder (Paralichthys olivaceus) plays a key role in the regulation of gonadal development. This study aimed to investigate molecular genetic characteristics of the FSHβ gene and elucidate the effects of single nucleotide polymorphisms (SNPs) of FSHβ on reproductive traits in Japanese flounder. We used polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) and sequencing of the FSHβ gene in 60 individuals. We identified only an SNP (T/C) in the coding region of exon3 of FSHβ. The SNP (T/C) did not lead to amino acid changes at the position 340 bp of FSHβ gene. Statistical analysis showed that the SNP was significantly associated with testosterone (T) level and gonadosomatic index (GSI) (P < 0.05). Individuals with genotype TC of the SNP had significantly higher serum T levels and GSI (P < 0.05) than that of genotype CC. Therefore, FSHβ gene could be a useful molecular marker in selection for prominent reproductive trait in Japanese Flounder.