Dynamic mRNA and miRNA expression of the head during early development in bighead carp (Hypophthalmichthys nobilis).

BACKGROUND: Head of fish species, an exquisitely complex anatomical system, is important not only for studying fish evolution and development, but also for economic values. Currently, although some studies have been made on fish growth and body shapes, very limited information is available on the molecular mechanism of head development. RESULTS: In this study, RNA sequencing (RNA-Seq) and small RNA sequencing (sRNA-Seq) technologies were used to conduct integrated analysis for the head of bighead carp at different development stages, including 1, 3, 5, 15 and 30 Dph (days post hatch). By RNA-Seq data, 26 pathways related to growth and bone formation were identified as the main physiological processes during early development. Coupling this to sRNA-Seq data, we picked out six key pathways that may be responsible for head development, namely ECM receptor interaction, TNF signaling pathway, osteoclast differentiation, PI3K-Akt signaling pathway, Neuroactive ligand-receptor interaction and Jak-STAT signaling pathway. Totally, 114 important candidate genes from the six pathways were obtained. Then we found the top 20 key genes according to the degree value by cytohubba, which regulated cell growth, skeletal formation and blood homeostasis, such as pik3ca, pik3r1, egfr, vegfa, igf1 and itga2b. Finally, we also acquired 19 key miRNAs playing multiple roles in the perfection of various tissues in the head (such as brain, eye and mouth) and mineralization of head bone system, such as let-7e, miR-142a-5p, miR-144-3p, miR-23a-3p and miR-223. CONCLUSIONS: Results of this study will be informative for genetic mechanisms of head development and also provide potential candidate targets for the interaction regulation during early growth in bighead carp.

Comparative transcriptomic analysis of hypothalamus-pituitary-liver axis in bighead carp (Hypophthalmichthys nobilis) with differential growth rate.

BACKGROUND: Growth rate is one of the most important features for aquaculture species and deciphering its regulation mechanism has great significance both in genetics and in economics. Hypothalamus-pituitary growth axis (HP growth axis) or neuro-endocrine axis plays a vital role in growth regulation in different aquaculture animals. RESULTS: In this study, the HP and liver transcriptomes of two female groups (H and L) with phenotypically extreme growth rate were sequenced using RNA-Seq. A total of 30,524 and 22,341 genes were found expressed in the two tissues, respectively. The average expression levels for the two tissues were almost the same, but the median differed significantly. A differential expression analysis between H and L groups identified 173 and 204 differentially expressed genes (DEGs) in HP and liver tissue, respectively. Pathway analysis revealed that DEGs in HP tissue were enriched in regulation of cell proliferation and angiogenesis while in liver tissue these genes were overrepresented in sterol biosynthesis and transportation. Genomic overlapping analyses found that 4 and 5 DEGs were within growth-related QTL in HP and liver tissue respectively. A deeper analysis of these 9 genes revealed 3 genes were functionally linked to the trait of interest. The expression of 2075 lncRNAs in HP tissue and 1490 in liver tissue were also detected, and some of lncRNAs were highly expressed in the two tissues. CONCLUSIONS: Above all, the results of the present study greatly contributed to the knowledge of the regulation of growth and then assisted the design of new selection strategies for bighead carp with improved growth-related traits.

Molecular characterization and expression regulation of the factor-inhibiting HIF-1 (FIH-1) gene under hypoxic stress in bighead carp (Aristichthys nobilis).

Factor-inhibiting HIF-1 (FIH-1) is an asparagine hydroxylase that interacts with hypoxia-inducible factor 1α (HIF-1α) to regulate transcriptional activity of HIF-1. Few studies of fish FIH-1 have been reported to date. In this study, the cDNA of FIH-1 gene was cloned and characterized for bighead carp, Aristichthys nobilis (AnFIH-1). The AnFIH-1 cDNA is 2065 bp in length, encoding a protein of 357 amino acid (aa) residues, which contains a JmjC homology region of the jumonji transcription factors. AnFIH-1 shares high identities with other vertebrate FIH-1 (79.1-96.4%), especially in the JmjC homology region, suggesting its conserved function. During the embryonic stages of A. nobilis, AnFIH-1 had significantly high expression levels in unfertilized egg and blastula. In healthy tissues, its predominant mRNA expression was detected in muscle. The mRNA levels of AnFIH-1 were significantly upregulated in the liver, gill, hypothalamus, and spleen after hypoxic treatment, and then decreased to pretreatment levels after 6-h re-oxygenation. However, in the muscle, continual increasing of mRNA expression was observed after hypoxic shock and re-oxygenation. These results indicate that FIH-1 may play an important role in physiological regulation for adapting to hypoxia stress in A. nobilis.

A high-resolution genetic linkage map and QTL fine mapping for growth-related traits and sex in the Yangtze River common carp (Cyprinus carpio haematopterus).

BACKGROUND: A high-density genetic linkage map is essential for QTL fine mapping, comparative genome analysis, identification of candidate genes and marker-assisted selection for economic traits in aquaculture species. The Yangtze River common carp (Cyprinus carpio haematopterus) is one of the most important aquacultured strains in China. However, quite limited genetics and genomics resources have been developed for genetic improvement of economic traits in such strain. RESULTS: A high-resolution genetic linkage map was constructed by using 7820 2b-RAD (2b-restriction site-associated DNA) and 295 microsatellite markers in a F2 family of the Yangtze River common carp (C. c. haematopterus). The length of the map was 4586.56 cM with an average marker interval of 0.57 cM. Comparative genome mapping revealed that a high proportion (70%) of markers with disagreed chromosome location was observed between C. c. haematopterus and another common carp strain (subspecies) C. c. carpio. A clear 2:1 relationship was observed between C. c. haematopterus linkage groups (LGs) and zebrafish (Danio rerio) chromosomes. Based on the genetic map, 21 QTLs for growth-related traits were detected on 12 LGs, and contributed values of phenotypic variance explained (PVE) ranging from 16.3 to 38.6%, with LOD scores ranging from 4.02 to 11.13. A genome-wide significant QTL (LOD = 10.83) and three chromosome-wide significant QTLs (mean LOD = 4.84) for sex were mapped on LG50 and LG24, respectively. A 1.4 cM confidence interval of QTL for all growth-related traits showed conserved synteny with a 2.06 M segment on chromosome 14 of D. rerio. Five potential candidate genes were identified by blast search in this genomic region, including a well-studied multi-functional growth related gene, Apelin. CONCLUSIONS: We mapped a set of suggestive and significant QTLs for growth-related traits and sex based on a high-density genetic linkage map using SNP and microsatellite markers for Yangtze River common carp. Several candidate growth genes were also identified from the QTL regions by comparative mapping. This genetic map would provide a basis for genome assembly and comparative genomics studies, and those QTL-derived candidate genes and genetic markers are useful genomic resources for marker-assisted selection (MAS) of growth-related traits in the Yangtze River common carp.

Transcriptomic Profiles of Brain Provide Insights into Molecular Mechanism of Feed Conversion Efficiency in Crucian Carp (Carassius auratus).

Feed efficiency is an economically crucial trait for cultured animals, however, progress has been scarcely made in the genetic analyses of feed conversion efficiency (FCE) in fish because of the difficulties in measurement of trait phenotypes. In the present investigation, we present the first application of RNA sequencing (RNA-Seq) combined with differentially expressed genes (DEGs) analysis for identification of functional determinants related to FCE at the gene level in an aquaculture fish, crucian carp (Carassius auratus). Brain tissues of six crucian carp with extreme FCE performances were subjected to transcriptome analysis. A total of 544,612 unigenes with a mean size of 644.38 bp were obtained from Low- and High-FCE groups, and 246 DEGs that may be involved in FCE traits were identified in these two groups. qPCR confirmed that genes previously identified as up- or down-regulated by RNA-Seq were effectively up- or down-regulated under the studied conditions. Thirteen key genes, whose functions are associated with metabolism (Dgkk, Mgst3 and Guk1b), signal transduction (Vdnccsa1b, Tgfα, Nr4a1 and Tacr2) and growth (Endog, Crebrtc2, Myh7, Myh1,Myh14 and Igfbp7) were identified according to GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) annotations. Our novel findings provide useful pathway information and candidate genes for future studies of genetic mechanisms underlying FCE in crucian carp.

Seasonal succession of bacterial communities in cultured Caulerpa lentillifera detected by high-throughput sequencing.

An increasing number of microorganisms are being identified as pathogens for diseases in macroalgae, but the species composition of bacteria related to Caulerpa lentillifera, fresh edible green macroalgae worldwide, remains largely unclear. The bacterial communities associated with C. lentillifera were investigated by high-throughput 16S rDNA sequencing, and the bacterial diversities in washed and control groups were compared in this study. A total of 4,388 operational taxonomic units were obtained from all the samples, and the predominant prokaryotic phyla were Proteobacteria, Bacteroidetes, Planctomycetes, Cyanobacteria, Actinobacteria, Verrucomicrobia, Chloroflexi, and Acidobacteria in C. lentillifera. The bacterial diversity changed with seasons and showed an increasing trend of diversity with the rising temperature in C. lentillifera. There were slight reductions in the abundance and diversity of bacteria after washing with tap water for 2 h, indicating that only parts of the bacterial groups could be washed out, and hidden dangers in C. lentillifera still exist. Although the reduction in the abundance of some bacteria revealed a positive significance of washing C. lentillifera with tap water on food safety, more effective cleaning methods still need to be explored.

Transcriptome analysis of the toxicity response of green macroalga Caulerpa lentillifera J. Agardh to high dissolved arsenite.

Arsenic (As) is a hazardous pollutant that negatively impacts the physiological functions of alga. So far, a detailed understanding of algal response to As stress is still lacking. In this study, a transcriptome analysis was performed to illustrate the toxicity response of Caulerpa lentillifera J. Agardh, an edible algae with rich nutrition, to arsenite [As(III)], a toxic form of As. Totally, 1913 differentially expressed genes (DEGs) were screened, of which 642 were up- and 1271 were downregulated in C. lentillifera under As(III) stress (30 mg·L-1) compared with control. As(III) stress promoted the growth of C. lentillifera at low concentration (0.1 mg·L-1) and inhibited the growth at high concentration (≥ 0.5 mg·L-1). Multiple DEGs involved in oxidoreductase activities were significantly affected by As(III), and several DEGs related to antioxidant enzyme activity were downregulated, resulting in suffering from oxidative stress in C. lentillifera. Results also showed that As(III) stress inhibited chlorophyll and carotenoid synthesis, destroyed the integrity of chloroplasts, and interfered with the absorption of light energy, thereby inhibiting photosynthesis in C. lentillifera. The highly enriched ABC transporter-related genes involved in the detoxification process were upregulated under As(III) stress, indicating their critical role in the resistance to As stress in C. lentillifera. The gene expressions for 10 selected DEGs were confirmed by qRT-PCR, showing the reliability of the data revealed by RNA sequencing. Our novel work illustrated the toxicity of C. lentillifera under As(III) stress at the molecular level, serving as a basis for future investigations on the prevention and treatment of such pollutants.

Genome-Wide Association Study Reveals SNPs and Candidate Genes Related to Growth and Body Shape in Bighead Carp (Hypophthalmichthys nobilis).

Growth is an economically important trait in bighead carp and other aquaculture species that affects production efficiency. Interestingly, the head of the bighead carp has a high market value in China; therefore, it is important to study the genetic bases of both growth and body shape traits. A genome-wide association study was performed based on 2b-RAD sequencing of 776 individuals to identify SNPs associated with growth and body shape traits, including body weight, body length, body height, and deheaded body length. In total, 26 significant and 19 suggestive SNPs were identified, and more than half of these significant SNPs were clustered in LG16. Two LGs (LG16 and LG21) contained QTLs associated with body weight. Fourteen SNPs of LG16 and two LG21 SNPs were found to be associated with body length. For body height, 12 significantly associated SNPs were identified in LG16. Additionally, 12 SNPs of LG16 and 3 SNPs of LG21 were found to be associated with deheaded body length. Forty-three genes were significantly or suggestively associated with body shape/growth traits based on GWAS results, 18 of which were candidate genes for all BW, BL, BH, and DBL traits. One of these genes, fndc5b, was selected for further analyses. Association analysis revealed that one SNP (g.245 C > T) in the introns of fndc5b was significantly associated with growth-related traits in growth-extreme samples. The mRNA levels of fndc5b in the brains of the lightweight group were significantly higher than those of the heavy-weight group. This study helps to reveal the genetic structure of growth and body development in fish and provides candidate genes for future molecular marker-assisted selection for fast growth and better body conformation in bighead carp.

Transcriptome sequencing provides insights into the mechanism of hypoxia adaption in bighead carp (Hypophthalmichthys nobilis).

Hypoxia negatively affects the behavior, immunology, physiology, and growth of fish. Therefore, uncovering the genetic mechanisms underlying hypoxia adaptation and tolerance in fish prior to any genetic improvement is essential. Bighead carp is one of the most important freshwater fish species in aquaculture worldwide; however, this species does not have a strong ability to tolerate hypoxia. In this study, the dissolved oxygen level (0.6 mg/L) was maintained above the asphyxiation point of bighead carp for a long time to simulate hypoxia stress. The liver, gills, and heart were sampled before (0 h) and after (1 h, 2 h, 4 h) the hypoxia tests. Glutathione peroxidase (GPx) and catalase (CAT) activities and malondialdehyde (MDA) levels in the liver were significantly (p < 0.05) elevated at 1 h after hypoxic stress. By observing tissue morphology, the cell structure of the liver and gill tissues was found to change to varying degrees before and after hypoxia stress. Transcriptome sequencing was performed on 36 samples of gill, liver, and heart at four time points, and a total of 293.55G of data was obtained. In the early phase (0-1 h), differentially expressed genes (DEGs, 807 genes upregulated, 654 genes downregulated) were mainly enriched in signal transduction, such as cytokine-cytokine receptor interactions and ECM-receptor interactions. In the middle phase (0-2 h), DEGs (1201 genes upregulated and 2036 genes downregulated) were mainly enriched in regulation and adaptation, such as the MAPK and FoxO signaling pathways. Finally, in the later phase (0-4 h), DEGs (3975 genes upregulated and 4412 genes downregulated) were mainly enriched in tolerance and apoptosis, such as the VEGF signaling pathway and apoptosis. The genes with the most remarkable upregulation at different time points in the three tissues had some similarities. Genetic differences in these genes may be responsible for the differences in hypoxia tolerance among individuals. Altogether, our study provides new insights into the molecular mechanisms of hypoxia adaptation in fish. Further, the key regulatory genes identified provide genetic resources for breeding hypoxia-tolerant bighead carp species.

Transcriptome sequencing and metabolite analysis reveal the toxic effects of nanoplastics on tilapia after exposure to polystyrene.

Plastic particles, which are formed from routinely used plastics and their fragments, have become a new pollutant raising widespread concern about their potential effects. Several studies have been conducted to examine their toxicity, but the effects of nano-sized plastic fragments on freshwater organisms remain largely unclear and need to be further investigated. In this study, larval tilapia were first exposed to 100 nm polystyrene nanoparticles (PS-NPs, 20 mg/L) for seven days and then returned to freshwater without PS-NPs for another seven days in order to determine the toxic effects of PS-NPs at both transcriptomic and metabolomic levels. A total of 203 significantly changed metabolites, and 2,152 differentially expressed unigenes were identified between control and PS-NP treatment groups, control and recovery groups, as well as treatment and recovery groups. Our data suggested that PS-NPs induced abnormal metabolism of glycolipids, energy, and amino acids in tilapia after short-term exposure. Additionally, PS-NPs caused disturbed signaling, as suggested by the transcriptomic results. Different transcriptomic and metabolomic levels between the treatment group and recovery group indicated a persistent impact of PS-NPs on tilapia. The presence of adhesion molecule-related differentially expressed genes (DEGs) suggested that PS-NPs might cause early inflammatory responses. Notably, the detection of chemical stimulus involved in the sensory perception of smell was the most severely impacted biological process. Our work systemically studied the ecotoxicity of nano-sized plastics in aquatic creatures at the molecular and genetic levels, serving as a basis for future investigations on the prevention and treatment of such pollutants.

[Effects of heavy metal cadmium on Caulerpa lentillifera based on transcriptome analysis]. / 基于转录组分析重金属镉对长茎葡萄蕨藻的影响.

With the acceleration of industrialization, the toxic effect of heavy metal cadmium (Cd) pollution has become prominent. In order to explore the molecular mechanism of the physiological regulation of Caulerpa lentillifera under Cd stress, we analyzed the transcriptome of Cd-stressed (Hcd2+) algae tissues using RNA-Seq. A total of 702 differentially expressed genes (DEGs) were screened between the control and Hcd2+ groups, out of which 257 genes were up-regulated and 445 genes were down-regulated in the Hcd2+ group. We conducted functional annotation and enrichment analysis of the obtained DEGs. The results showed that various biological functions of C. lentillifera were affected under Cd2+stress, which eventually showed growth inhibition. Results of GO enrichment analysis showed that the production and removal of reactive oxygen species (ROS) in C. lentillifera were out of balance and caused oxidative damage such as DNA damage. Results of KEGG enrichment analysis showed that many photosynthesis-related pathways were inhibited, indicating that Cd2+ stress led to disorder of photosynthetic reaction of C. lentillifera.

Construction of a high-density genetic linkage map and mapping of quantitative trait loci for growth-related traits in silver carp (Hypophthalmichthys molitrix).

High-density genetic map and quantitative trait loci (QTL) mapping are powerful tools for identifying genomic regions that may be responsible for such polygenic trait as growth. A high-density genetic linkage map was constructed by sequencing 198 individuals in a F1 family of silver carp (Hypophthalmichthys molitrix) in this study. This genetic map spans a length of 2,721.07 cM with 3,134 SNPs distributed on 24 linkage groups (LGs). Comparative genomic mapping presented a high level of syntenic relationship between silver carp and zebrafish. We detected one major and nineteen suggestive QTL for 4 growth-related traits (body length, body height, head length and body weight) at 6, 12 and 18 months post hatch (mph), explaining 10.2~19.5% of phenotypic variation. All six QTL for growth traits of 12 mph generally overlapped with QTL for 6 mph, while the majority of QTL for 18 mph were identified on two additional LGs, which may reveal a different genetic modulation during early and late muscle growth stages. Four potential candidate genes were identified from the QTL regions by homology searching of marker sequences against zebrafish genome. Hepcidin, a potential candidate gene identified from a QTL interval on LG16, was significantly associated with growth traits in the analyses of both phenotype-SNP association and mRNA expression between small-size and large-size groups of silver carp. These results provide a basis for elucidating the genetic mechanisms for growth and body formation in silver carp, a world aquaculture fish.

Molecular cloning, expression pattern of follistatin gene and association analysis with growth traits in bighead carp (Hypophthalmichthys nobilis).

Follistatin (FST) is a single-chain gonadal protein involving in various biological effects. FST plays important roles in not only ovary development but also body growth, whereas myostatin (MSTN) negatively regulates muscle growth. In this study, FST gene in bighead carp (HynFST) was cloned and characterized. A 5797 bp genomic sequence of HynFST, consisting six exons and five introns were cloned. The full-length cDNA of HynFST (2134 bp) has an open reading fragment encoding a polypeptide of 349 amino acids. Sequence comparison and phylogenetic analysis confirmed that FSTs are conserved throughout the vertebrates and HynFST belongs to FST-1 isoform. Nine single nucleotide polymorphisms (SNPs) of the HynFST were identified and three of them (g.2443 T > C, g.2852 T > C and g.5483A > G) were significantly associated with four growth-related traits. The average body weight of those fish with the combined genotype (CC CC GG) was 12.15-22.63% higher than that of triplotype (TT TT AA) in two bighead carp populations. HynFST was expressed in most of the development stages and various tissues with highest level in ovary. The co-expression results for FST and MSTN in brain and muscle of divergent weight groups showed that FST may inhibit MSTN expression, thus enhancing growth in bighead carp. Our results suggest that FST has significant genetic effects on the regulation of early growth in bighead carp. This study would facilitate the elucidation of multiple functions of FST gene in fish and exploration of the potentials as a gene marker in selective breeding programs for growth of bighead carp.

Sex-specific markers developed by next-generation sequencing confirmed an XX/XY sex determination system in bighead carp (Hypophthalmichehys nobilis) and silver carp (Hypophthalmichthys molitrix).

Sex-specific markers are powerful tools for identifying sex-determination system in various animals. Bighead carp (Hypophthalmichehys nobilis) and silver carp (Hypophthalmichthys molitrix) are two of the most important edible fish in Asia, which have a long juvenility period that can lasts for 4-5 years. In this study, we found one sex-specific marker by next-generation sequencing together with bioinformatics analysis in bighead carp. The male-specific markers were used to perform molecular sexing in the progenies of artificial gynogenetic diploids and found all progenies (n = 160) were females. Meanwhile, around 1 : 1 sex ratio was observed in a total of 579 juvenile offspring from three other families. To further extend the male-specific region, we performed genome walking and got a male-specific sequence of 8,661 bp. Five pairs of primers were designed and could be used to efficiently distinguish males from females in bighead carp and silver carp. The development of these male-specific markers and results of their molecular sexing in different populations provide strong evidence for a sex determination system of female homogametry or male heterogametry (XX/XY) in bighead carp and silver carp. To the best of our knowledge, this is the first report of effective sex-specific markers in these two large carp species.

A High-Density Genetic Linkage Map and QTL Fine Mapping for Body Weight in Crucian Carp (Carassius auratus) Using 2b-RAD Sequencing.

A high-resolution genetic linkage map is essential for a wide range of genetics and genomics studies such as comparative genomics analysis and QTL fine mapping. Crucian carp (Carassius auratus) is widely distributed in Eurasia, and is an important aquaculture fish worldwide. In this study, a high-density genetic linkage map was constructed for crucian carp using 2b-RAD technology. The consensus map contains 8487 SNP markers, assigning to 50 linkage groups (LGs) and spanning 3762.88 cM, with an average marker interval of 0.44 cM and genome coverage of 98.8%. The female map had 4410 SNPs, and spanned 3500.42 cM (0.79 cM/marker), while the male map had 4625 SNPs and spanned 3346.33 cM (0.72 cM/marker). The average recombination ratio of female to male was 2.13:1, and significant male-biased recombination suppressions were observed in LG47 and LG49. Comparative genomics analysis revealed a clear 2:1 syntenic relationship between crucian carp LGs and chromosomes of zebrafish and grass carp, and a 1:1 correspondence, but extensive chromosomal rearrangement, between crucian carp and common carp, providing evidence that crucian carp has experienced a fourth round of whole genome duplication (4R-WGD). Eight chromosome-wide QTL for body weight at 2 months after hatch were detected on five LGs, explaining 10.1-13.2% of the phenotypic variations. Potential candidate growth-related genes, such as an EGF-like domain and TGF-ß, were identified within the QTL intervals. This high-density genetic map and QTL analysis supplies a basis for genome evolutionary studies in cyprinid fishes, genome assembly, and QTL fine mapping for complex traits in crucian carp.

Quantitative trait loci mapping for feed conversion efficiency in crucian carp (Carassius auratus).

QTL is a chromosomal region including single gene or gene clusters that determine a quantitative trait. While feed efficiency is highly important in aquaculture fish, little genetic and genomic progresses have been made for this trait. In this study, we constructed a high-resolution genetic linkage map in a full-sib F1 family of crucian carp (Carassius auratus) consisting of 113 progenies with 8,460 SNP markers assigning onto 50 linkage groups (LGs). This genetic map spanned 4,047.824 cM (0.478 cM/marker) and covered 98.76% of the crucian carp genome. 35 chromosome-wide QTL affecting feed conversion efficiency (FCE, 8 QTL), relative growth rate (RGR, 9 QTL), average daily gain (ADG, 13 QTL) and average daily feed intake (ADFI, 5 QTL) were detected on 14 LGs, explaining 14.0-20.9% of the phenotypic variations. In LGs of LG16, LG25, LG36 and LG49, several QTL affecting different traits clustered together at the identical or close regions of the same linkage group. Seven candidate genes, whose biological functions may involve in the energy metabolism, digestion, biosynthesis and signal transduction, were identified from these QTL intervals by comparative genomics analysis. These results provide a basis for elucidating genetic mechanism of feed efficiency and potential marker-assisted selection in crucian carp.

Molecular characterization and expression of three preprosomatostatin genes and their association with growth in common carp (Cyprinus carpio).

Somatostatins (SSs) are a structurally diverse family of peptides that play important roles in the regulation of growth, development and metabolism in vertebrates. In this study, three preprosomatostatin genes (PSSs) in the common carp, Cyprinus carpio (Cc) were identified and characterized. Based on cloned sequences and genome BLAST, six isoforms of the PSS gene in C. carpio (CcPSS) were identified and included CcPSS1a and CcPSS1b, CcPSS2a and CcPSS2b, and finally, CcPSS3a and CcPSS3b. The open reading frames (ORF) of CcPSS1a, CcPSS2a and CcPSS3a consist of 345, 336 and 363 nucleotides. During embryonic development, the expressions of CcPSS2 and CcPSS3 were first observed at the stage of optic vesicle, and CcPSS1 mRNA was initially detected at the stage of muscular effect. The highest mRNA levels of CcPSS1, CcPSS2 and CcPSS3 were observed at 1-day post-hatch (dph), 2-dph and the stage of heart beating, respectively. In the adult brain, the distributions of three CcPSS mRNAs were differential but overlapping in the hypothalamus, telencephalon and medulla oblongata. For peripheral tissues, all three CcPSS mRNAs were detected in the mid-intestine, and CcPSS1 and CcPSS3 mRNAs were also expressed in the liver. Owing to the importance of somatostatins on regulating growth, functional mutations of CcPSSs were identified in a C. carpio population. A total of 23 polymorphic sites were detected in CcPSS1a and CcPSS3a. Of them, two SNPs (CcPSS1a-g.922C>T, and CcPSS3a-g.1125C>A) were significantly associated with growth traits, indicating their potential applications in gene (marker)-assisted selective breeding in C. carpio.