Eight single nucleotide polymorphisms and their association with food habit domestication traits and growth traits in largemouth bass fry (Micropterus salmoides) based on PCR-RFLP method.

Background: The largemouth bass (Micropterus salmoides), an economically important freshwater fish species widely farmed in China, is traditionally cultured using a diet of forage fish. However, given the global decline in forage fish fisheries and increasing rates of waterbody pollution and disease outbreaks during traditional culturing, there is a growing trend of replacing forage fish with formulated feed in the largemouth bass breeding industry. The specific molecular mechanisms associated with such dietary transition in this fish are, nevertheless, poorly understood. Methods: To identify single nucleotide polymorphisms (SNPs) related to food habit domestication traits and growth traits in largemouth bass fry, we initially genotyped fry using eight candidate SNPs based on polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method, with genetic parameters being determined using Popgen32 and Cervus 3.0. Subsequently, we assessed the associations between food habit domestication traits of largemouth bass fry and these SNPs using the Chi-square test or Fisher's exact test. Furthermore, we used a general linear model to assess the relationships between the growth traits of largemouth bass fry and these SNPs. The Pearson correlation coefficient between growth traits and the SNPs was also determined using bivariate correlation analysis in IBM SPSS Statistics 22. Finally, the phenotypic variation explained (PVE) by the SNPs was calculated by regression analysis in Microsoft Excel. Results: The genotyping results obtained based on PCR-RFLP analysis were consistent with those of direct sequencing. Five SNPs (SNP01, SNP02, SNP04, SNP05, and SNP06) were found to be significantly correlated with the food habit domestication traits of fry (P < 0.05); SNP01 (P = 0.0011) and SNP04 (P = 0.0055) particularly, had showed highly significant associations. With respect to growth traits, we detected significant correlations with the two SNPs (SNP01 and SNP07) (P < 0.05), with SNP01 being significantly correlated with body length, and height (P < 0.05), and SNP07 being significantly correlated with body height only (P < 0.05). Conclusions: Our findings indicated that the PCR-RFLP can be used as a low-cost genotyping method to identify SNPs related to food habit domestication and growth traits in largemouth bass, and that these trait-related SNPs might provide a molecular basis for the future breeding of new varieties of largemouth bass.

Effect of secondary attack by Aeromonas hydrophila on the expression level of hif genes in common carp (Cyprinus carpio).

Hypoxia-inducible factors (hifs) are involved in infectious diseases inflammatory reactions, and immune regulation. Common carp, a representative allotetraploid species that has undergone genome-wide replication events, has important research value. In this study, common carp were infected twice with Aeromonas hydrophila. Liver tissues of common carp were collected at 4 h, 12 h, 24 h, 48 h, 3 days, 7 days post-first infection and 4 h, 12 h, 24 h post-second infection. The mRNA levels of hif genes were determined at different time points. The hif2a-2, hif3a-2, hif3b-1 and hif3b-2 expression levels in the infected group were upregulated when compared with those in the control group, whereas the expression levels of other genes were downregulated after the second infection. This indicates that the effect of A. hydrophila infection on gene expression pattern is dependent on the host, pathogen, infected tissue and gene. Pressure analysis of the hif gene family revealed that the non-synonymous substitution to synonymous substitution ratio of 12 hif genes was <1, which indicated that they were in a state of purification and selection. Combined with the differences between copy genes, the polyclonal antibodies against Hif1b-1 and Hif1b-2 were successfully prepared in this study. Western blot analysis showed that the protein expression of Hif1b-1 and Hif1b-2 reached to the highest level 48 h after the first infection. After the second A. hydrophila infection, the protein expression levels of Hif1b-1 and Hif1b-2 reached the highest levels at 4 and 48 h, respectively. This may indicate that the Hif1b-1 and Hif1b-2 genes in common carp play an important role in the immune mechanism at the protein level. The findings of this study will lay the foundation for future studies on the immune regulatory function of common carp hif genes, which may aid in devising novel therapeutic strategies for common carp diseases, such as A. hydrophila infection.

Genome-wide mining of gpx gene family provides new insights into cadmium stress responses in common carp (Cyprinus carpio).

Glutathione peroxidase (Gpx) is an important member of antioxidant enzymes, which can play a vital role in metabolizing reactive oxygen species (ROS) and in maintaining cell homeostasis. In order to study the evolutionary dynamics of gpx gene family in allotetraploid fish species, we identified a total of 14 gpx genes in common carp Cyprinus carpio, while 9 gpx genes were discovered in the diploid progenitor-like species Poropuntius huangchuchieni. Comparative genomic analysis and phylogenetic analysis revealed that the common carp gpx genes had significant expansion and were divided into five distinct subclades. Exon-intron distribution patterns and conserved motif analysis revealed highly conserved evolutionary patterns. Transcript profiles suggested that different gpx genes had specific patterns of regulation during early embryonic development. In adult tissues, gpx genes had a relatively broad expression distribution, most of which were highly expressed in the gills, intestines, and gonads. RT-qPCR studies showed that most gpx genes were downregulated during the initial cd2+ treatment stage. Dietary supplementation of Bacillus coagulans at different concentrations (Group 2 of 1.0 × 107 cfu/g, Group 3 of 1.0 × 108 cfu/g, and Group 4 of 1.0 × 109 cfu/g) induced different regulatory responses of gpx subclades. This result suggested that the appropriate concentration of B. coagulans can improve gpx gene expression when exposed to heavy metal cadmium treatment, which may play a vital role in the resistance to oxidative stress and immune responses. This study has expanded our understanding of the functional evolution of the gpx gene family in common carp.

Phylogeny of the HO family in cyprinus carpio and the response of the HO-1 gene to adding Bacillus coagulans in feed under Cd2+ stress.

The heavy metal cadmium (Cd2+) is an environmental pollutant that poses serious health hazards. Due to the increasing contamination of aquatic systems with Cd2+, the increased accumulation of Cd2+ in fish has become a food safety and public health concern. Heme oxygenase (HO) is an important antioxidant enzyme that plays a key role in defending the body against oxidative damage, but little research has been done in common carp. In this study, 6 HO genes were identified in the common carp genome database. Comparative genomics analysis showed considerable expansion of the HO genes and verified the four-round whole genome duplication (WGD) event in common carp. Phylogenetic analysis revealed that all HO genes of common carp were clustered into orthologous groups, indicating high conservation during evolution. In addition, the tissue distribution results showed that most HO genes had extensive tissue distribution and showed tissue-specific expression patterns. Exposure to 0.5 mg/L Cd2+ significantly reduced the expression of TGF-ß and IL-10 in common carp, which may indicate that Cd2+ exposure can destroy the physical barrier function of the intestine, inhibit intestinal immune defense and induce intestinal inflammation. To find a suitable concentration of Bacillus coagulans that could activate HO-1 genes and the immunity of the organism, we investigated the changes in HO-1 gene expression levels in the intestinal tract of common carp under Cd2+ stress at 30 days and 60 days by adding different concentrations of B. coagulans to the feed. Compared with the Cd2+ stress group without supplementation, the expression levels of the HO-1 gene in the gut of three different concentrations of B. coagulans were almost increased. And B. coagulans with L2 concentrations had better activation effect on the HO-1 gene. Similarly, compared to the Cd2+ stressed group, adding B. coagulans to the diet can almost cause the early upregulation of IL-10 and TGF-ß genes. Therefore, the addition of appropriate concentrations of B. coagulans may be a good way to activate HO-1, IL-10, and TGF-ß genes, reduce oxidative damage, and encourage the immune.

Genome-wide identification and characterization of olfactory receptor genes in common carp (Cyprinus carpio).

The environment contains a large extent of chemical information, which could be detected as olfactory sense. Olfactory in vertebrates plays important roles on many aspects during life time, including localizing prey or food, avoiding predators, mating behavior and social communication. Considering the essential role of olfactory receptors in the specific recognition of diverse stimuli, understanding the evolutionary dynamics of olfactory receptors in teleost means a lot, especially in the allotetraploid common carp, who has undergone the fourth whole-genome duplication event. Here, we identified the whole set of olfactory receptor genes in representative teleosts and found a significant contraction in common carp when compared with other teleosts. Odorant receptor genes (OR) occupy the most among four groups of olfactory receptors, including 33 functional genes and 16 pseudogenes. Furthermore, 6 trace amine-associated receptor (TAAR) genes (including 1 pseudogene), 7 odorant-related-A receptor genes, and 10 olfactory C family receptor genes (including 3 pseudogenes) were identified in common carp. Phylogenetic and motif analysis were performed to illustrate the phylogenetic relationship and structural conservation of teleost olfactory receptors. Selection pressure analysis suggested that olfactory receptor groups in common carp were all under relaxed purifying-selection. Additionally, gene expression divergences for olfactory receptor genes were investigated during embryonic development stages of common carp. We aim to determine the abundance of common carp olfactory receptor genes, explore the evolutionary fate and expression dynamics, and provide some genomic clues for the evolution of polyploid olfactory after whole-genome duplication and for future studies of teleost olfactory.

Transcriptome analysis of common carp (Cyprinus carpio) provides insights into the ovarian maturation related genes and pathways in response to LHRH-A and dopamine inhibitors induction.

Luteinizing hormone-releasing hormone analog (LHRH-A) and dopamine inhibitors have been widely used to induce oocyte maturation and ovulation in domesticated fishes. Although this approach represents a reliable method for regulating fish reproduction, the underlying molecular mechanisms mediating LH action are largely unexplored. The objective of this study was to determine the transcriptional profile of gene programming in hormone-treated common carp. In the present study, female common carp were intraperitoneally injected with LHRH-A together with dopamine inhibitors, and control fish were injected with saline. Ovarian morphological changes were analysed by both light microscopy and scanning electron microscopy. Furthermore, gene expression profiling of the brain and ovarian tissues was performed by Illumina sequencing. Compared to the control carp, hormone treatment resulted in morphological changes including disappearance of nuclear membrane, breakdown of germinal vesicle (GVBD), and fusion of yolk globules, reflecting that hormones significantly promoted oocyte maturation. In comparison to control, we have identified 867 and 9,053 differentially expressed genes in the hormone-treated female brain and ovary, respectively. In the brain, most of the identified genes were significantly enriched in 18 KEGG pathways. In the ovarian tissue, the identified genes were significantly involved in 9 pathways. In the hormone-treated carp, genes were involved in calcium signalling pathway, cAMP signalling pathway, insulin secretion, and oxidative phosphorylation pathway, which showed obvious associations with ovarian maturation. The present study provides transcriptomic information for hormone-treated carp, which might be useful for studying the endocrine regulation and mechanisms of ovarian maturation in domesticated fishes.

Phylogeny of Slc15 family and response to Aeromonas hydrophila infection following Lactococcus lactis dietary supplementation in Cyprinus carpio.

Solute carrier 15 family (Slc15) are membrane proteins that utilize the proton gradient and negative membrane protential for the transmembrane transporter of di-/tripeptide and peptide-mimetic molecules, in addition, they also play important roles in immunoreaction. In this study, 10 Slc15 genes were identified in the common carp genome database. Comparative genomics analysis showed considerable expansion of the Slc15 genes and verified the four-round whole genome duplication (WGD) event in common carp. Phylogenetic analysis revealed all Slc15 genes of common carp were clustered into orthologous groups indicating the highly conservative during evolution. Besides, the tissues and temporal expression examined by RT-PCR and qRT-PCR showed that most of the Slc15 genes had a narrow tissue distribution and exhibited tissue-specific expression patterns. Expression divergences were observed between these copies proving function divergence after the WGD. Then, we investigated the dietary supplementation effects of three Lactococcus lactis strains on the expression of Slc15 genes in common carp infected by A. hydrophila to find an effective way to treat aquatic diseases. Almost all of the Slc15 genes had an increased expression trend in the early post-challenge stage, and reached the highest expression level at 12h post-challenge. Then, the expression level showed a bluff descent at the last two stages and the expression level reached the lowest at 48 h post-challenge. Slc15 genes expression is actively up-regulated when stimulated by inflammatory factors, which can "amplify" immune signals, and improve the body's defense against foreign invasion in the early stage of the inflammatory response. So activation of the Slc15 genes may be an effective way for infectious disease treatment. As expected, three strains improved the expression of Slc15 genes variously compared with the control/infection groups. The strain 3 of L. lactis had a better induction of Slc15 genes compared with strain 1 and strain 2. It might be applied as a potential activation of Slc15 genes for disease treatment and adding befitting L. lactis may be a good way to protect aquatilia from bacillosis.

Mitogenomic Perspectives on the Adaptation to Extreme Alkaline Environment of Amur ide (Leuciscus waleckii).

Amur ide (Leuciscus waleckii, Family Cyprinidae) is widely distributed in Northeast Asia. L. waleckii usually inhabits freshwater environments but can also survive in the Lake Dali Nur, one of the most extreme aquatic environments on the earth, with an alkalinity up to 50 mmol/L (pH 9.6). To investigate mechanisms of mitogenomic evolution underlying adaptation to extreme environments, we determined 30 complete mitogenomes that included Lake Dali Nur (alkaline environment, AL) population and Amur basin (freshwater environment, FW) population. Through phylogenetic and divergence time analysis, we found that AL and FW populations forming distinct two groups which were consistent with geographic divergence (the formation of Lake Dali Nur). In addition, we found that almost of the windows exhibited higher nucleotide diversity in FW population (avg 0.0046) than AL population (avg 0.0012). This result indicated that severe environment selection had remarkably reduced the genetic diversity of mitogenome in AL population and suggested that severe environment selection had remarkably reduced the genetic diversity of mitogenome in the AL population. Compared with the FW population (ω = 0.064), the AL population (ω = 0.092) had a larger mean ω (dN/dS ratios) value for the 13 concatenated mitochondrial protein-coding genes, indicating that the high alkaline tolerated group had accumulated more nonsynonymous mutations. These nonsynonymous mutations had resulted in slightly beneficial amino acid changes that allowed adaption to the severe conditions. This study provides an additional view to decipher the adaptive mitogenome evolution of L. waleckii of the high alkaline environment.

Genome-wide identification, evolution, and mRNA expression of complement genes in common carp (Cyprinus carpio).

Complement is a complex component of innate immune system, playing an important role in defense against pathogens and host homeostasis. The complement system has been comprehensively studied in mammals, however less is known about complement in teleost, especially in tetraploid common carp (Cyprinus carpio). In this study, a total of 110 complement genes were identified and characterized in common carp, which include almost all the homologs of mammalian complement genes. These genes were classified into three pathways (alternative pathways, lectin pathways and classical pathways), similar to those in mammals. Phylogenetic and selection pressure analysis showed that the complement genes were evolving-constrained and the function was conserved. Most of the complement genes were highly expressed in spleen, liver, brain and skin among the tested 12 health tissues of common carp. After Aeromonas hydrophila infection in the common carp, many members of complement genes were activated to bring about an immune response and expressed to against any pathogenic encroachment. Gene expression divergences which were found between two homoeologous genes suggested the functional divergences of the homoeologous genes after the 4R WGD event, revealing the evolutionary fate of the tetraploid common carp after the recent WGD.

The roles of two myostatins and immune effects after inhibition in Qi river crucian carp (Carassius auratus).

Myostatin, through type I receptor (kinase 4, 5, ALK4/5), functions to participate in the immune system and negatively regulate muscle growth in mammals. However, the role of myostatin (mstn) in the immune system of teleosts is largely unknown. In a previous study, we cloned the mstn1 cDNA encoding myostatin in Qi river crucian carp (Carassius auratus). In the present study, we have cloned mstn2 cDNA, which was characterized and analyzed together with mstn1. Tissue distribution analysis showed that both mstn genes are expressed in numerous tissues, with mstn1 dominantly expressed in the muscle and brain, whereas mstn2 is mainly expressed in the brain. During embryogenesis, mstn1 and mstn2 exhibit different expression patterns. Both mstn1 and mstn2 expression increased stepwise in the brain at different developmental stages. Furthermore, both genes are differentially regulated during different periods of fasting/re-feeding. Following the exposure of C. auratus to polyI:C, lipopolysaccharide (LPS), and Aeromonas hydrophila, both genes were upregulated in different tissues, which indicated that they might be involved in the immune response against pathogenic invasion. Blocking the Mstn signal pathway with SB-431542 (a chemical inhibitor of ALK4/5) resulted in significantly increased body length and weight. However, the mortality of SB-431542-treated fish was higher after A. hydrophila challenge. Moreover, decreased expression of lysozymes (lyz), complement component 3 (c3), ß-defensin 3 (defb3), and interferon γ (ifnγ) were exhibited in treated fish, compared with the controls. Furthermore, the expression of nf-κb1, three pro-inflammatory cytokines (il1ß, il6, and tnfα), and inflammatory cytokines (il8 and il10) were significantly increased in both the SB-431542-treated group and the control after A. hydrophila infection, suggesting that the NF-κB pathway was not suppressed in the SB-431542-treated fish. Taken together, our data suggest that both mstn1 and mstn2 play important roles in early body development, muscle growth, and the immune system by acting downstream of the NF-κB signal pathway.

A Drive to Driven Model of Mapping Intraspecific Interaction Networks.

Community ecology theory suggests that an individual's phenotype is determined by the phenotypes of its coexisting members to the extent at which this process can shape community evolution. Here, we develop a mapping theory to identify interaction quantitative trait loci (QTL) governing inter-individual dependence. We mathematically formulate the decision-making strategy of interacting individuals. We integrate these mathematical descriptors into a statistical procedure, enabling the joint characterization of how QTL drive the strengths of ecological interactions and how the genetic architecture of QTL is driven by ecological networks. In three fish full-sib mapping experiments, we identify a set of genome-wide QTL that control a range of societal behaviors, including mutualism, altruism, aggression, and antagonism, and find that these intraspecific interactions increase the genetic variation of body mass by about 50%. We showcase how the interaction QTL can be used as editors to reconstruct and engineer new social networks for ecological communities.

High-Density Linkage Map and Mapping for Sex and Growth-Related Traits of Largemouth Bass (Micropterus salmoides).

The largemouth bass is an important species, and its culture has risen sharply with the surge in fish aquaculture in China. Due to the lack of selective breeding technology for the largemouth bass, the growth rate and disease resistance are low, its sexual maturation is slow, and other serious problems are contributing to a sharp decline in the safety and quality of largemouth bass products in recent decades. Therefore, comprehensive breeding programs to improve the economic performance and promote the modern industrial development of largemouth bass must be considered a priority. Here, a total of 152 adult largemouth bass, including two parents and 150 progenies, were selected to produce the genetic mapping family. Then, a high-density linkage map was constructed based on restriction site-associated DNA sequencing using 6,917 single-nucleotide polymorphisms (SNPs) located in 24 linkage groups (LGs). The total genetic length of the linkage map was 1,261.96 cM, and the length of each LG varied from 24.72 cM for LG02 to 117.53 cM for LG16, with an average length of 52.58 cM and an average SNP number of 286. Thirteen significant quantitative trait loci (QTLs) for sex determination were located on LG04, LG05, LG08, LG12, LG15, LG21, and LG23. An informative QTL cluster that included six QTLs was detected on LG12. However, one notable QTL, which accounted for 71.48% of the total phenotypic variation, was located in the region of 1.85 cM on LG05. In addition, 32 identified QTLs were related to growth, including body weight, body length, body height, and head length. The QTLs for these growth-related traits are located in 13 LG regions and have little effect on phenotypic variation. This high-density genetic linkage map will enable the fine-mapping of economic traits and support the future genome assembly of the largemouth bass. Additionally, our study will be useful for future selective culture of largemouth bass and could potentially be used in molecular-assisted breeding of largemouth bass for aquaculture.

The allotetraploid origin and asymmetrical genome evolution of the common carp Cyprinus carpio.

Common carp (Cyprinus carpio) is an allotetraploid species derived from recent whole genome duplication and provides a model to study polyploid genome evolution in vertebrates. Here, we generate three chromosome-level reference genomes of C. carpio and compare to related diploid Cyprinid genomes. We identify a Barbinae lineage as potential diploid progenitor of C. carpio and then divide the allotetraploid genome into two subgenomes marked by a distinct genome similarity to the diploid progenitor. We estimate that the two diploid progenitors diverged around 23 Mya and merged around 12.4 Mya based on the divergence rates of homoeologous genes and transposable elements in two subgenomes. No extensive gene losses are observed in either subgenome. Instead, we find gene expression bias across surveyed tissues such that subgenome B is more dominant in homoeologous expression. CG methylation in promoter regions may play an important role in altering gene expression in allotetraploid C. carpio.

Patterns of Geographical and Potential Adaptive Divergence in the Genome of the Common Carp (Cyprinus carpio).

The common carp, Cyprinus carpio, is a cyprinid fish species cultured in Europe and Asia. It accounts for >70% of freshwater aquaculture production worldwide. We conducted a population genomics analysis on C. carpio using high-throughput SNP genotyping of 2,198 individuals from 14 populations worldwide to determine the genetic architecture of common carp populations and the genetic bases for environmental adaptation. Structure analyses including phylogeny and principal component analysis were also conducted, showing distinct geographical patterns in European and Asian populations. The linkage disequilibrium block average lengths of the 14 populations ranged from 3.94 kb to 36.67 kb. Genes within selective sweep regions were identified by genome scanning among the different populations, including gdf6a, bmpr1b, and opsin5. Gene Ontology and KEGG enrichment analyses revealed potential trait-related loci and genes associated with body shape, scaling patterns, and skin color. This population genomics analysis may provide valuable clues for future genome-assisted breeding of C. carpio.

Effects of dietary supplementation of three strains of Lactococcus lactis on HIFs genes family expression of the common carp following Aeromonas hydrophila infection.

HIFs (Hypoxia inducible factors) are the main regulators of the expression change of oxygen-dependent genes, in addition, they also play important roles in immune regulation. HIFs participate in infectious diseases and inflammatory responses, providing us a new therapeutic target for the treatment of diseases. In this study, 16 HIFs were identified in common carp genome database. Comparative genomics analysis showed large expansion of HIF gene family and approved the four round whole genome duplication (WGD) event in common carp. To further understand the function of HIFs, the domain architectures were predicted. All HIF proteins had the conserved HLH-PAS domain, which were essential for them to form dimer and bind to the downstream targets. The differences in domain of HIFα and HIFß might result in their different functions. Phylogenetic analysis revealed that all HIFs were divided into two subfamilies and the HIFs in common carp were clustered with their teleost counterparts indicating they are highly conservative during evolution. In addition, the tissue distribution was examined by RT-PCR showed that most of HIF genes had a wide range of tissue distribution but exhibited tissue-specific expression patterns. The expression divergences were observed between the copy genes, for example, HIF1A-1, HIF2A-1, ARNT-2 had wide tissue distribution while their copies had limited tissue distribution, proving the function divergence of copies post the WGD event. In order to find an effective activation of HIFs and apply to treatment of aquatic diseases, we investigate the dietary supplementation effects of different strains of Lactococcus lactis on the expression of HIFα subfamily members in kidney of common carp infected with A. hydrophila. In addition, all of the HIF genes have a high expression in the early stages of infection, and decreased in the treatment time point of 48 h in common carp. This phenomenon confirms that as a switch, the main function of HIFs is to regulate the production of immune response factors in early infection. So activation of the switch may be an effective method for infectious disease treatment. As expected, the treatment groups improved the expression of HIFs compared with the control group, and the effects of the three strains are different. The strain1 of L. lactis had a stronger induction on HIF genes than strain2 and strain3, and it might be applied as a potential activation of HIF genes for disease treatment. So, adding befitting L. lactis maybe a well method to activate the HIF genes to protect them from mycobacterial infection.

Genomic, Transcriptomic, and Epigenomic Features Differentiate Genes That Are Relevant for Muscular Polyunsaturated Fatty Acids in the Common Carp.

Polyunsaturated fatty acids (PUFAs) are a set of important nutrients that mainly include arachidonic acid (ARA4), docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), and α-linolenic acid (ALA). Recently, fish-derived PUFAs have been associated with cardiovascular health, fetal development, and improvement of brain functions. Studies have shown that fish muscular tissues are rich in PUFAs, which are influenced by various factors, including genetic variations, regulatory profiles, and methylation status of desaturase genes during fatty acid desaturation and elongation processes. However, the genetic mechanism and the pathways involved in fatty acid metabolism in fishes remain unclear. The overall aim of this study was to assess differences in gene expression responses among fishes with different fatty acid levels. To achieve this goal, we conducted genome-wide association analysis (GWAS) using a 250K SNP array in a population of 203 samples of common carp (Cyprinus carpio) and identified nine SNPs and 15 genes associated with muscular PUFA content. Then, RNA-Seq and whole genome bisulfite sequencing (WGBS) of different groups with high and low EPA, DHA, ARA4, and ALA contents in muscle, liver and brain tissues were conducted, resulting in 6,750 differentially expressed genes and 5,631 genes with differentially methylated promoters. Gene ontology and KEGG pathway enrichment analyses of RNA-Seq and WGBS results identified enriched pathways for fatty acid metabolism, which included the adipocytokine signaling pathway, ARA4 and linoleic acid metabolism pathway, and insulin signaling pathway. Integrated analysis indicated significant correlations between gene expression and methylation status among groups with high and low PUFA contents in muscular tissues. Taken together, these multi-level results uncovered candidate genes and pathways that are associated with fatty acid metabolism and paved the way for further genomic selection and carp breeding for PUFA traits.

Genome-wide identification of interleukin-17 (IL17) in common carp (Cyprinus carpio) and its expression following Aeromonas hydrophila infection.

Interleukin-17 (IL17) family cytokines are well known for having pro-inflammatory actions as important mediators of mucosal immune responses and are tightly regulated by various kinds of signals. However, most studies of IL17 genes have focused on mammals, and much less is known about IL17 genes in fish species. To better understand the scope and actions of the IL17 gene family in common carp, we characterized seven IL17 gene homologs from genomic and transcriptomic databases that could be classified into three subclasses according to different comparative genomic analyses. Phylogenetic analysis revealed that most IL17s are highly conserved, though recent gene duplication and gene loss events do exist. Through observation, we found that IL17D has undergone gene duplication in common carp and that all the IL17E genes were lost in vertebrates except mammals. The expression patterns of IL17 genes in common carp were examined during early developmental stages and in various healthy tissues, and the results indicated that most IL17 genes are ubiquitously expressed during early development and show particular tissue-specific expression in various healthy tissues, with relatively high levels in the spleen, liver, and kidney. To gain insights into the mucosal actions of inflammatory processes, the expression profiles of IL17 genes in gills from common carp were investigated after experimental challenge with Aeromonas hydrophila. After A. hydrophila infection, most IL17 genes were upregulated at 4 h postinfection in the gill and then gradually declined, while IL17A/F2 and IL17N were generally upregulated at 12 h postinfection, and IL17D2 maintained an increasing tendency. In contrast, IL17D showed the third phenomenon, rising expression, suggesting that immunogenes have different response strategies to bacterial invasion. Overall, the expression of IL17 in unstimulated tissues and toxicity attack test results demonstrated that these genes play critical roles under normal conditions and during bacterial infection. Moreover, this common carp IL17 gene family research provides a genomic resource for future studies on IL17 gene evolution, fish disease management and immune regulation.

Genome wide identification of taste receptor genes in common carp (Cyprinus carpio) and phylogenetic analysis in teleost.

Taste receptors (TRs) are seven trans-membrane G protein-coupled receptors as well as the interface between internal and external milieus, which playing pivotal roles in nutrient identification and acquisition. To better understand the scope and function of tr gene family in common carp, one of the most economic and important breeding fish species, which has undergone an additional round of whole genome duplication (WGD), we characterized 13 tr gene homologues including eight type I and five type II taste receptor genes from common carp genome, which were more than any other teleosts. Phylogenetic and syntenic analysis revealed the evolution dynamics of tr gene family, which was highly conserved, though gene duplication and gene loss do exist recently. Furthermore, the motif and dN/dS analyses indicated that these receptors were under different negative selection pressure. Additionally, the gene expression divergences were observed in 12 health tissues of common carp, with a relatively high level in barbel and head kidney, demonstrating tissue-specific expression of tr genes in the tetraploidized genome. The overarching goals of this study were to identify the abundance of tr genes in common carp, compare the gene divergence among species with varied feeding habits and provide genomic resources for future studies on teleost taste sensation.

Genome-Scale Association Study of Abnormal Scale Pattern in Yellow River Carp Identified Previously Known Causative Gene in European Mirror Carp.

Common carp (Cyprinus carpio) is one of the most widely studied fish species due to its great economic value and strong environmental adaptability. Scattered scale, a typical phenotype of the mirror carp that is derived from Europe, has never been observed in the Yellow River carp previously. We recently identified approximately one fourth of the F1 progenies displaying scattered scale in a full-sib Yellow River carp family in our breeding program, despite both parents that showed wild type with normal scale patterns. This family provides us unique materials to investigate the genetic basis underlying the abnormal scale mutant in Yellow River carp population. Genome-wide association study (GWAS) and association mapping were performed based on genome-wide single nucleotide polymorphisms (SNP) genotyped with common carp 250 K SNP genotyping array in 82 samples of the Yellow River carp family. We identified a 1.4 Mb genome region that was significantly associated with abnormal scattered scale patterns. We further identified a deletion mutation in fibroblast growth factor receptor 1 a1 (fgfr1a1) gene within this genome region. Amplification and sequencing analysis of this gene revealed a 311-bp deletion in intron 10 and exon 11, which proved that fgfr1a1 could be the causal gene responsible for abnormal scattered scale in the Yellow River carp family. Since similar fragment mutation with 306-bp and 310-bp deletions had been previously reported as causal mutation of scattered scale patterns in the mirror carp, we speculate that either the deletion mutation was introduced from Europe-derived mirror carp or the deletion independently occurred in the mutation hotspot in fgfr1a1 gene. The results provided insights into the genetic basis of scale pattern mutant in Yellow River carp population, which would help us to eliminate the recessive allele of the abnormal scale patterns in Yellow River carp population by molecular marker-assisted breeding.

Dynamic regulation of mRNA and miRNA associated with the developmental stages of skin pigmentation in Japanese ornamental carp.

The Japanese ornamental carp (Cyprinus carpio var. Koi) is famous for multifarious colors and patterns, making it commonly culture and trade across the world. Although functional genes and inheritance of color traits have been commonly studied, seldom attentions were focused on the genetic regulation during the developmental process of pigmentation. To better understand the mechanism of skin color development, we observed the morphogenesis of pigment cells during the post-embryonic stages and analysed the temporal expression pattern of mRNAs/miRNAs profiles in four distinct developmental stages. 59 and 103 differentially expressed genes/miRNAs (DEGs/DEMs) associated with pigmentation and skin were identified, including pax7, mitf, tyr, tyrp1, etc., and the highest DEGs were detected at 11 days post hatching (dph). In addition, the functional characteristics of mRNAs/miRNAs associated with pteridine and carotenoid pathway were also examined. Furthermore, 65 miRNA-mRNA interaction pairs related to pigmentation, pteridines and carotenoids metabolism were detected between different stages. Interestingly, the largest pairs appeared in the transition from 11 dph to 48 dph, which had the similar trend with DEGs further manifesting the importance of 11 dph. This study produced a comprehensive programme of DEGs/DEMs during color development, which will provide resources to understand the regulation mechanism in color formation. The understanding of genetic basis in color formation might promote the production and breeding of the Koi carp.