Comparative transcriptomic analyses of brain-liver-muscle in channel catfish (Ictalurus punctatus) with differential growth rate.

Growth is an important economically trait for aquatic animals. The popularity of farmed channel catfish (Ictalurus punctatus) in China has recently surged, prompting a need for research into the genetic mechanisms that drive growth and development to expedite the selection of fast-growing variants. In this study, the brain, liver and muscle transcriptomes of channel catfish between fast-growing and slow-growing groups were analyzed using RNA-Seq. Totally, 63, 110 and 86 differentially expressed genes (DEGs) were from brain, liver and muscle tissues. DEGs are primarily involved in growth, development, metabolism and immunity, which are related to the growth regulation of channel catfish, such as growth hormone receptor b (ghrb), fibroblast growth factor receptor 4 (fgfr4), bone morphogenetic protein 1a (bmp1a), insulin-like growth factor 2a (igf2a), collagen, type I, alpha 1a (col1a1a), acyl-CoA synthetase long chain family member 2 (acsl2) and caveolin 1 (cav1). This study advances our knowledge of the genetic mechanisms accounting for differences in growth rate and offers crucial gene resources for future growth-related molecular breeding programs in channel catfish.

Transcriptome analyses reveal differentially expressed genes associated with development of the palatal organ in bighead carp (Hypophthalmichthys nobilis).

The palatal organ is a filter-feeding related organ and occupies a considerable proportion of the head of bighead carp (Hypophthalmichthys nobilis), a large cyprinid fish intensive aquaculture in Asia. In this study, we performed RNA-seq of the palatal organ during growth periods of two (M2), six (M6) and 15 (M15) months of age after hatching. The numbers of differentially expressed genes (DEGs) were 1384, 481 and 1837 for M2 VS M6, M6 VS M15 and M2 VS M15 respectively. The following signaling pathways of energy metabolism and cytoskeleton function were enriched, including ECM-receptor interaction, Cardiac muscle contraction, Steroid biosynthesis and PPAR signaling pathway. Several members of collagen family (col1a1, col2a1, col6a2, col6a3, col9a2), Laminin gamma 1 (lamc1), integrin alpha 1 (itga1), Fatty acid binding protein 2 (fads2) and lipoprotein lipase (lpl), and Protein tyrosine kinase 7 (Ptk7) are candidate genes for growth and development of basic tissues of the palatal organ. Furthermore, taste-related genes such as fgfrl1, fgf8a, fsta and notch1a were also identified, which may be involved in the development of taste buds of the palatal organ. The transcriptome data obtained in this study provide insights into the understanding functions and development mechanisms of palatal organ, and potential candidate genes that may be related to the genetic modulation of head size of bighead carp.

Genetic responses in sexual diploid and unisexual triploid goldfish (Carassius auratus) introduced into a high-altitude environment.

Anthropogenic biological invasions represent major concerns but enable us to investigate rapid evolutionary changes and adaptation to novel environments. The goldfish Carassius auratus with sexual diploids and unisexual triploids coexisting in natural waters is one of the most widespread invasive fishes in Tibet, providing an ideal model to study evolutionary processes during invasion in different reproductive forms from the same vertebrate. Here, using whole-genome resequencing data of 151 C. auratus individuals from invasive and native ranges, we found different patterns of genomic responses between diploid and triploid populations during their invasion of Tibet. For diploids, although invasive individuals derived from two different genetically distinct sources had a relative higher diversity (π) at the population level, their individual genetic diversity (genome-wide observed heterozygosity) was significantly lower (21.4%) than that of source individuals. Population structure analysis revealed that the invasive individuals formed a specific genetic cluster distinct from the source populations. Runs of homozygosity analysis showed low inbreeding only in invasive individuals, and only the invasive population experienced a recent decline in effective population size reflecting founder events. For triploids, however, invasive populations showed no loss of individual genetic diversity and no genetic differentiation relative to source populations. Regions of putative selective sweeps between invasive and source populations of diploids mainly involved genes associated with mannosidase activity and embryo development. Our results suggest that invasive diploids deriving from distinct sources still lost individual genetic diversity resulting from recent inbreeding and founder events and selective sweeps, and invasive triploids experienced no change in genetic diversity owing to their reproduction mode of gynogenesis that precludes inbreeding and founder effects and may make them more powerful invaders.

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.

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.

Updated Genome Assembly of Bighead Carp (Hypophthalmichthys nobilis) and Its Differences Between Male and Female on Genomic, Transcriptomic, and Methylation Level.

Cyprinidae is one of the largest family in freshwater fishes, and it is most intensively cultured fish taxon of the world. However, studies about sex determination in this large family is still rear, and one of the reasons is lack of high quality and complete genome. Here, we used nanopore to sequence the genome of a male bighead carp, obtaining contig N50 = 24.25 Mb, which is one of the best assemblies in Cyprinidae. Five males and five females were re-sequenced, and a male-specific region on LG19 was confirmed. We find this region holds many male-specific markers in other Cyprinidae fishes, such as grass carp and silver carp. Transcriptome analyses of hypothalamus and pituitary tissues showed that several sex-specific differentially expressed genes were associated with steroid biosynthesis. The UCH64E gene, located in the male-specific region on LG19, showed higher expression levels in male than female tissues of bighead carp. The methyl-RAD of hypothalamus tissues between males and females indicated that the sexual methylation differences are significant in bighead carp. We also compared the methylation sites recognized using methyl-RAD and nanopore raw reads and found that approximately 73% of the methylation sites identified using methyl-RAD were within nanopore CpG sites.

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.

Comparative transcriptome analyses and identification of candidate genes involved in vertebral abnormality of bighead carp Hypophthalmichthys nobilis.

Body deformity occurs both in wild and farmed fishes, which is one of the most challenging problems for aquaculture industry. In most cases, such body deformities are linked to skeletal deformities. Currently, very limited information is available on skeletal deformities of farmed fish species which may be caused by genetic factor. In this study, we performed muscle and vertebra transcriptome analyses in body deformity and normality of bighead carp Hypophthalmichthys nobilis (from one meiotic gynogenesis family) using RNA-Seq. A total of 43,923 and 44,416 unigenes were predicted in muscles and vertebrae, respectively. Based on these data, we further explored the gene expression profiles in gynogenetic normal and abnormal bighead carp. No differentially expressed gene (DEG) was found in transcriptome data of muscles. Totally, 20 key DEGs were identified in transcriptome data of vertebrae, such as low density lipoprotein-related protein 2 (lrp2), bone morphogenetic protein 2B (bmp2b) and collagen alpha-1(IV) (col4a1). 12 potential pathways were also identified in vertebra transcriptome data, which were mainly involved in development, growth, cytoskeleton and energy metabolism, such as MAPK signaling pathway, regulation of actin cytoskeleton and TGF-beta signaling pathway. Results of this study will be informative for the understanding of genetic mechanisms for body shape formation and also provide potential candidate genes for selection program involved in body shape and skeletal development in H. nobilis.

Cardiac Transcriptomics Reveals That MAPK Pathway Plays an Important Role in Hypoxia Tolerance in Bighead Carp (Hypophthalmichthys nobilis).

As aquatic animals, fishes often encounter various situations of low oxygen, and they have evolved the ability to respond to hypoxia stress. Studies of physiological and molecular responses to hypoxia stress are essential to clarify genetic mechanisms underlying hypoxia tolerance in fish. In this study, we performed acute hypoxia treatment in juvenile bighead carp (Hypophthalmicthys nobilis) by decreasing water O2 from 6.5 mg/L to 0.5 mg/L in three hours. This hypoxia stress resulted in a significant increase in blood lactate and serum glucose. Comparisons of heart transcriptome among hypoxia tolerant (HT), hypoxia sensitive (HS), and normoxia control (NC) groups showed that 820, 273, and 301 differentially expressed genes (DEGs) were identified in HS vs. HT, NC vs. HS, and NC vs. HT (false discovery rate (FDR) < 0.01, Fold Change> 2), respectively. KEGG pathway enrichment showed that DEGs between HS and HT groups were mainly involved in mitogen-activated protein kinase (MAPK) signaling, insulin signaling, apoptosis, tight junction and adrenergic signaling in cardiomyocytes pathways, and DEGs in MAPK signaling pathway played a key role in cardiac tolerance to hypoxia. Combined with the results of our previous cDNA-amplified fragment length polymorphism (cDNA-AFLP) analysis of hypoxia stress in this species, such genes as stbp2, ttn, mapk, kcnh, and tnfrsf were identified in both studies, representing the significance of these DEGs in hypoxia tolerance in bighead carp. These results provide insights into the understanding of genetic modulations for fish heart coping with hypoxia stress and generate basic resources for future breeding studies of hypoxia resistance in bighead carp.

Identifying Candidate Genes Involved in the Regulation of Early Growth Using Full-Length Transcriptome and RNA-Seq Analyses of Frontal and Parietal Bones and Vertebral Bones in Bighead Carp (Hypophthalmichthys nobilis).

Growth, one of the most important traits monitored in domestic animals, is essentially associated with bone development. To date, no large-scale transcriptome studies investigating bone development in bighead carp have been reported. In this study, we applied Isoform-sequencing technology to uncover the entire transcriptomic landscape of the bighead carp (Hypophthalmichthys nobilis) in early growth stage, and obtained 63,873 non-redundant transcripts, 20,907 long non-coding RNAs, and 1,579 transcription factors. A total of 381 alternative splicing events were seen in the frontal and parietal bones with another 784 events simultaneously observed in the vertebral bones. Coupling this to RNA sequencing (RNA-seq) data, we identified 27 differentially expressed unigenes (DEGs) in the frontal and parietal bones and 45 DEGs in the vertebral bones in the fast-growing group of fish, when compared to the slow-growing group of fish. Finally, 15 key pathways and 20 key DEGs were identified and found to be involved in regulation of early growth such as energy metabolism, immune function, and cytoskeleton function and important cellular pathways such as the arginine and proline metabolic pathway (p4ha1), FoxO signaling pathway (sgk1), cell adhesion molecules (b2m, ptprc, and mhcII), and peroxisome proliferator-activated receptor signaling pathway (scd). We established a novel full-length transcriptome resource and combined it with RNA-seq to elucidate the mechanism of genetic regulation of differential growth in bighead carp. The key DEGs identified in this study could fuel further studies investigating associations between growth and bone development and serve as a source of potential candidate genes for marker-assisted breeding programs.

QTL Fine Mapping for Sex Determination Region in Bighead Carp (Hypophthalmichthys nobilis) and Comparison with Silver Carp (Hypophthalmichthys molitrix).

Bighead carp (Hypophthalmichthys nobilis) and silver carp (Hypophthalmichthys molitrix) are genetically close aquaculture fish in the Cyprinidae, which have been confirmed to hold XX/XY sex determination. However, genomic locations of potential sex-related loci in these two fishes are still unknown. In this study, a high-resolution genetic linkage map was constructed by using 2976 SNP and 924 microsatellite markers in a F1 full-sib family of bighead carp, the length of which spanned 2022.34 cM with an average inter-marker distance of 0.52 cM. Comparative genomics revealed a high level of genomic synteny between bighead carp and zebrafish as well as grass carp. QTL fine mapping for sex trait was performed based on this linkage map of bighead carp and an unpublished linkage map of silver carp. A map distance of 3.863 cM (69.787-73.650 cM) on LG19 of bighead carp and 4.705 cM (79.096-83.801 cM) on LG21 of silver carp was significantly associated with sex phenotypes, and these two LGs are homologous between two fish species. Fourteen markers harboring in these regions were in strong linkage disequilibrium with the sex phenotype variance explained (PVE) varying from 89 to 100%. Two common markers were mapped on the QTL regions of bighead carp and silver carp, suggesting that these two carp species may have similar genetic bases for sex determination. Eleven potentially sex-related genes were identified within or near the sex QTL markers in two species. This study provided insights into elucidating mechanisms and evolution of sex determination in cyprinid fishes.

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.

ß-elemene inhibits radiation and hypoxia-induced macrophages infiltration via Prx-1/NF-κB/HIF-1α signaling pathway.

Background: In cancers, tumor-associated macrophages (TAMs) play an important role in the progression, evasion of immunity and sensitivity to therapy. Unfortunately, radiation and hypoxia could induce the M2 macrophages infiltration and polarization. Materials and methods: In this study, we investigated the relevance of macrophage recruitment with radiation and hypoxia by transwell. We also evaluated the effect of ß-elemene on the infiltration of M2 macrophages and explored its underlying molecular mechanism by a series of in vitro and in vivo experiments. Results: Irradiated or hypoxia lung cancer cells recruit macrophages, and the recruitment is MCP-1 dependent. We also found that radiation and hypoxia-induced MCP-1 secretion follows upregulation of Prx-1, which leads to nuclear accumulation of NF-κB and HIF-1α expression. In addition, ß-elemene could effectively suppress this recruitment phenomenon through Prx-1/NF-κB/HIF-1α signaling. Conclusion: Our study showed that radiation and hypoxia significantly promoted the macrophages recruitment. ß-elemene could effectively suppress this recruitment phenomenon and MCP-1 expression via inhibiting Prx-1/NF-κB/HIF-1α pathways.

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.

Brain and intestine transcriptome analyses and identification of genes involved in feed conversion efficiency of Yellow River carp (Cyprinus carpio haematopterus).

Feed cost is one of the largest variable input costs in aquaculture. In general, dietary energy is directed toward protein deposition and muscle growth. However, most of the dietary energy will be used to support body maintenance if feed conversion efficiency (FCE) is relatively low. Thus, improving feed efficiency will make great contributions to the productivity, profitability, and sustainability of fish farming industry. In the present study, we performed comparative transcriptome analyses of brain and intestine tissues from extreme FCE groups and identified differentially expressed genes (DEGs) and regulatory pathways that may be involved in FCE and related traits in one of the important common carp strains of China, the Yellow River carp (Cyprinus carpio haematopterus). Totally, 557 and 341 DEGs between high and low FCE groups were found in brain and intestine tissues, respectively, including 66 up- and 491 down-regulated in brain of high FCE group and 282 up- and 59 down-regulated in intestine of high FCE group (p < 0.01, FDR < 0.05). These DEGs are mainly involved in metabolic pathway, organismal system and genetic information processing pathway. Finally, 20 key DEGs potentially involved in FCE of Yellow River carp were identified from these two tissues. Expression patterns (up or down regulation in the high or low FCE group) of these DEGs have been successfully validated by quantitative real-time PCR of 10 unigenes. This study provides insights into the genetic mechanisms underlying feed efficiency in Yellow River carp and supplies valuable FCE-related candidate gene resources for potential molecular breeding studies.

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.

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.

P44/42 MAPK signal pathway-mediated hyperphosphorylation of paxillin and redistribution of E-cadherin was involved in microcystin-LR-reduced cellular adhesion in a human liver cell line.

Microcystin-LR (MC-LR) is the most common and toxic variant of microcystins. We hypothesize that p44/42 MAPK (ERK1/2) signal pathway is involved in MC-LR-induced cell adhesion alteration in a human liver cell line-HL7702. We identified that MC-LR constantly activated MEK1/2-ERK1/2 signal pathway for 24 h, 48 h and 72 h in vitro. MC-LR reduced hepatocytes adhesion efficiency. Furthermore, as the focal adhesion biomarker, hyperphosphorylation of paxillin (ser83) was induced by MC-LR, which can be blocked by ERK1/2 pathway inhibitor (U0126) and was enhanced after hepatocytes transfected with pCMV6-MAPK plasmid. E-cadherin, as a biomarker which reflects the dynamic of cell-cell adhesion, its redistribution in hepatocytes was induced by MC-LR, and these redistribution and colocalization can be attenuated by U0126. Furthermore, MC-LR increased the co-localization efficiency of p-ERK1/2 with E-cadherin and paxillin. Finally, MC-LR-induced adhesive alteration of hepatocytes can be blocked by ERK1/2 signal pathway inhibitor. These data suggest ERK1/2-phospho-paxillin (ser83)/E-cadherin axis is involved in MC-LR toxic mechanism, which probably provides adaptive protection against MC-LR-induced hepatocytes adhesion changes.