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.

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.

ß-elemene inhibits tumor-promoting effect of M2 macrophages in lung cancer.

Macrophages in tumor are mostly M2-polarized and have been reported to promote tumorigenesis, which are also defined as tumor-associated macrophages (TAMs). ß-elemene has therapeutic effects against several cancers, however, it remains unknown whether ß-elemene could inhibit cancer by targeting TAMs. Herein, we examined the effect of ß-elemene on macrophages to elucidate a novel mechanism of ß-elemene in tumor therapy. We showed that the conditioned medium of M2 macrophages promoted lung cancer cells to migration, invasion and epithelial mesenchymal transition, which could be inhibited by ß-elemene. Moreover, ß-elemene regulated the polarization of macrophages from M2 to M1. ß-elemene also inhibited the proliferation, migration, invasion of lung cancer cells and enhanced its radiosensitivity. These results indicate ß-elemene suppresses lung cancer by regulating both macrophages and lung cancer cells, it is a promising drug for combination with chemotherapy or radiotherapy.

Microsatellite-centromere mapping in common carp through half-tetrad analysis in diploid meiogynogenetic families.

Gene-centromere (G-C) mapping provides insights into the understanding of the composition, structure, and evolution of vertebrate genomes. Common carp (Cyprinus carpio) is an important aquaculture fish and has been proposed to undertake tetraploidization. In this study, we selected 214 informative microsatellite markers across 50 linkage groups of a common carp genetic map to perform gene-centromere mapping using half-tetrad analysis. A total of 199 microsatellites were segregated under the Mendelian expectations in at least one of the three gynogenetic families and were used for G-C distance estimation. The G-C recombination frequency (y) ranged from 0 to 0.99 (0.43 on average), corresponding to a fixation index (F) of 0.57 after one generation of gynogenesis. Large y values for some loci together with significant correlation between G-C distances and genetic linkage map distances suggested the presence of high interference in common carp. Under the assumption of complete interference, 50 centromeres were localized onto corresponding linkage groups (LGs) of common carp, with G-C distances of centromere-linked markers per LG ranging from 0 to 10.3 cM (2.9 cM on average). Based on the information for centromere positions, we proposed a chromosome formula of 2n = 100 = 58 m/sm + 42 t/st with 158 chromosome arms for common carp, which was similar to a study observed by cytogenetic method. The examination of crossover distributions along 10 LGs revealed that the proportion of crossover chromatids was overall higher than that of non-crossover chromatids in gynogenetic progenies, indicating high recombination levels across most LGs. Comparative genomics analyses suggested that the chromosomes of common carp have undergone extensive rearrangement after genome duplication. This study would be valuable to elucidate the mechanism of genome evolution and integrate physical and genetic maps in common carp.

Molecular Characterization and Growth Association of Two Apolipoprotein A-Ib Genes in Common Carp (Cyprinus carpio).

Apolipoprotein A-I (ApoA-I) is functionally involved in the transportation and metabolism of lipids in vertebrates. In this study, two isoforms of apoA-Ib in common carp (Cyprinus carpio L.) were characterized. Sequence comparison and phylogenetic analysis showed that C. carpio ApoA-Ib is relatively conserved within cyprinid fishes. During embryonic development, C. carpio apoA-Ib was first expressed at the stage of multi-cells, and the highest mRNA level was observed at the stage of optic vesicle. A ubiquitous expression pattern was detected in various tissues with extreme predominance in the liver. Significantly different expression levels were observed between light and heavy body weight groups and also in the compensatory growth test. Seventeen and eight single-nucleotide polymorphisms (SNPs) were identified in matured mRNA of the C. carpio apoA-Ib.1 and apoA-Ib.2, respectively. Two of these SNPs (apoA-Ib.2-g.183A>T and apoA-Ib.2-g.1753C>T) were significantly associated with body weight and body length in two populations of common carp. These results indicate that apoA-Ib may play an important role in the modulation of growth and development in common carp.

Comparative mapping for bighead carp (Aristichthys nobilis) against model and non-model fishes provides insights into the genomic evolution of cyprinids.

Comparative mapping provides an efficient method to connect genomes of non-model and model fishes. In this study, we used flanking sequences of the 659 microsatellites on a genetic map of bighead carp (Aristichthys nobilis) to comprehensively study syntenic relationships between bighead carp and nine model and non-model fishes. Of the five model and two food fishes with whole genome data, Cyprinus carpio showed the highest rate of positive BLAST hits (95.3 %) with bighead carp map, followed by Danio rerio (70.9 %), Oreochromis niloticus (21.7 %), Tetraodon nigroviridis (6.4 %), Gasterosteus aculeatus (5.2 %), Oryzias latipes (4.7 %) and Fugu rubripes (3.5 %). Chromosomal syntenic analyses showed that inversion was the basic chromosomal rearrangement during genomic evolution of cyprinids, and the extent of inversions and translocations was found to be positively correlated with evolutionary relationships among fishes studied. Among the five investigated cyprinids, linkage groups (LGs) of bighead carp, Hypophthalmichthys molitrix and Ctenopharyngodon idella exhibited a one-to-one relationship. Besides, LG 9 of bighead carp and homologous LGs of silver carp and grass carp all corresponded to the chromosomes 10 and 22 of zebrafish, suggesting that chromosomal fission may have occurred in the ancestor of zebrafish. On the other hand, LGs of bighead carp and common carp showed an approximate one-to-two relationship with extensive translocations, confirming the occurrence of a 4th whole genome duplication in common carp. This study provides insights into the understanding of genome evolution among cyprinids and would aid in transferring positional and functional information of genes from model fish like zebrafish to non-model fish like bighead carp.

Novel single nucleotide polymorphisms of the insulin-like growth factor-I gene and their associations with growth traits in common carp (Cyprinus carpio L.).

Insulin-like growth factor-I (IGF-I) plays an important role in the growth and development of vertebrates. To study polymorphisms of IGF-I, we screened a total of 4555 bp of genomic sequences in four exons and partial introns for the discovery of single nucleotide polymorphism (SNP) in common carp (Cyprinus carpio). Three SNPs (g.3759T>G, g.7627T>A and g.7722T>C) in intron 2 and a nonsynonymous SNP (g.7892C>T) in exon 3 were identified in a pilot population including random parents and their progenies. 289 progenies were further genotyped for studying possible associations between genotypes or combined genotypes and growth traits. The results showed that the locus g.7627T>A was significantly associated with body weight and body length, and fish with genotype AA had a mean body weight 5.9% higher than those with genotype TT. No significant associations were observed between genotypes of other loci and growth traits. However, when both g.7627T>A and g.7722T>C were considered, the combined genotype TT/TT was extremely associated with the lowest values of body length and body weight and the highest K value in comparison with other diplotypes (p < 0.01). These results suggest that genotype AA at g.7627T>A and its combined genotypes with alleles from another locus have positive effects on growth traits, which would be a candidate molecular marker for further studies in marker-assisted selection in common carp.

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.

Molecular characterization of myostatin (MSTN) gene and association analysis with growth traits in the bighead carp (Aristichthys nobilis).

Myostatin (MSTN) is a member of the transforming growth factor-ß superfamily and functions as a negative regulator of skeletal muscle development and growth. In this study, the bighead carp MSTN gene (AnMSTN for short) was cloned and characterized. The 3,769 bp genomic sequence of AnMSTN consisted of three exons and two introns, and the full length cDNA (2,141 bp) of the gene had an open reading frame encoding a polypeptide of 375 amino acids. The deduced amino acid sequence of AnMSTN showed 67.1-98.7 % homology with MSTNs of avian, mammalian and teleostean species. Sequence comparison and phylogenetic analysis confirmed the MSTNs were conserved throughout the vertebrates and AnMSTN belonged to MSNT-1 isoform. AnMSTN was expressed in various tissues with the highest expression in muscle. Two single nucleotide polymorphisms, g.1668T > C in intron 2 and g.2770C > A in 3' UTR, were identified in AnMSTN by sequencing PCR fragments, and genotyped by SSCP. Association analysis showed that g.2770C > A genotypes were significantly associated with total length, body length and body weight (P < 0.01). These results suggest that AnMSTN involves in the regulation of growth, and this polymorphism would be informative for further studies on selective breeding in bighead carp.

Polymorphisms in Myostatin Gene and associations with growth traits in the common carp (Cyprinus carpio L.).

Myostatin (MSTN) is a member of the transforming growth factor-ß superfamily that negatively regulates skeletal muscle development and growth. In the present study, partial genomic fragments of MSTN were screened for single nucleotide polymorphisms (SNPs) in selected common carp individuals from wild populations, and two SNPs in intron 2 (c.371 + 749A > G, c.371 + 781T > C) and two synonymous SNPs in exon 3 (c.42A > G, c.72C > T) were identified. Genotyping by direct sequencing of polymerase chain reaction (PCR) products for these four SNPs were performed in 162 individuals from a commercial hatchery population. Association analysis showed that two SNPs in exon 3 were significantly associated with body weight (BW) and condition factor (K), and haplotype analyses revealed that haplotype H7H8 showed better growth performance. Our results demonstrated that some of the SNPs in MSTN may have positive effects on growth traits and suggested that MSTN could be a candidate gene for growth and marker-assisted selection in common carp.

Development and characterization of new single nucleotide polymorphism markers from expressed sequence tags in common carp (Cyprinus carpio).

The common carp (Cyprinus carpio) is an important aquaculture fish worldwide but only limited single nucleotide polymorphism (SNP) markers are characterized from expressed sequence tags (ESTs) in this species. In this study, 1487 putative SNPs were bioinformatically mined from 14,066 online ESTs mainly from the European common carp, with the occurrence rate of about one SNP every 173 bp. One hundred and twenty-one of these SNPs were selected for validation using PCR fragment sequencing, and 48 out of 81 primers could amplify the expected fragments in the Chinese common carp genome. Only 26 (21.5%) putative SNPs were validated, however, 508 new SNPs and 68 indels were identified. The ratios of transitions to transversions were 1.77 for exon SNPs and 1.05 for intron SNPs. All the 23 SNPs selected for population tests were polymorphic, with the observed heterozygosity (Ho) ranging from 0.053 to 0.526 (mean 0.262), polymorphism information content (PIC) from 0.095 to 0.357 (mean 0.246), and 21 SNPs were in Hardy-Weinberg equilibrium. These results suggest that different common carp populations with geographic isolation have significant genetic variation at the SNP level, and these new EST-SNP markers are readily available for genetics and breeding studies in common carp.

Microsatellite development for an endangered bream Megalobrama pellegrini (Teleostei, Cyprinidae) using 454 sequencing.

Megalobrama pellegrini is an endemic fish species found in the upper Yangtze River basin in China. This species has become endangered due to the construction of the Three Gorges Dam and overfishing. However, the available genetic data for this species is limited. Here, we developed 26 polymorphic microsatellite markers from the M. pellegrini genome using next-generation sequencing techniques. A total of 257,497 raw reads were obtained from a quarter-plate run on 454 GS-FLX titanium platforms and 49,811 unique sequences were generated with an average length of 404 bp; 24,522 (49.2%) sequences contained microsatellite repeats. Of the 53 loci screened, 33 were amplified successfully and 26 were polymorphic. The genetic diversity in M. pellegrini was moderate, with an average of 3.08 alleles per locus, and the mean observed and expected heterozygosity were 0.47 and 0.51, respectively. In addition, we tested cross-species amplification for all 33 loci in four additional breams: M. amblycephala, M. skolkovii, M. terminalis, and Sinibrama wui. The cross-species amplification showed a significant high level of transferability (79%-97%), which might be due to their dramatically close genetic relationships. The polymorphic microsatellites developed in the current study will not only contribute to further conservation genetic studies and parentage analyses of this endangered species, but also facilitate future work on the other closely related species.

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.

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.