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Interleukin-1 receptor-associated kinase 4 (IRAK4) plays a crucial role in the Toll-like receptor/IL-1R signal pathway, which mediates the downstream signal transduction involved in innate and adaptive immunity. In the present study, an IRAK4 homologue (named as MaIRAK4) from blunt snout bream (Megalobrama amblycephala) was cloned and characterized. The open reading frame (ORF) of MaIRAK4 contains 1422 nucleotides, encoding a putative protein of 473 amino acids. Protein structural analysis revealed that MaIRAK4 has an N-terminal death domain (DD) and a central kinase domain (S_TKc), similar to those of mammals and other fishes. Multiple sequence alignment demonstrated that MaIRAK4 is highly homologous with that of grass carp (97.67%). The qRT-PCR analysis showed that MaIRAK4 expressed widely in all examined tissues, including heart, liver, spleen, kidney, head-kidney, gill, intestine and muscle, with the highest expression in the liver and spleen. After stimulation with LPS, MaIRAK4 expression upregulated significantly and reached a peak at 6 h and 12 h post LPS stimulation in the spleen and head-kidney, respectively. After challenge with Aeromonas hydrophila, MaIRAK4 expression peaked at 48 h and 72 h in spleen/head-kidney and liver, respectively. These results implied that MaIRAK4 is involved in the host defense against bacterial infection. Subcellular localization analysis indicated that MaIRAK4 distributed in the cytoplasm. Co-immunoprecipitation and subcellular co-localization assay revealed that MaIRAK4 can combine with MaMyD88 through DD domain. MaIRAK4 overexpression can induce slightly the NF-κB promoter activity in HEK 293 cells. However, the activity of NF-κB promoter was dramatically enhanced after co-transfection with MaIRAK4 and MaMyD88 plasmids. The results showed that MaIRAK4 was involved in NF-κB signal pathway mediated by maMyD88. The expression level of pro-inflammatory cytokines (IL-1β, IL-6, IL-8 and TNF-α) decreased significantly after the siRNA-mediated knockdown of MaIRAK4. Together, these results suggest that MaIRAK4 plays an important function in the innate immunity of M. amblycephala by inducing cytokines expression.

Background Interspecific postzygotic reproduction isolation results from large genetic divergence between the subgenomes of established hybrids. Polyploidization immediately after hybridization may reset patterns of homologous chromosome pairing and ameliorate deleterious genomic incompatibility between the subgenomes of distinct parental species in plants and animals. However, the observation that polyploidy is less common in vertebrates raises the question of which factors restrict its emergence. Here, we perform analyses of the genome, epigenome, and gene expression in the nascent allotetraploid lineage (2.95 Gb) derived from the intergeneric hybridization of female goldfish (Carassius auratus, 1.49 Gb) and male common carp (Cyprinus carpio, 1.42 Gb), to shed light on the changes leading to the stabilization of hybrids. Results We firstly identify the two subgenomes derived from the parental lineages of goldfish and common carp. We find variable unequal homoeologous recombination in somatic and germ cells of the intergeneric F1 and allotetraploid (F22 and F24) populations, reflecting high plasticity between the subgenomes, and rapidly varying copy numbers between the homoeolog genes. We also find dynamic changes in transposable elements accompanied by genome merger and duplication in the allotetraploid lineage. Finally, we observe the gradual decreases in cis-regulatory effects and increases in trans-regulatory effects along with the allotetraploidization, which contribute to increases in the symmetrical homoeologous expression in different tissues and developmental stages, especially in early embryogenesis. Conclusions Our results reveal a series of changes in transposable elements, unequal homoeologous recombination, cis- and trans-regulations (e.g. DNA methylation), and homoeologous expression, suggesting their potential roles in mediating adaptive stabilization of regulatory systems of the nascent allotetraploid lineage. The symmetrical subgenomes and homoeologous expression provide a novel way of balancing genetic incompatibilities, providing a new insight into the early stages of allopolyploidization in vertebrate evolution.

Background Hybridization, which can quickly merge two or more divergent genomes and form new allopolyploids, is an important technique in fish genetic breeding. However, the merged subgenomes must adjust and coexist with one another in a single nucleus, which may cause subgenome interaction and dominance at the gene expression level and has been observed in some allopolyploid plants. In our previous studies, newly formed allodiploid hybrid fish derived from herbivorous Megalobrama amblycephala (♀) × carnivorous Culter alburnus (♂) had herbivorous characteristic. It is thus interesting to further characterize whether the subgenome interaction and dominance derive dietary adaptation of this hybrid fish. Results Differential expression, homoeolog expression silencing and bias were investigated in the hybrid fish after 70 days of adaptation to carnivorous and herbivorous diets. A total of 2.65 × 108 clean reads (74.06 Gb) from the liver and intestinal transcriptomes were mapped to the two parent genomes based on specific SNPs. A total of 2538 and 4385 differentially expressed homoeologous genes (DEHs) were identified in the liver and intestinal tissues between the two groups of fish, respectively, and these DEHs were highly enriched in fat digestion and carbon metabolism, amino acid metabolism and steroid biosynthesis. Furthermore, subgenome dominance were observed in tissues, with paternal subgenome was more dominant than maternal subgenome. Moreover, subgenome expression dominance controlled functional pathways in metabolism, disease, cellular processes, environment and genetic information processing during the two dietary adaptation processes. In addition, few but sturdy villi in the intestine, significant fat accumulation and a higher concentration of malondialdehyde in the liver were observed in fish fed carnivorous diet compared with fish fed herbivorous diet. Conclusions Our results indicated that diet drives phenotypic and genetic variation, and the asymmetric expression of homoeologous genes (including differential expression, expression silencing and bias) may play key roles in dietary adaptation of hybrid fish. Subgenome expression dominance may contribute to uncovering the mechanistic basis of heterosis and also provide perspectives for fish genetic breeding and application.

Meiosis is the key process for producing mature gametes. A natural fertile triploid Carassius auratus population (3nDTCC) and an artificially derived sterile triploid crucian carp (3nCC) have been previously observed, providing suitable model organisms for investigating meiosis characteristics in triploid fish. In the present study, the microstructures and ultrastructures of spermatogenesis were studied in these fishes. TdT-mediated dUTP nick end labeling detection was performed to investigate the apoptosis of spermatocytes. Fluorescence in situ hybridization was employed to trace chromatin pairing. In addition, the mRNA expressions of cell cycle-related genes (i.e., cell division control 2 and cell cycle protein B) were determined by quantitative realtime polymerase chain reaction to illustrate the molecular mechanism of abnormal meiosis in the 3nCC. The results showed that the 3nCC undergoes an irregular prophase I, with the chromosomes distributed in a unipolar radial manner and exhibiting partial pairing, hindered metaphase I, and degenerated cells in the subsequent stages. Meanwhile, the 3nDTCC presented a relatively regular meiotic prophase I with complete conjugate chromosome pairs and chromosomes distributed along the karyotheca, which were presented as a ring structure by slicing. Only the spreads with 130-150 irregular chromosomes can be easily detected in the 3nDTCC, suggesting that it may undergo an abnormal metaphase I. This study provides new insights into the meiosis of fertile and sterile triploid cyprinid fish.

Triploid crucian carp (TCC) is obtained by hybridization of female diploid red crucian carp (Carassius auratus red var., RCC) and male allotetraploid hybrids. In this study, high-throughput sequencing was used to conduct the transcriptome analysis of the female hypothalamus of diploid RCC, diploid common carp (Cyprinus carpio L., CC) and TCC. The key functional expression genes of the hypothalamus were obtained through functional gene annotation and differential gene expression screening. A total of 71.56 G data and 47,572 genes were obtained through sequencing and genome mapping, respectively. The Fuzzy Analysis Clustering assigned the differentially expressed genes (DEGs) into eight groups, two of which, overdominance expression (6005, 12.62%) and underdominance expression (3849, 8.09%) in TCC were further studied. KEGG enrichment analysis showed that the DEGs in overdominance were mainly enriched in four pathways. The expression of several fertility-related genes was lower levels in TCC, whereas the expression of several growth-related genes and immune-related genes was higher levels in TCC. Besides, 15 DEGs were verified by quantitative real-time PCR (qPCR). The present study can provide a reference for breeding sterility, fast-growth, and disease-resistant varieties by distant hybridization.

Distant hybridization is an important technique in fish genetic breeding. In this study, based on the establishment of an allodiploid fish lineage (BT, 2n=48, F1–F6) derived from distant hybridization between female Megalobrama amblycephala (BSB, 2n=48) and male Culter alburnus (TC, 2n=48), and the backcross progeny (BTB, 2n=48) derived by backcrossing female F1 of BT to male BSB, an improved hybrid bream (BTBB, 2n=48) was obtained by backcrossing BTB (♀) to BSB (♂). Moreover, the morphological and genetic characteristics of BTBB individuals were investigated; BTBB was similar to BSB in appearance but had a higher body height than BSB. The study results regarding chromosome numbers and DNA content indicated that BTBB is a diploid hybrid fish. The 5S rDNA and Hox gene of BTBB were inherited from the original parents. Gonadal development in BTBB was normal. On the other hand, BTBB had a faster growth rate, higher muscle protein level, and lower muscle carbohydrate level than BSB. Hence, bisexual fertile BTBB is promoted and can be applied as a high-quality fish, and it can also be used as a new fish germplasm resource to develop high-quality fish further. Thus, this study is of great significance for fish genetic breeding.

Homoeologous recombination (HR), the exchange of homoeologous chromosomes, contributes to subgenome adaptation to diverse environments by producing various phenotypes. However, the potential relevance of HR and innate immunity is rarely described in triploid cyprinid fish species. In our study, two allotriploid genotypes (R2C and RC2), whose innate immunity was stronger than their inbred parents (Carassius auratus red var. and Cyprinus carpio L.), were obtained from backcrossing between male allotetraploids of C. auratus red var.×C. carpio L. and females of their two inbred parents, respectively. The work detected 140 HRs shared between the two triploids at the genomic level. Further, transcriptions of 54 homoeologous recombinant genes (HRGs) in R2C and 65 HRGs in RC2 were detected using both Illumina and PacBio data. Finally, by comparing expressed recombinant reads to total expressed reads in each of the genes, a range of 0.1%–10% was observed in most of the 99–193 HRGs, of which six recombinant genes were classified as “response to stimulus”. These results not only provide a novel way to predict HRs in allopolyploids based on cross prediction at both genomic and transcriptional levels, but also insight into the potential relationship between HRs related to innate immunity and adaptation of the triploids and allotetraploids.

Ganoderma lucidum has salutary effects on tumor treatment, including pancreatic cancer and hepatocellular carcinoma. However, the molecular mechanisms underlying Ganoderma lucidum therapy is obscure. In this study, the Hepa1-6-bearing C57 BL/6 mouse model was utilized to explore the therapeutic efficacy of Ganoderma lucidum extract (GLE), documenting that it could effectively inhibit tumor growth. The microRNA (miRNA) profiles of GLE-treated and untreated mice were detected, and 25 differentially expressed (DE) miRNAs were determined, including 24 up-expressed and one down-expressed miRNAs. Using the ClusterOne algorithm, 8 hub miRNAs were isolated from the established miRNA-target network. The qRT-PCR assay demonstrated that these 8 miRNAs were up-expressed in the GLE treated tumor mice. Furthermore, the mRNA profiles showed that there are 76 DE mRNAs between GLE treated and model groups. The protein-protein interaction (PPI) network shows that Cntn1, Irs1, Nfkbia, Rybp and Ywhaz playing important roles, and qRT-PCR further revealed they were down-expressed in GLE treated Hepa1-6-bearing C57 BL/6 mice. The rebuilt miRNA-target network was shown that these 5 mRNAs were regulated by mmu-mir-23a-5p, -3102-3p, -337-3p, and -467a-3p, respectively. This study suggested that these 4 interesting miRNAs were potential biomarkers for evaluation of GLE efficacy, which may down-regulate the expression of Cntn1, Irs1, Nfkbia, Rybp and Ywhaz, and mediate many signaling pathways occurring in tumor treatment.

Background Hybridization and polyploidization are regarded as the major driving forces in plant speciation, diversification, and ecological adaptation. Our knowledge regarding the mechanisms of duplicated-gene regulation following genomic merging or doubling is primarily derived from plants and is sparse for vertebrates. Results We successfully obtained an F1 generation (including allodiploid hybrids and triploid hybrids) from female Megalobrama amblycephala Yih (BSB, 2n = 48) × male Xenocypri davidi Bleeker (YB, 2n = 48). The duplicated-gene expression patterns of the two types of hybrids were explored using RNA-Seq data. In total, 5.44 × 108 (69.32 GB) clean reads and 499,631 assembled unigenes were obtained from the testis transcriptomes. The sequence similarity analysis of 4265 orthologs revealed that the merged genomes were dominantly expressed in different ploidy hybrids. The differentially expressed genes in the two types of hybrids were asymmetric compared with those in both parents. Furthermore, the genome-wide expression level dominance (ELD) was biased toward the maternal BSB genome in both the allodiploid and triploid hybrids. In addition, the dosage-compensation mechanisms that reduced the triploid expression levels to the diploid state were determined in the triploid hybrids. Conclusions Our results indicate that divergent genomes undergo strong interactions and domination in allopolyploid offspring. Genomic merger has a greater effect on the gene-expression patterns than genomic doubling. The various expression mechanisms (including maternal effect and dosage compensation) in different ploidy hybrids suggest that the initial genomic merger and doubling play important roles in polyploidy adaptation and evolution. Electronic supplementary material The online version of this article (10.1186/s12864-018-4883-7) contains supplementary material, which is available to authorized users.

Rediploidization is considered to be a part of the evolutionary history of allotetraploids, and resulted in the emergence of novel epigenetic regulatory activities. To study the changing patterns of gene expression following the reduction of a genome by 50%, we used RNA-seq and quantitative real-time PCR (qPCR) to investigate total gene expression and homoeolog expression in three hybrids of a C. auratus red var. (2n = 100, ♀) (R) and C. carpio (2n = 100, ♂) (C) (i.e., F1, F18, and G4) and their original parents. A comparison of homoeolog expression between G4 and F18 identified 7 genes (0.22%) that exhibited novel R/C homoeolog expression patterns in G4, while 4 genes (0.12%) were affected by R/C homoeolog silencing. We determined the direction and extent of the homoeolog expression bias (HEB). The C-HEB genes (i.e., nrp1a and igf1) and R-HEB genes (i.e., fgf23 and esm1) provided insights into the effects of the dominance of one parental homoeolog expression on growth regulation. This dominance may contribute to the rapid growth of G4 fish. Our findings may be relevant for clarifying the relationship between growth heterosis and differences in homoeolog expression patterns.

Hybridization drives rapid speciation by shaping novel genotypic and phenotypic profiles. Genomic incompatibility and transcriptome shock have been observed in hybrids, although this is rarer in animals than in plants. Using the newly sequenced genomes of the blunt snout bream (Megalobrama amblycephala [BSB]) and the topmouth culter (Culter alburnus [TC]), we focused on the sequence variation and gene expression changes in the reciprocal intergeneric hybrid lineages (F1–F3) of BSB × TC. A genome-wide transcriptional analysis identified 145–974 expressed recombinant genes in the successive generations of hybrid fish, suggesting the rapid emergence of allelic variation following hybridization. Some gradual changes of gene expression with additive and dominance effects and various cis and trans regulations were observed from F1 to F3 in the two hybrid lineages. These asymmetric patterns of gene expression represent the alternative strategies for counteracting deleterious effects of the subgenomes and improving adaptability of novel hybrids. Furthermore, we identified positive selection and additive expression patterns in transforming growth factor, beta 1b (tgfb1b), which may account for the morphological variations of the pharyngeal jaw in the two hybrid lineages. Our current findings provide insights into the evolution of vertebrate genomes immediately following hybridization.

Onychostoma brevibarba is a new discovered species which is distributed in Xiang Jiang River of the middle Chang Jiang basin in Hunan Province, South China. In this study, the ploidy levels of O. brevibarba were confirmed by counting chromosomal numbers and analyzing karyotype. The complete mitochondrial genome of O. brevibarba was determined and analyzed. Besides, we firstly performed the full-length transcriptome of O. brevibarba derived from 5 different tissues using the PacBio SMRT sequencing. The result shows that O. brevibarba was a diploid with 50 chromosomes [corrected]. The complete mitogenome of O. brevibarba was 16,602 bp in size and very similar (89.1-91.3%) to that of other Onychostoma species but was distinct from all congeners. The full-length transcriptome dataset of O. brevibarba comprised 120,239 unigenes. Among the unigenes, 91,542 were functionally annotated, whereas 26,794 were found to have two or more isoforms. This study could provide many new insights into cytology and molecular characteristics of O. brevibarba; it laid the foundation for further exploration of the genomic signatures of species of Onychostoma.

Hybridization is widely used. However, for a long time, systematic theories and technologies related to hybridization in fish have been lacking. In this study, through long-term systematic research, we investigated and obtained the main rules regarding inheritance and reproduction related to fish distant hybridization. Furthermore, we established one-step and multistep breeding technologies that were suitable for interspecific hybridization and intraspecific hybridization. Simultaneously, we used these two breeding technologies to produce a batch of diploid fish lineages and tetraploid fish lineages and improved fishes. In addition, we widely discuss the methods, technologies and results of hybridization breeding, referring to the domestic and foreign literature on fish hybridization. We hope that this paper will be beneficial for the research and application of fish hybrid breeding.

Background Polyploidy and hybridization are both recognized as major forces in evolution. Most of our current knowledge about differences in gene regulation in polyploid hybrids comes from plant studies. The gene expression of diverged genomes and regulatory interactions are still unclear in lower vertebrates. Results We generated 229 million cleaned reads (42.23 Gbp) from triploid of maternal grass carp (Ctenopharyngodon idellus, Cyprininae, 2n = 48) × paternal blunt snout bream (Megalobrama amblycephala, Cultrinae, 2n = 48) and their diploid parents using next-generation sequencing. In total, 157,878 contigs were assembled and 15,444 genes were annotated. We examined gene expression level changes among the parents and their triploid offspring. The mechanisms of dosage compensation that reduced triploid expression levels to the diploid state were determined in triploid fish. In this situation, novel gene expression and gene silencing were observed. Then, we established a model to determine the extent and direction of expression level dominance (ELD) and homoeolog expression bias (HEB) based on the relative expression level among the parents and their triploid offspring. Conclusions Our results showed that the genome-wide ELD was biased toward maternal genome in triploid. Extensive alterations in homoeolog expression suggested a combination of regulatory and epigenetic interactions through the transcriptome network. Additionally, the expression patterns of growth genes provided insights into the relationship between the characteristics of growth and underlying mechanisms in triploids. Regulation patterns of triploid state suggest that various expression levels from the initial genomic merger have important roles in adaptation. Electronic supplementary material The online version of this article (doi:10.1186/s12864-016-3424-5) contains supplementary material, which is available to authorized users.

Hybridization and polyploidization are considered important driving forces that form new epigenetic regulations. To study the changing patterns of expression accompanying hybridization and polyploidization, we used RNA-seq and qRT-PCR to investigate global expression and homoeologue expression in diploid and tetraploid hybrids of Carassius auratus red var. (♀) (R) and Cyprinus carpio (♂) (C). By comparing the relative expression levels between the hybrids and their parents, we defined the expression level dominance (ELD) and homoeologue expression bias (HEB) in liver tissue. The results showed that polyploidization contributed to the conversion of homoeologue ELD. In addition, hybridization had more effect on the change in HEB than polyploidization, while polyploidization had more effect on the change of global gene expression than hybridization. Meanwhile, similar expression patterns were found in growth-related genes. The results suggested that hybridization and polyploidization result in differential degrees of maternal HEB in three tissues (liver, muscle and ovary) tested. The results of this study will increase our understanding of the underlying regulation mechanism of rapid growth in diploid hybrids and allotetraploids. The differential degrees of global expression and homoeologue expression contribute to growth heterosis in newly formed hybrids, ensuring the on-going success of allotetraploid speciation.

Genome-wide association studies (GWAS) have shown that Kruppel-like factor 14 (KLF14) is associated with type 2 diabetes mellitus (T2DM). However, no report has demonstrated a relationship between KLF14 and glucose metabolism. The aim of this study was to determine whether KLF14 is associated with glucose metabolism and insulin signaling in vitro. The mRNA and protein expressions of KLF14 were determined by Real-time PCR and Western blotting. Glucose uptake was assessed by 2-[(3)H]-deoxyglucose (2-DG) uptake. Western blotting was used to identify the activation of insulin signaling proteins. KLF14 mRNA and protein in fat and muscle were significantly decreased in HFD-fed mice, db/db mice and T2DM patients. Overexpression of KLF14 enhanced insulin-stimulated glucose uptake and the activation of Akt kinase in Hepa1-6 cells. The phosphorylation of insulin receptor (InsR), insulin receptor substrate-1(IRS-1), glycogen synthase kinase-3β (GSK-3β) and Akt also elevated significantly by up-regulation of KLF14. KLF14 overexpression in Hepa1-6 cells prevented the inhibition of glucose uptake and Akt phosphorylation induced by high glucose and/or high insulin, or T2DM serum. However, KLF14's ability to increase glucose uptake and Akt activation was significantly attenuated by LY294002, a PI3-kinase inhibitor. These data suggested that KLF14 could increase insulin sensitivity probably through the PI3K/Akt pathway.