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In terms of taxonomic status, common carp ( Cyprinus carpio , Cyprininae) and crucian carp ( Carassius auratus , Cyprininae) are different species; however, in this study, a newborn homodiploid crucian carp-like fish (2 n =100) (2 n NCRC) lineage (F1–F3) was established from the interspecific hybridization of female common carp (2 n =100)×male blunt snout bream ( Megalobrama amblycephala , Cultrinae, 2 n =48). The phenotypes and genotypes of 2 n NCRC differed from those of its parents but were closely related to those of the existing diploid crucian carp. We further sequenced the whole mitochondrial (mt) genomes of the 2 n NCRC lineage from F1 to F3. The paternal mtDNA fragments were stably embedded in the mt-genomes of F1–F3 generations of 2 n NCRC to form chimeric DNA fragments. Along with this chimeric process, numerous base sites of F1–F3 generations of 2 n NCRC underwent mutations. Most of these mutation sites were consistent with the existing diploid crucian carp. Moreover, the mtDNA organization and nucleotide composition of 2 n NCRC were more similar to those of the existing diploid crucian carp than those of the parents. The inheritable chimeric DNA fragments and mutant loci in the mt-genomes of different generations of 2 n NCRC provided important evidence of the mtDNA change process in the newborn lineage derived from hybridization of different species. Our findings demonstrated for the first time that the paternal mtDNA were transmitted into the mt-genomes of homodiploid lineage, which provided new insights into the existence of paternal mtDNA in the mtDNA inheritance.

Doublesex and mab-3-related transcription factor (dmrt) genes are widely distributed across various biological groups and play critical roles in sex determination and neural development. Here, we applied bioinformatics methods to exam cross-species changes in the dmrt family members and evolutionary relationships of the dmrt genes based on genomes of 17 fish species. All the examined fish species have dmrt1–5 while only five species contained dmrt6. Most fish harbored two dmrt2 paralogs (dmrt2a and dmrt2b), with dmrt2b being unique to fish. In the phylogenetic tree, 147 DMRT are categorized into eight groups (DMRT1–DMRT8) and then clustered in three main groups. Selective evolutionary pressure analysis indicated purifying selections on dmrt1–3 genes and the dmrt1–3–2(2a) gene cluster. Similar genomic conservation patterns of the dmrt1–dmrt3–dmrt2(2a) gene cluster with 20-kb upstream/downstream regions in fish with various sex-determination systems were observed except for three regions with remarkable diversity. Synteny analysis revealed that dmrt1, dmrt2a, dmrt2b, and dmrt3–5 were relatively conserved in fish during the evolutionary process. While dmrt6 was lost in most species during evolution. The high conservation of the dmrt1–dmrt3–dmrt2(2a) gene cluster in various fish genomes suggests their crucial biological functions while various dmrt family members and sequences across fish species suggest different biological roles during evolution. This study provides a molecular basis for fish dmrt functional analysis and may serve as a reference for in-depth phylogenomics.

Largemouth bass (LMB; Micropterus salmoides) has been an economically important fish in North America, Europe, and China. This study obtained a chromosome-level genome assembly of LMB using PacBio and Hi-C sequencing. The final assembled genome is 964 Mb, with contig N50 and scaffold N50 values of 1.23 Mb and 36.48 Mb, respectively. Combining with RNA sequencing data, we annotated a total of 23,701 genes. Chromosomal assembly and syntenic analysis proved that, unlike most Perciformes with the popular haploid chromosome number of 24, LMB has only 23 chromosomes (Chr), among which the Chr1 seems to be resulted from a chromosomal fusion event. LMB is phylogenetically closely related to European seabass and spotted seabass, diverging 64.1 million years ago (mya) from the two seabass species. Eight gene families comprising 294 genes associated with ionic regulation were identified through positive selection, transcriptome and genome comparisons. These genes involved in iron facilitated diffusion (such as claudin, aquaporins, sodium channel protein and so on) and others related to ion active transport (such as sodium/potassium-transporting ATPase and sodium/calcium exchanger). The claudin gene family, which is critical for regulating cell tight junctions and osmotic homeostasis, showed a significant expansion in LMB with 27 family members and 68 copies for salinity adaptation. In summary, we reported the first high-quality LMB genome, and provided insights into the molecular mechanisms of LMB adaptation to fresh and brackish water. The chromosome-level LMB genome will also be a valuable genomic resource for in-depth biological and evolutionary studies, germplasm conservation and genetic breeding of LMB.

The white crucian carp (Carassius auratus cuvieri, WCC) not only is one of the most economically important fish in Asia, characterized by strong reproductive ability and rapid growth rates, but also represents a good germplasm to produce hybrid progenies with heterosis. Gene knockout technique provides a safe and acceptant way for fish breeding. Achieving gene knockout in WCC and its hybrid progeny will be of great importance for both genetic studies and hybridization breeding. Tyrosinase (TYR) is a key enzyme in melanin synthesis. Depletion of tyr in zebrafish and mice results in mosaic pigmentation or total albinism. Here, we successfully used CRISPR-Cas9 to target tyr in WCC and its hybrid progeny (WR) derived from the cross of WCC (♀) and red crucian carp (Carassius auratus red var., RCC, ♂). The level of TYR protein was significantly reduced in mutant WCC. Both the mutant WCC and the mutant WR showed different degrees of melanin reduction compared with the wild-type sibling control fish, resulting from different mutation efficiency ranging from 60% to 90%. In addition, the transcriptional expression profiles of a series of pivotal pigment synthesis genes, i.e. tyrp1, mitfa, mitfb, dct and sox10, were down-regulated in tyr-CRISPR WCC, which ultimately caused a reduction in melanin synthesis. These results demonstrated that tyr plays a key role in melanin synthesis in WCC and WR, and CRISPR-Cas9 is an effective tool for modifying the genome of economical fish. Furthermore, the tyr-CRISPR models could be valuable in understanding fundamental mechanisms of pigment formation in non-model fish.

Focusing on adaptation of aquatic organisms, especially fish, can help elucidate complex dynamics in freshwater ecology. The differences in genetic and epigenetic regulation between diploid and triploid Carassius auratus affect survival under eutrophication. To identify the underlying mechanisms that lead to better adaption of triploids than diploids, we compared mRNA and microRNA (miRNA) expressions in liver tissue of diploid and triploid individuals obtained from the Dongting lake water system in central China. Differential expression analysis revealed that 566 transcripts were significantly up-regulated, whereas 758 were down-regulated in triploids; of these differentially expressed transcripts, 33 transcripts including cacna1d, nfkb2, hspa1 and fgfr4 were involved in the MAPK signaling pathway, and eight transcripts were determined to be regulated by seven miRNAs. Additionally, four of 25 differential expressed (DE) transcripts (mhc1, irf7, nfkb2 and pik3c) involving the viral carcinogenesis pathway were regulated by four miRNAs. Furthermore, genetic polymorphisms analysis showed that more heterozygous mutations were detected in triploids than diploids. The dN/dS results revealed that 21 genes were under positive selection (dN/dS>1) in C. auratus complex. We hypothesize that these changes related to genetic and epigenetic regulation may be caused by abiotic stresses, and facilitate adaptation to environmental changes.

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.

Hybridization can combine the genomes of different strains or species, which leads to changes of genotype and phenotype in the hybrids. In this study, we aimed to investigate the genetic variations of hybrids (WR-F1 and WR-F2) derived from the intraspecific hybridization of white crucian carp (Carassius auratus cuvieri, WCC, ♀) and red crucian carp (Carassius auratus red var., RCC, ♂). Here, we compared the orthologous genes in the liver transcriptomes of hybrids with those of WCC and RCC, and classified the orthologous genes into eight gene patterns within three categories (chimera, mutant, and biparental origin genes). The results revealed 19.04%, 4.17% chimeric genes and 6.90%, 5.05% mutations of orthologous genes in WR-F1 and WR-F2 respectively. Seventeen of twenty-three characterized genes (77%) were confirmed to be the chimeras at the genomic DNA level. The GO classification discovered that some chimeric and mutant genes were related to metabolic process, immune system and developmental process in WR-F1. Our results provide the new evidence that hybridization can combine the parental genomes, leading to changes in the genotype of the resultant hybrids. This is the first report on the formation of chimeric genes from fish intraspecific hybridization, which is potentially interesting from the context of both evolution and the genetic breeding of fish.

Distant hybridization occurs widely in fishes and is a useful strategy to produce different ploidy offsprings. In this study, we obtained androgenetic blunt snout bream (2n = 48, ADBSB), triploid hybrids (3n = 124, 3nJB) and tetraploid hybrids (4n = 148, 4nJB) from the hybridization of two species from different subfamilies: Carassius cuvieri (♀, 2n = 100, JCC) and Megalobrama amblycephala (♂, 2n = 48, BSB). The ploidy levels of ADBSB, 3nJB and 4nJB hybrids were confirmed by counting chromosomal numbers, forming chromosomal karyotype, and measuring DNA content. In the phenotypes and reproductive traits, 3nJB and 4nJB exhibited significantly divergences from JCC and BSB, but ADBSB took after BSB. Fluorescence in situ hybridization (FISH) and 5S rDNA analyses revealed that the genetic traits of the offsprings compared with those of their parents. This is the first report on the coexistence in vertebrates of viable androgenetic, triploid and tetraploid hybrids being produced by crossing JCC and BSB. The formation of the different ploidy offspring is great significance in both evolution and fish genetic breeding and it also provides a good model for studying the genomic variation in the first generation of interspecific hybrids.

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.

BackgroundDistant hybridization can generate changes in phenotypes and genotypes that lead to the formation of new hybrid lineages with genetic variation. In this study, the establishment of two bisexual fertile carp lineages, including the improved diploid common carp (IDC) lineage and the improved diploid scattered mirror carp (IDMC) lineage, from the interspecific hybridization of common carp (Cyprinus carpio, 2n = 100) (♀) × blunt snout bream (Megalobrama amblycephala, 2n = 48) (♂), provided a good platform to investigate the genetic relationship between the parents and their hybrid progenies.ResultIn this study, we investigated the genetic variation of 12Hoxgenes in the two types of improved carp lineages derived from common carp (♀) × blunt snout bream (♂).Hoxgene clusters were abundant in the first generation of IDC, but most were not stably inherited in the second generation. In contrast, we did not find obvious mutations inHoxgenes in the first generation of IDMC, and almost all theHoxgene clusters were stably inherited from the first generation to the second generation of IDMC. Interestingly, we found obvious recombinant clusters ofHoxgenes in both improved carp lineages, and partially recombinant clusters ofHoxgenes were stably inherited from the first generation to the second generation in both types of improved carp lineages. On the other hand, someHoxgenes were gradually becoming pseudogenes, and some genes were completely pseudogenised in IDC or IDMC.ConclusionsOur results provided important evidence that distant hybridization produces rapid genomic DNA changes that may or may not be stably inherited, providing novel insights into the function of hybridization in the establishment of improved lineages used as new fish resources for aquaculture.

Polyploidization can significantly alter the size of animal gametes. Autotetraploid fish (RRRR, 4nRR = 200) (4nRR) possessing four sets of chromosomes were derived from whole-genome duplication in red crucian carp (RR, 2n = 100) (RCC). The diploid eggs of the 4nRR fish were significantly larger than the eggs of RCC. To explore the differences between the ovaries of these two ploidies of fishes at the molecular level, we compared the ovary transcriptome profiles of 4nRR fish and RCC and identified differentially expressed genes (DEGs). A total of 19,015 unigenes were differentially expressed between 4nRR fish and RCC, including 12,591 upregulated and 6,424 downregulated unigenes in 4nRR fish. Functional analyses revealed that eight genes (CDKL1, AHCY, ARHGEF3, TGFβ, WNT11, CYP27A, GDF7, and CKB) were involved in the regulation of cell proliferation, cell division, gene transcription, ovary development and energy metabolism, suggesting that these eight genes were related to egg size in 4nRR fish and RCC. We validated the expression levels of these eight DEGs in 4nRR fish and RCC using quantitative PCR. The study results provided insights into the regulatory mechanisms underlying the differences in crucian carp egg sizes.

Gut microbiota play critical roles in host nutrition and metabolism. However, little is known about the genetic effects on the gut microbiota assemblages because a suitable model for investigation is lacking. In the present study, we established the reciprocal hybrid fish lineages derived from the parents with different feeding habits, namely, herbivorous blunt snout bream (Megalobrama amblycephala, BSB, 2n = 48) and carnivorous topmouth culter (Culter alburnus, TC, 2n = 48). We investigated the genetic effects on gut microbiota assemblages by using 16S rRNA gene sequencing. The results showed that the gut characteristics (structure, relative gut length, relative gut mass, and Zihler’s index) differed between the two types of hybrids and the two parents. In particular, a strong correlation between genotype and gut microbial assemblages indicated that host genetic (subgenome) significantly altered the gut microbial communities. In addition, the microbial structures (composition and abundance) in the two types of hybrids were more similar to those in BSB parent (P > 0.05) than to those in TC parent (P < 0.05), and the cellulase contents in the gut (produced by gut microbes) also showed the similar results. The results suggested that the host genomic interaction (mainly subgenome domination) had a sizeable effect on shaping the gut microbiota assemblages in reciprocal hybrid fish. This study enriches our understanding of the relationship between host genetic and gut microbiota assemblages, and provides insight into gut microbiota and metabonomics.

Based on the formation of an autotetraploid fish line (4nAUT, 4n = 200; F2–F11) derived from the distant hybridization of female Carassius auratus red var. (RCC, 2n = 100) × male Megalobrama amblycephala (BSB, 2n = 48), we produced autotriploid hybrids (3nAUT) by crossing females of RCC with males of 4nAUT and allotriploid hybrids (3nALT) by crossing females of Cyprinus carpio (CC, 2n = 100) with males of 4nAUT. The aim of this study was to comparatively investigate the reproductive characteristics of 3nALT and 3nAUT. We investigated morphological traits, chromosomal numbers, DNA content and gonadal development in 3nAUT and 3nALT. The results indicated both 3nAUT and 3nALT possessed 150 chromosomes and were triploid hybrids. The females and males of 3nALT and males of 3nAUT had abnormal gonadal development and could not generate mature eggs or sperm, but the females of 3nAUT had normal gonadal development and generated mature eggs at 2 years old. The females of 3nAUT generated different sizes of eggs, which fertilized with haploid sperm from RCC and formed viable diploid, triploid, and tetraploid offspring. The formation of these two kinds of triploid hybrids provides an ideal model for studying the reproductive traits of triploid hybrids, which is of great value in animal genetics and reproductive biology.

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.

Background Nucleolar dominance is an epigenetic phenomenon that occurs in interspecific hybrids and involves the expression of 45S rRNA genes inherited from one progenitor due to the silencing of the other progenitor’s rRNA genes. In this paper, changes in the genetics and expression of 45S rRNA genes in F1 and F2 hybrid progeny of blunt snout bream (BSB, Megalobrama amblycephala) × topmouth culter (TC, Culter alburnus) are investigated. Results The 45S rDNA loci were analyzed by cloning, RT-PCR and sequencing methods. The results show that nucleolar dominance patterns differ in the F1 and F2 hybrids. In the F1 hybrids of BSB × TC, all the tested individuals inherited and expressed the 45S rRNA genes of both BSB and TC, indicating that nucleolar dominance is not established in the F1 hybrids. However, in the F2 hybrids of BSB × TC, five patterns are observed. Pattern 1 inherits and expresses only the 45S rRNA gene of BSB. Pattern 2 inherits the 45S rRNA gene from both BSB and TC, but only expresses the 45S rRNA of BSB. Pattern 3 inherits and expresses the 45S rRNA gene from both BSB and TC. Pattern 4 inherits the 45S rRNA gene from both BSB and TC, but only expresses the 45S rRNA gene of TC. Pattern 5 inherits and expresses only the 45S rRNA gene of TC. Conclusions Nucleolar dominance shows distinctive patterns in intergeneric hybrids of BSB × TC. It is not established in F1 hybrids and is random in F2 hybrids. This study provides new insights into the phenomenon of nucleolar dominance in genetic hybrids in vertebrates. Electronic supplementary material The online version of this article (doi:10.1186/s12863-016-0457-3) 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.