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Goldfish (Carassius auratus) have long fascinated evolutionary biologists and geneticists because of their diverse morphological and color variations. Recent genome-wide association studies have provided a clue to uncover genomic basis underlying these phenotypic variations, but the causality between phenotypic and genotypic variations have not yet been confirmed. Here, we edited proposed candidate genes to recreate phenotypic traits and developed a rapid biotechnology approach which combines gene editing with high-efficiency breeding, artificial gynogenesis, and temperature-induced sex reversal to establish homozygous mutants within two generations (approximately eight months). We first verified that low-density lipoprotein receptor-related protein 2B (lrp2aB) is the causal gene for the dragon-eye variation and recreated the dragon-eye phenotype in side-view Pleated-skirt Lion-head goldfish. Subsequently, we demonstrated that the albino phenotype was determined by both homeologs of oculocutaneous albinism type II (oca2), which has subfunctionalized to differentially govern melanogenesis in the goldfish body surface and pupils. Overall, we determined two causal genes for dragon-eye and albino phenotypes, and created four stable homozygous strains and more appealing goldfish with desirable traits. The developed biotechnology approach facilitates precise genetic breeding, which will accelerate re-domestication and recreation of phenotypically desirable goldfish.

Triploids are rare in nature because of difficulties in meiotic and gametogenic processes, especially in vertebrates. The Carassius complex of cyprinid teleosts contains sexual tetraploid crucian carp/goldfish (C. auratus) and unisexual hexaploid gibel carp/Prussian carp (C. gibelio) lineages, providing a valuable model for studying the evolution and maintenance mechanism of unisexual polyploids in vertebrates. Here we sequence the genomes of the two species and assemble their haplotypes, which contain two subgenomes (A and B), to the chromosome level. Sequencing coverage analysis reveals that C. gibelio is an amphitriploid (AAABBB) with two triploid sets of chromosomes; each set is derived from a different ancestor. Resequencing data from different strains of C. gibelio show that unisexual reproduction has been maintained for over 0.82 million years. Comparative genomics show intensive expansion and alterations of meiotic cell cycle-related genes and an oocyte-specific histone variant. Cytological assays indicate that C. gibelio produces unreduced oocytes by an alternative ameiotic pathway; however, sporadic homologous recombination and a high rate of gene conversion also exist in C. gibelio. These genomic changes might have facilitated purging deleterious mutations and maintaining genome stability in this unisexual amphitriploid fish. Overall, the current results provide novel insights into the evolutionary mechanisms of the reproductive success in unisexual polyploid vertebrates.

Assembly of a complete Y chromosome is a significant challenge in animals with an XX/XY sex-determination system. Recently, we created YY-supermale yellow catfish by crossing XY males with sex-reversed XY females, providing a valuable model for Y-chromosome assembly and evolution. Here, we assembled highly homomorphic Y and X chromosomes by sequencing genomes of the YY supermale and XX female in yellow catfish, revealing their nucleotide divergences with only less than 1% and with the same gene compositions. The sex-determining region (SDR) was identified to locate within a physical distance of 0.3 Mb by FST scanning. Strikingly, the incipient sex chromosomes were revealed to originate via autosome–autosome fusion and were characterized by a highly rearranged region with an SDR downstream of the fusion site. We found that the Y chromosome was at a very early stage of differentiation, as no clear evidence of evolutionary strata and classical structure features of recombination suppression for a rather late stage of Y-chromosome evolution were observed. Significantly, a number of sex-antagonistic mutations and the accumulation of repetitive elements were discovered in the SDR, which might be the main driver of the initial establishment of recombination suppression between young X and Y chromosomes. Moreover, distinct three-dimensional chromatin organizations of the Y and X chromosomes were identified in the YY supermales and XX females, as the X chromosome exhibited denser chromatin structure than the Y chromosome, while they respectively have significantly spatial interactions with female- and male-related genes compared with other autosomes. The chromatin configuration of the sex chromosomes as well as the nucleus spatial organization of the XX neomale were remodeled after sex reversal and similar to those in YY supermales, and a male-specific loop containing the SDR was found in the open chromatin region. Our results elucidate the origin of young sex chromosomes and the chromatin remodeling configuration in the catfish sexual plasticity.

Chedrinae fish, which belong to Danionidae, have important ornamental, economic, and scientific value. At present, however, their mitogenomic features are unclear and their phylogenetic relationships remain controversial. In this study, we presented five new Chedrinae mitochondrial genomes (mitogenomes) and analyzed the conserved and variable mitogenomic characteristics of 17 Chedrinae fish. The gene composition and arrangement and secondary structure of transfer RNAs (tRNAs) were highly conserved among the Chedrinae mitogenomes. However, the length of the control region and base composition were variable. Interestingly, the mitogenome of Barilius barila was unusual, with lower A+T content in the first codon of protein-coding genes (PCGs) (47.32% versus average of 54.47%) and distinct pattern of codons per thousand codons (CDspT). Three Chedrinae fish had a long tandem repeat (>291 bp) in the 5'-end of the control region, which may increase their adaptability. In addition, tRNALys had notably larger DHU and TΨC loops than other tRNAs. The phylogenetic trees of the Chedrinae fish suggested that the Barilius genus was not a monophyletic group but could be divided into two main groups based on significant differences in A+T content. This study provides insights into the mitogenomic features and phylogenetic implications of Chedrinae fish, which should benefit their systematics and conservation.

Viral co-infection has been found in animals; however, the mechanisms of co-infection are unclear. The abundance and diversity of viruses in water make fish highly susceptible to co-infection. Here, we reported a co-infection in fish, which resulted in reduced host lethality and illustrated the intracellular molecular mechanism of viral co-infection. The spring viremia of carp virus (SVCV) is a highly lethal virus that infects Cyprinidae, such as zebrafish. The mortality of SVCV infection was significantly reduced when co-infected with the grass carp reovirus (GCRV). The severity of tissue damage and viral proliferation of SVCV was also reduced in co-infection with GCRV. The transcriptome bioinformatics analysis demonstrated that the effect on the host transcripts in response to SVCV infection was significantly reduced in co-infection. After excluding the extracellular interactions of these two viruses, the intracellular mechanisms were studied. We found that the GCRV NS38 remarkably decreased SVCV infection and viral proliferation. The interaction between GCRV NS38 and SVCV nucleoprotein (N) and phosphoprotein (P) proteins was identified, and NS38 downregulated both N and P proteins. Further analysis demonstrated that the N protein was degraded by NS38 indispensable of the autophagy receptor, sequestosome 1 (p62). Meanwhile, K63-linked ubiquitination of the P protein was reduced by NS38, leading to ubiquitinated degradation of the P protein. These results reveal that the intracellular viral protein interactions are a crucial mechanism of co-infection and influence the host pathology and expand our understanding in intracellular viral interactions co-infection.

Unisexual animals are commonly found in some polyploid species complexes, and most of these species have had a long evolutionary history. However, their method for avoiding genomic decay remains unclear. The polyploid Carassius complex naturally comprises the sexual amphidiploid C. auratus (crucian carp or goldfish) (AABB) and the gynogenetic amphitriploid C. gibelio (gibel carp) (AAABBB). Recently, we developed a fertile synthetic amphitetraploid (AAAABBBB) male from C. gibelio by incorporating a C. auratus genome. In this study, we generated novel amphitriploids (AAABBB) by backcrossing the amphitetraploid male with the amphidiploid C. auratus. Whole-genome resequencing revealed the genomic changes, including recombination and independent assortment between homologs of C. gibelio and C. auratus. The fertility, sex determination system, oocyte development, and fertilization behaviors of the novel amphitriploids were investigated. Approximately 80% of the novel amphitriploid females recovered the unisexual gynogenesis ability. Intriguingly, two types of primary oocyte (with and without homolog synapsis) were discovered, and their distinct development fates were observed. Type I oocytes entered apoptosis due to improper synaptonemal complex assembly and incomplete double-strand break repair, whereas subsequent type II oocytes bypassed meiosis through an alternative ameiotic pathway to develop into mature eggs. Moreover, gynogenesis was stabilized in their offspring, and a new array of diverse gynogenetic amphitriploid clones was produced. These revealed genomic changes and detailed cytological data provide comprehensive evidence that changes in ploidy drive unisexual and sexual reproduction transition, thereby resulting in genomic diversity and allowing C. gibelio avoid genomic decay.

How unisexual animals eliminate deleterious mutations to avoid dead ends is one of the most interesting puzzles in evolutionary genetics. Incorporation of microchromosomes derived from exogenous sperm had been observed in gynogenetic animals, but little is known about their detailed process and hereditary fate. Here, we show a stable genome incorporation case in an artificial clone F of gynogenetic gibel carp (Carassius gibelio). A total of 12 exogenous DNA fragments were screened through a read depth-dependent comparison strategy and confirmed to be specific to the clone F and the paternal blunt snout bream (Megalobrama amblycephala Yin) by SCAR (sequence characterized amplified regions) marker detection. Moreover, these sperm-derived DNA fragments were not detected in some samples in early gynogenetic generations, but they were found to exist in all examined individuals through artificial gynogenetic selections of 13 generations, implying that they might have stably incorporated into the genome of clone F. Furthermore, chromosome localization and sequence characterization indicate that the largest fragment CgA22_34 is derived from blunt snout bream non-LTR retrotransposon and durably incorporated into only one of three homologous chromosomes of gibel carp clone F. Our results suggest that the incorporated sperm-derived DNA fragments by allogynogenesis should increase genetic diversity and introduce new traits into unisexual animals which will benefit genetic breeding of gibel carp. During the process, transposable elements (TEs) may play significant roles in shaping the genome structures. Simultaneously, the incorporated DNA fragments are able to be used as genetic markers to perform selective breeding programs in aquaculture practices of gibel carp.

Aquaculture is one of the most efficient modes of animal protein production and plays an important role in global food security. Aquaculture animals exhibit extraordinarily diverse sexual phenotypes and underlying mechanisms, providing an ideal system to perform sex determination research, one of the important areas in life science. Moreover, sex is also one of the most valuable traits because sexual dimorphism in growth, size, and other economic characteristics commonly exist in aquaculture animals. Here, we synthesize current knowledge of sex determination mechanisms, sex chromosome evolution, reproduction strategies, and sexual dimorphism, and also review several approaches for sex control in aquaculture animals, including artificial gynogenesis, application of sex-specific or sex chromosome-linked markers, artificial sex reversal, as well as gene editing. We anticipate that better understanding of sex determination mechanisms and innovation of sex control approaches will facilitate sustainable development of aquaculture.

Background Fatty liver has become a main problem that causes huge economic losses in many aquaculture modes. It is a common physiological or pathological phenomenon in aquaculture, but the causes and occurring mechanism are remaining enigmatic. Methods Each three liver samples from the control group of allogynogenetic gibel carp with normal liver and the overfeeding group with fatty liver were collected randomly for the detailed comparison of histological structure, lipid accumulation, transcriptomic profile, latent pathway identification analysis (LPIA), marker gene expression, and hepatocyte mitochondria analyses. Results Compared to normal liver, larger hepatocytes and more lipid accumulation were observed in fatty liver. Transcriptomic analysis between fatty liver and normal liver showed a totally different transcriptional trajectory. GO terms and KEGG pathways analyses revealed several enriched pathways in fatty liver, such as lipid biosynthesis, degradation accumulation, peroxidation, or metabolism and redox balance activities. LPIA identified an activated ferroptosis pathway in the fatty liver. qPCR analysis confirmed that gpx4, a negative regulator of ferroptosis, was significantly downregulated while the other three positively regulated marker genes, such as acsl4, tfr1 and gcl, were upregulated in fatty liver. Moreover, the hepatocytes of fatty liver had more condensed mitochondria and some of their outer membranes were almost ruptured. Conclusions We reveal an association between ferroptosis and fish fatty liver for the first time, suggesting that ferroptosis might be activated in liver fatty. Therefore, the current study provides a clue for future studies on fish fatty liver problems.

Evolutionary fates of duplicated genes have been widely investigated in many polyploid plants and animals, but research is scarce in recurrent polyploids. In this study, we focused on foxl2, a central player in ovary, and elaborated the functional divergence in gibel carp (Carassius gibelio), a recurrent auto-allo-hexaploid fish. First, we identified three divergent foxl2 homeologs (Cgfoxl2a-B, Cgfoxl2b-A, and Cgfoxl2b-B), each of them possessing three highly conserved alleles and revealed their biased retention/loss. Then, their abundant sexual dimorphism and biased expression were uncovered in hypothalamic–pituitary–gonadal axis. Significantly, granulosa cells and three subpopulations of thecal cells were distinguished by cellular localization of CgFoxl2a and CgFoxl2b, and the functional roles and the involved process were traced in folliculogenesis. Finally, we successfully edited multiple foxl2 homeologs and/or alleles by using CRISPR/Cas9. Cgfoxl2a-B deficiency led to ovary development arrest or complete sex reversal, whereas complete disruption of Cgfoxl2b-A and Cgfoxl2b-B resulted in the depletion of germ cells. Taken together, the detailed cellular localization and functional differences indicate that Cgfoxl2a and Cgfoxl2b have subfunctionalized and cooperated to regulate folliculogenesis and gonad differentiation, and Cgfoxl2b has evolved a new function in oogenesis. Therefore, the current study provides a typical case of homeolog/allele diversification, retention/loss, biased expression, and sub-/neofunctionalization in the evolution of duplicated genes driven by polyploidy and subsequent diploidization from the recurrent polyploid fish.

Polyploids in vertebrates are generally associated with unisexual reproduction, but the direct consequences of polyploidy on sex determination system and reproduction mode remain unknown. Here, we synthesized a group of artificial octoploids between unisexual gynogenetic hexaploid Carassius gibelio and sexual tetraploid Carassius auratus. The synthetic octoploids were revealed to have more than 200 chromosomes, in which 50 chromosomes including the X/Y sex determination system were identified to transfer from sexual tetraploid C. auratus into the unisexual gynogenetic hexaploid C. gibelio. Significantly, a few synthetic octoploid males were found to be fertile, and one octoploid male was confirmed to regain sexual reproduction ability, which exhibits characteristics that are the same to sexual reproduction tetraploid males, such as 1:1 sex ratio occurrence, meiosis completion and euploid sperm formation in spermatogenesis, as well as normal embryo development and gene expression pattern during embryogenesis. Therefore, the current finding provides a unique case to explore the effect of sex determination system incorporation on reproduction mode transition from unisexual gynogenesis to sexual reproduction along with genome synthesis of recurrent polyploidy in vertebrates.

Viperin is increasingly thought as a key protein in antiviral response. However, the in vivo antiviral effects of fish viperin remains unresolved. Hexaploid gibel carp (Carassius gibelio) is an important aquaculture species in China and its culture industry has suffered enormous economic losses due to an epizootic disease caused by C. auratus herpesvirus (CaHV) since 2012. In our previous studies, two duplicated viperin homeologs (Cgviperin-A and Cgviperin-B) were identified and their divergent antiviral mechanisms were revealed through in vitro overexpression in CAB (C. auratus blastulae embryonic) cell. Here, we tried to explore their function difference in vivo by using CRISPR/Cas9 editing. Firstly, we uncovered their biased expression patterns and differential promoter activities. Then we mutated them singly or simultaneously in resistant clone H of gibel carp by using CRISPR/Cas9 and finally confirmed that they cooperatively participated in the antiviral battle between gibel carp and CaHV through virus challenge and comparative transcriptome analysis. CgViperin-A depletion mainly affected immune-related pathways and Cgviperin-B deficiency influenced autophagy-associated pathways. Simultaneous disruption of them resulted in complete loss of resistance ability of gibel carp against CaHV. Significantly, the deficiency of Cgviperin-A influenced the expression of Cgviperin-B in Cgviperin-A−/−/− mutant, but Cgviperin-A transcripts kept a high level of expression in the Cgviperin-B−/−/− mutant, same as in the WT. Therefore, the current findings are not only the first time to clarify the antiviral activities of Viperin in the battle between teleost and virus in vivo, but also explain the detailed evolutionary divergence of a duplicated gene in a recurrent polyploid fish.

Sex is prevalent in nature and sex determination is one of the most fundamental biological processes, while the way of initiating female and male development exhibits remarkable diversity and variability across vertebrates. The knowledge on why and how sex determination mechanisms evolve unusual plasticity remains limited. Here, we summarize sex determination systems, master sex-determining genes and gene-regulatory networks among vertebrates. Recent research advancements on sex determination system transition are also introduced and discussed in some non-model animals with multiple sex determination mechanisms. This review will provide insights into the origin, transition and evolutionary adaption of different sex determination strategies in vertebrates, as well as clues for future perspectives in this field.

Various genes have been screened on extra chromosomes, but their molecular characterization, expression pattern and biological function are still unclear. Here, we utilized a male-specific sequence of polyploid gibel carp (Carassius gibelio) to identify a novel male-specific SET (Su(var)3–9, Enhancer-of-zeste, Trithorax) domain-containing gene setdm on extra microchromosomes of gibel carp males. And setdm was characterized in molecule and expression aspects, in which its expression was specific to testis, and had relative high transcription during middle/late stages of testis development. Moreover, prominent expression of Setdm in spermatogenic cells was observed in testis through immunofluorescence co-localization analysis. These results suggest that biological function of setdm might be related to testis development and spermatogenesis of gibel carp. Additionally, the homeologous gene setdmf of setdm, was also characterized, and its expression was gonad-specific, in which its expressed product was detected to mainly distribute in gametogenic cells of testis and ovary, and to have dynamic expression pattern similar to that of setdm. Based on the current results, we propose that the novel male-specific setdm on extra microchromosomes might be functional divergence gene of the gonad-specific setdmf. Therefore, these findings will help us to further understand evolutionary fate and functional role of genes on extra microchromosomes.

Lanzhou catfish (Silurus lanzhouensis), an important commercial fish in China, takes three years to be sexually mature and males display faster growth performance than females. Thus, shortening of breeding cycle and production of all-male population in Lanzhou catfish have great significance in aquaculture. Here, we first developed an optimal method for artificial gynogenesis, through which the male heterogametic XX/XY sex determination system was revealed in Lanzhou catfish. Subsequently, sex-specific markers were identified via 2b-RAD-seq and verified in artificial breeding and wild populations. Intriguingly, a hermaphrodite Lanzhou catfish with XY genotype was occasionally obtained, whose gonad contained both mature ovarian and testicular tissues. The eggs and sperm from the intersexual gonad were used for artificial fertilization to establish a self-fertilization family, which contained 21 XX females, 25 XY males, and 12 YY males in the offspring. And the YY males are able to mate with XX females to produce all-male progenies with XY genotype. Compared with the conventional way of expending two-generation time, self-fertilization of the XY hermaphrodite saved one-generation time to produce YY males. Therefore, this study not only reveals XX/XY sex determination system in Lanzhou catfish, but also directly obtains YY males for future production of all-male population in aquaculture.

In East Asia, the dark sleeper, Odontobutis obscura (O. obscura) is a crucial commercial species of freshwater fish; however, its molecular biology research is still undeveloped, including its innate immune system, which is pivotal to antiviral responses. In this study, we cloned and identified the characterization and kinase function of dark sleeper TANK-binding kinase 1 (TBK1), supplementing the evidence of the conservation of this classical factor in fish. First, the ORF of Odontobutis obscurus (O. obscura) TBK1 (OdTBK1) was cloned from liver tissue by RACE-PCR. Subsequent nucleic acid and amino acid sequence analysis suggested that OdTBK1 is homologous with other fish TBK1, and the N-terminal Serine/Threonine protein kinases catalytic domain (S_TKc) and C-terminal coiled coil domain (CCD) are conserved. Subsequently, the cellular distribution demonstrated that OdTBK1 was located in the cytoplasm region. With regard to the identification of functions, OdTBK1 activated several interferon (IFN) promoters' activity and induced downstream IFN-stimulated genes (ISGs) expression. In a canonical manner, wild-type OdTBK1 significantly phosphorylated interferon regulatory factor 3 (IRF3) but failed when the N-terminal region was truncated. Furthermore, overexpression of OdTBK1 decreased viral proliferation remarkably. Collectively, these data systematically analyzed the characterization and function of OdTBK1, initiating the study of the innate antiviral response of dark sleeper.

The dark sleeper, Odontobutis obscura (O. obscura), is a commercially important species of freshwater sleeper native to East Asia. However, its molecular biology system is unexplored, including the interferon (IFN) signaling pathway, which is crucial to the antiviral response. In this study, we characterised the IFN regulation pattern of dark sleeper interferon regulatory factor 3 (OdIRF3), supplementing evidence of the conservation of this classical pathway in fish. First, the open reading frame (ORF) of OdIRF3 was cloned from the liver tissue by Rapid amplification of cDNA ends (RACE). Amino acid sequence analysis suggested that OdIRF3 is homologous with other fish IRF3 and that the N-terminal DNA-binding domain (DBD) and the C-terminal IRF-association domain (IAD) are conserved. Then, the cellular distribution demonstrated that OdIRF3 is located in the cytoplasm region and transfers into the nuclear region under stimulation. For the function identification, OdIRF3 activated several types of IFN promoters and induced downstream interferon stimulated genes (ISGs) expression. Finally, the overexpression of OdIRF3 significantly decreased viral proliferation. Taken together, these data systematically characterised the sequence, cellular location, and function in IFN expression of OdIRF3, shedding light on the molecular biology mechanism of the dark sleeper.

Allopolyploidization plays an important role in speciation, and some natural or synthetic allopolyploid fishes have been extensively applied to aquaculture. Although genetic and epigenetic inheritance and variation associated with plant allopolyploids have been well documented, the relative research in allopolyploid animals is scarce. In this study, the genome constitution and DNA methylation inheritance in a newly synthetic allopolyploid of gynogenetic gibel carp were analyzed. The incorporation of a whole genome of paternal common carp sperm in the allopolyploid was confirmed by genomic in situ hybridization, chromosome localization of 45S rDNAs, and sequence comparison. Pooled sample-based methylation sensitive amplified polymorphism (MSAP) revealed that an overwhelming majority (98.82%) of cytosine methylation patterns in the allopolyploid were inherited from its parents of hexaploid gibel carp clone D and common carp. Compared to its parents, 11 DNA fragments in the allopolyploid were proved to be caused by interindividual variation, recombination, deletion, and mutation through individual sample-based MSAP and sequencing. Contrast to the rapid and remarkable epigenetic changes in most of analyzed neopolyploids, no cytosine methylation variation was detected in the gynogenetic allopolyploid. Therefore, the newly synthetic allopolyploid of gynogenetic gibel carp combined genomes from its parents and maintained genetic and epigenetic stability after its formation and subsequently seven successive gynogenetic generations. Our current results provide a paradigm for recurrent polyploidy consequences in the gynogenetic allopolyploid animals.

Background Coexistence and transition of diverse sex determination strategies have been revealed in some ectothermic species, but the variation between males caused by different sex determination strategies and the underlying mechanism remain unclear. Here, we used the gynogenetic gibel carp (Carassius gibelio) with both genotypic sex determination (GSD) and temperature-dependent sex determination (TSD) strategies to illustrate this issue. Results We found out that males of GSD and TSD in gibel carp had similar morphology, testicular histology, sperm structure and sperm vitality. However, when maternal individuals were mated with males of GSD, sperm nucleus swelling and fusing with the female pronucleus were observed in the fertilized eggs. On the contrary, when maternal individuals were mated with males of TSD, sperm nucleus remained in the condensed status throughout the whole process. Subsequently, semen proteomics analysis unveiled that DNA replication and gene expression-related pathways were inhibited in the sperm from males of TSD compared to males of GSD, and most differentially expressed proteins associated with DNA replication, transcription and translation were down-regulated. Moreover, via BrdU incorporation and immunofluorescence detection, male nucleus replication was revealed to be present in the fertilized eggs by the sperm from males of GSD, but absent in the fertilized eggs by the sperm from males of TSD. Conclusions These findings indicate that DNA replication and gene expression-related pathways are associated with the distinct sperm nucleus development behaviors in fertilized eggs in response to the sperm from males of GSD and TSD. And this study is the first attempt to screen the differences between males determined via GSD and TSD in gynogenetic species, which might give a hint for understanding evolutionary adaption of diverse sex determination mechanisms in unisexual vertebrates.

Comprehending sex determination mechanism is a first step for developing sex control breeding biotechnologies in fish. Pseudobagrus ussuriensis, one of bagrid catfishes in Bagridae, had been observed to have about threefold size dimorphism between males and females, but its sex determination mechanism had been unknown. In this study, we firstly used the amplified fragment length polymorphism (AFLP)-based screening approach to isolate a male-specific DNA fragment and thereby identified a 10,569 bp of male-specific sequence and a 10,365 bp of female-related sequence by genome walking in the bagrid catfish, in which a substantial genetic differentiation with 96.35 % nucleotide identity was revealed between them. Subsequently, a high differentiating region of 650 bp with only 70.26 % nucleotide identity was found from the corresponding two sequences, and three primer pairs of male-specific marker, male and female-shared marker with different length products in male and female genomes, and female-related marker were designed. Significantly, when these markers were used to identify genetic sex of the bagrid catfish, only male individuals was detected to amplify the male-specific marker fragment, and female-related marker was discovered to produce dosage association in females and in males. Our current data provide significant genetic evidence that P. ussuriensis has heterogametic XY sex chromosomes in males and homogametic XX sex chromosomes in females. Therefore, sex determination mechanism of P. ussuriensis is male heterogametic XX/XY system.

Most vertebrates reproduce sexually, and plastic sex determination mechanisms including genotypic sex determination (GSD) and environmental sex determination (ESD) have been extensively revealed. However, why sex determination mechanisms evolve diversely and how they correlate with diverse reproduction strategies remain largely unclear. Here, we utilize the superiority of a hexaploid gibel carp (Carassius gibelio) that is able to reproduce by unisexual gynogenesis and contains a rare but diverse proportion of males to investigate these puzzles. A total of 2248 hexaploid specimens were collected from 34 geographic wild populations throughout mainland China, in which 24 populations were revealed to contain 186 males with various incidences ranging from 1.2 to 26.5%. Subsequently, the proportion of temperature-dependent sex determination (TSD) was revealed to be positively correlated to average annual temperature in wild populations, and male incidence in lab gynogenetic progenies was demonstrated to increase with the increasing of larval rearing temperature. Meanwhile, extra microchromosomes were confirmed to play genotypic male determination role as previously reported. Thereby, GSD and TSD were found to coexist in gibel carp, and the proportions of GSD were observed to be much higher than that of TSD in sympatric wild populations. Our findings uncover a potential new mechanism in the evolution of sex determination system in polyploid vertebrates with unisexual gynogenesis ability, and also reveal a possible association of sex determination mechanism transition between TSD and GSD and reproduction mode transition between unisexual gynogenesis and bisexual reproduction.

Gibel carp ( Carassius auratus gibelio) is a uniquely gynogenetic species with a minor ratio of males in natural habitats, but its male origin and sex determination mechanisms have been unknown. In this study, a male-biased mutant family was discovered from the gynogenetic gibel carp, and a male-specific SCAR marker was identified from the mutant family. Normal spermatogenesis was observed in the male testes by immuno. fluorescence histochemistry. Nearly identical AFLP profiles were observed between males and females, but a male-specific 86 bp AFLP fragment was screened by sex-pool bulked segregant analysis and individual screening. Based on the male-specific AFLP fragment, a total of 579 bp sequences were cloned by genome walking. Subsequently, a male-specific SCAR marker was designed, and the male-specific DNA fragment was confirmed to be steadily transmitted to the next generation and consistently detected only in males. (C) 2009 National Natural Science Foundation of China and Chinese Academy of Sciences. Published by Elsevier Limited and Science in China Press. All rights reserved.

A dynamic continuous ultrasound-assisted extraction with high intensity ultrasonic probe (CUAE-HIUP) combined with solid-phase extraction (SPE) for preconcentration and clean-up of the extract prior to high-performance liquid chromatography (HPLC) determination of the main biological active ingredients, sodium Danshensu and four tanshinones (dihydrotanshione I, tanshinone I, cryptotanshinone and tanshinone IIA) from root of Salvia miltiorrhiza bunge has been developed. An experimental design (Plackett-Burman design, 2(6)x3/16) was used to optimize the CUAE-HIUP procedure and the SPE step. Detection limits of 1.2-4.2 x 10(-6) mg mL(-1) were achieved with the preconcentration efficiency of more than 10-folds by the SPE procedure. The repeatability and within-laboratory reproducibility of the whole process, expressed as relative standard deviations (RSDs), were 1.5-3.6%, 1.6-3.1%, respectively. As compared with the conventional extraction techniques such as room temperature extraction (24 h), Soxhlet (4 h) and even microwave-assisted extraction (2 min), CUAE-HIUP achieved the highest extraction yield (98.9%) and the least amount (10 mL) of solvent compared with the other techniques (16 mL) in only 3 min. The method was successfully applied to the determination of the five biological active ingredients in root of S. miltiorrhiza bunge and Danshen-containing pharmaceutical formulations.

Sex is generally determined by sex chromosomes in vertebrates, and sex chromosomes exhibit the most rapidly-evolving traits. Sex chromosome evolution has been revealed previously in numerous cases, but the association between sex chromosome origin and the reproduction mode transition from unisexual to sexual reproduction remains unclear. Here, we have isolated a male-specific sequence via analysis of amplified fragment length polymorphism from polyploid gibel carp (Carassius gibelio), a species that not only has the ability to reproduce unisexually but also contains males in wild populations. Subsequently, we have found through FISH analysis that males have several extra microchromosomes with repetitive sequences and transposable elements when compared to females. Moreover, we produced sex-reversed physiological females with a male-specific marker by using estradiol hormone treatment, and two gynogenetic families were established from them. In addition, the male incidence rates of two gynogenetic families were revealed to be closely associated with the extra microchromosome number of the sex-reversed physiological females. These results suggest that the extra microchromosomes in males might resemble a common feature of sex chromosomes and might play a significant role in male determination during the evolutionary trajectory of the reproduction mode transition from unisexual to sexual reproduction in the polyploid fish.

Background Gynogenesis is one of unisexual reproduction modes in vertebrates, and produces all-female individuals with identical genetic background. In sexual reproduction vertebrates, the roles of primordial germ cells on sexual dimorphism and gonadal differentiation have been largely studied, and two distinct functional models have been proposed. However, the role of primordial germ cells remains unknown in unisexual animals, and it is also unclear whether the functional models in sexual reproduction animals are common in unisexual animals. Results To solve these puzzles, we attempt to utilize the gynogenetic superiority of polyploid Carassius gibelio to create a complete germ cell-depleted gonad model by a similar morpholino-mediated knockdown approach used in other examined sexual reproduction fishes. Through the germ cell-depleted gonad model, we have performed comprehensive and comparative transcriptome analysis, and revealed a complete alteration of sex-biased gene expression. Moreover, the expression alteration leads to up-regulation of testis-biased genes and down-regulation of ovary-biased genes, and results in the occurrence of sterile all-males with testis-like gonads and secondary sex characteristics in the germ cell-depleted gynogenetic Carassius gibelio. Conclusions Our current results have demonstrated that unisexual gynogenetic embryos remain keeping male sex determination information in the genome, and the complete depletion of primordial germ cells in the all-female fish leads to sex-biased gene expression alteration and sterile all-male occurrence. Electronic supplementary material The online version of this article (doi:10.1186/s12864-015-2130-z) contains supplementary material, which is available to authorized users.

Unisexual polyploid vertebrates are commonly known to reproduce by gynogenesis, parthenogenesis, or hybridogenesis. One clone of polyploid Carassius gibelio has been revealed to possess multiple modes of unisexual gynogenesis and sexual reproduction, but the cytological and developmental mechanisms have remained unknown. In this study, normal meiosis completion was firstly confirmed by spindle co-localization of β-tubulin and Spindlin. Moreover, three types of various nuclear events and development behaviors were revealed by DAPI staining and BrdU-incorporated immunofluorescence detection during the first mitosis in the fertilized eggs by three kinds of different sperms. They include normal sexual reproduction in response to sperm from the same clone male, typical unisexual gynogenesis in response to sperm from the male of another species Cyprinus carpio and an unusual hybrid-similar development mode in response to sperm from another different clone male. Based on these findings, we have discussed cytological and developmental mechanisms on multiple reproduction modes in the polyploid fish and highlighted evolutionary significance of meiosis completion and evolutionary consequences of reproduction mode diversity in polyploid vertebrates.

Dmrt1 has been suggested to play significant roles in sex determination and differentiation, but various expression patterns and cell types have been observed in the testis of vertebrates. Polyploid gibel carp, because of the multiple modes of unisexual gynogenesis and sexual reproduction, has become a unique case to explore the evolution of sex determination and differentiation. However, the sex-determination related genes in gibel carp have remained unknown. In this study, we identified and characterized 4 cDNAs of Dmrt1 genes. Subsequently, a polyclonal antibody specific to CagDMRT1 was prepared to examine its expression and distribution patterns at protein level. Significantly, both relative real-time PCR and Western blot detection confirmed predominant expression of CagDmrt1 in the adult testis of gibel carp. Moreover, the intensive expression of CagDMRT1 around spermatogenic cysts was revealed during spermatogenesis. And, following immunofluorescence co-localization of CagDMRT1 and CagVASA, a prominent CagDMRT1 expression in Sertoli cells and a mild CagDMRT1 expression in spermatogenic cells including spermatogonia and primary spermatocytes were clearly characterized. The CagDMRT1 signal in Sertoli cells is extensively distributed in both nuclei and cytoplasm, while the CagDMRT1 in spermatogonia and primary spermatocytes is mainly expressed in nuclei, and there is only the remained CagDMRT1 signal in the cytoplasm of secondary spermatocytes. These findings suggest that DMRT1 should be related to testis differentiation and spermatogenesis in gibel carp. (C) 2014 Elsevier B.V. All rights reserved.