Formation of Different Polyploids Through Disrupting Meiotic Crossover Frequencies Based on cntd1 Knockout in Zebrafish.

Polyploidy, a significant catalyst for speciation and evolutionary processes in both plant and animal kingdoms, has been recognized for a long time. However, the exact molecular mechanism that leads to polyploid formation, especially in vertebrates, is not fully understood. Our study aimed to elucidate this phenomenon using the zebrafish model. We successfully achieved an effective knockout of the cyclin N-terminal domain containing 1 (cntd1) using CRISPR/Cas9 technology. This resulted in impaired formation of meiotic crossovers, leading to cell-cycle arrest during meiotic metaphase and triggering apoptosis of spermatocytes in the testes. Despite these defects, the mutant (cntd1-/-) males were still able to produce a limited amount of sperm with normal ploidy and function. Interestingly, in the mutant females, it was the ploidy not the capacity of egg production that was altered. This resulted in the production of haploid, aneuploid, and unreduced gametes. This alteration enabled us to successfully obtain triploid and tetraploid zebrafish from cntd1-/- and cntd1-/-/- females, respectively. Furthermore, the tetraploid-heterozygous zebrafish produced reduced-diploid gametes and yielded all-triploid or all-tetraploid offspring when crossed with wild-type (WT) or tetraploid zebrafish, respectively. Collectively, our findings provide direct evidence supporting the crucial role of meiotic crossover defects in the process of polyploidization. This is particularly evident in the generation of unreduced eggs in fish and, potentially, other vertebrate species.

Symmetric subgenomes and balanced homoeolog expression stabilize the establishment of allopolyploidy in cyprinid fish.

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.

The subgenomes show asymmetric expression of alleles in hybrid lineages of Megalobrama amblycephala × Culter alburnus.

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.

Comparative analyses of hypothalamus transcriptomes reveal fertility-, growth-, and immune-related genes and signal pathways in different ploidy 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.

Elevated expression of inhibin α gene in sterile allotriploid crucian carp.

Inhibin and Activin, belong to the transforming growth factor ß superfamily (TGF-ß), which associate with the regulation of the reproductive process by the modulation of the hypothalamic-pituitary-gonad (HPG) axis. In this study, we reported the molecular cloning and tissue expression of inhibin α in allotriploid crucian carp and its parent- diploid red crucian carp. The full-length cDNA of inhibin α were respectively 1632 bp and 1642 bp in allotriploids and diploids, which both consisted of a 1044 bp open reading frame (ORF) encoding 347 amino acids. Real-time quantitative PCR (RT-qPCR) showed that allotriploids and diploids had significant expression of inhibin α in testis and ovary, and the expression of inhibin α in the gonads of allotriploids was higher than that of diploids. The immunohistochemistry indicated that the ovarian development of allotriploids was abnormal, and the expression of Inhibin α in the ovary of allotriploids was higher than that of diploids. Results of co-immunoprecitation (co-IP) demonstrated that the Inhibin α and Activin ßA, Inhibin α and Activin ßB can form dimers. These findings suggested that the elevated expression of inhibin α and the competitive binding of Inhibin α subunit with Activin ß subunits in allotriploids may be releted to the sterility of allotriploids. Furthermore, these results will facilitate the investigation of reproduction characteristics in allotriploids and provide theoretical basis for the study of polyploid breeding in the future.

Hox genes reveal variations in the genomic DNA of allotetraploid hybrids derived from Carassius auratus red var. (female) × Cyprinus carpio L. (male).

BACKGROUND: Hox transcription factors are master regulators of animal development. Although highly conserved, they can contribute to the formation of novel biological characteristics when modified, such as during the generation of hybrid species, thus potentially serving as species-specific molecular markers. Here, we systematically studied the evolution of genomic sequences of Hox loci in an artificial allotetraploid lineage (4nAT, 4n = 200) derived from a red crucian carp (♀, RCC, 2n = 100) × common carp (♂, CC, 2n = 100) cross and its parents (RCC and CC). RESULTS: PCR amplification yielded 23 distinct Hox gene fragments from 160 clones in 4nAT, 22 fragments from 90 clones in RCC, and 19 fragments from 90 clones in CC. Sequence alignment of the HoxA3a and HoxC10a genes indicated both the inheritance and loss of paternal genomic DNA in 4nAT. The HoxA5a gene from 4nAT consisted of two subtypes from RCC and two subtypes from CC, indicating that homologous recombination occurred in the 4nAT hybrid genome. Moreover, 4nAT carried genomic pseudogenization in the HoxA10b and HoxC13a loci. Interestingly, a new type of HoxC9a gene was found in 4nAT as a hybrid sequence of CC and RCC by recombination in the intronic region. CONCLUSION: The results revealed the influence of Hox genes during polyploidization in hybrid fish. The data provided insight into the evolution of vertebrate genomes and might be benefit for artificial breeding programs.

Diploid hybrid fish derived from the cross between female Bleeker's yellow tail and male topmouth culter, two cyprinid fishes belonging to different subfamilies.

BACKGROUND: Bleeker's yellow tail (Xenocypris davidi Bleeker, YT) and topmouth culter (Culter alburnus Basilewsky, TC) are both famous and important economic freshwater fish in China. YT, a kind of omnivorous fish, has strong resistance. TC, a kind of carnivorous fish, has high-quality meat but poor resistance. Distant hybridization can integrate the advantages of both parents. There has been no previous report regarding hybrid fish derived from female YT × male TC. It is expected that hybridization of these two kinds of fish will result in F1 hybrids with improved characteristics, such as faster growth rate, stronger resistance, and high-quality meat, which are of great significance in fish genetic breeding. RESULTS: In this study, we investigated the main biological characteristics of diploid hybrid fish derived from female YT × male TC. The hybrids had an intermediate number of upper lateral line scales between those for YT and TC. The hybrids were diploids with 48 chromosomes and had the same karyotype formula as their parents. The hybrids generated variations in 5S rDNA (designated class IV: 212 bp) and lost specific 5S rDNA derived from the maternal parent (designated class II: 221 bp), which might be related to hybridization. In terms of reproductive traits, all the tested female hybrids exhibited normal gonadal development, and the two-year-old F1 females produced mature eggs. However, all the tested testes of the male hybrids could not produce mature sperm. It is possible that the hybrid lineage will be established by back-crossing the fertile female hybrids and their parents. CONCLUSIONS: Obtaining a fertile female hybrid fish made the creation of a new type of fish possible, which was significant in fish genetic breeding.

Comparative analysis of testis transcriptomes associated with male infertility in triploid cyprinid fish.

Spermatogenesis involves a series of cellular transformations and thousands of regulated genes. Previously, we showed that the triploid fish (3nBY) cannot produce mature spermatozoa. In the present study, evaluation of the testis microstructure revealed that germ cells of 3nBY could develop into round spermatids, but then degenerated, resulting in male infertility. In this study we comparatively analysed the testis transcriptomes from 3nBY and its diploid parent YB and identified a series of differentially expressed genes (DEGs) that were enriched in the Wnt signalling pathway and the apoptotic and ubiquitin-mediated proteolysis processes in 3nBY. Gene ontology functional analyses revealed that some DEGs in 3nBY were directly associated with the process of gamete generation, development and sperm flagellum assembly. In addition, the expression of a number of genes related to meiosis (Inhibitor Of DNA Binding 2 (ID2), Ovo Like Transcriptional Repressor 1 (OVOL1)), mitochondria (ATP1b (ATPase Na+/K+ Transporting Subunit Beta 1), ATP2a (ATPase, Ca++ Transporting, Cardiac Muscle, Slow Twitch 2), ATP5a (ATP Synthase F1 Subunit Alpha), Mitochondrially Encoded Cytochrome C Oxidase I (COX1), NADH Dehydrogenase Subunit 4 (ND4)) and chromatin structure (Histone 1 (H1), Histone 2a (H2A), Histone 2b (H2B), Histone 3 (H3), Histone 4 (H4)) was lower in the testes of 3nBY, whereas the expression of genes encoding ubiquitin (Ubiquitin Conjugating Enzymes (UBEs), Ring Finger Proteins (RNFs)) and apoptosis (CASPs (Caspase 3, Caspase 7,Caspase 8), BCLs (B-Cell Lymphoma 3, B-Cell CLL/Lymphoma 2, B Cell CLL/Lymphoma 10)) proteins involved in spermatid degeneration was higher. These data suggest that the disrupted expression of genes associated with spermatogenesis and the increased expression of mitochondrial ubiquitin, which initiates cell apoptosis, may result in spermatid degeneration in male 3nBY. This study provides information regarding the potential molecular regulatory mechanisms underlying male infertility in polyploid fish.

Genomic incompatibilities in the diploid and tetraploid offspring of the goldfish × common carp cross.

Polyploidy is much rarer in animals than in plants but it is not known why. The outcome of combining two genomes in vertebrates remains unpredictable, especially because polyploidization seldom shows positive effects and more often results in lethal consequences because viable gametes fail to form during meiosis. Fortunately, the goldfish (maternal) × common carp (paternal) hybrids have reproduced successfully up to generation 22, and this hybrid lineage permits an investigation into the genomics of hybridization and tetraploidization. The first two generations of these hybrids are diploids, and subsequent generations are tetraploids. Liver transcriptomes from four generations and their progenitors reveal chimeric genes (>9%) and mutations of orthologous genes. Characterizations of 18 randomly chosen genes from genomic DNA and cDNA confirm the chimera. Some of the chimeric and differentially expressed genes relate to mutagenesis, repair, and cancer-related pathways in 2nF1. Erroneous DNA excision between homologous parental genes may drive the high percentage of chimeric genes, or even more potential mechanisms may result in this phenomenon. Meanwhile, diploid offspring show paternal-biased expression, yet tetraploids show maternal-biased expression. These discoveries reveal that fast and unstable changes are mainly deleterious at the level of transcriptomes although some offspring still survive their genomic abnormalities. In addition, the synthetic effect of genome shock might have resulted in greatly reduced viability of 2nF2 hybrid offspring. The goldfish × common carp hybrids constitute an ideal system for unveiling the consequences of intergenomic interactions in hybrid vertebrate genomes and their fertility.

Differential expression of activin ßA and ßB genes in female allotriploid and diploid red crucian carp Carassius auratus red var.

activin ßA and ßB from diploid and allotriploid crucian carp were cloned.The differential expression of activin ßA and ßB genes in female allotriploid and diploid red crucian carp Carassius auratus red var. were studied and found to be expressed in all the tested tissues; particularly, the expression of activin ßA and ßB was elevated in the ovaries of allotriploids and differential expression in pituitaries during the non-breeding season and the breeding season period. The immunohistochemistry indicated that the abnormal triploid ovaries were dominated by small oogonium-like cells with dense signals and that the elevated expression of activin ßA and ßB in the ovaries of allotriploids may be related to allotriploid sterility.

Asymmetric expression patterns reveal a strong maternal effect and dosage compensation in polyploid hybrid fish.

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.

Different expression patterns of sperm motility-related genes in testis of diploid and tetraploid cyprinid fish†.

Sperm motility is an important standard to measure the fertility of male. In our previous study, we found that the diploid spermatozoa from allotetraploid hybrid (4nAT) had longer durations of rapid and slow progressive motility than haploid spermatozoa from common carp (COC). In this study, to explore sperm motility-related molecular mechanisms, we compared the testis tissues transcriptomes from 2-year-old male COC and 4nAT. The RNA-seq data revealed that 2985 genes were differentially expressed between COC and 4nAT, including 2216 upregulated and 769 downregulated genes in 4nAT. Some differentially expressed genes, such as tubulin genes, dynein, axonemal, heavy chain(dnah) genes, mitogen-activated protein kinase(mapk) genes, tektin 4, FOX transcription factors, proteasome genes, and ubiquitin carboxyl-terminal hydrolase(uchl) genes, are involved in the regulation of cell division, flagellar and ciliary motility, gene transcription, cytoskeleton, energy metabolism, and the ubiquitin-proteasome system, suggesting that these genes were related to sperm motility of the 4nAT. We confirmed the differential expression of 12 such genes in 4nAT by quantitative PCR. By western blotting, we also confirmed increased expression of Uchl3 in 4nAT testis. In addition, we identified 1915 and 2551 predicted long noncoding RNA (lncRNA) transcripts from testis tissue transcriptomes of COC and 4nAT, respectively. Of these, 1575 lncRNAs were specifically expressed in 4nAT and 939 were specifically expressed in COC. This study provides insights into the transcriptome profile of testis tissues from diploid and tetraploid, which are useful for research on regulatory mechanisms behind sperm motility in male polyploidy.

Comparative Proteomic, Physiological, Morphological, and Biochemical Analyses Reveal the Characteristics of the Diploid Spermatozoa of Allotetraploid Hybrids of Red Crucian Carp (Carassius auratus) and Common Carp (Cyprinus carpio).

The generation of diploid spermatozoa is essential for the continuity of tetraploid lineages. The DNA content of diploid spermatozoa from allotetraploid hybrids of red crucian carp and common carp was nearly twice as great as that of haploid spermatozoa from common carp, and the durations of rapid and slow progressive motility were longer. We performed comparative proteomic analyses to measure variations in protein composition between diploid and haploid spermatozoa. Using two-dimensional electrophoresis followed by liquid chromatography tandem mass spectrometry, 21 protein spots that changed in abundance were analyzed. As the common carp and the allotetraploid hybrids are not fully sequenced organisms, we identified proteins by Mascot searching against the National Center for Biotechnology Information non-redundant (NR) protein database for the zebrafish (Danio rerio), and verified them against predicted homologous proteins derived from transcriptomes of the testis. Twenty protein spots were identified successfully, belonging to four gene ontogeny categories: cytoskeleton, energy metabolism, the ubiquitin-proteasome system, and other functions, indicating that these might be associated with the variation in diploid spermatozoa. This categorization of variations in protein composition in diploid spermatozoa will provide new perspectives on male polyploidy. Moreover, our approach indicates that transcriptome data are useful for proteomic analyses in organisms lacking full protein sequences.

YY super sperm lead to all male triploids and tetraploids.

BACKGROUND: Androgenesis is a unique and rarely encountered reproductive mode in which the offspring only inherit the paternal nuclear genome, resulting in relatively few viable individuals. RESULTS: In this study, a super male (YY) crucian carp was obtained by androgenesis with the diploid sperm of autotetraploid crucian carp (4n = 200). Flow cytometry assay confirmed the fish was diploid. The scanning electron microscopy and flow cytometry analysis results of sperm revealed that the YY crucian carp produced unreduced diploid sperm. To prove the special reproductive characteristic and homozygosity of the YY crucian carp, three rounds of hybridization experiments were performed. First, self-crossing between female androgenic progenies and YY crucian carp generated all male tetraploids. Then, hybridization of female red crucian carp (2n = 100) and female autotetraploid fish (4n = 200) with YY crucian carp produced all male triploids and all male tetraploids, respectively. CONCLUSIONS: This is the first time reported producing a viable diploid homozygous YY fish with unreduced diploid sperm of the autotetraploid fish, which were derived from distant hybridization. These results will not only help explaining the sex determination mechanism in teleost fish, but also play a significant role in genetic breeding in aquaculture.

Formation of the synaptonemal complex in a gynogenetic allodiploid hybrid fish.

Introduction: The correct pairing and separation of homologous chromosomes during meiosis is crucial to ensure both genetic stability and genetic diversity within species. In allodiploid organisms, synapsis often fails, leading to sterility. However, a gynogenetic allodiploid hybrid clone line (GDH), derived by crossing red crucian carp (Carassius auratus ♀) and common carp (Cyprinus carpio ♂), stably produces diploid eggs. Because the GDH line carries 100 chromosomes with 50 chromosomes from the red crucian carp (RCC; ♀, 2n = 2x = 100) and 50 chromosomes from the common carp (CC; C. carpio L., ♂, 2n = 2x = 100), it is interesting to study the mechanisms of homologous chromosome pairing during meiosis in GDH individuals. Methods: By using fluorescence in situ hybridization (FISH) with a probe specific to the red crucian carp to label homologous chromosomes, we identified the synaptonemal complex via immunofluorescence assay of synaptonemal complex protein 3 (SCP3). Results: FISH results indicated that, during early ovarian development, the GDH oogonium had two sets of chromosomes with only one set from Carassius auratus, leading to the failure formation of normal bivalents and the subsequently blocking of meiosis. This inhibition lasted at least 5 months. After this long period of inhibition, pairs of germ cells fused, doubling the chromosomes such that the oocyte contained two sets of chromosomes from each parent. After chromosome doubling at 10 months old, homologous chromosomes and the synaptonemal complex were identified. Discussion: Causally, meiosis proceeded normally and eventually formed diploid germ cells. These results further clarify the mechanisms by which meiosis proceeds in hybrids.

A Novel Allotriploid Hybrid Derived From Female Goldfish × Male Bleeker's Yellow Tail.

Hybridization is a traditional and effective strategy to alter the genotypes and phenotypes of the offspring, and distant hybridization is a useful strategy to generate polyploids in fish. In this study, goldfish (Carassius auratus, GF, 2n = 100) and Bleeker's yellow tail (Xenocypris davidi Bleeker, YT, 2n = 48), which belong to different subfamilies, were crossed with each other. The cross of female GF × male YT successfully obtained hybrid offspring (GFYT hybrids), while the cross of female YT × male GF was lethal, and all the fertilized eggs stopped developing before the neurula stage of embryogenesis. All GFYT hybrids possessed 124 chromosomes (3n = 124) with two sets from GF and one set from YT. The measurable and countable traits of GFYT hybrids were identified, and the genetic characteristics of 5S rDNA between GFYT hybrids and their parents were also revealed. There were, respectively, four and three different 5S rDNA types in GF (assigned as GF-Ⅰ∼Ⅳ) and YT (assigned as YT-Ⅰ∼Ⅲ), and GFYT hybrids specifically inherited YT-Ⅰ and YT-Ⅱ 5S rDNA types from YT and GF-Ⅲ and GF-Ⅳ from GF. In addition, there were only testis-like and fat-like gonads been found in GFYT hybrids. Interestingly, there were pyknotic and heteromorphous chromatin and invaginated cell membrane observed in the spermatids of testis-like gonads, but no mature sperm were found. Furthermore, TUNEL assays indicated that, compared with control, apparent apoptotic signals, which were mainly distributed around spermatid regions, were detected in the testis-like gonads, and the expression of apoptosis pathway-related genes including p53, bcl-2, bax, and caspase9 was significantly upregulated. Moreover, the expression of meiosis-related genes including spo11, dmc1, and rad51 showed an abnormally high expression, but mns1 and meig1, two key genes involved in the maturation of spermatid, were extremely downregulated. In brief, this is the first report of allotriploid via distant hybridization between GF and YT that possessing different chromosome numbers in vertebrates. The obtainment of GFYT hybrids not only harbors potential benefits and application in aquaculture but also further extends the understanding of the influence of hybridization and polyploidization on the genomic constitution of the hybrid offspring. Furthermore, they can be used as a model to test the origin and consequences of polyploidization and served as a proper resource to study the underlying mechanisms of spermatogenesis dysfunctions.

Coexistence of diploid, triploid and tetraploid crucian carp (Carassius auratus) in natural waters.

BACKGROUND: Crucian carp (abbreviated CC) belongs to the genus of Carassius within the family of Cyprinidae. It has been one of the most important freshwater species for Chinese aquaculture and is especially abundant in the Dongting water system of Hunan province. CC used to be considered as all diploid forms. However, coexistence of diploid (abbreviated 2nCC), triploid (abbreviated 3nCC) and tetraploid crucian carp (abbreviated 4nCC) population of the Dongting water system was first found by our recently researches. RESULTS: We examined the ploidy level and compared biological characteristics in different ploidy CC. In reproductive mode, 2nCC was bisexual generative and 4nCC generated all-female offspring by gynogenesis. However, 3nCC generated progenies in two different ways. 3nCC produced bisexual triploid offspring fertilized with 3nCC spermatozoa, while it produced all-female triploid offspring by gynogenesis when its ova were activated by heterogenous spermatozoa. The complete mitochondrial DNA of three different ploidy fishes was sequenced and analyzed, suggesting no significant differences. Interestingly, microchromosomes were found only in 3nCC, which were concluded to be the result of hybridization. Allogenetic DNA fragments of Sox genes were obtained in 3nCC and 4nCC, which were absent in 2nCC. Phylogenetics analysis based on Sox4 gene indicated 3nCC and 4nCC formed a separate group from 2nCC. CONCLUSIONS: In summary, this is the first report of the co-existence of three types of different ploidy crucian carps in natural waters in China. It was proved that the coexistence of different ploidy CC was reproductively maintained. We further hypothesized that 3nCC and 4nCC were allopolyploids that resulted from hybridization. The different ploidy CC population we obtained in this study possesses great significance for the study of polyploidization and the evolution of vertebrates.

Structural Abnormalities of Spermatozoa in Triploid Gynogenetic Crucian Carp (Carassius auratus).

The spermatozoa of triploid gynogenetic crucian carp (Carassius auratus) (3nDTCC) possess a spermatogenesis process with a normal genetic background. However, the genetic materials of these spermatozoa do not completely inherit gynogenetic progeny in general. Understanding the intrinsic mechanism may be helpful for developing breeding strategies of gynogenetic fishes. In this study, the spermatozoa ultrastructure was systematically studied in diploid red crucian carp and 3nDTCC to demonstrate their cytological structural differences. In addition, the artificial breeding tests of 3nDTCC(♀) with different ploidy spermatozoa were performed to verify the contributions of genetic materials from 3nDTCC spermatozoa to the gynogenesis progeny. Furthermore, the mRNA expression of centriole-related genes (i.e., cep57, cetn1, rootletin, and nek2) involved in spermatozoa packaging was also determined by quantitative real-time PCR (qPCR) to illustrate the molecular expression characteristics of the spermatozoa packaging process in 3nDTCC. The results reveal the adaptive features of spermatozoa in 3nDTCC, including the loose midpiece structure, abnormal head structure, and abnormal expression of centriole-related genes, which may influence the motility of spermatozoa and make it not involved normally in the genetic composition of the gynogenesis offspring.

Localization of RNA Pol II CTD (S5) and Transcriptome Analysis of Testis in Diploid and Tetraploid Hybrids of Red Crucian Carp (♀) × Common Carp (♂).

Polyploidy occurs naturally in fish; however, the appearance of these species is an occasional and gradual process, which makes it difficult to trace the changes in phenotypes, genotypes, and regulation of gene expression. The allotetraploid hybrids (4nAT) of red crucian carp (RCC; ♀) × common carp (CC; ♂) generated from interspecies crossing are a good model to investigate the initial changes after allopolyploidization. In the present study, we focused on the changes in the active sites of the testicular transcriptome of the allotetraploid by localization of RNA Pol II CTD YSPTSPS (phospho S5) using immunofluorescence and RNA-seq data via bioinformatic analysis. The results showed that there was no significant difference in signal counts of the RNA Pol II CTD (S5) between the different types of fish at the same stages, including RCC, CC, 2nF1, and 4nAT, which means that the number of transcriptionally active sites on germ cell chromosomes was not affected by the increase in chromosome number. Similarly, RNA-seq analysis indicated that in the levels of chromosomes and 10-kb regions in the genome, there were no significant changes in the highly active sites in RCC, 2nF1, and 4nAT. These findings suggest that at the beginning of tetraploid origin, the active transcriptome site of 4nAT in the testis was conserved in the regions of the genome compared to that in RCC and 2nF1. In conclusion, 4nAT shared a similar gene expression model in the regions of the genome with RCC and 2nF1 with significantly different expression levels.

Abnormal meiosis in fertile and sterile triploid cyprinid fish.

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.