Rurong Zhao

The formation of the polyploid hybrids from different subfamily fish crossings and its evolutionary significance.

This study provides genetic evidences at the chromosome, DNA content, DNA fragment and sequence, and morphological levels to support the successful establishment of the polyploid hybrids of red crucian carp × blunt snout bream, which belonged to a different subfamily of fish (Cyprininae subfamily and Cultrinae subfamily) in the catalog. We successfully obtained the sterile triploid hybrids and bisexual fertile tetraploid hybrids of red crucian carp (RCC) (♀) × blunt snout bream (BSB) (♂) as well as their pentaploid hybrids. The triploid hybrids possessed 124 chromosomes with two sets from RCC and one set from BSB; the tetraploid hybrids had 148 chromosomes with two sets from RCC and two sets from BSB. The females of tetraploid hybrids produced unreduced tetraploid eggs that were fertilized with the haploid sperm of BSB to generate pentaploid hybrids with 172 chromosomes with three sets from BSB and two sets from RCC. The ploidy levels of triploid, tetraploid, and pentaploid hybrids were confirmed by counting chromosomal number, forming chromosomal karyotype, and measuring DNA content and erythrocyte nuclear volume. The similar and different DNA fragments were PCR amplified and sequenced in triploid, tetraploid hybrids, and their parents, indicating their molecular genetic relationship and genetic markers. In addition, this study also presents results about the phenotypes and feeding habits of polyploid hybrids and discusses the formation mechanism of the polyploid hybrids. It is the first report on the formation of the triploid, tetraploid, and pentaploid hybrids by crossing parents with a different chromosome number in vertebrates. The formation of the polyploid hybrids is potentially interesting in both evolution and fish genetic breeding.

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

BackgroundCrucian 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.ResultsWe 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.ConclusionsIn 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.

The fertility of the hybrid lineage derived from female Megalobrama amblycephala × male Culter alburnus.

Distant hybridization can combine together the genomes of different species, which leads to changes of the offspring in phenotypes and genotypes. In this study, we successfully establish a fertile hybrid lineage by intergeneric hybridization of female blunt snout bream (BSB, Megalobrama amblycephala) × male topmouth culter (TC, Culter alburnus) and investigate some important biological traits of this lineage including the morphological traits, chromosomal number, karyotype, DNA content, gonadal development, egg and milt yield, sperm shape and density, fertilization rate and early survival rate. The results show that: (1) the diploid and triploid hybrids coexist in F1 and only diploid hybrids are found in F2, in which the diploid hybrids of F1 and F2 possess 48 chromosomes with one chromosome set of BSB and one chromosome set of TC, and the triploid hybrids of F1 possess 72 chromosomes with two chromosome sets of BSB and one chromosome set of TC. (2) All the tested males and females of the diploid F1 and F2 hybrids have the normal gonadal development and produce mature sperm and egg, respectively, which are fertilized with each other to form F2 and F3 hybrids, respectively, and finally form a diploid hybrid lineage (F1-F3). (3) The good fertility of the F1 and F2 hybrids of female BSB × male TC potentially provides reproductive base to make the hybrid lineage propagate from one generation to another. The formation of the hybrid lineage (F1-F3) also provides an ideal model to research the reproductive rules of distant hybrid progeny.

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

Significance Why is polyploidization rarer in animals than in plants? This question remains unanswered due to the absence of a suitable system in animals for studying instantaneous polyploidization and the crucial changes that immediately follow hybridization. RNA-seq analyses discover extensive chimeric genes and immediate mutations of orthologs in both diploid and tetraploid offspring of the goldfish (♀) × common carp (♂) hybrids. Overall, diploid offspring show paternal-biased expression, yet tetraploids show maternal-biased expression. Some chimeric and differentially expressed genes relate to crucial functions of normal cell cycle activities, and cancer-related pathways in 2nF1. The discovery of fast changes at the levels of chromosomes, genomic DNA, and transcriptomes suggests that allopolyploidization hinders genomic functions in vertebrates, and this conclusion may extend to all animals. 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.

Evidence for the Formation of the Male Gynogenetic Fish

The females and unexpected males of gynogenetic red crucian carps (GRCC) with the 1:1 sex ratio were found in the progeny of the distant crossing of red crucian carp (RCC; ♀, 2n = 100) × blunt snout bream (BSB; ♂, 2n = 48). The females and males of GRCC were fertile, and they mated each other to generate the red crucian carps (GRCC1) and another variational gray crucian carps (GGCC). The GRCC and their offspring were proved to be diploids (2n = 100) with one to three microchromosomes by examining the chromosomal metaphases. The evidences for the male’s genetic effect in GRCC were provided by means of fluorescence in situ hybridization, Sox-HMG DNA markers, and microsatellite DNA markers. The genotypic variances of GRCC resulted in their phenotypic variances which were quite different from their maternal parent. It was concluded that the formation of the male gynogenetic fish in GRCC resulted from the genetic leakage of the paternal fish in the form of the microchromosomes including the paternal male-determining gene. After being activated by the sperm of BSB, which was inactivated and finally degraded but left the microchromosomes, the egg of RCC, in which the 50 chromosomes were spontaneously doubled to 100 chromosomes, developed into the diploid male gynogenetic fish. The formation of the bisexual GRCC and their progeny indicated that the distant hybridization could generate the bisexual diploid gynogenetic fish with genetic variation derived from the paternal fish, which is of great significance in both fish genetic breeding and evolutionary biology.

A new type of homodiploid fish derived from the interspecific hybridization of female common carp × male blunt snout bream

It is commonly believed that hybridization might lead to the formation of new polyploidy species, but it is unclear whether hybridization can produce a new homodiploid species. Here, we report the spontaneous occurrence of a new crucian carp-like homodiploid fish (2n = 100) that originated from the interspecific hybridization of female common carp (Cyprinus carpio, Cyprininae, 2n = 100) × male blunt snout bream (Megalobrama amblycephala, Cultrinae, 2n = 48). The phenotype and reproductive traits of this new crucian carp-like homodiploid fish were found to be very similar to those of the existing diploid species (diploid crucian carp; Carassius auratus). FISH and 5S rDNA analyses revealed that the genotype of the crucian carp-like homodiploid fish differs from those of its parents but is closely related to that of diploid crucian carp. The results provide evidence of the existence of a possible route through which the distant hybridization of this cross can generate crucian carp. The new type of homodiploid fish is of great value in fish genetic breeding and for studying the early evolutionary process.

Cell fusion as the formation mechanism of unreduced gametes in the gynogenetic diploid hybrid fish

The gynogenetic diploid hybrid clone line (GDH) derived from red crucian carp (♀ RCC) × common carp (♂ CC) possesses the unusual reproductive trait of producing unreduced diploid eggs. To identify the mechanism underlying this phenomenon, we examined the structure, in vivo developmental process and in vitro dynamic development of the GDH gonad. In summary, compared with RCC and CC, GDH showed certain special straits. First, a high frequency (84.7%) of germ cell fusion occurred in gonadal tissue culture in vitro as observed by time-lapse microscopy. Second, microstructural and ultrastructural observation showed numerous binucleated and multinucleated germ cells in the gonad, providing evidence of germ cell fusion in vivo. By contrast, in the diploid RCC and CC ovaries, neither cell fusion nor multinucleated cells were observed during the development of gonads. Third, the ovary of GDH remained at stage I for 10 months, whereas those of RCC and CC remained at that stage for 2 months, indicating that the GDH germ cells underwent abnormal development before meiosis. This report is the first to demonstrate that cell fusion facilitates the formation of unreduced gametes in vertebrates, which is a valuable finding for both evolutionary biology and reproductive biology.

Microsatellite analysis of different ploidy offspring of artificial gynogenesis in Cyprinus carpio

Gynogenesis was induced by using UV-irradiated spermatozoa of blunt snout bream Megalobrama amblycephala to activate eggs of common carp Cyprinus carpio. The maternal genome was then duplicated by cold shock in 0 to 4° C cold water to retain the second polar body. Two kinds of fry, normal fry and abnormal tortuous fry, were hatched. Their DNA content was measured by flow cytometry. The normal fry were identified as diploid, representing the successful gynogenesis in C. carpio whereas the abnormal tortuous fry were haploid. Ten microsatellite loci were used to study the genetic diversity among C. carpio, diploid gynogenetic C. carpio and unduplicated haploid tortuous fry. The results indicated that the genetic homozygosity of gynogenetic C. carpio was significantly higher than that of C. carpio. The genetic homozygosity of the haploid C. carpio was intermediate between that of gynogenetic C. carpio and C. carpio. It might be easier for the allogenetic DNA fragments to be integrated into the haploid genome than into diploid gynogenetic genome.

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

Abstract 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 regulatorymechanisms behind sperm motility in male polyploidy. Summary Sentence Clues of variations in tetraploid testis are characterized by comparative transcriptome analysis between tetraploid hybrid and common carp.

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)

ABSTRACT 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.