bo Qin

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.

Analysis of 5S rDNA organization and variation in polyploid hybrids from crosses of different fish subfamilies

In this article, sequence analysis of the coding region (5S) and adjacent nontranscribed spacer (NTS) were conducted in red crucian carp (RCC), blunt snout bream (BSB), and their polyploid offspring. Three monomeric 5S rDNA classes (designated class I: 203 bp; class II: 340 bp; and class III: 477 bp) of RCC were characterized by distinct NTS types (designated NTS-I, II, and III for the 83, 220, and 357 bp monomers, respectively). In BSB, only one monomeric 5S rDNA was observed (designated class IV: 188 bp), which was characterized by one NTS type (designated NTS-IV: 68 bp). In the polyploid offspring, the tetraploid (4nRB) hybrids partially inherited 5S rDNA classes from their female parent (RCC); however, they also possessed a unique 5S rDNA sequence (designated class I-L: 203 bp) with a novel NTS sequence (designated NTS-I-L: 83 bp). The characteristic paternal 5S rDNA sequences (class IV) were not observed. The 5S rDNA of triploid (3nRB) hybrids was completely inherited from the parental species, and generally preserved the parental 5S rDNA structural organization. These results first revealed the influence of polyploidy on the organization and evolution of the multigene family of 5S rDNA of fish, and are also useful in clarifying aspects of vertebrate genome evolution.

Organization and Variation Analysis of 5S rDNA in Different Ploidy-level Hybrids of Red Crucian Carp × Topmouth Culter

Through distant crossing, diploid, triploid and tetraploid hybrids of red crucian carp (Carassius auratus red var., RCC♀, Cyprininae, 2n = 100) × topmouth culter (Erythroculter ilishaeformis Bleeker, TC♂, Cultrinae, 2n = 48) were successfully produced. Diploid hybrids possessed 74 chromosomes with one set from RCC and one set from TC; triploid hybrids harbored 124 chromosomes with two sets from RCC and one set from TC; tetraploid hybrids had 148 chromosomes with two sets from RCC and two sets from TC. The 5S rDNA of the three different ploidy-level hybrids and their parents were sequenced and analyzed. There were three monomeric 5S rDNA classes (designated class I: 203 bp; class II: 340 bp; and class III: 477 bp) in RCC and two monomeric 5S rDNA classes (designated class IV: 188 bp, and class V: 286 bp) in TC. In the hybrid offspring, diploid hybrids inherited three 5S rDNA classes from their female parent (RCC) and only class IV from their male parent (TC). Triploid hybrids inherited class II and class III from their female parent (RCC) and class IV from their male parent (TC). Tetraploid hybrids gained class II and class III from their female parent (RCC), and generated a new 5S rDNA sequence (designated class I–N). The specific paternal 5S rDNA sequence of class V was not found in the hybrid offspring. Sequence analysis of 5S rDNA revealed the influence of hybridization and polyploidization on the organization and variation of 5S rDNA in fish. This is the first report on the coexistence in vertebrates of viable diploid, triploid and tetraploid hybrids produced by crossing parents with different chromosome numbers, and these new hybrids are novel specimens for studying the genomic variation in the first generation of interspecific hybrids, which has significance for evolution and fish genetics.

Biological characteristics of an improved triploid crucian carp

An improved triploid crucian carp (ITCC) was produced by crossing improved tetraploids (G1×AT, ♂) with improved red crucian carp (IRCC, ♀), which were obtained by distant crossing and gynogenesis. The biological characteristics of ITCC, including the number and karyotype of chromosomes, gonadad and pituitary structures, phenotype, and growth rate are reported. ITCC possessed 150 chromosomes with the karyotype 33m+51sm+33st+33t. In the breeding season, both ovary-like and testis-like gonads of ITCC were unable to produce normal mature gametes. The ultrastructure of the pituitary of ITCC showed that most of the endocrine granules in gonadotrophic hormone (GTH) cells had not been released, providing endocrinological evidence for the sterility of ITCC. Compared with triploid crucian carp (TCC) produced by mating Japanese crucian carp with allotetraploid hybrids, ITCC not only retained the excellent traits of fast growth rate and sterility, but also acquired improved morphological characteristics, including higher body, shorter tail and smaller head.

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.

Research advances in animal distant hybridization

Distant hybridization refers to crosses between two different species or higher-ranking taxa that enables interspecific genome transfer and leads to changes in phenotypes and genotypes of the resulting progeny. If progeny derived from distant hybridization are bisexual and fertile, they can form a hybrid lineage through self-mating, with major implications for evolutionary biology, genetics, and breeding. Here, we review and summarize the published literature, and present our results on fish distant hybridization. Relevant problems involving distant hybridization between orders, families, subfamilies, genera, and species of animals are introduced and discussed, with an additional focus on fish distant hybrid lineages, genetic variation, patterns, and applications. Our review serves as a useful reference for evolutionary biology research and animal genetic breeding.

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.

Novel genetic markers derived from the DNA fragments of Sox genes.

With the pair of degenerate primers designed against the conserved regions of HMG-box of Sox gene family, DNA fragments of different sizes were obtained by amplifying the whole genome DNA samples of many animals, including natural fish, artificial hybrid fish, Aves, reptiles, amphibians and hexapods. Each sample was identified by the specific DNA-band pattern formed by the DNA fragments with defined number and size which marked the samples genetic characteristics. In addition, 50 DNA fragments from 22 kinds of animals were sequenced and comparatively analyzed so as to study their genetic relationships, especially that between artificial hybrids and their original parents. Based on the specific DNA-band pattern and the specific DNA sequence obtained in tissue DNA sample, we established the novel genetic DNA markers derived from the DNA fragments of Sox genes. The present results proved that the novel DNA markers provided fast and accurate markers for different animal phylogenetic branches and that these convenient markers can also distinguish closely related species and hybrids using a single gene family tool.

Formation and biological characterization of three new types of improved crucian carp

The improved tetraploids (G1×AT) were obtained by distant crossing and gynogenesis and the high-body individuals accounted for 2% among G1×AT. After mating with each other, the high-body individuals produced three kinds of bisexual fertile diploid fishes: high-body red crucian carp, high-body fork-like-tails goldfish and gray common carp. The high-body red crucian carp mating with each other formed three types of improved crucian carp (ICC) including improved red crucian carp (IRCC), improved color crucian carp (ICCC) and improved gray crucian carp (IGCC). The phenotypes, chromosome numbers, gonadal structure and fertility of the three kinds of ICC and their offspring were observed. All the three kinds of ICC possessed some improved phenotypes such as higher body, smaller head and shorter tail. The ratios of the body height to body length of IRCC, ICCC and IGCC were 0.54, 0.51 and 0.54, respectively. All of them were obviously higher than that of red crucian carp 0.41 (P<0.01). Three kinds of ICC had the same chromosome number as red crucian carp with 100 chromosomes. All the ICC possessed normal gonads producing mature eggs or sperm, which was important for the production of an improved diploid population. Compared with red crucian carp, all the ICC had stronger fertility such as higher gametes production, higher fertilization rate and higher hatchery rate. Three types of improved diploid fish population were generated from the three kinds of ICC by self-crossing, respectively. The ICC can serve as ornamental fish and edible fish. They are also ideal parents to produce triploids by mating with tetraploids. The new ICC plays an important role in biological evolution and fish genetic breeding.