A novel nucleo-cytoplasmic hybrid clone formed via androgenesis in polyploid gibel carp
© Gui et al; licensee BioMed Central Ltd. 2011
Received: 3 November 2010
Accepted: 28 March 2011
Published: 28 March 2011
Unisexual vertebrates have been demonstrated to reproduce by gynogenesis, hybridogenesis, parthenogenesis, or kleptogenesis, however, it is uncertain how the reproduction mode contributes to the clonal diversity. Recently, polyploid gibel carp has been revealed to possess coexisting dual modes of unisexual gynogenesis and sexual reproduction and to have numerous various clones. Using sexual reproduction mating between clone D female and clone A male and subsequent 7 generation multiplying of unisexual gynogenesis, we have created a novel clone strain with more than several hundred millions of individuals. Here, we attempt to identify genetic background of the novel clone and to explore the significant implication for clonal diversity contribution.
Several nuclear genome markers and one cytoplasmic marker, the mitochondrial genome sequence, were used to identify the genetic organization of the randomly sampled individuals from different generations of the novel clone.
Chromosome number, Cot-1 repetitive DNA banded karyotype, microsatellite patterns, AFLP profiles and transferrin alleles uniformly indicated that nuclear genome of the novel clone is identical to that of clone A, and significantly different from that of clone D. However, the cytoplasmic marker, its complete mtDNA genome sequence, is same to that of clone D, and different from that of clone A.
The present data indicate that the novel clone is a nucleo-cytoplasmic hybrid between the known clones A and D, because it originates from the offspring of gonochoristic sexual reproduction mating between clone D female and clone A male, and contains an entire nuclear genome from the paternal clone A and a mtDNA genome (cytoplasm) from the maternal clone D. It is suggested to arise via androgenesis by a mechanism of ploidy doubling of clone A sperm in clone D ooplasm through inhibiting the first mitotic division. Significantly, the selected nucleo-cytoplasmic hybrid female still maintains its gynogenetic ability. Based on the present and previous findings, we discuss the association of rapid genetic changes and high genetic diversity with various ploidy levels and multiple reproduction modes in several unisexual and sexual complexes of vertebrates and even other invertebrates.
Since Amazon molly Poecilia formosa, the first clonal reproduction vertebrate, was found in 1932, about 90 all-female unisexual complexes have been reported in fish, amphibians and reptiles. These unisexual animals have been revealed to reproduce by gynogenesis, hybridogenesis, parthenogenesis, or kleptogenesis[3–6], but it has remained uncertain how the reproduction mode contributes to the clonal diversity. Recently, unique dual modes of unisexual gynogenesis and sexual reproduction have been discovered in polyploid gibel carp[7, 8], which gives a significant clue to the formation of clone diversity in unisexual vertebrates.
Gibel carp, also commonly known as silver crucian carp or Prussian carp, which was previously nominated as a subspecies (Carassius auratus gibelio) of goldfish, has been currently suggested as a separate species Carassius gibelio. It was preliminarily uncovered to have unisexual reproduction ability of allogynogenesis early in the last century[10, 11]. Allogynogenesis is a form of gynogenesis stimulated by heterologous sperm from other fish species. In allogynogenesis, some supernumerary minichromosomes or chromosomal fragments can be accidently incorporated into the maternal genomes[12, 13], but the heterologous sperm does not decondense and fuse with the female pronucleus[8, 11]. Diverse karyotypes with 156 or 162 chromosomes and triploid origin relative to goldfish with 100 chromosomes were confirmed in wild gibel carp populations. Moreover, numerous different clones and genetic diversity were discriminated by serum transferrin phenotypes, RAPD and SCAR markers[17, 18], microsatellite markers, transferrin allele polymorphism[20, 21], and mtDNA control region sequences. Significantly, an approximate 1%-10% of males identical to the females in genetic background were found in natural habitats, and genetic recombination evidence was obtained in the offspring produced by bisexual mating between different clones. Therefore, genetic evidence indicates that gibel carp have coexisting dual modes of unisexual gynogenesis and gonochoristic sexual reproduction, as it can reproduce all-female offspring through unisexual gynogenesis when the eggs are activated by heterologous sperm from other fish species, and also produces female and male offspring through sexual reproduction when the eggs are inseminated by homologous sperm from the gibel carp males.
Theoretically, the dual reproduction modes provide a unique way for creating novel clones in laboratory, because numerous genetic recombination offspring can be obtained by sexual reproduction mating between different clones, and some better and valuable clones can be selected and proliferated by unisexual gynogenesis. To exploit the new breeding potential, we performed numerous sexual mating experiments between different clones since 1996. Clone A and clone D are very diverse among the identified clones. Karyotype of clone A individuals contains 156 chromosomes, which are composed of 36 metacentric(m), 54 submetacentric(sm), 36 subtelocentric(st), 24 acrocentric(t), and six small chromosomes, whereas karyotype of clone D individuals has 162 chromosomes, with 42 m, 54 sm, 36 st, 24 t, and six small chromosomes. Through sexual reproduction mating between clones A and D, we found a few of fast-growing individuals. Significantly, the fast-growing individuals still possess its unisexual reproduction ability of gynogenesis. Thereby, the novel clone A+ was created by originally sexual mating between clone D female and clone A male, and rapidly multiplied up to several hundred millions by subsequent 7 generations of unisexual gynogenesis. Here, we summarize formation process of the novel clone strain and attempt to identify the genetic organization and background by nuclear and cytoplasmic markers including chromosome number count, Cot-1 DNA fluorescent banding karyotype analysis, microsatellite electrophoretic pattern, AFLP profile, transferrin allele identification and mitochondrial genome sequence comparison, because most of them had been proven to be particularly valuable for clone discrimination, diversity evaluation and genealogical relationship analysis in several unisexual vertebrates, such as gynogenetic Amazon molly Poecilia formosa[23, 24], gynogenetic Phoxinus eos-neogaeus, hybridogenetic Poeciliopsis[26–28], hybridogenetic water frog Rana esculenta , kleptogenetic salamanders[30–35], parthenogenetic lizards, and the gynogenetic gibel carp[19–22, 37]. Based on these studies, we explore and discuss the significant implication for clonal diversity contribution.
Source of samples
Clone D female and clone A male were used as the maternal and paternal for the propagation experiments of gonochoristic sexual reproduction mating. Control gynogenetic individuals of clone D were inseminated by sperm from red common carp (Cyprinus carpio) to activate the eggs. As described previously, spawning was artificially induced by two intraperitoneal injections with a mixture of acetone-dried carp pituitary, hCG and LRH-A. Ovulated eggs from clone D were divided into two parts and respectively inseminated with sperm from a clone A male and from a red common carp male. The produced offspring were respectively cultured in separate fishponds. After they reached to adults for one year, the phenotype, size and sex were determined. Subsequently, one fast-growing clone A-like individual was selected as the clonal maternal and its unisexual reproduction ability of gynogenesis was demonstrated by Xingguo red common carp (Cyprinus carpio) sperm stimulation, because the offspring are all-females, and identical to the maternal. Moreover, they have been proliferated by 7 successive generations of gynogenesis with Xingguo red common carp (Cyprinus carpio) sperm stimulation, and more than several hundred millions of clonal individuals have been produced. From the fifth generation, the clonal strain as a new aquatic variety has been introduced into more than 10 hatcheries, and their gynogenetic offspring were cultured throughout China. As a stock hatchery, the successively gynogenetic offspring of 7 generations have been maintained in the Guanqiao Experimental Station of the Hydrobiology Institute in Wuhan, Hubei Province, and all the samples for genetic analysis were collected from the Station. Besides the maternal and paternal, generally, ten individuals were randomly sampled from each analyzed clone and generation, and their fin tissues and blood cells were sampled for DNA extraction and DNA content measurement respectively. The fin tissues were stored in 95% ethanol at -20°C and blood cells were fixed by 70% ethanol and stored at 4°C.
Genomic DNA extraction and Cot-1 DNA isolation
Genomic DNA were extracted individually from fin tissues as described previously. Cot-1 DNA was isolated as described previously, and labeled with biotin-16-dUTP by a nick translation reaction for FISH.
Chromosome preparation and fluorescence in situ hybridization
Chromosome metaphases were prepared from kidney cells of at least 5 individuals for each clone by the method of kidney cell-phytohemagglutinin (PHA) culture in vivo and then were counterstained with DAPI. Fluorescent in situ hybridization (FISH) of Cot-1 DNA was performed according to the method described previously, and images were acquired using a Leica inverted DMIRE2 microscope and a Leica LCS SP2 confocal image system (Leica, Germany).
Genetic marker application
Ten microsatellite primers were applied to amplify microsatellites in genomic DNA and PCR amplification was performed as the described method. As for AFLP analysis, genome DNA was digested with EcoR I and Mse I and then selective amplification was performed using ten primer combinations. Products of microsatellite and AFLP were separated using 6% denaturing polyacrylamide electrophoresis and were visualized by silver staining. Transferrin alleles were amplified using special primer (Tf760 and TF1162) and then were cloned and sequenced as described previously[20, 21]. To obtain the complete mitochondrial genome, twenty conserved primers were used to amplify contiguous and overlapping fragments. According to the aligned sequences, another two pairs of primers were designed to amplify the fragments including the varied sites for further validation.
Sperm DNA content measurement
Clone A sperms were sampled from 8 individuals in reproduction season and 20 μl of them were respectively dropped into 1 ml of 70% ethanol. The fixed sperms at 4°C overnight were washed with PBS buffer for 3 times at 1000 rpm for 5 min. The pellets were resuspended in 0.5 ml of 0.5% pepsin in 0.1 M HCl. After the solution were incubated at room temperature for 30 min with gentle shaking, 100 μl of them was stained by adding 2 ml of propidium iodide solution (40 μgPI/ml) containing 4 kU/ml RNase (DNase-free ) at room temperature for 3 h in the dark. After the mixed cells were filtered through a special nylon mesh, the DNA content measurement was performed as described previously by Phoenix Flow Systems (Beckman and Coulter). The blood cells of clone A were also sampled and detected from the same individuals as controls under the same conditions.
Microsatellite and AFLP amplification bands in the gibel carp clones were evaluated based on the electrophoretic patterns and the genetic diversities were calculated by Arlequin 3.11 software. The gene regions of mitochondrial genome were identified with the homologous regions of the complete mitochondrial DNA sequence of triploid hybrids of tetraploid and Japanese crucian carp (GenBank accession number: AY771781). All sequences of transferrin and mitochondrial DNA were aligned and exported with CLUSTAL X  and Mega 4.0 software .
Formation process of novel clone A+
Identical chromosome number and banded karyotype to clone A
Moreover, the Cot-1 repetitive DNA fluorescence banding shows homologous chromosome-specifically patterns, and non-homologous chromosomes have different banded patterns. According to the various fluorescent intensities that locate at centromeric regions and other interstitial regions and the differential chromosome size and shape, we compiled the triploid karyotypes of clones A+, clone A and clone D with three homologous chromosomes. In all the analyzed three karyotypes, there are 9 small chromosomes that are wholly labeled by the Cot-1 repetitive DNA fluorescence, in which three of them are submetacentric chromosomes, and others are six small chromosomes. Also, the short arms of three same size subtelocentric chromosomes are entirely stained by the Cot-1 repetitive DNA fluorescence in all examined three clones. Significantly, clones A+ (Figure 2 g) and clone A (Figure 2 h) possess identical karyotypes that are comprised of 36 m, 54 sm, 36 st, 24 t, and six small chromosomes, whereas the karyotype of clone D (Figure 2i) is different from that of clones A+ and clone A, which contains 42 m, 54 sm, 36 st, 24 t and six small chromosomes.
Identical nuclear genome to clone A
Primer pair sequences and allele distribution of 10 microsatellites among clone A, clone D and clone A+ of gibel carp*
Primer pair sequence
Allele size (bp)
84, 94, 100
88, 92, 96
84, 94, 100
270, 288, 302
260, 290, 292
270, 288, 302
50 - 55°C
210, 220, 224
218, 222, 226
210, 220, 224
50 - 55°C
226, 230, 234
226, 230, 234
50 - 55°C
50 - 55°C
138, 150, 156
136, 138, 156
138, 150, 156
50 - 55°C
110, 114, 116
110, 112, 118
110, 114, 116
50 - 55°C
150, 156, 166
152, 160, 162
150, 156, 166
140, 162, 170
Comparison of amplification bands by AFLP among clone A, clone D and clone A+ of gibel carp*
No. of amplified bands
No. of clone-specific bands
No. of shared bands
A and A+
Genetic divergences among the five transferrin alleles
Same mitochondrial genome to clone D
Genetic uniformity among different generations
Clone A sperm DNA content
In this study, we reported a detailed formation process of the novel clone strain, and analyzed and identified the genetic organization and background by nuclear and cytoplasmic genome markers. The current data clearly indicate that the nucleus of clone A+ comes from that of clone A, because its chromosome number, Cot-1 repetitive DNA banded karyotype, and nuclear genome markers including microsatellite patterns, AFLP profiles and transferrin alleles are identical to that of clone A, and significantly different from that of clone D, whereas the cytoplasm comes from that of clone D, as the cytoplasmic marker-mtDNA genome is completely same to that of clone D, and different from that of clone A. Therefore, the major finding in this study is that the novel strain is clonal and represents a novel hybrid genotype with an entire nuclear genome from the paternal clone A and a mtDNA genome (cytoplasm) from the maternal clone D. And, the selected fast-growing individual as clonal maternal might be a nucleo-cytoplasmic hybrid between clone A and clone D, which might be formed via androgenesis of the clone A sperm in the clone D ooplasm.
As far as we know, androgenesis has been observed to occur naturally in interspecific hybrids of Sicilian stick insects[47, 48] and triploid Asian clams (genus Corbicula)[49, 50]. In fish, natural androgenesis has never been reported, but viable androgenetic fishes have been artificially induced in some ancient polyploid species, such as rainbow trout, common carp, loach[53, 54], and sturgeon, and in a artificial allotetraploid hybrid of common carp and red crucian carp. Generally, androgenesis involves loss or inactivation of the female genome and doubling of the paternal genome. In the sexual mating between clone D female and clone A male, most of the fertilized eggs might undergo the fusion of clone D female pronucleus and clone A male pronucleus, and resulted in high mortality during embryo hatching and fingerling cultivation owing to the significant difference of chromosome number and karyotypes between them. The surviving offspring differ from clone D and clone A in morphological phenotype. Indeed, fusion failure of the female and male pronuclei might also occur in a few of the fertilized eggs, in which some developed into clone D-identical individuals, some developed into the clone A-like individuals. The clone D-identical individual might occur through the mechanism of gynogenesis, because the sperm was occasionally recognized as heterologous sperm, just like that from other fish species owing to the significant genetic difference between clones A and D. The clone A-like individual should be suggested to form via androgenesis, because it has an entire nuclear genome from the paternal clone A and a mtDNA genome from the maternal clone D. In contrast to gynogenesis, the entire female nuclear genome of clone D might be extruded, and the offspring might be produced by two possible mechanisms, such as by ploidy doubling of clone A sperm through inhibiting the first mitotic division or by inclusion of clone A unreduced sperm into clone D ooplasm. If the latter is true, it is impossible to differentiate females and males in the androgenetic offspring (Figure 1) according to the XY sex determination mechanism, even though the direct evidence for existence of a few proportion of unreduced sperms have been obtained by flow cytometry in the paternal clone A sperms (Figure 6). Therefore, we suggest that the clone A-like androgenetic individuals should be formed by the mechanism of ploidy doubling of clone A sperm through inhibiting the first mitotic division, and thereby the nucleo-cytoplasmic hybrids can differentiate into females and males.
Significantly, the selected nucleo-cytoplasmic hybrid female still maintains its gynogenetic ability, and thereby forms a nucleo-cytoplasmic clone with over several hundred millions of clonal individuals. In the past 20 years, we also obtained one allotetraploid clone with additional genome incorporation[58, 59] and one supertriploid clone with subgenomic incorporation in the gibel carp. Actually, unisexual clones were early synthesized in 1973 from Poeciliopsis monacha-lucida, and the stably inherited clone with minichromosome incorporation was also found from gynogenetic Amazon molly (Poecilia formosa)[12, 61]. However, this androgenetic nucleo-cytoplasmic hybrid clone should be the first case in vertebrates, and might be related to the high polyploidy level and the diverse reproduction modes. In invertebrate freshwater clams of the genus Corbicula that reproduce by androgenesis, ploidy changes including diploid, triploid, and tetraploid exist widely among different species and populations, and the higher ploidy levels have been suggested to be related to the origin of androgenetic reproduction[49, 62]. In interspecific hybrids of Sicilian stick insects, various reproduction modes including parthenogenesis, hybridogenesis and androgenesis have been revealed to be associated with hybridization, polyploidy and karyotype re-diploidization, and these modes have been believed to be a noteworthy ability to overcome species-specific reproduction isolation mechanisms[48, 63]. Similarly, various ploidy including triploid, supertriploid and tetraploid and different reproduction modes including gynogenesis, androgenesis and sexual reproduction also exist extensively in gibel carp. Therefore, gibel carp and even other unisexual vertebrates may also possess the ability for overcoming species-specific reproduction isolation mechanisms. In unisexual salamanders (genus Ambystoma), Bogart et al. revealed a new reproductive mode of kleptogenesis by which unisexual salamanders steal sperm genome from donors of normally bisexual species[33, 34]. Perhaps, various ploidy levels and different reproduction modes are common characteristics for the unisexual animal complexes, regardless of whatever they are invertebrates or vertebrates, and it is these characteristics to result in rapid incorporation of nuclear genome, chromosome, chromosomal segment or mtDNA genome and to cause rapid genetic changes. Moreover, they achieve remarkable ecological success, even though some are rare and localized, and others are abundant . For example, in comparison with the related sexual species Carassius auratus, polyploid gibel carp have wide geographic distribution and occupy more multiple niches from northern Europe to Asia.
Indeed, the coexisting dual modes of unisexual gynogenesis and sexual reproduction in gibel carp might be indicative case of a change in reproductive mode from complete unisexual reproduction towards sexual reproduction. In green toads (Bufo viridis)[64, 65] and water frogs (Pelophylax esculentus) involving various ploidy levels, sexual reproduction triploids have been observed. Recently, a male-biased mutant family that contains 97.2% males was also found from the gynogenetic progeny of a gibel carp clone. Therefore, various ploidy levels and highly diverse reproductive modes might be the causes for novel clone formation and clonal diversity in unisexual vertebrates, and also make them excellent models for the studies of evolutionary genetics and ecology, both theoretically and empirically. Additionally, as the nucleo-cytoplasmic hybrid clone escapes the genetic and developmental destruction caused by drastic treatments of irradiation and physical shocks in induced androgenesis and nuclear transplantation[67, 68], the novel finding will be of great significance for exploiting the genetic breeding approaches in gibel carp. Of course, this finding of nucleo-cytoplasmic hybrid clone can help us to understand some reasons for clonal and genetic diversity production of unisexual animals, but it remains unknown whether similar clones also exist in natural populations of gibel carp. Therefore, further genetic resource survey will be required for elucidating the evolutionary mechanisms of polyploid gibel carp through the nuclear and cytoplasmic markers.
In conclusion, we here identify a novel clone in polyploid gibel carp, and suggest that the novel clone might be a nucleo-cytoplasmic hybrid between the known clones A and D, because it originates from the offspring of gonochoristic sexual reproduction mating between clone D female and clone A male, and contains an entire nuclear genome from the paternal clone A and a mtDNA genome (cytoplasm) from the maternal clone D. It is suggested to arise via androgenesis by a mechanism of ploidy doubling of clone A sperm in clone D ooplasm through inhibiting the first mitotic division. Significantly, the selected nucleo-cytoplasmic hybrid female still maintains its gynogenetic ability, and thereby forms a novel clone with over several hundred millions of clonal individuals proliferated by 7 successive generations of gynogenesis. The androgenetic nucleo-cytoplasmic hybrid clone should be the first case in vertebrates, and might be related to the high polyploidy level and the diverse reproduction modes in gibel carp. Based on the present and previous findings, we discuss the association of rapid genetic changes and high genetic diversity with various ploidy levels and multiple reproduction modes in several unisexual and sexual complexes of vertebrates and even other invertebrates. Therefore, the novel finding will be of great significance not only for exploiting the genetic breeding approaches in gibel carp but also for the studies of evolutionary genetics and ecology in other unisexual animals.
This work was supported by the earmarked fund for Modern Agro-industry Technology Research System (NYCYTX-49), the National Key Basic Research Program (2010CB126301), the National Natural Science Foundation of China (30630050), the Autonomous Project of State Key Laboratory of Freshwater Ecology and Biotechnology (2008FBZ15), and Innovation Project of Institute of Hydrobiology, Chinese Academy of Sciences (075A011301, 085A021301).
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