Gynogenetic Hybrid Research Articles

Challenges and Costs of Asexuality: Variation in Premeiotic Genome Duplication in Gynogenetic Hybrids from Cobitis taenia Complex.

The transition from sexual reproduction to asexuality is often triggered by hybridization. The gametogenesis of many hybrid asexuals involves premeiotic genome endoreplication leading to bypass hybrid sterility and forming clonal gametes. However, it is still not clear when endoreplication occurs, how many gonial cells it affects and whether its rate differs among clonal lineages. Here, we investigated meiotic and premeiotic cells of diploid and triploid hybrids of spined loaches (Cypriniformes: Cobitis) that reproduce by gynogenesis. We found that in naturally and experimentally produced F1 hybrids asexuality is achieved by genome endoreplication, which occurs in gonocytes just before entering meiosis or, rarely, one or a few divisions before meiosis. However, genome endoreplication was observed only in a minor fraction of the hybrid’s gonocytes, while the vast majority of gonocytes were unable to duplicate their genomes and consequently could not proceed beyond pachytene due to defects in bivalent formation. We also noted that the rate of endoreplication was significantly higher among gonocytes of hybrids from natural clones than of experimentally produced F1 hybrids. Thus, asexuality and hybrid sterility are intimately related phenomena and the transition from sexual reproduction to asexuality must overcome significant problems with genome incompatibilities with a possible impact on reproductive potential.

Genomic and epigenetic alterations in diploid gynogenetic hybrid fish

Gynogenesis, as an effective technology to manipulate genome, is used fairly widely in aquaculture-related research. However, the knowledge regarding gynogenesis in allodiploid hybrid fish with the hybrid genome producing haploid gametes is sparse. This study focused on the analyses on the genomic and epigenetic alterations of the gynogenetic hybrids with improved trait. The mature eggs of the fourth generation (BTF4, 2n = 48) of allodiploid hybrid lineage of blunt snout bream (BSB, 2n = 48, Megalobrama amblycephala, ♀) × topmouth culter (TC, 2n = 48, Culter alburnus, ♂) were activated by UV-irradiated sperm from koi carp (Koi, 2n = 100, Cyprinus carpio haematopterus, ♂) and doubled by cold shock. The analyses of the DNA content and morphological observation confirmed that all of the progenies were gynogenetic diploids (GBTF4, 2n = 48), suggesting that the eggs of BTF4 possessed the hybrid genome. A genomic survey resulted from microsatellite indicated that 87.72% of the GBTF4 genome was the same as that of BTF4, 12.28% of the GBTF4 genome exhibited polymorphism compared with that of BTF4, and 0.03% of the GBTF4 genome was inherited from Koi. On the other hand, the results of methylation sensitive amplification polymorphism (MSAP) showed that the DNA methylation level (38.61%) in the GBTF4 genome was lower than that (40.99%) in the BTF4 genome. In addition, MSAP profiling also showed 30.1% methylation pattern changes in GBTF4 compared with BTF4 at 588 randomly selected CCGG sites. These results enriched our knowledge regarding the combination of hybridization and gynogenesis and the genomic and epigenetic alterations in allodiploid hybrid fish following process of gynogenesis, providing new insights into fish genetic breeding.

Characterization of H3 methylation in regulating oocyte development in cyprinid fish.

Histone post-modifications are important epigenetic markers involved in multiple cellular processes via regulation of gene transcription or remodeling of chromatin structure. Oocyte development is a critical process under rigorous control to prevent the generation of aberrant gametes. However, the regulatory mechanism of oocyte early development is not well-understood due to the tiny size and poor distinguishability of the gonad in juvenile stages. Here, two cyprinid hybrid fishes, a sterile allotriploid fish and a gynogenetic hybrid fish with delayed oocyte development, provided research models to investigate the mechanisms involved. We used cytogenetic and molecular methods to confirm the pachytene arrest of oocytes in allotriploid fish and gynogenetic hybrid fish. On the basis of these developmental differences, we screened 21 different histone H3 modifications by ELISA and found that four modifications (H3K4me3, H3K9me3, H3K79me, and H3K79me3) differed significantly in the two cyprinid hybrid fishes. Changes in histone methylation at the three residues (H3K4, K9, K79) were caused by specific methyltransferases and demethylases. Our results provide new insights into the epigenetic regulation of oocyte early development in fish, a process critical for understanding of reproductive biology and with practical applications in the aquacultural breeding industry.

Rapid genomic changes in allopolyploids of Carassius auratus red var. (♀) × Megalobrama amblycephala (♂).

To better understand genomic changes in the early generations after polyploidisation, we examined the chromosomal consequences of genomic merger in allotetraploid hybrids (4 nF1) (AABB, 4n = 148) of Carassius auratus red var. (RCC) (AA, 2n = 100) (♀) × Megalobrama amblycephala (BSB) (BB, 2n = 48) (♂). Complete loss of the paternal 5S rDNA sequence and the expected number of maternal chromosomal loci were found in 4 nF1, suggesting directional genomic changes occurred in the first generations after polyploidisation. Recent studies have reported instability of newly established allotetraploid genomes. To assess this in the newly formed 4 nF1 genome, we performed fluorescence in situ hybridisation on an allotetraploid gynogenetic hybrid (4 nG) (AABB, 4n = 148) and an allopentaploid hybrid (5 nH) (AABBB, 5n = 172) from 4 nF1 (♀) × BSB (♂) with 5S rDNA gene and centromere probes from RCC, the original diploid parent. The expected numbers of maternal chromosomal loci were found in 4 nG, while chromosomal locus deletions and chromosome recombinations were detected in 5 nH. These observations suggest that abnormal meiosis did not lead to obvious genomic changes in the newly established allotetraploid genomes, but hybridisation with the original diploid parent resulted in obvious genomic changes in the newly established allotetraploid genomes, as was found for the maternal genome.

Ants with Attitude: Australian Jack-jumpers of the Myrmecia pilosula species complex, with descriptions of four new species (Hymenoptera: Formicidae: Myrmeciinae).

The six known "Jack-jumper species Myrmecia pilosula Fr. Smith 1858, M. croslandi Taylor 1991, M. banksi, M. haskinsorum, M. imaii and M. impaternata spp.n. are reviewed, illustrated and keyed. Myrmecia imaii is known only from southwest Western Australia, the others variously from southeastern Australia and Tasmania. These taxa were previously confused under the name M. pilosula (for which a lectotype is designated). Previous cytogenetical findings, which contributed importantly to current taxonomic understanding, are summarized for each species. Eastern and Western geographical races of the widespread M. pilosula are recognized. Myrmecia croslandi is one of only two eukaryote animals known to possess a single pair of chromosomes (2n=2 3 or 4). Myrmecia impaternata is evidentially an allodiploid (n=5 or 14, 2n=19) sperm-dependent gynogenetic hybrid between M. banksi and an element of the eastern race of M. pilosula, or their immediate ancestry. The sting-injected venom of these ants can induce sometimes fatal anaphylaxis in sensitive humans.

The cloning of cdc2 cDNAs and a comparative study of its expression in different ploidy fishes including the diploid gynogenetic hybrid of red crucian carp×common carp

Cdc2(Cyclin Dependent Kinase,namely CDK1)encoded by cdc2 gene and CyclinB combination regulates G2/M transition.To find out molecular mechanism that diploid hybrid fish could produce diploid gamete,the full length cDNAs of cdc2 in the third gynogenetic generation(G3),red crucian carp(Carassius auratus red var.),triploid crucian carp and allotetraploid were obtained by PCR and rapid amplification of cDNA ends.Our data showed that all the cDNAs of cdc2 gene in the four different ploidy fishes encode a protein of 302 amino acids containing a domain(PSTAVRE)which combine Cyclins.A high homology of 97.6% of the Cdc2 protein can be drawn by comparing the amino acid sequences in these four fishes,which indicates the higher conservative function and evolution of Cdc2 protein in these four fishes.A comparative expression pattern of cdc2 in early-stage gonads of G3 and different ploidy fishes was carried out by Real-time PCR using specific primers against the same sequences of coding regions in the four fishes.The results showed that the expression of cdc2 in the ovary of G3 was higher than those of red crucian carp and triploid crucian carp,while lower than that of allotetraploid,which,at the molecular level,indicates existence of polyploid oogonia in early-stage gonads of G3.The higher expression of cdc2 in G3 suggests that consecutive S-phase replication may occur without intervening mitosis,which might be related to the formation mechanisms for the diploid eggs generated by diploid hybrids.

Distant hybridization leads to different ploidy fishes

Distant hybridization makes it possible to transfer the genome of one species to another, which results in changes in phenotypes and genotypes of the progenies. This study shows that distant hybridization or the combination of this method with gynogenesis or androgenesis lead to different ploidy fishes with genetic variation, including fertile tetraploid hybrids, sterile triploid hybrids, fertile diploid hybrids, fertile diploid gynogenetic fish, and their derived progenies. The formations of the different ploidy fishes depend on the genetic relationship between the parents. In this study, several types of distant hybridization, including red crucian carp (Carassius auratus red var.) (2n=100, abbreviated as RCC) (female) x common carp (Cyprinus carpio L.) (2n=100, abbreviated as CC) (male), and RCC (2n=100) (female) x blunt snout bream (Megalobrama amblycephala) (2n=48, abbreviated as BSB) (male) are described. In the distant hybridization of RCC (female) x CC (male), bisexual fertile F(3)-F(18) allotetraploid hybrids (4n=200, abbreviated as 4nAT) were formed. The diploid hybrid eggs and diploid sperm generated by the females and males of 4nAT developed into diploid gynogenetic hybrids and diploid androgenetic hybrids, respectively, by gynogenesis and androgenesis, without treatment for doubling the chromosome. Improved tetraploid hybrids and improved diploid fishes with genetic variation were derived from the gynogenetic hybrid line. The improved diploid fishes included the high-body RCC and high-body goldfish. The formation of the tetraploid hybrids was related to the occurrence of unreduced gametes generated from the diploid hybrids, which involved in premeiotic endoreduplication, endomitosis, or fusion of germ cells. The sterile triploid hybrids (3n=150) were produced on a large scale by crossing the males of tetraploid hybrids with females of diploid fish (2n=100). In another distant hybridization of RCC (female) x BSB (male), different ploidy fishes were obtained, including diploid bisexual fertile natural gynogenetic fish (2n=100), sterile triploid hybrids (3n=124), and bisexual fertile tetraploid hybrids (4n=148). Furthermore, two kinds of pentaploid hybrids (5n=172 and 5n=198) were formed. The biological characteristics and the mechanisms of formation of the different ploidy fish were compared and discussed at the cellular and molecular level. The results indicated distant hybridization or the combination of this method with gynogenesis or androgenesis affects the formation of different ploidy fish with genetic variation.

Diversity and distribution of gynogenetic hybrids between Fundulus diaphanus and Fundulus heteroclitus in Porter's Lake (Nova Scotia) in relation to salinity

Unisexual hybrids between Fundulus diaphanus and Fundulus heteroclitus were found in low proportions at intermediate salinity in Porter's Lake, Nova Scotia. One clone accounted for 72% of the hybrids, with most other hybrids being different at a single microsatellite allele. This clone thrives over a wide range of salinities, suggesting a general-purpose genotype.

Identification and Expression Analysis of Genes Involved in Early Ovary Development in Diploid Gynogenetic Hybrids of Red Crucian Carp × Common Carp

Diploid eggs of allotetraploid hybrids (red crucian carp female symbol x common carp male symbol), when activated by UV-irradiated sperm of scatter scale carp, can develop into diploid progenies without chromosome duplication treatment. Diploid progenies produce diploid eggs, which develop into diploid population by the same way. To understand the molecular mechanism underlying the production of diploid eggs by the diploid fish, we constructed a forward suppression subtractive hybridization complementary DNA (cDNA) library. The cDNAs from the ovary in proliferation phase were employed as the "tester," and those in growth phase were used as the "driver." Seventy-three cDNA clones that are specifically expressed in proliferation phase were detected by dot-blot hybridization. Sequencing analyses revealed that several of these cDNAs have high homologies to the known sequences in the NCBI database. Their encoded proteins include the protein preventing mitosis catastrophe (PMC), the signal recognition particle 9, the ATP-binding cassette transporter, the glucanase-xylanase fusion protein, and others. These genes were confirmed by reverse transcriptase-polymerase chain reaction. The expression profile of the PMC gene at different time points was analyzed by quantitative real-time polymerase chain reaction. The results indicated that the expression of this suppression subtractive hybridization-identified gene changed during the time course, corresponding with the cellular phenomenon in the ovary development. Our studies provide insights into the molecular mechanism underlying the ovary development of diploid gynogenetic fish.

Establishment of the diploid gynogenetic hybrid clonal line of red crucian carp × common carp

This study investigated the gynogenetic cytobiological behavior of the third gynogenetic generation (G(3)), which was generated from the diploid eggs produced by the second gynogenetic generation (G(2)) of red crucian carp x common carp, and determined the chromosomal numbers of G(3), G(2)xscatter scale carp and G(2)xallotetraploid hybrids of red crucian carp x common carp. The results showed that the diploid eggs of G(2) with 100 chromosomes, activated by UV-irradiated sperm from scatter scale carp and without the treatment for doubling the chromosomes, could develop into G(3) with 100 chromosomes. Similar to the first and second gynogenetic generations (G(1) and G(2)), G(3) was also diploid (2n=100) and presented the hybrid traits. The triploids (3n=150) and tetraploids (4n=200) were produced by crossing G(2) with scatter scale carp, and crossing G(2) with allotetraploids, respectively. The extrusion of the second polar body in the eggs of G(2) ruled out the possibility that the retention of the second polar body led to the formation of the diploid eggs. In addition, we discussed the mechanism of the formation of the diploid eggs generated by G(2). The establishment of the diploid gynogenesis clonal line (G(1), G(2) and G(3)) provided the evidence that the diploid eggs were able to develop into a new diploid hybrid clonal line by gynogenesis. By producing the diploid eggs as a unique reproductive way, the diploid gynogenetic progeny of allotetraploid hybrids of red crucian carp x common carp had important significances in both biological evolution and production application.

Asexual and sexual hybrids betweenFundulus diaphanusandF. heteroclitusin the Canadian Atlantic region

Levels and origins of clonal diversity in asexual hybrid animals are critical to understanding how they can coexist with their sexual progenitor species. In this study, asexual gynogenetic hybrids between Fundulus diaphanus and Fundulus heteroclitus known from two sites in Nova Scotia (Canada) were characterized using discriminant morphological traits, eight microsatellite loci, and mitochondrial DNA. Fifteen clonal genotypes were uncovered, all bearing the same F. diaphanus maternal haplotye. Each site harboured a different dominant clone along with several rarer clones that all appear to be of recent origin. Unexpectedly, highly introgressed sexually reproducing hybrids (0.25 > q > 0.75) were also detected. Sexual hybrids with maternal ascendance in either species were also found at three other sites in the Atlantic region. Based on a single meristic trait (scale counts), it is shown that asexual clones can be significantly more variable than populations of sexual parental species. Also, species are morphologically more alike when living in sympatry, suggesting that introgression may occur via sexual hybrids. Altogether, these results confirm and refine the available knowledge on this hybrid system, and indicate that hybridization is probably a more widespread phenomenon than suspected, with implications for the phenotypic variability of a widely used model species, F. heteroclitus.

Black spots and female association preferences in a sexual/asexual mating complex (Poecilia, Poeciliidae, Teleostei)

We investigated whether female association preferences for males are influenced by black spot disease (BSD), a parasite induced change of the host phenotype. We compared three different species of fish: a gynogenetic hybrid species, Poecilia formosa (amazon molly) and two sexual species (Poecilia latipinna and Poecilia mexicana), which were involved in the natural hybridisation leading to the amazon molly. Contrary to their sexual relatives, asexual amazon mollies significantly avoided images of males infected with black spot disease. We propose that amazon molly females have direct fitness benefits from choosing healthy males. The adaptive significance of the preference for BSD-uninfected males in the asexual amazon molly is yet unclear but may involve avoidance of predation or parasite infection as well as increased sperm availability.

PHENOTYPIC CONSEQUENCES OF GENETIC VARIATION IN A GYNOGENETIC COMPLEX OF PHOXINUS EOS-NEOGAEUS CLONAL FISH (PISCES: CYPRINIDAE) INHABITING A HETEROGENEOUS ENVIRONMENT

We examined the genetic composition, habitat use, and morphological variation of a Phoxinus eos-neogaeus unisexual hybrid complex and its sexually reproducing progenitor species inhabiting beaver-modified drainages of Voyageurs National Park, Minnesota. In addition to the single diploid P. eos-neogaeus gynogenetic clone, triploid and diploid-triploid mosaic biotypes were present at our study sites. Both P. eos and P. neogaeus, and all three hybrid biotypes were ubiquitous throughout one intensively surveyed drainage, but abundances and relative frequencies of the parental species and hybrids varied considerably within and among successional environments. Data from a large number of additional sites indicated that the proportion of polyploid hybrids within an environment was negatively related to hybrid relative frequency, implying that the genomic constitution of hybrids is an important determinant of clonal fitness among successional environments. Statistical comparisons of variation along size-free multivariate body shape axes indicated that despite its genetic uniformity, the P. eos-neogaeus clone is no less variable than its sexual progenitors, suggesting that a single genotype may actually respond to environmental variation with as much phenotypic variation as a genetically variable sexual population. The incorporation and expression of a third genome in triploid and diploid-triploid mosaic biotypes derived from the gynogenetic clone significantly expanded phenotypic variation of the clone. This additional variation results in greater similarities in habitat use and morphological overlap with the parental species, primarily P. eos, the predominant sperm donor for gynogenetic hybrid females in this complex. Polyploid augmentation of a diploid gynogenetic clone appears to be typical in the P. eos-neogaeus complex, and the additional genetic and phenotypic variation that it generates has potentially significant ecological and evolutionary consequences for the success and persistence of a single genotype in highly variable environments.

Production of gynogenetic progeny from allotetraploid hybrids red crucian carp×common carp

Following activation by UV-irradiated sperm, diploid eggs of the allotetraploid hybrids (red crucian carp ♀×common carp ♂) developed into normal live gynogens. Cold-shocking these activated diploid eggs failed to induce the expected tetraploidy in them. All these gynogenetic progenies, generated with or without the cold shock, were female diploids with 100 chromosomes, indicating the XXXX genotype of the tetraploid females. The gynogenetic females matured at the age of 2+ suggesting delayed maturity, in comparison to the diploid females of the red crucian carp. Thirty percent of these gynogens were fertile with normal ovary and mature eggs. These gynogens were not morphologically distinguishable from the F 2 hybrids. Like the diploid F 2 hybrids, these diploid gynogens produced diploid eggs, for these eggs developed into triploids with 150 chromosomes, when crossed with red crucian carp. Without the treatment for doubling the chromosome number, the diploid eggs, produced by the F 1 gynogens, developed into the F 2 gynogens following activation by UV-irradiated sperm. Production of these diploid eggs from the diploid gynogens may have resulted from the pre-meiotic endoreduplication of chromosomes. For aquaculture, these diploid eggs remain an important gamete source for the production of tetraploids and triploids, and establishment of diploid gynogenetic hybrid line.

Factors necessary for restoring an evolutionary change in an anural ascidian embryo

The ascidian Molgula oculata has a tailed (or urodele) larva, whereas Molgula occulta develops directly via a tailless (or anural) embryo. Interspecific hybrid embryos produced by fertilizing M. occulta eggs with M. oculata sperm ( M. occulta × M. oculata hybrids) can develop urodele larval structures, including a brain pigment cell and a short tail containing a small notochord. Development of larval features differs in individual M. occulta clutches: some eggs develop into hybrids with both a brain pigment cell and a tail, some into hybrids with either a brain pigment cell or a tail, and others into hybrids without urodele features. The expression of a 58-kDa protein (p58), which is present in eggs and embryos of urodele ascidians but lacking in those of most anural species, also varies in expression between different clutches of M. occulta eggs. Western blot and immunofluorescence studies show that p58 expression is correlated with the ability of hybrid embryos to express urodele features. For example, clutches of M. occulta eggs containing relatively high levels of p58 produce many hybrids with both a brain pigment cell and a tail. Differential expression of p58 occurs during oogenesis in M. occulta individuals: p58 is found at similar levels in previtellogenic oocytes, but in some animals it disappears during vitellogenesis, while in others it persists throughout oogenesis and is present in mature eggs. When M. occulta eggs are extracted with Triton X-100, p58 remains in the detergent-insoluble fraction, suggesting that it is associated with the cytoskeleton. In most unfertilized M. occulta eggs, p58 is uniformly distributed, but after fertilization it is localized in the uncleaved zygote and then concentrated in embryonic ectoderm, notochord, and muscle lineage cells. Despite containing high levels of p58, gynogenetic hybrid embryos, produced by fertilizing M. occulta eggs with uv-irradiated M. oculata sperm, develop into hybrids without a brain pigment cell or a tail. The results suggest that both a functional paternal genome and p58 are necessary for restoration of larval features in M. occulta × M. oculata hybrids. The cytoskeletal complex containing p58 may mediate the localization of key maternal factors in the egg or may be involved in cellular interactions during embryogenesis which are responsible for development of urodele cell fates.

Notch is required for successive cell divisions in the developing Drosophila retina

Following activation by UV-irradiated sperm, diploid eggs of the allotetraploid hybrids (red crucian carp ♀×common carp ♂) developed into normal live gynogens. Cold-shocking these activated diploid eggs failed to induce the expected tetraploidy in them. All these gynogenetic progenies, generated with or without the cold shock, were female diploids with 100 chromosomes, indicating the XXXX genotype of the tetraploid females. The gynogenetic females matured at the age of 2+ suggesting delayed maturity, in comparison to the diploid females of the red crucian carp. Thirty percent of these gynogens were fertile with normal ovary and mature eggs. These gynogens were not morphologically distinguishable from the F2 hybrids. Like the diploid F2 hybrids, these diploid gynogens produced diploid eggs, for these eggs developed into triploids with 150 chromosomes, when crossed with red crucian carp. Without the treatment for doubling the chromosome number, the diploid eggs, produced by the F1 gynogens, developed into the F2 gynogens following activation by UV-irradiated sperm. Production of these diploid eggs from the diploid gynogens may have resulted from the pre-meiotic endoreduplication of chromosomes. For aquaculture, these diploid eggs remain an important gamete source for the production of tetraploids and triploids, and establishment of diploid gynogenetic hybrid line.