Gynogenetic Diploids Research Articles

First successful inducing gynogenesis in Crassostrea angulata by inactivated tetraploid Crassostrea gigas sperm

Artificial induction of gynogenesis is widely practiced in aquatic animals. However, there have been few reports of obtaining adult gynogenetic marine bivalves. This study established a new and effective method for inducing gynogenesis in C. angulata using inactivated tetraploid C. gigas sperm, ultimately obtaining gynogenetic diploids. The optimal induction conditions were as follows: sperm were irradiated with ultraviolet light (254 nm) at an intensity of 1035 μW/cm2 for 60 s before fertilization with the eggs. Fifteen minutes post-fertilization, zygotes were treated with cytochalasin B (0.5 mg/L) for a duration of 20 min under a condition of 28 °C. Six different ploidy levels (2n, 3n, 4n, 5n, 6n and aneuploid) of larvae were found by karyotype analysis and flow cytometry methods, with a diploid rate of 63.57 ± 2.80 % and an induction efficiency of 7.47 ± 0.33 %. At 6 months after fertilization, induced progenies of 4 different ploidy levels (2n, 3n, 4n and aneuploid) were examined from 3 replicate groups. The proportions of diploids, triploids, tetraploids and aneuploids were 31.67 ± 3.68 %, 57 ± 3.74 %, 7.67 ± 0.94 % and 3.67 ± 0.94 %, respectively. Diploidy was determined by microsatellite analysis, which did not contain any paternal genes, suggesting them as true gynogenetic progenies. The growth traits (shell length, width, height and whole weight) of the gynogenetic diploids did not show any significant difference (P < 0.05) compared to the control diploids. Meanwhile, triploids demonstrated a significant advantage in growth. Gynogenetic diploids underwent sexual differentiation and consisted of females (31.40 ± 1.52 %), males (67.67 ± 2.75 %), and hermaphrodites (0.93 ± 1.31 %). Interestingly, the gonads of diploids developed well and produced functional gametes. This research provides robust technical support for chromosome engineering breeding and the creation of pure lines of bivalves.

A new method for creating androgenetic diploid fish from tetraploid carp sperm and their genetic composition analysis

The application of cold shock to inhibit the extrusion of the second polar body in fertilized eggs is one of the main methods to induce triploid in diploid fish. In this study, we originally planned to induce hexaploid by this method in tetraploid carp. However, among the surviving offspring, we observed only 50.7% tetraploid and 49.3% diploid individuals, which could potentially result from either androgenetic diploids or gynogenetic diploids. The absence of irradiation and the high proportion of diploids attracted us. However, the lack of sex-specific markers for tetraploid carp, makes it difficult to determine their origin. Fortunately, we also carried out the cross of yellow carp (Cyprinus carpio yellow var., 2n, ♀) × tetraploid carp (♂) and red crucian carp (Carassius auratus red var., 2n, ♀) × tetraploid carp (♂) with cold shock inhibiting the second meiosis, diploid offspring have also been found, and whether they have undergone gynogenesis or androgenesis can be judged based on phenotype and genotype. In terms of phenotypes, the key phenotypes of two diploids, such as the whisker length, number of lateral scales, and presence of melanin, are consistent with tetraploid carp. In terms of genotypes, through single-copy nuclear gene cloning and fluorescence in situ hybridization (FISH), it is supported that both diploids are androgenetic offspring. In addition, 14.9% of tetraploid offspring were found in red crucian carp (♀) × tetraploid carp (♂) with cold shock inhibiting the second meiosis. By the above-mentioned means of detection, it was found that they were created by inhibiting the extrusion of the second polar body of the fertilized eggs. From this it will be seen that the application of cold shock before the extrusion of the second polar body of fertilized eggs cause not only polyploidy, but also the elimination of both second polar body and female nucleus. Previous studies have found that cold shock can induce lethal androgenetic haploids in diploid fish, but these androgenetic diploid fish produced by tetraploid carp sperm were surviving. We summarize the mechanism behind the above phenomenon, which is mainly related to meiosis occurring at the edge of the cell membrane. Cold shock may disrupt the function of the spindle, resulting in the simultaneous retention or extrusion of female nucleus and second polar body. This study will provide valuable insights into androgenesis, polyploidization, and hybridization in fish genetic breeding.

Induction of Gynogenetic Diploids the Tropical Oyster, Crassostrea belcheri 1873 (Ostreids: Ostreoidea)

The induction of diploid gynogenesis in the Tropical oyster, Crassostrea belcheri 1873, was carried out through two main experiments: 1) the destruction of spermatozoa DNA using ultraviolet (UV) irradiation, and 2) the induction of gynogenetic diploids. UV light source was placed 30 cm over the sperm for various durations, including 0 (control), 30, 60, 90, and 120 seconds. The study revealed that the highest survival rate was observed in trochophore at 90 minutes (42.5 ± 2.50%) compared to the normal control C. belcheri (p&gt;0.05). Based on cytogenetic study, haploid (n=10), diploid (2n=20), and aneuploid were observed in all trials, while only diploid (2n=20) could be seen in the control group. Diploid gynogenesis was induced using 100 µmol 6-DMAP for 10 minutes. The study found that the survival rate and developmental stages in both trochophore and D-shape were gradually decreasing as the duration of UV rays increased. The highest survival rate of trochophore in the control group was 75.20 ± 4.84%, while survival rates in other experimental groups ranged from 52.22 ± 4.82% to 59.33 ± 13.57%. However, regarding the survival rate of D-shape larvae, their survival rates were relatively low across all trials. They displayed abnormal embryo development, distorted shape, unnatural swimming, and were smaller than normal (40-50 µm). Moreover, haploid (n=10), diploid (2n=20), triploid (3n=30), tetraploid (4n=40), and aneuploid were observed in all trials, while only diploid (2n=20) could be discovered in the control group. However, gynogenesis in this tropical oyster is still limited at this time. Therefore, suitable conditions for inducing gynogenesis in oysters should be further developed.

Genome-wide analysis facilitates estimation of the amount of male contribution in meiotic gynogenetic three-spined stickleback (Gasterosteus aculeatus).

Gynogenetic embryos - those inheriting only maternal DNA - can be experimentally created by fertilizing eggs with radiation-treated sperm containing inactivated paternal chromosomes. Diploidy in the zygotes can be maintained through prevention of the second meiosis or restored by preventing the first mitosis after the maternal chromosome complement has been replicated. These gynogenetic organisms are useful in many fields including aquaculture, evolutionary biology and genomics. Although gynogenetic organisms have been created in numerous species, the completeness of uni-parental inheritance has often been assumed rather than thoroughly quantified across the genome. Instead, when tests of uni-parental inheritance occur, they typically rely on well-studied genetically determined phenotypes that represent a very small sub-set of the genome. Only assessing small genomic regions for paternal inheritance leaves the question of whether some paternal contributions to offspring might still have occurred. In this study, the authors quantify the efficacy of creating gynogenetic diploid three-spined stickleback fish (Gasterosteus aculeatus). To this end, the authors mirrored previous assessments of paternal contribution using well-studied genetically determined phenotypes including sex and genetically dominant morphological traits but expanded on previous studies using dense restriction site-associated DNA sequencing (RAD-seq) markers in parents and offspring to assess paternal inheritance genome-wide. In the gynogenetic diploids, the authors found no male genotypes underlying their phenotypes of interest - sex and dominant phenotypic traits. Using genome-wide assessments of paternal contribution, nevertheless, the authors found evidence of a small, yet potentially important, amount of paternally "leaked" genetic material. The application of this genome-wide approach identifies the need for more widespread assessment of paternal contributions to gynogenetic animals and promises benefits for many aspects of aquaculture, evolutionary biology and genomics.

Study on artificial induction and early development of gynogenetic fat greenling Hexagrammos otakii

Fat greenling Hexagrammos otakii, belonging to Scorpaeniformes, is an important commercial marine fish species distributed in coast waters of China, Japan, and Korea. Artificial gynogenesis induction is one of the important techniques of chromosome manipulation and genetic breeding. The eggs are fertilized with genetic inactivated sperm, and genome information of the offspring is totally contributed by maternal genome. Until now, no gynogenesis induction was reported in H. otakii and other Scorpaeniformes species. The present study developed an effective gynogenesis induction method in H. otakii by applying hydrostatic pressure to the eggs activated with UV-irradiated homologous sperm. The optimal inducing conditions were 36,000 erg mm−2 for UV-irradiated and pressure of 65 MPa for 6 min at 20 min post fertilization. The relative hatching rates could reach 92%. The early development and growth before 6 month post hatching showed no difference between the gynogenetic and control diploid. The all-maternal inheritance was confirmed with 4 microsatellite loci. The genetic diversity of the control and gynogenetic diploid revealed with 20 microsatellite loci showed that the homozygosity of the gynogenetic diploid stocks was 0.84 in average, which was nearly double the value of the control diploid stocks (0.45 in average). Under scanning electron microscope, the egg envelop had a gelatinous-honeycomb before 0.5 h post fertilization (hpf). Then all or part of the gelatinous-honeycomb layer disappeared before 24 hpf. The uniform pores distributed to hexagonal pattern on the egg envelop were found in the control diploid and haploid groups. However, no pore on the egg envelop was found in the gynogenetic diploid from fertilization to hatching. These results would provide a valuable reference for gynogenesis induction in fish with viscous ovarian fluid coated eggs, although special incubation process to increase the survival rate should be further developed based on the characteristics of gynogenetic H. otakii.

Gynogenetic diploids, tetraploids, or octoploids, and a path to polyploidy in anuran amphibians.

Unreduced gametes have been implicated in the evolution of polyploid species of plants and animals and are normally produced by female anuran amphibians. Such eggs may initiate the evolution of polyploid species that have arisen independently in several anuran families. Polyploid females could also produce unreduced eggs that might lead to species with higher ploidy levels, or their eggs may develop gynogenetically to reduce the ploidy level. Diploid Hyla chrysoscelis (2n = 24) and tetraploid H. versicolor (4n = 48) are sibling cryptic species of North American grey treefrogs. Artificial crosses using H. versicolor females and genetically distant diploid males were performed to produce haploid H. versicolor and to assess the production of unreduced eggs in this tetraploid species. Gynogenetic diploid (haploid H. versicolor), allotriploid, gynogenetic tetraploid, allopentaploid, autohexaploid, and gynogenetic octoploid tadpoles were confirmed using chromosome counts from tadpole tail tip squashes. The transformation and survival of different ploidies varied. Gynogenetic diploids transformed but expressed aspects of haploid syndrome and died before or shortly after transformation.

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.

Weighted Correlation Network Analysis (WGCNA) of Japanese Flounder (Paralichthys olivaceus) Embryo Transcriptome Provides Crucial Gene Sets for Understanding Haploid Syndrome and Rescue by Diploidization

Artificial gynogenesis is of great research value in fish genetics and breeding technology. However, existing studies did not explain the mechanism of some interesting phenomena. Severe developmental defects in gynogenetic haploids can lead to death during hatching. After diploidization of chromosomes, gynogenetic diploids may dispense from the remarkable malformation and restore the viability, although the development time is longer and the survival rate is lower compared with normal diploids. The aim of this study was to reveal key mechanism in haploid syndrome of Japanese flounder, a commercially important marine teleost in East Asia. We measured genome-scale gene expression of flounder haploid, gynogenetic diploid and normal diploid embryos using RNA-Seq, constructed a module-centric co-expression network based on weighted correlation network analysis (WGCNA) and analyzed the biological functions of correlated modules. Module gene content analysis revealed that the formation of gynogenetic haploids was closely related to the abnormality of plasma proteins, and the up-regulation of p53 signaling pathway might rescue gynogenetic embryos from haploid syndrome via regulating cell cycle arrest, apoptosis and DNA repair. Moreover, normal diploid has more robust nervous system. This work provides novel insights into molecular mechanisms in haploid syndrome and the rescue process by gynogenetic diploidization.

Sex-specific markers developed by next-generation sequencing confirmed an XX/XY sex determination system in bighead carp (Hypophthalmichthys nobilis) and silver carp (Hypophthalmichthys molitrix).

Sex-specific markers are powerful tools for identifying sex-determination system in various animals. Bighead carp (Hypophthalmichehys nobilis) and silver carp (Hypophthalmichthys molitrix) are two of the most important edible fish in Asia, which have a long juvenility period that can lasts for 4-5 years. In this study, we found one sex-specific marker by next-generation sequencing together with bioinformatics analysis in bighead carp. The male-specific markers were used to perform molecular sexing in the progenies of artificial gynogenetic diploids and found all progenies (n = 160) were females. Meanwhile, around 1 : 1 sex ratio was observed in a total of 579 juvenile offspring from three other families. To further extend the male-specific region, we performed genome walking and got a male-specific sequence of 8,661 bp. Five pairs of primers were designed and could be used to efficiently distinguish males from females in bighead carp and silver carp. The development of these male-specific markers and results of their molecular sexing in different populations provide strong evidence for a sex determination system of female homogametry or male heterogametry (XX/XY) in bighead carp and silver carp. To the best of our knowledge, this is the first report of effective sex-specific markers in these two large carp species.

Performance and characteristics of the progenies from the reciprocal crosses of Pangasianodon hypophthalmus (Sauvage, 1878) and Clarias gariepinus (Burchell, 1822)

This study was designed to evaluate the culture performance and characteristics of the offspring from the reciprocal crosses of Pangasianodon hypophthalmus and Clarias gariepinus. The hybrid ♀C. gariepinus × ♂P. hypophthalmus (Clariothalmus) gave better hatchability than ♀P. hypophthalmus × ♂C. gariepinus (Pangapinus), similar to P. hypophthalmus but lower than the value recorded in the C. gariepinus. Cytogenetic analysis of the Clariothalmus reveals the presence of diploid hybrid (2n = 58 representing 60% of the population) and gynogenetic diploid offspring (2n = 56 representing 40% of the population), while, Pangapinus offspring were all gynogenetic diploid (2n = 60). Hence, the diploid hybrid exhibited phenotypic divergence from both parents, while the gynogenetic diploids were phenotypically indistinguishable from the maternal parent and had similar meristic counts. After 112 days of culture, gynogenetic diploid of the Clariothalmus offspring had better growth than the other crosses, however, cannibalism and aggressive behaviour was higher in this group of fish compared to other. Hence, culturing the diploid hybrid Clariothalmus separately from its gynogenetic diploid may be a management practice of interest as demonstrated in this study. This study provides evidence to support the success of hybridization between African and Asian catfish in one direction and demonstrates their potential for culture.

Sex-specific markers developed by next-generation sequencing confirmed an XX/XY sex determination system in bighead carp (Hypophthalmichehys nobilis) and silver carp (Hypophthalmichthys molitrix).

Sex-specific markers are powerful tools for identifying sex-determination system in various animals. Bighead carp (Hypophthalmichthys nobilis) and silver carp (Hypophthalmichthys molitrix) are two of the most important edible fish in Asia, which have a long juvenility period that can lasts for 4–5 years. In this study, we found one sex-specific marker by next-generation sequencing together with bioinformatics analysis in bighead carp. The male-specific markers were used to perform molecular sexing in the progenies of artificial gynogenetic diploids and found all progenies (n = 160) were females. Meanwhile, around 1 : 1 sex ratio was observed in a total of 579 juvenile offspring from three other families. To further extend the male-specific region, we performed genome walking and got a male-specific sequence of 8,661 bp. Five pairs of primers were designed and could be used to efficiently distinguish males from females in bighead carp and silver carp. The development of these male-specific markers and results of their molecular sexing in different populations provide strong evidence for a sex determination system of female homogametry or male heterogametry (XX/XY) in bighead carp and silver carp. To the best of our knowledge, this is the first report of effective sex-specific markers in these two large carp species.

Ploidy Manipulation of Zebrafish Embryos with Heat Shock 2 Treatment.

Manipulation of ploidy allows for useful transformations, such as diploids to tetraploids, or haploids to diploids. In the zebrafish Danio rerio, specifically the generation of homozygous gynogenetic diploids is useful in genetic analysis because it allows the direct production of homozygotes from a single heterozygous mother. This article describes a modified protocol for ploidy duplication based on a heat pulse during the first cell cycle, Heat Shock 2 (HS2). Through inhibition of centriole duplication, this method results in a precise cell division stall during the second cell cycle. The precise one-cycle division stall, coupled to unaffected DNA duplication, results in whole genome duplication. Protocols associated with this method include egg and sperm collection, UV treatment of sperm, in vitro fertilization and heat pulse to cause a one-cell cycle division delay and ploidy duplication. A modified version of this protocol could be applied to induce ploidy changes in other animal species.

Characterization of Embryo Transcriptome of Gynogenetic Olive Flounder Paralichthys olivaceus.

Olive flounder Paralichthys olivaceus is an important commercially cultured marine flatfish in China, Korea, and Japan. Gynogenesis, via meiogynogenesis and mitogynogenesis, shows advantages in breeding and sex control, but the low survival rate, especially for mitogynogenesis, limits its application. In this study, we sequenced the embryo transcriptomes of gynogenetic haploid, meiogynogenetic diploid, mitogynogenetic diploid, and common diploid flounder and investigated their respective genetic characteristics by analyzing differentiated expressed genes. Compared with common diploid, the gynogenetic haploid showed significant downregulation in notch signaling and wingless-related integration site (Wnt) signaling pathways, which may be the source of haploid syndrome. In both meiogynogenesis and mitogynogenesis, several upregulated genes including complement C3, formin-2, and intelectin may be related to increased survival compared to the haploid. The downregulation of immune system and energy metabolism-related genes caused retarded development of gynogenetic diploids compared with the common diploid. These data provided new and important information for application of artificially induced gynogenesis to aquaculture.

Microsatellite-centromere mapping in Japanese scallop (Patinopecten yessoensis) through half-tetrad analysis in gynogenetic diploid families

Gene-centromere mapping is an essential prerequisite for understanding the composition and structure of genomes. Half-tetrad analysis is a powerful tool for mapping genes and understanding chromosomal behavior during meiosis. The Japanese scallop (Patinopecten yessoensis), a cold-tolerant species inhabiting the northwestern Pacific coast, is a commercially important marine bivalve in Asian countries. In this study, inheritance of 32 informative microsatellite loci was examined in 70-h D-shaped larvae of three induced meiogynogenetic diploid families of P. yessoensis for centromere mapping using half-tetrad analysis. The ratio of gynogenetic diploids was proven to be 100%, 100% and 96% in the three families, respectively. Inheritance analysis in the control crosses showed that 51 of the 53 genotypic ratios observed were in accordance with Mendelian expectations at the 5% level after Bonferroni correction. Seven of the 32 microsatellite loci showed the existence of null alleles in control crosses. The second division segregation frequency (y) of the microsatellite loci ranged from 0.07 to 0.85 with a mean of 0.38, suggesting the existence of positive interference after a single chiasma formation in some chromosomes in the scallop. Microsatellite-centromere distances ranged from 4 cM to 42 cM under the assumption of complete interference. Information on the positions of centromeres in relation to the microsatellite loci will represent a contribution towards the assembly of genetic maps in the commercially important scallop species.

Ploidy manipulation and induction of alternate cleavage patterns through inhibition of centrosome duplication in the early zebrafish embryo.

Whole genome duplication is a useful genetic tool because it allows immediate and complete genetic homozygosity in gynogenetic offspring. A whole genome duplication method in zebrafish, Heat Shock, involves a heat pulse in the period 13-15 min postfertilization (mpf) to inhibit cytokinesis of the first mitotic cycle. However, Heat Shock produces a relatively low yield of gynogenotes. A heat pulse at a later time point during the first cell cycle (22 mpf, HS2) results in a high (>80%) frequency of embryos exhibiting a precise one-cell division stall during the second cell cycle, inducing whole genome duplication. Coupled with haploid production, HS2 generates viable gynogenetic diploids with yields up to 4 times higher than those achieved through standard Heat Shock. The cell cycle delay also causes blastomere cleavage pattern variations, supporting a role for cytokinesis in spindle orientation during the following cell cycle. Our studies provide a new tool for whole genome duplication, induced gynogenesis, and cleavage pattern alteration in zebrafish, based on a time period before the initiation of cell division that is sensitive to temperature-mediated interference with centrosome duplication. Targeting of this period may also facilitate genetic and developmental manipulations in other organisms.

Functional Sperm of the Yellowtail (Seriola quinqueradiata) Were Produced in the Small-Bodied Surrogate, Jack Mackerel (Trachurus japonicus).

Production of xenogeneic gametes from large-bodied, commercially important marine species in closely related smaller surrogates with short generation times may enable rapid domestication of the targeted species. In this study, we aimed to produce gametes of Japanese yellowtail (Seriola quinqueradiata) using jack mackerel (Trachurus japonicus) as a surrogate with a smaller body size and shorter maturation period. Donor spermatogonia were collected from the testes of yellowtail males and transferred into the peritoneal cavity of 10- and 12-day-old jack mackerel larvae. Twenty days later, 59.5% of the recipients survived of which 88.2% had donor-derived germ cells in their gonads. One year later, genomic DNA templates were prepared from the semen of 96 male recipients and subjected to polymerase chain reaction (PCR) analyses using primers specific for the yellowtail vasa sequence, resulting in the detection of positive signals in semen from two recipients. The milt collected from the recipients was used for fertilization with yellowtail eggs. Of eight hatchlings obtained from the crosses, two were confirmed to be derived from donor yellowtail by DNA markers, although the others were gynogenetic diploids. These findings indicate that it is possible to produce donor-derived sperm in xenogeneic recipients with a smaller body size and shorter generation time by transplanting spermatogonia. Thus, the xenogeneic transplantation of spermatogonia might be a potential tool to produce gametes of large-bodied, commercially important fish, although the efficiency of the method requires further improvement. This is the first report demonstrating that donor-derived sperm could be produced in xenogeneic recipient via spermatogonial transplantation in carangid fishes.

Genetic contribution of parents in sex determination of honmoroko Gnathopogon caerulescens

The honmoroko has been inferred to have an XX/XY sex determination system, but the parental genome can also affect the sex ratio of the offspring. The extent of parental effects on sex determination was examined by checking the sex ratios of F1 and F2 gynogenetic diploids and control diploids. Eleven gynogenetic broods from different females consisted of all or nearly all females, but eight broods showed a variable proportion of males (<50 %). One second-generation brood of gynogenetic diploids consisted wholly of females, but others produced some males. In crosses with a control diploid female, four males from a high-percentage male brood of gynogenetic diploids produced offspring with a balanced sex ratio. Sib-mating between a gynogenetic female and three gynogenetic males from the brood produced predominantly male progeny. These results suggest that there are at least four possible genotypes: genotypic female (XX), phenotypic female carrying a silent Y chromosome, genotypic male (XY), and genotypic supermale (YY). These inferences suggest that this fish has an XY system but a relatively high proportion of females possess a mutated, silent Y chromosome which does not lead to testis formation.

A Dense Linkage Map for Chinook salmon (Oncorhynchus tshawytscha) Reveals Variable Chromosomal Divergence After an Ancestral Whole Genome Duplication Event

Comparisons between the genomes of salmon species reveal that they underwent extensive chromosomal rearrangements following whole genome duplication that occurred in their lineage 58−63 million years ago. Extant salmonids are diploid, but occasional pairing between homeologous chromosomes exists in males. The consequences of re-diploidization can be characterized by mapping the position of duplicated loci in such species. Linkage maps are also a valuable tool for genome-wide applications such as genome-wide association studies, quantitative trait loci mapping or genome scans. Here, we investigated chromosomal evolution in Chinook salmon (Oncorhynchus tshawytscha) after genome duplication by mapping 7146 restriction-site associated DNA loci in gynogenetic haploid, gynogenetic diploid, and diploid crosses. In the process, we developed a reference database of restriction-site associated DNA loci for Chinook salmon comprising 48528 non-duplicated loci and 6409 known duplicated loci, which will facilitate locus identification and data sharing. We created a very dense linkage map anchored to all 34 chromosomes for the species, and all arms were identified through centromere mapping. The map positions of 799 duplicated loci revealed that homeologous pairs have diverged at different rates following whole genome duplication, and that degree of differentiation along arms was variable. Many of the homeologous pairs with high numbers of duplicated markers appear conserved with other salmon species, suggesting that retention of conserved homeologous pairing in some arms preceded species divergence. As chromosome arms are highly conserved across species, the major resources developed for Chinook salmon in this study are also relevant for other related species.

Amplified fragment length polymorphism analysis to assess crossover interference and homozygosity in gynogenetic diploid Pacific abalone (Haliotis discus hannai)

Recombination analysis in gynogenetic diploids is a powerful tool for assessing the degree of inbreeding, investigating crossover events and understanding chiasma interference during meiosis. To estimate the marker-centromere recombination rate, the inheritance pattern of 654 amplified fragment length polymorphism (AFLP) markers was examined in the 72-h veliger larvae of two meiogynogenetic diploid families in the Pacific abalone (Haliotis discus hannai). The second-division segregation frequency (y) of the AFLP loci ranged from 0.00 to 0.96, with 23.9% of loci showing y-values higher than 0.67, evidencing the existence of interference. The average recombination frequency across the 654 AFLP loci was 0.45, allowing estimation of the fixation index of 0.55, indicating that meiotic gynogenesis could provide an effective means of rapid inbreeding in the Pacific abalone. The AFLP loci have a small proportion (4.4%) of y-values greater than 0.90, suggesting that a relatively low or intermediate degree of chiasma interference occurred in the abalone chromosomes. The information obtained in this study will enhance our understanding of the abalone genome and will be useful for genetic studies in the species.

Induction of diploid gynogenesis in southern flounder (Paralichthys lethostigma) with heterologous sperm of Pagrosomus major

Female southern flounder(Paralichthys lethostigma) grow faster than males.Therefore,farming all-female progenies could maximize profits from cultured fish.In the present study,diploid gynogenesis was induced by activat-ing flounder egg development with UV-irradiated(72 mJ/cm2) Pagrosomus major sperm for 3 min in sea water,and then subjecting the eggs to a cold-shock(0–2℃) treatment for 45 min.Cold shock was used to prevent extrusion of the second polar body.In the control treatments,one or more of these steps was omitted to separately assess the effective-ness of UV irradiation and cold shock.When the initial treatment was applied 3 min after hatching(treatment duration,45 min),the fertilization rate of eggs activated by UV-irradiated heterologous sperm was(37.2 ± 5.1)%,the malforma-tion rate of newly hatched larvae was(8.3 ± 2.5)%,and the hatching rate was up to(9.4 ± 0.71)%.Flow cytometry and chromosome karyotyping were used to examine the ploidy level of larvae experienced to the cold-shock treatment.All of the gynogenetic larvae were diploid(2n = 48),and no haploid or aneuploid phenomena were detected.The duration of hatching of haploid embryos and gynogenetic embryos was significantly different from that of control normal em-bryos.There were no significant differences in the morphological index between gynogenetic diploids and normal lar-vae(P0.05).All of the haploid larvae(100%) showed abnormal external morphology(P0.05) while normal diploid larvae and gynogenetic diploid larvae showed normal morphologies.In summary,these methods resulted in successful diploid gynogenesis in southern flounder using heterologous sperm.This technique could serve as a useful tool to pro-duce monosex progenies of southern flounder.