Induction of Gynogenesis of Silurus astus Using Cold Shock

[ Objective] The cold shock method was used to induce diploid gynogenesis of Silurus astus. [ Mthod ] The Silurus astus sperms were irradiated by ultraviolet, then conducted for fertilization. The different cold shock starting time and duration were set to observe diploid gynogenesis of Silurus astus. [ Result] Under such inducing condition that ultraviolet irradiated 15 min,cold shock started 5 rain after fertilization, and cold shock duartion was 40 rain,the survival rate of diploid Silurus astus reached the highest（8.5% ）. [ Conclusion] The experiment laid foundation for culturing new excellent Silurus astus variety and accumulated original files tor further study of gynogenesis development mechanism.

Haploid Induction via In vitro Gynogenesis in Tomato(Solanum lycopersicum L.)

In order to determine the potential for haploid induction via in vitro gynogenesis in tomato, the ovules and protoplasts of embryo sacs from the hybrids Zhongza 101 and Zhongza 105 were cultured. An efficient method of ovule isolation was established in this study. Using this method, 100-150 ovules could be isolated from one ovary. Isolated ovules were cultured on three induction media to induce gynogenesis in vitro. During culture, ovules were enlarged markedly, with opaque white color. When observed microscopically, there were cell divisions and cell clumps in embryo sacs. Subsequently, the cell clumps in embryo sacs ceased growth, likely because the integument grew faster than embryo sacs did and hindered the fiarther development of embryo sacs. Therefore, subsequent callus morphogenesis might be originated from the integument. Thousands ofcalli from the two tomato varieties were obtained. Five diploid plants were regenerated after 15 months of subculturing. To eliminate the hindering effect of integument on embryo sac cells, the protoplasts of embryo sacs were prepared and cultured. After 48 hours of culture, the protoplasts of embryo sacs doubled in size and gradually formed clusters of cells. These results suggested that gynogenesis might be a potential way for haploid induction in tomato.

Comparative Microsatellite Analysis of Grass Carp Genomes of Two Gynogenetic Groups and the Xiangjiang River Group

The genomes of three groups of grass carp, namely the Xiangjiang River grass carp group （Xiangjiang group）, a one-generation artificially induced meio-gynogenetic grass carp group （meio-gynogenetic-1 group）, and a two-generation artificially induced meio-gynogenetic grass carp group （meio-gynogenetic-2 group）, were comparatively analyzed with microsatellite markers. Genetic polymorphism had been observed in the Xiangjiang group and most of the examined loci had more than two alleles. But the degree of genetic diversity was not very high. Although all the examined genetic loci in the analyzed individuals were in homozygous state, the genotypes of different individuals of the group were not identical in the meio-gynogenetic-1 group. In the meio-gynogenetic-2 group, not only the examined genetic loci of each individual were homozygous but also the genotypes of all the analyzed individuals of the group were the same. These results suggested that the examined meio-gynogenetic-2 group is a homozygous group and homozygous clone could be produced by continuous artificial induction of gynogenesis for two generations. It was found that the polymorphism existed not only at the allele level but also at the locus level; many alleles of the microsatellite loci and some of the microsatellite loci had been lost during the process of artificial gynogenesis. Therefore, both protection of the diversity of natural grass carp resource and selection of homozygous traits with desired economic genotypes are very important aspects for grass carp breeding.

In vitro Culture of Unfertilized Ovary of Strawberry(Fragaria spp.)

[Objective] This study was to investigate the in vitro culture of unfertilized ovary of strawberry.[Methods] Employing single factor experiment,we investigated six key factors including genotype,extrinsic hormone,sucrose concentration,low temperature pretreatment,growth environment and development status,and illumination condition on induction of gynogenesis in vitro of unfertilized ovary,on the induction of gynogenesis in vitro of unfertilized ovary.[Results] The optimal conditions for in vitro culture of unfertilized ovary of strawberry were as follows：the primary flower buds cultured on bare land as explants,selection of appropriate genotype,2,4-D as external hormone,sucrose at the concentration of 6%,low temperature pretreatment for 48 hours and dark culture under alternated temperature.[Conclusion] The research provided reference for ploidy breeding in strawberry.

Differential screening and characterization analysis of the egg envelope glycoprotein ZP3 cDNAs between gynogenetic and gonochoristic cruciancarp

Gynogenetic silver crucian carp, Carassius auratus gibelio, is an intriguing model system. In the present work, a systemic study has been initiated by introducing suppression subtractive hybridization technique into this model system to identify the differentially expressed genes in oocytes between gynogenetic silver crucian carp and its closely related gonochoristic color crucian carp. Five differential cDNA fragments were identified from the preliminary screening, and two of them are ZP3 homologues. Moreover, the full length ZP3 cDNAs were cloned from their oocyte cDNA libraries. The length of ZP3 cDNAs were 1378 bp for gyno-carp and 1367 bp for gono-carp, and they can be translated into proteins with 435 amino acids. Obvious differences are not only in the composition of amino acids, but also in the number of potential O-linked oligosaccharide sites. In addition, gyno-carp ZP3 amino acid sequence has an unexpected higher identity value with common carp (83.5%) than that with the closely related gono-carp (74.7%). The unique homology may be originated from the ancient hybridization. Northern blot analysis confirmed that expression of the ZP3 gene occurred exclusively in the oocytes. Because O-linked oligosaccharides on ZP3 have been demonstrated to play very important roles in fertilization, it is suggested that the extra O-linked glycosylation sites may be related to the unique sperm-egg recognition mechanism in gynogenesis.

Comparative studies on in vitro sperm decondensation and pronucleus formation in egg extracts between gynogenetic and bisexual fish

A cell-free system based upon the egg extracts from gynogenetic gibel carp (Carassius auratus gibelio)or bisexual red common carp (Cyprinus carpio red variety) was developed to investigate developmentalbehaviors of the demembranated sperm nuclei. Both red common carp and gibel carp sperm nuclei coulddecondense fully and form pronuclei in the red common carp egg extracts. Gibel carp sperm nuclei couldalso decondense fully and form pronuclei in the gibel carp egg extracts, but red common carp sperm nucleicould not decondense sufficiently in the same extracts. The significant differences of morphological changeswere further confirmed by ultrastructural observation of transmission electron microscopy. The data furtheroffer cytological evidence for gonochoristic reproduction in the gynogenetically reproducing gibel carp. Inaddition, the sperm nuclei in vitro decondensation is dependent on the pH in the extracts, and the decon-densed efficiency is optimal at pH 7. However, no DNA replication was observed in the two kinds of eggextracts during the incubation period of the sperm nuclei. It is suggested that the egg extracts preparedfrom the gynogenetic gibel carp should be a valid in vitro system for studying molecular mechanism ongynogenesis and reproduction mode diversity in fish.

Comparative investigation on spindle behavior and MPF activity changes during oocyte maturation between gynogenetic and amphimictic crucian carp

The spindle behavior and MPF activity changes in the progression of oocyte maturation were investigated and compared with cytological observation and kinase assay between gynogenetic silver crucian carp and amphimictic colored crucian carp. MPF activity was measured by using histone H1 as phosphorylation substrate. There were two similar oscillatory MPF kinase activity changes during oocyte maturation in two kinds of fishes with different reproductive modes, but there existed some subtle difference between them. The subtle difference was that the first peak of MPF kinase activity was kept to a longerlasting time in the gynogenetic silver crucian carp than in the amphimictic colored crucian carp. It was suggested that the difference may be related to the spindle behavior changes, such as tripolar spindle formation and spindle rearrangement in the gynogenetic crucian carp.

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.

Genetic discrimination for three gynogenetic clones of silver carp Hypophthalmichthys molitrix, based on restriction endonuclease analysis of Nd5-Nd6 region of mitochondrial DNA

Three artificial gynogenetic clones of silver carp were produced for the analysis of restriction enzyme digestion patterns of ND5-ND6 region from mtDNA of the clones. It is revealed that all intraclonal individuals shared completely the same digestion patterns but among interclonal individuals did not. The three clones were mixed and cultured in a pond together for two years, and restriction endonuclease digestion patterns of ND5-ND6 were used as genetic markers to assess the growth performance of each clone.

Induction of gyno-tetraploidy in Japanese fl ounder Paralichthys olivaceus

Tetraploid fish are important for mass production of triploids in polyploid breeding.In this study,we reported a novel protocol for artificial induction of tetraploidy by a combination of cold shock and hydrostatic pressure administered to gynogenetically developed eggs in Japanese flounder Paralichthys olivaceus.The induction was carried out by activating the eggs with UV-irradiated sperm of red sea bream Pagrus major,administering a cold shock(0℃,5 to 45 min)3 min after fertilization to inhibit second polar body exclusion,incubating the eggs for 60 min at 17℃,and treating them with a 650 kg/cm2 hydrostatics pressure shock for 6 min.We named the embryos gyno-tetraploids that developed from eggs after such treatments.The hatching rate of the gyno-tetraploids ranged from 20.99%±3.66%to 36.01%±2.79%,and the tetraploid rate ranged from 80.00%to 100.00%.All-maternal inheritance was verified using 6 high-recombination-rate microsatellite markers.This method successfully induced gyno-tetraploidy.The successful induction of gyno-tetraploidy lays the foundation for triploidization of new varieties with improved economic traits of interest that can benefit commercial culture.

Artificial induction of mito-gynogenetic diploids in large yellow croaker(Pseudosciaena crocea) by hydrostatic pressure

The present study investigated conditions for inducing mito-gynogenetic(endomitosis) diploids by hydrostatic pressure in the large yellow croaker Pseudosciaena crocea.In haploid control groups,the development of eggs was activated with ultraviolet radiated semen.All fry presented typical haploid syndrome in the haploid control groups,and were verified as haploids using cytometry.After hydrostatic pressure treatment,morphologically normal fry reappeared at different frequencies according to the intensity and time of pressure shock.Fry with normal appearance in the pressure treated groups were verified as gynogenetic double haploids(GDHs),containing only one allele from the female parent at all four diagnostic microsatellite loci.For a fixed duration of 3 min,the optimal intensity of blocking the first mitosis was determined to be 40 Mpa,which was similar to that of blocking the second meiosis.There was a "window" of starting time,from 36.1 min to 38.1 min post-insemination at 25.0±1.0°C,within which the production of GDHs was not significantly different.Maximum production of morphologically normal fries,9.36%±2.97% of developed eggs,was found when the eggs were shocked with hydrostatic pressure at 40 Mpa for 3 min,starting from 38.1 min post insemination at 25.0±1.0°C.

Sperm Nuclear Expansion and Meiotic Maturation in Normal and Gynogenetic Eggs of the Scallop,Chlamys farreri

Sperm nuclear expansion, meiosis and the association of the male and female pronuclei leading to the four-cell stage in normal Chlamys farreri eggs were observed under a fluorescence microscope. The effects of ultraviolet (UV) irradiation on the fer- tilizing sperm were also examined. Both normal and UV-irradiated sperm nuclei enlarged at three distinct phases (phase A, meta- phase I; phase B, polar body formation; and phase C, female pronuclear development and expansion) that were temporally correlated with meiotic process of the maternal chromosomes. Sperm nuclei underwent a rapid, initial enlargement during phase A, but con- densed slightly during phase B, then re-enlarged during phase C. The effects of UV irradiation were not apparent during transforma- tion of the sperm nucleus into a male pronucleus, and there was not any apparent effect on meiotic maturation and development of the female pronucleus. However, the rate of expansion of the UV-irradiated sperm nuclei and the size of male pronuclei were reduced apparently. Unlike the female pronucleus, the male pronucleus derived from sperm genome inactivated by UV irradiation did not form chromosomes, but became a dense chromatin body (DCB). At mitotic anaphase, DCB did not participate in the karyokinesis of the first cleavage as evidenced by chromosomal nondisjunction, demonstrating the effectiveness of using UV irradiation to induce gynogenetic scallop embryos.

Selection of Tolerant Lines to Salinity Derived from Durum Wheat (<i>Triticum durum</i>Desf.) in Vitro Culture

The genetic variability is considered as the major principle of plant breeding for durum wheat. This variability can be induced in vitro by selection pressure exerted by stress factors such as salinity in order to regenerate the vitro plantlets tolerant. This study aims in the first step in the regeneration of plantlets tolerant to salinity from mature embryos culture derived from two Tunisian durum wheat varieties: improved (Razzek) and landrace (Jenah Khotifa (JK)) varieties. The tolerance evaluation to salt stress was applied in vitro (100 mmol&middotl-1 NaCl) and was based on various parameters. Our results showed that JK variety was distinguished by a stable response for all parameters tested: average weight of callus (368.1 mg for control and 307 mg under salt stress), callus regenerated percentage (36.6% for control and 35.7% under salt stress) and green shoots number/callus (17 for control and 17 under salt stress). This stability of response translates the adaptability of this variety to salinity. In order to fix regenerated JK plantlets in single generation and obtain HDs homozygous stable lines, in vitro gynogenesis technical is tested for this genotype. The Evaluation of gynogenetic capacity focused on about 1200 unfertilized ovaries of JK and was based on its ability to induction, differentiation, development of green shoots, and haploid plantlets regeneration. JK showed good tolerance to salinity and a relatively good response to gynogenesis.

Chromosome Transfer from Pink Salmon (Oncorhynchus gorbuscha) to Masu Salmon (O. masou)

Sperms from Pink salmon were subjected to incomplete irradiation by γ-ray to cause partial breakageof their chromosome. Normal eggs from masu salmon were fertilized by these damaged sperms andput under hydrostatic pressure to suppress the release of second polar bodies. The resultant eggscontained complete sets of genome from masu salmon and some chromosomes and chromosomefragments from pink salmon. Karyotype and isozyme of the embryos were analyzed. The resultsdemonstrate that the chromosomes from pink salmon showed genetic activities. Variations in hatchedindividuals were observed.

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) (♀)×common carp (Cyprinus carpio L.) (2n=100, abbreviated as CC) (♂), and RCC (2n=100) (♀)×blunt snout bream (Megalobrama amblycephala) (2n=48, abbreviated as BSB) (♂) are described. In the distant hybridization of RCC (♀)×CC (♂), bisexual fertile F3–F18 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 (♀)×BSB (♂), 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.

Genetic basis and breeding application of clonal diversity and dual reproduction modes in polyploid Carassius auratus gibelio

A unisexual species is generally associated with polyploidy, and reproduced by a unisexual reproduction mode, such as gyno- genesis, hybridogenesis or parthenogenesis. Compared with other unisexual and polyploid species, gibel carp (Carassius au- ratus gibelio) has a higher ploidy level of hexaploid. It has undergone several successive rounds of genome polyploidy, and experienced an additional, more recent genome duplication event. More significantly, the dual reproduction modes, including gynogenesis and sexual reproduction, have been demonstrated to coexist in the polyploid gibel carp. This article reviews the genetic basis concerning polyploidy origin, clonal diversity and dual reproduction modes, and outlines the progress in new va- riety breeding and gene identification involved in the reproduction and early development. The data suggests that gibel carp are under an evolutionary trajectory of diploidization. As a novel evolutionary developmental (Evo-Devo) biology model, this work highlights future perspectives about the functional divergence of duplicated genes and the sexual origin of vertebrate animals.

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 (G3), which was generated from the diploid eggs produced by the second gynogenetic generation (G2) of red crucian carp × common carp, and determined the chromosomal numbers of G3, G2×scatter scale carp and G2×allotetraploid hybrids of red crucian carp × common carp. The results showed that the diploid eggs of G2 with 100 chromosomes, activated by UV-irradiated sperm from scatter scale carp and without the treatment for doubling the chromosomes, could develop into G3 with 100 chromosomes. Similar to the first and second gynogenetic generations (G1 and G2), G3 was also diploid (2n=100) and presented the hybrid traits. The triploids (3n=150) and tetraploids (4n=200) were produced by crossing G2 with scatter scale carp and crossing G2 with allotetraploids, respectively. The extrusion of the second polar body in the eggs of G2 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 G2. The establishment of the diploid gynogenesis clonal line (G1, G2 and G3) 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 gyno- genetic progeny of allotetraploid hybrids of red crucian carp × common carp had important signifi- cances in both biological evolution and production application.

Discovery of Multiple Tetraploids in Artificially Propagated Populations of Allogynogenetic Silver Crucian Carp and Their Breeding Potentialities

1 Introduction Gynogenesis is a rare reproductive mode in fish. Among a few of species reproduced by this method, silver crucian carp (Carassius auratus gibelio) is the most special one. The specialities mainly include the following two aspects: (ⅰ)The crucian carp exists as a bisexual population which can be reproduced by natural gynogenesis, because there are both gynogenetic function and certain proportion of males in the offsprings; (ⅱ)

Differential gene expression of protein kinases in oocytes between natural gynogenetic silver crucian carp and amphimictic crucian carp

A modified mRNA differential display method has been applied to studying differential expression of protein kinase genes in oocytes between natural gynogenetic silver crucian carp and amphimictic crucian carp. Total RNA was reverse transcribed using downstream 3′primers T12MA, T12MG and T12MC respectively. Then the reverse transcription products were amplified using upstream 5′kinase-specific primer designed according to protein kinase conserved sequence. The PCR products had different patterns and numbers of cDNA bands on polyacrylamide gel. Totally 21 cDNAs fragments were recovered and cloned. Two of them were confirmed to be particularly expressed in oocytes of amphimictic crucian carp, and another was specific for gynogenetic silver crucian carp.

Preliminary Confirmation of Gynogenetic Reproductive Mode in Artificial Multiple Tetraploid Ailogynogenetic Silver Crucian Carp

I Introduction Artificial multiple tetraploid allogynogenetic silver crucian carp is a special individual, which was discovered from artificially propagated populations of allogynogenetic silver crucian carp and has the obviously enhanced growth rates. One of its main specialities is that the fish maintains the whole chromosomes (162) of silver crucian carp (Carassius auratus gibelio) and fuses one chromosome set of haploid sperm (50 chromosomes ) of