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.

Screen for stage-specific expression genes between tail bud stage and heartbeat beginning stage in embryogenesis of gynogenetic silver crucian carp

A systemic study was initiated to identify stage-specific expression genes in fish embryogenesis by using suppression subtractive hybridization (SSH) technique. In this study, we presented a preliminary result on screen for stage-specific expression genes between tail bud stage (TBS) and heartbeat beginning stage (HBS) in gynogenetic silver crucian carp (Carassius auratus gibelio). Two SSH plasmid libraries specific for TBS embryos and HBS embryos were constructed, and stage-specific expression genes were screened between the two stages. 1963 TBS positive clones and 2466 HBS positive clones were sampled to PCR amplification, and 1373 TBS and 1809 HBS PCR positive clones were selected to carry out dot blots. 169TBS dot blot positive clones and 272 HBS dot blot positive clones were sequenced. Searching GenBank by using these nucleotide sequences indicated that most of the TBS dot blot positive clones could not be found homologous sequences in the database, while known genes were mainly detected from HBS dot blot positive clones. Of the 79 known genes, 20 were enzymes or kinases involved in important metabolism of embryonic development. Moreover, specific expressions of partial genes were further confirmed by virtual northern blots. This study is the first step for making a large attempt to study temporal and spatial control f gene expression in the gynogenetic fish embryogenesis.

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.

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.

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.

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.

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.

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

Two unisexual artificial polyploid clones constructed by ge-nome addition of common carp(Cyprinus carp)and crucian carp(Carassius auratus)

A polyploid hybrid fish with natural gynogenesis can prevent segregation and maintain their hybrid vigor in their progenies. Supposing the reproduction mode of induced polyploid fish being natural gynogenesis, allopolyploid hybrid between common carp and crucian carp into allo-polyploid was performed. The purpose of this paper is to describe a lineage from sexual diploid carp transforming into allotriploid and allotetraploid unisexual clones by genome addition. The dip-loid hybrid between common carp and crucian carp reproduces an unreduced nucleus consisting of two parental genomes. This unreduced female pronucleus will fuse with male pronucleus and form allotriploid zygote after penetration of related species sperms. Allotriploid embryos grow nor-mally, and part of female allotriploid can produce unreduced mature ova with three genomes. Ma-ture ova of most allotriploid females are provided with natural gynogenetic trait and their nuclei do not fuse with any entrance sperm. All female offspring are produced by gynogenesis of allotriploid egg under activation of penetrating sperms. These offspring maintain morphological traits of their allotriploid maternal and form an allotetraploid unisexual clone by gynogenetic reproduction mode. However, female nuclei of rare allotriploid female can fuse with penetrating male pronuclei and result in the appearance of allotetraploid individuals by means of genome addition. All al-lotetraploid females can reproduce unreduced mature eggs containing four genomes. Therefore, mature eggs of allotetraploid maintain gynogenetic trait and allotetraploid unisexual clone is pro-duced under activation of related species sperms.

Genetic Analysis of Gynogenetic and Common Populations of Verasper moseri Using SSR Markers

The genetic structure and variation of the artificial meio- gynogenetic population and common population of barfin flounder （Verasper moseri） were analyzed using eight microsatellite markers. A total of 29 alleles were detected, of which 23 alleles were in the artificial gynogenetic population while 29 alleles were in the control group. The average observed heterozygosity （Ho） of eight loci in the control group （0.526 8） was several times higher than that （0.185 8） in the gynogenetic population. The results indicate that the genetic diversity of the control group was much higher than that of the gynogenetic population ofbarfin flounder （Verasper moseri）. Most loci significantly deviated from Hardy-Weinberg equilib- rium （HWE） after Bonferroni correction （p 〈 0.005 56） in the gynogenetic population, while four loci deviated from HWE in the control group. The coefficient of gene differentiation （GsT） was 0.131 0, and the genetic distance was 0.171 8 between the two populations, suggesting a significant genetic differentiation between the two populations.

Further evidence for paternal DNA transmission in gynogenetic grass carp

Gynogenesis is an important breeding method in aquaculture and has been widely applied to many fish species.If gynogenetic progenies are to inherit paternal partial genomic DNA,this will increase genetic variation and will provide a useful outcome for breeding.In this study,we investigated the genetic variation in homeobox(Hox)gene clusters(Hox A4 a,Hox A9 a,Hox A11 b,Hox B1 b,Hox C4 a,Hox C6 b,and Hox D10 a)among koi carp(Cyprinus carpio haematopterus,KOC;the stimulation sperm source),grass carp(Ctenopharyngodon idellus),and gynogenetic grass carp(GGC).We found paternal DNA(a special DNA fragment and Hox C6 b)derived from KOC and a recombinant gene belonging to Hox C6 b in GGC.We are the first to report the recombinant Hox C6 b in GGC.Our study provides further evidence for paternal DNA transmission to gynogenetic progenies,which is a finding with great significance for the genetic breeding of fish.

STUDY ON THE MODE OF PRIMARY CONTROL IN THE EGG OF GYNOGENETIC CRUCIAN CARP FOR INHIBITING THE DEVELOPMENT OF TWO TYPES OF SPERM NUCLEI

By comparing the different developmental characteristics of two types of sperm nucleiwhich were from gynogenetic fish (crucian carp) and amphimictic fishes (red carp, redgoldfish and sex--reversal red carp) respectively in the eggs of gynogenetic crucian carp, itwas preliminarily revealed that there existed selective inhibiting actions of the primarycontrol in the eggs of crucian carp for inhibiting the development of the two types of spermnuclei. To homologous sperms, the primary control showed weak effect, thus leading tothe decondensation of homologous sperm nuclei at different degrees in the eggs of cruciancarp. But to heterologous sperms, the primary control showed strong effects, resulting in thetotal inhibition of the development of heterologous sperm nuclei. Moreover, our experimentalresults also showed that the different developmental behavior of the two types of spermnuclei might have a great relationship to the changes or the sex ratio in the population ofgynogenetic crucian carp. The