Search Results (29)

Unpollinated ovary culture is an effective approach for generating haploid and doubled haploid lines, but its application in Chinese chive (Allium tuberosum) breeding has been constrained by strong genotype dependence and low regeneration efficiency. This study evaluated an efficient gynogenesis induction system and analyzed transcriptomic changes associated with embryogenesis. Among 20 evaluated genotypes, the commercial cultivar ‘21-CJ46’ showed the strongest response. The optimized induction conditions utilized ovaries collected 1 day before anthesis, cultured on Murashige and Skoog medium supplemented with 90 g/L sucrose, 1.0 mg/L 6-BA, and 0.2 mg/L 2,4-D at 25 °C. Under this system, ‘21-CJ46’ achieved a maximum embryo induction rate of 86.67%. Flow cytometry and chromosome counting indicated frequent spontaneous chromosome doubling, with regenerants mainly distributed as diploids and tetraploids. RNA-seq analysis comparing pre-induction (0 d) and 14 d ovaries showed extensive transcriptional reprogramming, including significant enrichment of phenylpropanoid biosynthesis, plant–pathogen interaction, and plant hormone signal transduction pathways. Differential expression analysis demonstrated that key embryogenesis regulators, such as BBM2, WUSCHEL9, LEC, PLT2, and ABI3, were regulated at 14 d. These results provide an induction protocol and molecular indications relevant to accelerating Chinese chive breeding. Full article

WW-type super-female broodstock are essential for all-female breeding in sturgeons under the ZZ/ZW sex-determination system, but their practical use is constrained by high mortality. This study investigates the underlying physiological and molecular mechanisms contributing to the reduced viability of WW-type super-female sterlet (Acipenser ruthenus) by comparing three genotypes (ZZ, ZW and WW) in terms of survival rates, oxidative stress levels, and gonadal gene expression. F₂ gynogenetic diploid juvenile sterlet with three genotypes were reared for 100 days under controlled conditions. Survival rates were recorded, and oxidative stress markers, including SOD, CAT, MDA and GSH-Px, were measured using commercially available assay kits. Gonadal gene expression profiles were analyzed using transcriptomic analysis. The results revealed that WW-type juveniles exhibited a significantly lower survival rate (64.2%) compared to ZZ-type and ZW-type fish (both 94.2%, p < 0.0001). While hepatic SOD and CAT activities did not differ among genotypes, MDA and GSH-Px levels were significantly higher in WW-type fish, suggesting enhanced lipid peroxidation and an insufficient compensatory antioxidant response. Transcriptome analysis revealed 747 significantly differentially expressed genes between WW-type super-females and normal ZZ/ZW individuals (p < 0.05), with significant enrichment in pathways related to immune regulation, receptor activity, lipid metabolism, and ferroptosis. Notably, downregulation of arachidonic acid metabolism genes (PTGS2, PTGES, PTGDS) was observed, while ferroptosis-related genes GPX4 and SLC3A2 were upregulated, suggesting that disturbed arachidonic acid metabolism, along with lipid peroxidation and ferroptosis activation, contribute to the reduced survival of WW-type super-females. These findings provide integrative physiological and transcriptomic evidence for the mechanistic basis of poor fitness in gynogenetic WW-type super-females and offer foundational data for improving the feasibility of all-female breeding in sturgeon. Full article

Gynogenesis offers a promising route for doubled haploid (DH) production in Cucurbita, yet efficient protocols remain scarce. This study established a reproducible ovary culture system for Cucurbita pepo and evaluated zeatin riboside (ZR) as an alternative cytokinin. Ovaries collected at anthesis and one day before were cultured to screen nine media with different cytokinin–auxin combinations. Subsequently, four optimized ZR-based formulations were evaluated. Both floral stages showed morphogenic activity, but embryo formation occurred almost exclusively in pre-anthesis ovaries. Among ZR treatments, E6.1 (1 mg·L⁻¹ ZR + 3 mg·L⁻¹ NAA, 30 g·L⁻¹ sucrose) achieved the highest embryogenic output (approximately 97 embryos per 100 explants), while high-sucrose media (120 g·L⁻¹) induced abundant swollen ovules but poor conversion, suggesting that excessive osmotic pressure promotes morphogenesis but hampers embryogenic transition. In total, 415 embryos were obtained, and 52 regenerants were analyzed by flow cytometry, confirming haploid, diploid, and mixoploid plants and evidencing spontaneous chromosome doubling during in vitro development. A categorical A–D scoring system enabled early prediction of embryogenic potential. This represents the first successful application of ZR in cucurbit gynogenesis and highlights its value as a biologically compatible cytokinin for DH production. The findings open new avenues for testing ZR-based formulations in other Cucurbita species under different auxin and sucrose regimes. Full article

Artificially induced gynogenesis, a technique that utilizes UV-irradiated sperm to activate eggs while excluding paternal genetic contribution, has been instrumental in the genetic improvement of aquaculture species. Although the allo-sperm effect has been observed in some freshwater fish and suggests the integration of paternal DNA, its occurrence and mechanisms in marine fish remain unclear. In this study, a 795.23 Mb chromosome-level genome assembly for red sea bream (Pagrus major) was presented, with a scaffold N50 of 32.03 Mb, encompassing 29,083 protein-coding genes. Furthermore, the allo-sperm effect was investigated on the artificial gynogenesis of Japanese flounder (Paralichthys olivaceus) induced by UV-irradiated P. major sperm. Whole-genome sequencing of gynogenetic and normal fertilized offspring revealed eight representative genomic sequences with >96.88% nucleotide identity to P. major, including six Sparidae-specific centromeric satellite DNA sequences. PCR validation and Sanger sequencing confirmed that these sequences were present exclusively in gynogenetic groups and absent in normally fertilized offspring, providing direct evidence of the allo-sperm effect. Our findings extend the allo-sperm effect to marine fish and demonstrate its potential across taxonomically distant taxa, P. olivaceus (Pleuronectiformes) × P. major (Spariformes). These results offer valuable genomic information for P. major, and provide important insights for future genetic breeding programs in aquaculture. Full article

Gynogenesis is a reproductive mode where offspring inherit exclusively maternal chromosomes. Gynogenetic development in fish may be induced intentionally by activating eggs with the UV-irradiated, inactive spermatozoa. In the meiotic variant of gynogenesis, the resultant haploid gynogenetic zygote is then exposed to a physical shock to inhibit the release of the 2nd polar body and to reconstitute the diploid state of the embryo. Here, meiotic gynogenesis was induced in the rainbow trout eggs from different clutches to find any differences in terms of gene expression and antioxidant enzyme activity between eggs with high and low ability for gynogenetic development. The survival rates of the gynogenotes after hatching from the eggs originating from five females varied from 16.6 ± 4.3% to 53.8 ± 9.8%. Biochemical and molecular examination revealed that eggs with higher developmental potential for meiotic gynogenesis exhibited significantly greater glutathione peroxidase (GPx) activity than eggs with lower efficiency of gynogenesis. Moreover, eggs exhibiting the highest ability for gynogenetic development showed increased transcription of the keratin 8 gene and decreased abundance of keratin 18 and tubulin β mRNA transcripts. Since keratins protect oocytes from physical stress after ovulation, the high abundance of keratin 8 in the rainbow trout eggs may increase their resilience to the physical shock applied for the zygote diploidization during gynogenesis. On the other hand, a low level of tubulin-building microtubules may increase the efficiency of high hydrostatic pressure (HHP) shock used for diploidization of the gynogenetic zygotes. Full article

Grass carp (Ctenopharyngodon idella) is one of the most economically important cyprinid species cultured in China. The diploid gynogenetic grass carp (2nGGC, 2n = 48) was generated from the hybrid of female grass carp (GC, 2n = 48) and male topmouth culter (TC, 2n = 48, Culter alburnus). This study obtained the full-length transcriptome of 2nGGC from five tissues using Pacific Biosciences (Pacbio) single-molecule real-time long-read isoform sequencing. Following the mapping of long reads to GC and TC reference genomes, a total of 1848 fusion isoforms were identified. Among them, 775 were distributed across different genomes, indicating that chimeric DNA fragments of TC were embedded in the 2nGGC genome. After removing the fusion genes and redundant isoforms, 107,721 full-length transcripts were obtained from 2nGGC, providing important full-length reference sequences for further research. Finally, comparative analysis of homologous gene variation identified 34 fragments in 2nGGC containing recombinant SNPs derived from both GC and TC. These results provide evidence that natural gynogenesis represents a form of “micro-hybridization” characterized by heterogeneous DNA fragments, distinct from traditional hybridization involving chromosome-level recombination. These findings offer valuable reference for fish genetic breeding. Full article

PIWI-interacting RNAs (piRNAs) are small non-coding RNAs that interact with PIWI proteins and play essential roles in genome stability, gonadal development, and gametogenesis in animals. The Japanese flounder (Paralichthys olivaceus) is an important marine culture teleost in North Asia, showing pronounced sexual size dimorphism, where gynogenetic induction of all-female cohorts can markedly enhance production. However, the PIWI/piRNA pathway in gynogenetic diploid P. olivaceus, which often exhibit gonadal dysgenesis, poor gamete quality, and low fertilization rates, remains poorly understood. In this study, RNA-seq and small RNA-seq data from 11 tissues and 6 developmental stages of common P. olivaceus, as well as the gonads of gynogenetic P. olivaceus, were analyzed to characterize the PIWI/piRNA pathway and its roles in transposon and gene regulation within the germline. The results showed that PIWI/piRNA genes were predominantly expressed in gonads and early embryogenesis in common P. olivaceus, with the highest expression in testis. Clustered piRNAs were identified in the testis and early embryos of common P. olivaceus, which targeted multiple transposon and gene families. Intriguingly, gynogenetic P. olivaceus gonads harbored abundant clustered piRNAs not only in the testes but also in the ovaries, both targeting similar transposon families as that in common P. olivaceus. Notably, the DNA transposon Tc1/Mariner family and pim genes were the most heavily targeted by piRNAs in gynogenetic P. olivaceus, with testis-biased expression. Expanded pim genes were identified in P. olivaceus, overlapping with piRNA clusters, and the in vitro test in P. olivaceus testes revealed that the expanded pim genes may be pseudogenes as a piRNA cluster reference to generate piRNAs regulating the conventional pim members. These unique features of the PIWI/piRNA pathway in gynogenetic diploid P. olivaceus may underline their impaired reproductive ability, and have important theoretical and practical implications for teleost gynogenetic breeding. Full article

Artificial gynogenesis is an essential technique for aquaculture breeding. Fertile offspring of the WuLi carp (Cyprinus carpio var. Quanzhounensis, 2n = 100, WLC) were successfully produced via gynogenesis using ultraviolet-irradiated sperm from the blunt snout bream (Megalobrama amblycephala, 2n = 48, BSB). As anticipated, gonadal section examination confirmed that all gynogenetic WuLi carp (2n = 100, GWB) were female. To investigate whether paternal DNA fragments from BSB were integrated into the GWB genome, comparative analyses of morphological traits, DNA content, chromosomal numbers, 5S rDNA sequences, microsatellite DNA markers, fluorescence in situ hybridization (FISH), growth performance and nutritional composition were systematically conducted between GWB and maternal WLC. The results revealed pronounced maternal inheritance patterns across morphological characteristics, DNA quantification, chromosomal configurations, 5S rDNA sequences and FISH signals, while microsatellite detection unequivocally confirmed paternal BSB DNA fragment integration into the GWB genome. Remarkably, GWB demonstrated significantly superior growth performance and elevated unsaturated fatty acid content relative to the maternal line. This approach not only addressed germplasm degradation in WLC but also provided valuable theoretical foundations for breeding programs in this commercially significant species. Full article

Background/Objectives: Fish eggs activated with UV-irradiated spermatozoa and exposed to the High Hydrostatic Pressure (HHP) shock to inhibit first cell cleavage develop as gynogenetic Doubled Haploids (DHs) that are fully homozygous individuals. Due to the expression of the recessive genes and side effects of the gamete treatment, survival of fish DHs is rather low, and most of the mitotic gynogenotes die before hatching. Nevertheless, as maternal gene products provided during oogenesis control the initial steps of embryonic development in fish, a maternal effect on the survival of gynogenotes needs to be also considered to affect efficiency of gynogenesis. Thus, the objective of this research was to apply an RNA-seq approach to discriminate transcriptional differences between rainbow trout (Oncorhynchus mykiss) eggs with varied abilities to develop after gynogenetic activation. Methods: Gynogenetic development of rainbow trout was induced in eggs originated from eight females. Maternal RNA was isolated and sequenced using RNA-Seq approach. Survival rates of gynogenotes and transcriptome profiles of eggs from different females were compared. Results: RNA-seq analysis revealed substantial transcriptional differences between eggs originated from different females, and a significant correlation between the ability of the eggs for gynogenesis and their transcriptomic profiles was observed. Genes whose expression was altered in eggs with the increased survival of DHs were mostly associated (GO BP) with the following biological processes: development, cell differentiation, cell migration and protein transport. Some of the genes are involved in the oocyte maturation (RASL11b), apoptosis (CASPASE 6, PGAM5) and early embryogenesis, including maternal to zygotic transition (GATA2). Conclusions: Inter-individual variation of the transcription of maternal genes correlated with the competence of eggs for gynogenesis suggest that at least part of the mortality of the rainbow trout DHs appear before activation of zygotic genome and expression of the lethal recessive traits. Full article

Polyploidy plays a significant role in loquat breeding, particularly in triploid breeding for seedless fruit production. Currently, loquat polyploid breeding primarily relies on natural seedling selection and sexual hybridization approaches. In this study, unfertilized ovules from four loquat varieties were in vitro cultured. Gynogenesis and embryoid regeneration were achieved in ‘Xingning 1’ and ‘Huabai 1’, with ‘Xingning 1’ demonstrating the highest gynogenesis efficiency (21.63%). Flow cytometry and chromosome counting revealed that the obtained embryoid lines included haploid, diploid, tetraploid, hexaploid, and chimeric ploidy types. Further characterization of ‘Xingning 1’-derived embryoid lines through SSR markers and whole-genome resequencing confirmed that the haploid, diploid, tetraploid, and hexaploidy embryoid originated from haploid–somatic chimeras, diploid, doubled diploid and tripled diploid, respectively. Metabolic analysis showed a positive correlation between ploidy level and the content of both soluble sugars and organic acids. This study explored a novel platform for polyploid induction in loquat and may provide methodological insights for improvement of other perennial fruit trees. Full article

Artificial gynogenesis is an effective means of producing pure lines and is widely used for genetic analysis of fish and for sex control. In this study, inactivated sperm from heterogenous blunt snout bream (Megalobrama amblycephala, MA) were used to activate Kohaku koi (Cyprinus carpio var. koi, CK) and produce high-quality female offspring. To determine whether the offspring were gynogenetic fish, the karyotype and DNA content of the CK, MA and the induced offspring (IO) were first compared and it was found that the IO were diploid with 100 chromosomes and their karyotype was 22m + 34sm + 22st + 22t. The DNA content of the IO was not significantly different from that of the CK. Subsequently, the amplified band profiles of CK, MA and IO were analyzed with species-specific microsatellite markers. The results showed that there were no amplified MA microsatellite bands in IO. The size of the amplified bands and the sequence of the 5S rDNA in CK, MA and IO were also analyzed. It was found that the amplified 5S rDNA gene fragments in IO contained two fragments that were both the same size as those of CK and matched more than 90% with those of CK. Finally, the sex of IO was verified using gonadal tissue sections. The result showed that IO was not an all-female population; males were also present (36.7%). In summary, a series of validation methods confirmed that the induced offspring were gynogenetic fish, which is the basis for the subsequent genetic improvement of pure lines of high-quality koi. Full article

Miscanthus × giganteus (Greef and Deuter ex Hodkinson and Renvoize) is a perennial, rhizomatous grass that has gained significant attention as an industrial crop, particularly as a bioenergy feedstock. It is a natural interspecific hybrid with 57 chromosomes (2n = 3x = 57). Due to its sterility, M. × giganteus has limited genetic variability, making traditional breeding methods ineffective for its improvement. Consequently, alternative approaches are being explored to enhance its cultivation and utility. The study aimed to investigate the potential for M. × giganteus plant regeneration through ovary and flower bud cultures. Indirect in vitro regeneration of M. × giganteus plants was successfully achieved using flower bud cultures. Embryogenic-like callus was derived from explants originating from inflorescences that had undergone a four-day pretreating at 10 °C. The most effective medium for callus induction was a modified MS medium supplemented with 5 mg·dm⁻³ dicamba, 0.2 mg dm⁻³ 6-benzylaminopurine, 30 g dm⁻³ sucrose, and solidified with 8 g dm⁻³ agar or agarose. The optimal conditions for callus induction were achieved by culturing in the dark. The regenerated plants exhibited the characteristic chromosome number of the species, confirming that the regenerants did not develop from embryo sac cells. In contrast, ovary culture failed to produce callus or regenerated plants, highlighting its ineffectiveness for M. × giganteus regeneration. These findings underscore the potential of flower bud culture as a successful in vitro regeneration method while demonstrating the limitations of ovary culture for this species. Full article

Qihe crucian carp Carassius auratus is one of the major economic aquacultural fish species in China. Due to environmental degradation and the impact of the reproduction mode of gynogenesis, the wild population of Qihe crucian carp exhibits an extremely low proportion of males. Therefore, it is imperative to develop the sperm preservation technology for Qihe crucian carp. In this study, the indicators, including sperm motility, sperm movement time, and sperm lifetime, were applied to evaluate the preservation effects of various extenders, cryoprotectant types and concentrations, and preservation manners, thus establishing the techniques of sperm short-term storage and cryopreservation for Qihe crucian carp. The results showed that HBSS was the most suitable extender solution, in which the sperm motility reached 93.00%. Under room temperature (26 °C), the sperm motility showed no significant differences across six different cryopreservation solutions. Under low temperature (4 °C), a 15% MeOH solution presented the most effective preservation effects of sperm in the short-term low-temperature storage. For cryopreservation in liquid nitrogen, the sperm activity in 20% DMSO was significantly higher than that in other solutions, representing the optimal cryopreservation solution. Furthermore, it was found that the sperm activity preserved by liquid nitrogen was significantly higher than that preserved by the ultra-freezer (−80 °C), indicating that the ultra-freezer cannot substitute for liquid nitrogen in cryopreservation. This is the first study to investigate the preservation of Qihe crucian carp sperm, providing valuable technical support for both genetic resource conservation and artificial breeding programs. Full article

The assessment of genetic improvement was comprehensively analyzed using the mtDNA Cyt b gene and SSR markers among three farmed grass carp populations caught in May 2024. The results of an mtDNA Cyt b gene analysis in 198 individuals showed that the haplotype diversity index (H_i) and nucleotide diversity index (P_i) were 0.555 and 0.00058, respectively. The results of the analysis of SSR marker data in 196 individuals indicated that the unequal dosage amplification at the same locus was found in the CC population. Moreover, the total number of alleles (A: 338), number of alleles per locus (Na: 15.36), observed heterozygosity (Ho: 0.8391), expected heterozygosity (He: 0.8380), and polymorphic information content (PIC: 0.8191) in the KC population was relatively higher than that in the CC (A: 129; Na: 5.86; Ho: 0.0025; He: 0.6191; PIC: 0.5747) and CY (A: 293; Na: 8.77; Ho: 0.821; He: 0.7483; and PIC: 0.5747) populations. The FST and AMOVA analysis showed the existence of a significant differentiation (p < 0.001), with a high genetic differentiation between the CC and CY populations. In summary, a high genetic variability exists in the KC population, while the father (CY) and mother (CC) populations have relatively low genetic variability. This study reveals evidence of the existence of a “micro-hybrid”. Moreover, the results demonstrated that combining both gynogenesis and backcross breeding technology is vital for the genetic improvement of grass carp. Moreover, continuous research into the genetic health of these populations is required as well as support for the protection of germplasm resources and artificial breeding. Full article

Doubled haploid technology is, so far, the fastest route to induce a true homozygous state in plants. True homozygous plants are particularly important for breeders, as they can facilitate hybrid breeding and are useful in fixing traits in a breeding line. Fabaceae species are of great importance in food and feed production; however, they are far behind other families with respect to the development of effective haploidization protocols. Here, we present the most recent status of research on haploidization protocols in cool-season grain legume crops, including dry peas, chickpeas, faba beans, lentils, lupines, and grass peas. The first four species are primarily for human consumption; the latter are utilized as forage. All the mentioned species have been subject to haploidization trials; however, repeatable protocols, including the regeneration of confirmed haploid or doubled haploid plants, have not been elaborated. Research in field pea, chickpea, grass pea, and lupine is promising, with the reported regeneration of microspore-derived embryos in all four species. Repeatable plant regeneration has been reported only in field peas and chickpeas. The most recent achievements on haploidization through male and female gametophytes in faba bean are also presented. The key factors for the effective stimulation of haploid cell development in cool-season legumes are reviewed, providing a useful basis for future efforts toward haploidization in this group. Full article