Androgenetic haploid Japanese pufferfish (Takifugu rubripes) induced by cold shock

Abstract

Here, we explored cold-shock-induced haploid androgenesis in Japanese pufferfish (Takifugu rubripes) and analysed chromosomes by fluorescence in situ hybridization (FISH), single nucleotide polymorphism (SNP) in sex determination locus and microsatellite genotypes to verify all-male inheritance. We also observed early embryonic development to have an insight into the cytological mechanism of cold-shock-induced gynogenesis. The chromosome number of control group was diploid (2n = 44), while in cold-shock group haploid (1n = 22) and triploid (3n = 66) embryos were detected. Cellular DNA content flow cytometry showed that the rate of haploid induction was 90%. One signal of nucleolar organizing region (NOR) was detected by silver nitrate staining and FISH using rDNA probe in androgenetic haploids from cold-shock group.SNP analysis revealed that about half of haploid embryos examined had the G genotype indicating male-specific Y chromosome. Microsatellite genotyping showed that 26 out of 29 haploid embryos from cold-shock group exclusively inherited paternally derived genotypes. Cytological observation revealed that both second polar body and egg nucleus were located on the blastodisc surface at 60 min post fertilization in cold-shock group, suggesting simultaneous extrusion of both nuclei. K E Y W O R D Sall-male inheritance, chromosome manipulation, microsatellite, sex determination, SNP, Takifugu rubripes | 3803 ZHOU et al. How to cite this article: Zhou H, Wang Q, Liu H-J, et al. Androgenetic haploid Japanese pufferfish (Takifugu rubripes) induced by cold shock. Aquac Res. 2019;50:3802-3811. https ://doi.

Genome-wide DNA methylation and gene expression patterns of androgenetic haploid tiger pufferfish (Takifugu rubripes) provide insights into haploid syndrome

“…We previously established the method of producing androgenetic haploid T. rubripes by cold shock treatment, and found that both second polar body and egg nucleus were located on the blastodisc surface in haploid tiger pufferfish embryo, suggesting the simultaneous extrusion of both nuclei 12 . According to our analysis on the haploid and diploid methylome profiles, we found that about 10.80% of C was methylated in both genetic female and male genomes, which is consistent with our previous reports in adult tiger pufferfish 26 and Chinese tongue sole ( Cynoglossus semilaevis ) 27 .…”

“…Embryos were developed until 4 day-post-fertilization (dpf) (the stage around eyespots appeared), and thirty embryos were randomly sampled from the cold-treated and control groups, respectively. Each embryo was treated by cold‐dropping with DAPI staining to confirm the chromosome number and to evaluate the cold treatment effect as previously described 12 . The rest embryos were kept until 6 dpf before hatching, and another thirty embryos were randomly sampled from the cold-treated and control groups, respectively.…”

“…One-third of each embryo (the tail part) was used for DNA relative content analysis by using Partec PA Flow Cytometry (Munich, Germany) as previously reported 12 .…”

Genome-wide DNA Methylation and Gene Expression Patterns of Androgenetic Haploid Tiger Pufferfish (Takifugu Rubripes) Provide Insights into Haploid Syndrome

“…Each embryo was dissected into three parts, and one-third of each embryo was used for DNA relative content analysis. DNA relative content of each embryo was measured using Partec PA Flow Cytometry (Munich, Germany) as previously reported [12]. After removing chorion and yolk, the embryos were placed into Cystain DNA 1 Step Staining Solution (Partec, Germany) at 4°C without exposure to light.…”