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.