DNA methylation has frequently been implicated in sex determination and differentiation in teleost species. In order to detect the DNA methylation patterns established during sexual differentiation in tiger pufferfish T . rubripes , we performed comprehensive whole genome methylation sequencing and analyses of the gonads of male, female, and pseudo male. We obtained a total of 33.12, 32.44, and 31.60 Gb clean data for male, female, and pseudo male, with a sequencing depth of 66.44×, 60.47× and 54.86×, respectively. The methylation level of cytosine (C) residues in the genomic DNA from gonads was 11.016%, 10.428%, and 11.083% in male, female, and pseudo male, respectively. More than 65% of C methylation was at CpG sites, and less than 1% was at CHG and CHH sites. In each regulatory element, there were low methylation levels on both sides of the transcription start site, and higher methylation levels in exons, introns, and downstream of genes. The highest mCpG was on chromosome 8 and the lowest mCpG was on chromosome 5. Comparisons of whole-genome DNA methylation between pairs of samples revealed that there were 3,173 differentially methylated regions (DMRs) between female and male, and 3,037 DMRs between male and pseudo male, corresponding to 0.232% and 0.223% of the length of the genome, respectively. There were only 1,635 DMRs between female and pseudo male, representing 0.127% of the length of the genome. A number of differentially methylated genes (DMGs) related to sex determination and differentiation were selected, such as amhr2 and pfcyp19a . After Bisulfite Sequencing PCR (BSP) verification, amhr2 was exhibited low methylation level in normal males and pseudo male, and high methylation level in normal females but pfcyp19a showed low methylation level in normal females and high methylation level in normal males and pseudo males. These results provide information about the molecular epigenetic mechanisms of DNA methylation during low-temperature induced masculinization of tiger pufferfish, and increase our understanding of the mechanisms of sex determination and differentiation in this important aquaculture fish species.

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Hybrid triploid loaches (Misgurnus anguillicaudatus) were generated from natural tetraploid and diploid loaches. We studied the gonads of parents and offspring from direct and reciprocal crosses through histological and transcriptome analyses. The triploid offspring had inferior ability to form sperm and egg cells compared with diploid forms. After sequencing the transcriptomes, 655,109,158 clean reads were obtained, and 62,821 unigenes and 178,962 transcripts were assembled. Of these unigenes, 23,189 were annotated in the GO database, 18,525 in the KEGG database and 24,661 in the KOG database. 36 fertility-related genes were found to be differentially expressed between the direct cross (2n × 4n) progenies and their parents, while 53 fertility-related genes between the reciprocal cross (4n × 2n) progenies and their parents. Following protein-protein interaction network analyses, 54 differentially expressed genes, including PLCB4, cyp17a1 and Pla2g4d, were mined, yielding candidate genes involved in the poor fertility of hybrid triploid loaches. This is the first report of transcriptomes of gonads of hybrid triploid loaches and their parents, offering a substantial contribution to sequence resources for this species and providing a deep insight into the molecular mechanism controlling the fertility of hybrid triploid fish.

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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.