Sex-Associated SNP Confirmation of Sex-Reversed Male Farmed Japanese Flounder Paralichthys olivaceus.

Female Japanese flounder Paralichthys olivaceus grow more rapidly than the male. The goal of all-female commercial production requires an efficient method of genetic sex identification. We conducted genome-wide association analysis of female and male farmed Japanese flounder (n = 24 per phenotypic sex) and found all regions of chromosome 24 to be significantly associated with phenotypic sex, suggesting it as the sex chromosome. Genetic sex was identified based on single nucleotide polymorphisms (SNP) on chromosome 24 (n = 3568) using multidimensional scaling analysis, and individuals were clearly separated according to sex by the first dimension. The 61 SNPs most highly associated with sex were selected, and an amplicon-based SNP panel was developed. This was used to determine genetic sex of 39 females and 40 males. Eleven phenotypic males were assigned as female with XX genotype, suggesting sex reversal. Genetic sex was also assessed based on the indel of the amh gene promoter, which is the major candidate sex gene of Japanese flounder. We found four SNPs perfectly associated with genotypic sex in the sex-associated SNP panel, one of which was located in exon 2 of the amh gene. Along with the indel of the amh gene promoter, the sex-associated SNP panel will be of value in identifying genetic sex of farmed Japanese flounder. Molecular sexing will facilitate all-female production by breeding sex-reversed males.

Pituitary luteinizing hormone synthesis starts in aromatase (cyp19a1b)-positive cells expressing esr1 and esr2b at the onset of puberty in Takifugu rubripes (fugu).

Unlike mammals, teleost fish have high aromatase activity (AA) in the pituitary. However, the cells responsible for oestradiol synthesis and the local physiological roles of this hormone remain unclear. Hence, we investigated the effects of age and development on steroidogenic activity, mRNA expression, and cyp19a1b localization in the pituitary gland of the Japanese pufferfish Takifugu rubripes. Under aquaculture conditions, AA was highest after puberty, and the mRNA expression levels of cyp19a1b and the oestrogen receptors esr1 and 2b and the level of serum testosterone (T) were significantly increased after puberty compared with the other developmental stages in male and female pufferfish. Immunohistochemistry using multiple antibodies and in situ hybridization analysis revealed that Cyp19a1b colocalizes with luteinizing hormone (LH) in pituitary cells. Furthermore, Esr1 was localized in the nuclei of all hormone-producing cells, whereas Esr2b was localized only in the nuclei of Cyp19- and LH-positive cells. The administration of an aromatizable androgen (T) or oestrogen (E2) to reproductively inactive females induced LH synthesis in vivo. We prepared spheroids from pituitary cells to investigate the role of local E2 in LH synthesis in vitro. Immunohistochemical analysis of spheroids showed that T-induced LH synthesis could be blocked by an aromatase inhibitor and/or an ER antagonist but not an AR antagonist. Taken together, these findings suggest that LH synthesis is initiated in cyp19a1b-, esr1-, and esr2b-expressing cells at the onset of puberty under the control of steroidal feedback, and both feedback and local oestrogen may be involved in controlling LH synthesis in these cells.

Genomic prediction for testes weight of the tiger pufferfish, Takifugu rubripes, using medium to low density SNPs.

Aquaculture production is expected to increase with the help of genomic selection (GS). The possibility of performing GS using only a small number of SNPs has been examined in order to reduce genotyping costs; however, the practicality of this approach is still unclear. Here, we tested whether the effects of reducing the number of SNPs impaired the prediction accuracy of GS for standard length, body weight, and testes weight in the tiger pufferfish (Takifugu rubripes). High values for predictive ability (0.563-0.606) were obtained with 4000 SNPs for all traits under a genomic best linear unbiased predictor (GBLUP) model. These values were still within an acceptable range with 1200 SNPs (0.554-0.588). However, predictive abilities and prediction accuracies deteriorated using less than 1200 SNPs largely due to the reduced power in accurately estimating the genetic relationship among individuals; family structure could still be resolved with as few as 400 SNPs. This suggests that the SNPs informative for estimation of genetic relatedness among individuals differ from those for inference of family structure, and that non-random SNP selection based on the effects on family structure (e.g., site-FST, principal components, or random forest) is unlikely to increase the prediction accuracy for these traits.

Gonadal sex differentiation and early ovarian/testicular development in cultured Pacific bluefin tuna, Thunnus orientalis (Temminck et Schlegel).

Pacific bluefin tuna (PBT), Thunnus orientalis, is one of the most important species for aquaculture in Japan. Recently, the reduction in muscle fat content associated with sexual maturation in farmed PBT has become a serious problem. To develop technologies for inducing sterility, detailed and reliable data on gonadal development in PBT are needed. Here, we demonstrated the process of gonadal sex differentiation, and of early ovarian and testicular development during the immature stages in PBT. Gonadal sex differentiation was first characterized by the formation of the ovarian cavity in female and of the efferent ducts in male 57 days post hatching (dph). The gonads then differentiated into ovaries or testes according to the genotypic sex until 83 dph. During this period, primordial germ cells, oogonia, and type-A spermatogonia were solitarily distributed in the gonads, and the number of germ cells did not differ between sexes. After gonadal sex differentiation, gonads of PBTs developed in a sexually dimorphic manner: proliferation and differentiation of germ cells occurred earlier in the ovaries than in the testes. The oogonia in ovaries formed cysts at 185 dph, but the type-A spermatogonia were solitarily distributed in testes at this stage, and cysts of type-A spermatogonia were first observed at 247 dph. Moreover, the oogonia entered meiosis and differentiated into chromatin-nucleolus stage oocytes until 247 dph, and subsequently into peri-nucleolus stage oocytes until 285 dph, whereas the type-A spermatogonia differentiated into type-B spermatogonia, spermatocytes, spermatids, and spermatozoa from 446 dph onwards. We believe the results of this study provide the necessary basis for future studies on sterile PBT production.

Genetic Dissection of a Precocious Phenotype in Male Tiger Pufferfish (Takifugu rubripes) using Genotyping by Random Amplicon Sequencing, Direct (GRAS-Di).

The novel non-targeted PCR-based genotyping system, namely Genotyping by Random Amplicon Sequencing, Direct (GRAS-Di), is characterized by the simplicity in library construction and robustness against DNA degradation and is expected to facilitate advancements in genetics, in both basic and applied sciences. In this study, we tested the utility of GRAS-Di for genetic analysis in a cultured population of the tiger pufferfish Takifugu rubripes. The genetic analyses included family structure analysis, genetic map construction, and quantitative trait locus (QTL) analysis for the male precocious phenotype using a population consisting of four full-sib families derived from a genetically precocious line. An average of 4.7 million raw reads were obtained from 198 fish. Trimmed reads were mapped onto a Fugu reference genome for genotyping, and 21,938 putative single-nucleotide polymorphisms (SNPs) were obtained. These 22 K SNPs accurately resolved the sibship and parent-offspring pairs. A fine-scale linkage map (total size: 1,949 cM; average interval: 1.75 cM) was constructed from 1,423 effective SNPs, for which the allele inheritance patterns were known. QTL analysis detected a significant locus for testes weight on Chr_14 and three suggestive loci on Chr_1, Chr_8, and Chr_19. The significant QTL was shared by body length and body weight. The effect of each QTL was small (phenotypic variation explained, PVE: 3.1-5.9%), suggesting that the precociousness seen in the cultured pufferfish is polygenic. Taken together, these results indicate that GRAS-Di is a practical genotyping tool for aquaculture species and applicable for molecular breeding programs, such as marker-assisted selection and genomic selection.

Expression profile of kalliklectin, a soluble-type mannose receptor, during embryogenesis and early larval development in fugu (Takifugu rubripes).

Kalliklectin is a unique fish-specific lectin, whose sequence is similar to the heavy chain of mammalian plasma kallikrein and coagulation factor XI. In this study, we aimed to evaluate dynamic expression profiles of the lectin gene, during early developmental stages, in fugu, Takifugu rubripes. Reverse transcription-polymerase chain reaction (RT-PCR) showed that the kalliklectin gene was not expressed until 14 h post-fertilization (hpf), while the mRNA was detected after 30 hpf. In real-time quantitative PCR (qPCR), the gene was first expressed at 10.5 hpf; then, the expression level increased with a peak at 30 hpf and then gradually decreased. On the other hand, western blotting with specific antibody detected the lectin protein at all tested stages, including the unfertilized egg, which suggests that the lectin detected in the early stages was a maternal factor. Immunohistochemistry demonstrated that kalliklectin was localized at the basement membranes of the newly hatched larvae, while the lectin was widely detected in epidermal cells in larva at 5 dph. A 40-kDa lectin was partially purified from unfertilized eggs using mannose-affinity chromatography, and the lectin was determined as kalliklectin by liquid chromatography with quadrupole time-of-flight tandem mass spectrometry (LC/Q-TOF-MS) analysis, which indicated that the lectin is functional in the eggs. The egg lectin can bind to Gram-positive bacterial pathogens of fish, such as Lactococcus garvieae and Streptococcus iniae. We conclude that fugu kalliklectin might be an important immunocomponent, transferred from mother to offspring.

A highly flexible and repeatable genotyping method for aquaculture studies based on target amplicon sequencing using next-generation sequencing technology.

Studies using genome-wide single nucleotide polymorphisms (SNPs) have become commonplace in genetics and genomics, due to advances in high-throughput sequencing technologies. Since the numbers of required SNPs and samples vary depending on each research goal, genotyping technologies with high flexibility in the number of SNPs/samples and high repeatability have been intensively investigated. For example, the ultrahigh-multiplexed amplicon sequencing, Ion AmpliSeq, has been used as a high-throughput genotyping method mainly for diagnostic purposes. Here, we designed a custom panel targeting 3,187 genome-wide SNPs of fugu, Takifugu rubripes, and applied it for genotyping farmed fugu to test its feasibility in aquaculture studies. We sequenced two libraries consisting of different pools of individuals (n = 326 each) on the Illumina MiSeq sequencer. Consequently, over 99% target regions (3,178 SNPs) were amplified and 2,655 SNPs were available after filtering steps. Strong correlation was observed in the mean depth of coverage of each SNP between duplicate runs (r = 0.993). Genetic analysis using these genotype data successfully detected the known population structure and the sex determining locus of fugu. These results show the method is superior in repeatability and flexibility, and suits genetic studies including molecular breeding, such as marker assisted and genomic selection.