Takashi Sakamoto

A breeding program of Asian seabass (Lates calcarifer, also called barramundi) was established for sustainable aquaculture in Thailand. Estimated breeding values (EBVs) for growth of the base population (G0, 51 families, N = 1655) were evaluated. Fish exhibited either high (HEBV, averaged body weight = 1036.80 ± 250.80 g, N = 133) or low (LEBV, averaged body weight = 294.50 ± 167.20 g, N = 147) growth EBVs, and their parental fish (N = 26) were analyzed by Specific Locus Amplified Fragment Sequencing (SLAF-Seq). An average of 159,769 SLAF tags/sample was generated, covering 13.79-fold of the genome size, and 225,498 SNPs were applied for population genomics. Observed (H_o) and expected (H_e) heterozygosity values were 0.224 and 0.308, 0.178 and 0.246, and 0.184 and 0.305, respectively. Polymorphic information content (PIC) ranged from 0.205–0.251. A selective sweep was performed based on Fst, and nucleotide polymorphism (π) revealed significant differences between allelic contents of growth- and immune-related genes in HEBV and LEBV populations. Kinship analysis revealed that 84.38% of examined fish showed r values < 0.2, and population admixture analysis revealed three subpopulations in HEBV and four subpopulations in LEBV groups. Fish that possessed a single cluster were found in each subgroup of both populations, along with those exhibiting mixed ancestral clusters. This information is critically important for further applications in our ongoing seabass improvement breeding program.

Objective: The ayu or sweetfish Plecoglossus altivelis is ray-finned fish that is widely distributed in East Asia. The genome size of ayu was estimated at approximately 420 Mb. Previously, we reported on ayu draft genome assembly by whole-genome shotgun using Illumina short reads and PacBio long reads; however, the assembly was not to chromosome level. Therefore, to improve the draft genome sequence of ayu to chromosome level, we performed in situ Hi-C sequencing as a source of linkage information. Results: The ayu genome assembly yielded 28 large scaffolds that corresponded to the karyotype of ayu (n = 28). The resulting ayu genome assembly has a N50 scaffold length of 17.0 Mb, improved from 4.3 Mb. The high-quality reference genome will be helpful for phylogenetic research on bony fishes and for breeding programs in ayu aquaculture.

Japanese flounder (Paralichthys olivaceus) is an important marine fish species of both fisheries and aquaculture in Northeast Asia. The commercial interest for all-female progenies due to several sex-related traits has prompted basic research on the mechanisms of sex determination in this species. By conducting a linkage analysis of the sex-determining locus, we initially identified 12 microsatellite markers linked to sex in 11 scaffolds, whose localization was restricted to a specific region of linkage group 9. Sequence analysis of this region identified 181 genes based on the UniProt database annotations. Among them, the amh gene was considered a potential candidate for sex determination because this gene is known to have taken over the role of sex determination in many teleosts. An in-depth sequence analysis of both the coding and non-coding regions of amh in XX and XY individuals detected nine SNPs linked with maleness. However, because these substitutions were synonymous, the upstream and downstream regions of amh were also investigated and a male-specific variant with deletions in the promoter region was detected. This truncated Y-specific amh variant was named amhy, and the amh shared by both sexes was named amhx. The association analysis using both females and males of the genotypic sex inferred by the presence/absence of amhy found complete association with phenotypic sex and genotype. Gene expression analysis in larvae derived from a single-pair progeny by quantitative real-time PCR detected amhy transcripts in the larval trunks between 20 and 100 days after hatching only in XY larvae. Localization of amhy by in situ hybridization was detected in presumptive Sertoli cells of XY gonads. Expression of amhx was almost undetectable in both XX and XY genotypes. Loss of Amh function by CRISPR-Cas9 induced male-to-female sex reversal, indicating that this gene was necessary for the masculinization of XY individuals. In conclusion, the complete linkage of amhy with males, its early expression in XY gonads before testicular differentiation, and the induction of sex reversal by loss-of-function mutation support the view that amhy is the sex-determining gene in this species.

Whole-genome duplication and genome compaction are thought to have played important roles in teleost fish evolution. Ayu (or sweetfish), Plecoglossus altivelis, belongs to the superorder Stomiati, order Osmeriformes. Stomiati is phylogenetically classified as sister taxa of Neoteleostei. Thus, ayu holds an important position in the fish tree of life. Although ayu is economically important for the food industry and recreational fishing in Japan, few genomic resources are available for this species. To address this problem, we produced a draft genome sequence of ayu by whole-genome shotgun sequencing and constructed linkage maps using a genotyping-by-sequencing approach. Syntenic analyses of ayu and other teleost fish provided information about chromosomal rearrangements during the divergence of Stomiati, Protacanthopterygii and Neoteleostei. The size of the ayu genome indicates that genome compaction occurred after the divergence of the family Osmeridae. Ayu has an XX/XY sex-determination system for which we identified sex-associated loci by a genome-wide association study by genotyping-by-sequencing and whole-genome resequencing using wild populations. Genome-wide association mapping using wild ayu populations revealed three sex-linked scaffolds (total, 2.03 Mb). Comparison of whole-genome resequencing mapping coverage between males and females identified male-specific regions in sex-linked scaffolds. A duplicate copy of the anti-Müllerian hormone type-II receptor gene (amhr2bY) was found within these male-specific regions, distinct from the autosomal copy of amhr2. Expression of the Y-linked amhr2 gene was male-specific in sox9b-positive somatic cells surrounding germ cells in undifferentiated gonads, whereas autosomal amhr2 transcripts were detected in somatic cells in sexually undifferentiated gonads of both genetic males and females. Loss-of-function mutation for amhr2bY induced male to female sex reversal. Taken together with the known role of Amh and Amhr2 in sex differentiation, these results indicate that the paralog of amhr2 on the ayu Y chromosome determines genetic sex, and the male-specific amh-amhr2 pathway is critical for testicular differentiation in ayu.

Greater amberjack (Seriola dumerili) is an important fishery resource with a circumglobal distribution from tropical to temperate waters. Here, we investigated the spawning migration and habitat utilization of S. dumerili in the East China Sea (ECS). Archival tags were attached to 22 adult fish to examine their horizontal and vertical movements and estimate the spawning ground. S. dumerili were captured and released in the coastal waters of eastern Taiwan on November of 2016 and 2017. Information from seven pop‐up satellite archival tags and seven depth–temperature recorders was recovered. Almost all of the fish stayed in the Taiwanese exclusive economic zone. Most individuals moved from released site to the southern edge of the ECS and showed behavior associated with the topographic features in winter (November to December). These phenomena may be related to foraging and be driven by oceanographic features such as the seasonal monsoon and the Kuroshio. The fish then migrated to the south offshore area of Taiwan in January and February. During their southward migration, the fish experienced a slowly elevated water temperature regime (SETR), which is one of the environmental factors that induce final oocyte maturation. In the spawning season (February to April), tagged females exhibited continuous diel vertical movements (DVMs) after experiencing the SETR. These continuous DVMs were observed over a wide geographic range from north to south in the Kuroshio off eastern Taiwan. Our study demonstrated that the putative spawning ground of S. dumerili must extend further in a north–south direction than predicted in a previous study.

The study of sex determination and sex chromosome organisation in non-model species has long been technically challenging, but new sequencing methodologies are now enabling precise and high-throughput identification of sex-specific genomic sequences. In particular, Restriction Site-Associated DNA Sequencing (RAD-Seq) is being extensively applied to explore sex determination systems in many plant and animal species. However, software designed to specifically search for sex-biased markers using RAD-Seq data is lacking. Here, we present RADSex, a computational analysis workflow designed to study the genetic basis of sex determination using RAD-Seq data. RADSex is simple to use, requires few computational resources, makes no prior assumptions about type of sex-determination system or structure of the sex locus, and offers convenient visualization through a dedicated R package. To demonstrate the functionality of RADSex, we re-analyzed a published dataset of Japanese medaka, Oryzias latipes , where we uncovered a previously unknown Y chromosome polymorphism. We then used RADSex to analyze new RAD-Seq datasets from 15 fish species spanning multiple systematic orders. We identified the sex determination system and sex-specific markers in six of these species, five of which had no known sex-markers prior to this study. We show that RADSex greatly facilitates the study of sex determination systems in non-model species and outperforms the commonly used RAD-Seq analysis software STACKS. RADSex in speed, resource usage, ease of application, and visualization options. Furthermore, our analysis of new datasets from 15 species provides new insights on sex determination in fish.