Chromosomal DNA sequences of the Pacific saury genome: versatile resources for fishery science and comparative biology.

Pacific saury (Cololabis saira) is a commercially important small pelagic fish species in Asia. In this study, we conducted the first-ever whole genome sequencing of this species, with single molecule, real-time (SMRT) sequencing technology. The obtained high-fidelity (HiFi) long-read sequence data, which amount to ~30-folds of its haploid genome size that was measured with quantitative PCR (1.17 Gb), were assembled into contigs. Scaffolding with Hi-C reads yielded a whole genome assembly containing 24 chromosome-scale sequences, with a scaffold N50 length of 47.7 Mb. Screening of repetitive elements including telomeric repeats was performed to characterize possible factors that need to be resolved towards 'telomere-to-telomere' sequencing. The larger genome size than in medaka, a close relative in Beloniformes, is at least partly explained by larger repetitive element quantity, which is reflected in more abundant tRNAs, in the Pacific saury genome. Protein-coding regions were predicted using transcriptome data, which resulted in 22,274 components. Retrieval of Pacific saury homologs of aquaporin (AQP) genes known from other teleost fishes validated high completeness and continuity of the genome assembly. These resources are available at https://treethinkers.nig.ac.jp/saira/ and will assist various molecular-level studies in fishery science and comparative biology.

A Cryptic Sex-Linked Locus Revealed by the Elimination of a Master Sex-Determining Locus in Medaka Fish.

AbstractSex chromosomes rapidly turn over in several taxonomic groups. Sex chromosome turnover is generally thought to start with the appearance of a new sex-determining gene on an autosome while an old sex-determining gene still exists, followed by the fixation of the new one. However, we do not know how prevalent the transient state is, where multiple sex-determining loci coexist within natural populations. Here, we removed a Y chromosome with a master male-determining gene DMY from medaka fish using high temperature-induced sex-reversed males. After four generations, the genomic characteristics of a sex chromosome were found on one chromosome, which was an autosome in the original population. Thus, the elimination of a master sex-determining locus can reveal a cryptic locus with a possible sex-determining effect, which can be the seed for sex chromosome turnover. Our results suggest that populations that seem to have a single-locus XY system may have other chromosomal regions with sex-determining effects. In conclusion, the coexistence of multiple sex-determining genes in a natural population may be more prevalent than previously thought. Experimental elimination of a master sex-determining locus may serve as a promising method for finding a locus that can be a protosex chromosome.

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.

Integrative genomic phylogeography reveals signs of mitonuclear incompatibility in a natural hybrid goby population.

Hybridization between divergent lineages generates new allelic combinations. One mechanism that can hinder the formation of hybrid populations is mitonuclear incompatibility, that is, dysfunctional interactions between proteins encoded in the nuclear and mitochondrial genomes (mitogenomes) of diverged lineages. Theoretically, selective pressure due to mitonuclear incompatibility can affect genotypes in a hybrid population in which nuclear genomes and mitogenomes from divergent lineages admix. To directly and thoroughly observe this key process, we de novo sequenced the 747-Mb genome of the coastal goby, Chaenogobius annularis, and investigated its integrative genomic phylogeographics using RNA-sequencing, RAD-sequencing, genome resequencing, whole mitogenome sequencing, amplicon sequencing, and small RNA-sequencing. Chaenogobius annularis populations have been geographically separated into Pacific Ocean (PO) and Sea of Japan (SJ) lineages by past isolation events around the Japanese archipelago. Despite the divergence history and potential mitonuclear incompatibility between these lineages, the mitogenomes of the PO and SJ lineages have coexisted for generations in a hybrid population on the Sanriku Coast. Our analyses revealed accumulation of nonsynonymous substitutions in the PO-lineage mitogenomes, including two convergent substitutions, as well as signals of mitochondrial lineage-specific selection on mitochondria-related nuclear genes. Finally, our data implied that a microRNA gene was involved in resolving mitonuclear incompatibility. Our integrative genomic phylogeographic approach revealed that mitonuclear incompatibility can affect genome evolution in a natural hybrid population.

Genomic selection for heterobothriosis resistance concurrent with body size in the tiger pufferfish, Takifugu rubripes.

Parasite resistance traits in aquaculture species often have moderate heritability, indicating the potential for genetic improvements by selective breeding. However, parasite resistance is often synonymous with an undesirable negative correlation with body size. In this study, we first tested the feasibility of genomic selection (GS) on resistance to heterobothriosis, caused by the monogenean parasite Heterobothrium okamotoi, which leads to huge economic losses in aquaculture of the tiger pufferfish Takifugu rubripes. Then, using a simulation study, we tested the possibility of simultaneous improvement of parasite resistance, assessed by parasite counts on host fish (HC), and standard length (SL). Each trait showed moderate heritability (square-root transformed HC: h2 = 0.308 ± 0.123, S.E.; SL: h2 = 0.405 ± 0.131). The predictive abilities of genomic prediction among 12 models, including genomic Best Linear Unbiased Predictor (GBLUP), Bayesian regressions, and machine learning procedures, were also moderate for both transformed HC (0.248‒0.344) and SL (0.340‒0.481). These results confirmed the feasibility of GS for this trait. Although an undesirable genetic correlation was suggested between transformed HC and SL (rg = 0.228), the simulation study suggested the desired gains index can help achieve simultaneous genetic improvements in both traits.

The Genetic Basis of Scale-Loss Phenotype in the Rapid Radiation of Takifugu Fishes.

Rapid radiation associated with phenotypic divergence and convergence provides an opportunity to study the genetic mechanisms of evolution. Here we investigate the genus Takifugu that has undergone explosive radiation relatively recently and contains a subset of closely-related species with a scale-loss phenotype. By using observations during development and genetic mapping approaches, we show that the scale-loss phenotype of two Takifugu species, T. pardalis Temminck & Schlegel and T. snyderi Abe, is largely controlled by an overlapping genomic segment (QTL). A search for candidate genes underlying the scale-loss phenotype revealed that the QTL region contains no known genes responsible for the evolution of scale-loss phenotype in other fishes. These results suggest that the genes used for the scale-loss phenotypes in the two Takifugu are likely the same, but the genes used for the similar phenotype in Takifugu and distantly related fishes are not the same. Meanwhile, Fgfrl1, a gene predicted to function in a pathway known to regulate bone/scale development was identified in the QTL region. Since Fgfr1a1, another memebr of the Fgf signaling pathway, has been implicated in scale loss/scale shape in fish distantly related to Takifugu, our results suggest that the convergence of the scale-loss phenotype may be constrained by signaling modules with conserved roles in scale development.

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.

Stem cell activity of type A spermatogonia is seasonally regulated in rainbow trout.

Spermatogonial stem cells (SSCs) support continuous production of sperm throughout the male's life. However, the biological characteristics of SSCs are poorly understood in animals exhibiting seasonal reproduction, even though most wild animals are seasonal breeders. During the spermiation season in rainbow trout, the lumen of the testes contains only spermatozoa and scattered type A spermatogonia (ASG) along the walls of the testicular lobules. These few remaining ASG, designated "residual ASG," are the only germ cells capable of supporting the next spermatogenesis, suggesting that the residual ASG are true SSCs. However, whether residual ASG can behave as SSCs in any teleost species is unknown. In this study, we attempted to clarify the biological characteristics of SSCs associated with seasonal reproduction in rainbow trout using spermatogonial transplantation. We found that the stem cell activity was clearly regulated seasonally during the annual reproductive cycle. Although the residual ASG exhibited moderate transplantability and colony-forming ability at the beginning of the spermiation season, these parameters decreased dramatically later and remained low until the next spermatogenesis was initiated. Furthermore, no clear correlations were observed between these qualitative changes and previously described morphologic characteristics of ASG or plasma sex steroid levels. Our results suggest that the biological properties of SSC populations in rainbow trout are seasonally regulated by a novel mechanism.

A 45,X/46,XY DSD (Disorder of Sexual Development) case with an extremely uneven distribution of 46,XY cells between lymphocytes and gonads.

In 45,X/46,XY DSDs, the proportion of the two cell lineages is uneven in different organs and tissues, and 45,X and 46,XY cells can be found throughout the body. The gonadal development of 45,X/46,XY patients depends on the population of 46,XY cells in the gonads and the clinical features are variable. We had a 45,X/46,XY DSD patient whose 46,XY population in peripheral blood was extremely low, less than 0.2%, and was not detected by FISH analysis. However, the patient showed bilateral testicular development and more than 50% of the cells in the gonads had the 46,XY karyotype. This case suggests that a drastically imbalanced distribution could occur in 45,X/46,XY DSD cases.

Combining next-generation sequencing with microarray for transcriptome analysis in rainbow trout gonads.

Microarray technology is a powerful tool for studying genome-wide gene expression. As the genome of many fish has not yet been determined, however, cDNA microarrays can only be designed from limited expressed sequence tag data. In this study, we designed a microarray based on the sequencing data (337,466 reads) obtained by next-generation sequencing of RNA extracted from rainbow trout (Oncorhynchus mykiss) embryonic genital ridge, testis, and ovary. These data (307,264 reads) were assembled into 28,668 contigs; 3,298 reads could not be assembled and 26,904 reads were unique sequences that did not cluster with other reads. Based on this information, 55,928 microarray probes were designed for a microarray, which was validated by hybridization experiments with RNA extracted from type A spermatogonia (A-SG) and testicular somatic cells. Expression of known spermatogonial markers was confirmed to be higher in A-SG than in testicular somatic cells whereas supporting-cell markers were expressed at higher levels in testicular somatic cells. This microarray analysis revealed that 8,068 transcripts showed at least fourfold higher signal in A-SG than testicular somatic cells. Fourteen of 17 randomly selected transcripts were expressed at significantly higher-levels in A-SG than somatic cells, by quantitative RT-PCR. In addition, three transcripts analyzed with in situ hybridization showed A-SG-specific signals in immature trout testis, with one of them exhibiting a heterogeneous expression pattern in A-SG. The rainbow trout gonad microarray developed in this study therefore appears to be a useful tool to understand gametogenesis in rainbow trout.

Flow-cytometric isolation and enrichment of teleost type A spermatogonia based on light-scattering properties.

The transplantation of germ cells is a powerful tool both for studying their development and for reproductive biotechnology. An intraperitoneal germ cell transplantation system was recently developed for use in several teleost species. Donor germ cells transplanted into the peritoneal cavity of hatchlings migrated toward and were incorporated into the recipient's genital ridges, where they underwent gametogenesis. Among male germ cells, only type A spermatogonia were capable of colonizing the recipient gonads, unlike those at more advanced stages. The enrichment of type A spermatogonia is therefore important to achieve efficient donor-cell incorporation and subsequent donor-derived gametogenesis. Here we established a simple and rapid system of isolation and enrichment for fish type A spermatogonia, using flow cytometry. Type A spermatogonia were found to have distinctive forward and side light scatter properties compared to that with other types of testicular cell. Based on these characteristics, we were able to isolate and enrich type A spermatogonia by using flow cytometry. After intraperitoneal transplantation, the enriched type A spermatogonia could be successfully incorporated into the recipient genital ridges. This flow cytometry approach using forward and side light scatter was also found to be applicable to other salmonid and sciaenid species, suggesting that it could be a powerful tool for isolating and enriching transplantable type A spermatogonia in a wide range of teleosts. We expect this method to contribute significantly to germ cell biology and biotechnology.