Draft genome sequencing and assembly of Risso's dolphin, Grampus griseus.

The Risso's dolphin (Grampus griseus) is one of the migratory marine mammals and they have commonly dispersed in tropical and temperate seas. It is a least concerned species in the IUCN red list of threatened species. However, their population size and factors affecting their population structure are unknown. Due to the wide distribution of this species, their populations might be genetically stable and less structured. To support genetic studies on dolphins and other marine mammals, we assembled the draft genome of Risso's dolphin that was found in Japan. The tissue samples were used to extract high molecular DNA and subjected to sequencing by Illumina HiSeq X, Oxford Nanopore MinION, and Bionano Saphyr. The assembled hybrid genome was 75.9% of complete eukaryotic BUSCOs and the genome size was 2.256 Gb with 2.042 Mb of scaffold N50. De novo assembly of this genome by Bionano Saphyr recovered 2.036 Gb total genome map length and structural variations. The gene structures of this draft genome were identified by BRAKER2, and 9947 genes were recovered. The data will be useful for future studies of cetaceans.

Age-Associated Different Transcriptome Profiling in Zebrafish and Rats: an Insight into the Diversity of Vertebrate Aging.

Most mammals, including humans, show obvious aging phenotypes, for example, loss of tissue plasticity and sarcopenia. In this regard, fish can be attractive models to study senescence because of their unique aging characteristics. The lifespan of fish varies widely, and several species can live for over 200 years. Moreover, some fish show anti-aging features and indeterminate growth throughout their life. Therefore, exploring the aging mechanism in fish could provide new insights into vertebrate aging. To this end, we conducted RNA sequencing (RNA-seq) assays for various organs and growth stages of zebrafish and compared the data with previously published RNA-seq data of rats. Age-associated differentially expressed genes (DEGs) for all zebrafish tissue samples reveal the upregulation of circadian genes and downregulation of hmgb3a. On one hand, a comparative analysis of DEG profiles associated with aging between zebrafish and rats identifies upregulation of circadian genes and downregulation of collagen genes as conserved transcriptome changes. On the other hand, in zebrafish, upregulation of autophagy-related genes in muscles and AP-1 transcription factor genes in various tissues is observed, which may imply fish-specific anti-aging characteristics. Consistent with our knowledge of mammalian aging, DEG profiles related to tissue senescence are observed in rats. We also detect age-associated downregulation of muscle homeostasis and differentiation-related genes in zebrafish gills, indicating a fish-specific senescence phenotype. Our results indicate both common and different aging profiles between fish and mammals, which could be used for future translational research.

Induction of endoplasmic reticulum stress markers in an acromegaly model.

Acromegaly is a growth hormone (GH) excess pathological condition in humans. Acromegaly is associated with somatic disfigurement and a wide range of systemic manifestations such as arthritis, neuropathy, carpal tunnel syndrome, reproductive disorders, metabolic disorders, and gastrointestinal complications. The influence of excess GH on the cellular level could aid in understanding the root causes of acromegaly-related health complications. Previously, we found that GH excess induces DNA damage to somatic cells and reduces the stem cells number and causes premature aging. In this study, an in-depth analysis of the acromegaly RNAseq data revealed the disruption of important biological cellular processes. Gene set enrichment analysis, heatmap, and enrichment analysis of acromegaly RNAseq data revealed induction of endoplasmic reticulum (ER) stress markers in various organs. Interestingly, the induction of ER stress was even more apparent than in aged zebrafish. Splicing of box-binding protein-1 (XBP1) mRNA is a hallmark of ER stress. Therefore, we quantified spliced XBP1 mRNA in different organs of our acromegaly model. Thus, our study emphasizes the importance of ER stress in GH oversecretion, which is important for understanding the health complications of acromegaly.

Selection of a reference gene for studies on lipid-related aquatic adaptations of toothed whales (Grampus griseus).

Toothed whales are one group of marine mammals that has developed special adaptations, such as echolocation for predation, to successfully live in a dynamic aquatic environment. Their fat metabolism may differ from that of other mammals because toothed whales have acoustic fats. Gene expression in the metabolic pathways of animals can change with respect to their evolution and environment. A real-time quantitative polymerase chain reaction (RT-qPCR) is a reliable technique for studying the relative expressions of genes. However, since the accuracy of RT-qPCR data is totally dependent on the reference gene, the selection of the reference gene is an essential step. In this study, 10 candidate reference genes (ZC3H10, FTL, LGALS1, RPL27, GAPDH, FTH1, DCN, TCTP, NDUS5, and UBIM) were initially tested for amplification efficiency using RT-qPCR. After excluding DCN, the remaining nine genes, which are nearly 100% efficient, were selected for the gene stability analysis. Stable reference genes across eight different fat tissue, liver, and muscle samples from Grampus griseus were identified by four algorithms, which were provided in Genorm, NormFinder, BestKeeper, and Delta CT. Finally, a RefFinder comprehensive ranking was performed based on the stability values, and the nine genes were ranked as follows: LGALS1 > FTL > GAPDH > ZC3H10 > FTH1 > NDUS5 > TCTP > RPL27 > UBIM. The LGALS1 and FTL genes were identified as the most stable novel reference genes. The third-ranked gene, GAPDH, is a well-known housekeeping gene for mammals. Ultimately, we suggest the use of LGALS1 as a reliable novel reference gene for genomics studies on the lipid-related aquatic adaptations of toothed whales.

Metagenomic analysis provides functional insights into seasonal change of a non-cyanobacterial prokaryotic community in temperate coastal waters.

The taxonomic compositions of marine prokaryotic communities are known to follow seasonal cycles, but functional metagenomic insights into this seasonality is still limited. We analyzed a total of 22 metagenomes collected at 11 time points over a 14-month period from two sites in Sendai Bay, Japan to obtain seasonal snapshots of predicted functional profiles of the non-cyanobacterial prokaryotic community. Along with taxonomic composition, functional gene composition varied seasonally and was related to chlorophyll a concentration, water temperature, and salinity. Spring phytoplankton bloom stimulated increased abundances of putative genes that encode enzymes in amino acid metabolism pathways. Several groups of functional genes, including those related to signal transduction and cellular communication, increased in abundance during the mid- to post-bloom period, which seemed to be associated with a particle-attached lifestyle. Alternatively, genes in carbon metabolism pathways were generally more abundant in the low chlorophyll a period than the bloom period. These results indicate that changes in trophic condition associated with seasonal phytoplankton succession altered the community function of prokaryotes. Our findings on seasonal changes of predicted function provide fundamental information for future research on the mechanisms that shape marine microbial communities.

Another polymorphic mitochondrial genome of Grampus griseus and phylogeny of family Delphinidae.

Risso's dolphin (Grampus griseus Cuvier, 1812) is the only species of genus Grampus and a cosmopolitan marine inhabitant. Here, we report a polymorphic complete mitochondrial genome of G. griseus. The size of the total mitochondrial genome was 16,386 bp in length and contains 13 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA genes, and a control region. 37 single nucleotide polymorphic sites (SNPs) were identified compared to the references. Based on the available total mitochondrial dolphin genomes' phylogenetics, G. griseus has formed a clade with 0.1415 distance, sister to the following species of the subfamily Globicephalinae and the taxonomy of Orcinus orca still needs further investigations.

Development of a time-series shotgun metagenomics database for monitoring microbial communities at the Pacific coast of Japan.

Although numerous metagenome, amplicon sequencing-based studies have been conducted to date to characterize marine microbial communities, relatively few have employed full metagenome shotgun sequencing to obtain a broader picture of the functional features of these marine microbial communities. Moreover, most of these studies only performed sporadic sampling, which is insufficient to understand an ecosystem comprehensively. In this study, we regularly conducted seawater sampling along the northeastern Pacific coast of Japan between March 2012 and May 2016. We collected 213 seawater samples and prepared size-based fractions to generate 454 subsets of samples for shotgun metagenome sequencing and analysis. We also determined the sequences of 16S rRNA (n = 111) and 18S rRNA (n = 47) gene amplicons from smaller sample subsets. We thereafter developed the Ocean Monitoring Database for time-series metagenomic data ( http://marine-meta.healthscience.sci.waseda.ac.jp/omd/ ), which provides a three-dimensional bird's-eye view of the data. This database includes results of digital DNA chip analysis, a novel method for estimating ocean characteristics such as water temperature from metagenomic data. Furthermore, we developed a novel classification method that includes more information about viruses than that acquired using BLAST. We further report the discovery of a large number of previously overlooked (TAG)n repeat sequences in the genomes of marine microbes. We predict that the availability of this time-series database will lead to major discoveries in marine microbiome research.

Skim-Sequencing Based Genotyping Reveals Genetic Divergence of the Wild and Domesticated Population of Black Tiger Shrimp (Penaeus monodon) in the Indo-Pacific Region.

The domestication of a wild-caught aquatic animal is an evolutionary process, which results in genetic discrimination at the genomic level in response to strong artificial selection. Although black tiger shrimp (Penaeus monodon) is one of the most commercially important aquaculture species, a systematic assessment of genetic divergence and structure of wild-caught and domesticated broodstock populations of the species is yet to be documented. Therefore, we used skim sequencing (SkimSeq) based genotyping approach to investigate the genetic structure of 50 broodstock individuals of P. monodon species, collected from five sampling sites (n = 10 in each site) across their distribution in Indo-Pacific regions. The wild-caught P. monodon broodstock population were collected from Malaysia (MS) and Japan (MJ), while domesticated broodstock populations were collected from Madagascar (MMD), Hawaii, HI, USA (MMO), and Thailand (MT). After various filtering process, a total of 194,259 single nucleotide polymorphism (SNP) loci were identified, in which 4983 SNP loci were identified as putatively adaptive by the pcadapt approach. In both datasets, pairwise FST estimates high genetic divergence between wild and domesticated broodstock populations. Consistently, different spatial clustering analyses in both datasets categorized divergent genetic structure into two clusters: (1) wild-caught populations (MS and MJ), and (2) domesticated populations (MMD, MMO and MT). Among 4983 putatively adaptive SNP loci, only 50 loci were observed to be in the coding region. The gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses suggested that non-synonymous mutated genes might be associated with the energy production, metabolic functions, respiration regulation and developmental rates, which likely act to promote adaptation to the strong artificial selection during the domestication process. This study has demonstrated the applicability of SkimSeq in a highly duplicated genome of P. monodon specifically, across a range of genetic backgrounds and geographical distributions, and would be useful for future genetic improvement program of this species in aquaculture.

An Acromegaly Disease Zebrafish Model Reveals Decline in Body Stem Cell Number along with Signs of Premature Aging.

In our previous publication, it was shown that growth hormone (GH) excess in acromegaly affects the cell integrity of somatic cells through increased DNA damage throughout the body and impaired DNA repair pathways. Acromegaly is a hormone disorder pathological condition that develops as a result of growth hormone over-secretion from the pituitary gland. We produced a zebrafish acromegaly model to gain a better understanding of the excess GH effects at the cellular level. Here we show that the acromegaly zebrafish model progressively reduced the number of stem cells in different organs and increased oxidative stress in stem cells. Importantly, the decline in the stem cells was even more apparent than in aged fish. The controversy and debate over the use of GH as an anti-aging therapy have been going on for several years. In this study, excess GH induced aging signs such as increased senescence-associated (SA)-ß-galactosidase staining of abdominal skin and similarity of the pattern of gene expression between aged and acromegaly zebrafish. Thus, this study highlights the role of excess GH in acromegaly stem cells.

Fine-scale population structure and ecotypes of anadromous Hilsa shad (Tenualosa ilisha) across complex aquatic ecosystems revealed by NextRAD genotyping.

The anadromous Hilsa shad (Tenualosa ilisha) live in the Bay of Bengal and migrate to the estuaries and freshwater rivers for spawning and nursing of the juveniles. This has led to two pertinent questions: (i) do all Hilsa shad that migrate from marine to freshwater rivers come from the same population? and (ii) is there any relationship between adults and juveniles of a particular habitat? To address these questions, NextRAD sequencing was applied to genotype 31,276 single nucleotide polymorphism (SNP) loci for 180 individuals collected from six strategic locations of riverine, estuarine and marine habitats. FST OutFLANK approach identified 14,815 SNP loci as putatively neutral and 79 SNP loci as putatively adaptive. We observed that divergent local adaptations in differing environmental habitats have divided Hilsa shad into three genetically structured ecotypes: turbid freshwater (Western Riverine), clear freshwater (Eastern Riverine) and brackish-saline (Southern Estuarine-Marine). Our results also revealed that genes involved in neuronal activity may have facilitated the juveniles' Hilsa shad in returning to their respective natal rivers for spawning. This study emphasized the application of fundamental population genomics information in strategizing conservation and management of anadromous fish such as Hilsa shad that intersect diverse ecotypes during their life-history stages.

Identification and Characterization of microRNAs and Their Predicted Functions in Biomineralization in the Pearl Oyster (Pinctada fucata).

The biological process of pearl formation is an ongoing research topic, and a number of genes associated with this process have been identified. However, the involvement of microRNAs (miRNAs) in biomineralization in the pearl oyster, Pinctada fucata, is not well understood. In order to investigate the divergence and function of miRNAs in P. fucata, we performed a transcriptome analysis of small RNA libraries prepared from adductor muscle, gill, ovary, and mantle tissues. We identified 186 known and 42 novel miRNAs in these tissues. Clustering analysis showed that the expression patterns of miRNAs were similar among the somatic tissues, but they differed significantly between the somatic and ovary tissues. To validate the existence of the identified miRNAs, nine known and three novel miRNAs were verified by stem-loop qRT-PCR using U6 snRNA as an internal reference. The expression abundance and target prediction between miRNAs and biomineralization-related genes indicated that miR-1990c-3p, miR-876, miR-9a-3p, and novel-3 may be key factors in the regulatory network that act by controlling the formation of matrix proteins or the differentiation of mineralogenic cells during shell formation in mantle tissue. Our findings serve to further clarify the processes underlying biomineralization in P. fucata.

A Preliminary Metagenome Analysis Based on a Combination of Protein Domains.

Metagenomic data have mainly been addressed by showing the composition of organisms based on a small part of a well-examined genomic sequence, such as ribosomal RNA genes and mitochondrial DNAs. On the contrary, whole metagenomic data obtained by the shotgun sequence method have not often been fully analyzed through a homology search because the genomic data in databases for living organisms on earth are insufficient. In order to complement the results obtained through homology-search-based methods with shotgun metagenomes data, we focused on the composition of protein domains deduced from the sequences of genomes and metagenomes, and we utilized them in characterizing genomes and metagenomes, respectively. First, we compared the relationships based on similarities in the protein domain composition with the relationships based on sequence similarities. We searched for protein domains of 325 bacterial species produced using the Pfam database. Next, the correlation coefficients of protein domain compositions between every pair of bacteria were examined. Every pairwise genetic distance was also calculated from 16S rRNA or DNA gyrase subunit B. We compared the results of these methods and found a moderate correlation between them. Essentially, the same results were obtained when we used partial random 100 bp DNA sequences of the bacterial genomes, which simulated raw sequence data obtained from short-read next-generation sequences. Then, we applied the method for analyzing the actual environmental data obtained by shotgun sequencing. We found that the transition of the microbial phase occurred because the seasonal change in water temperature was shown by the method. These results showed the usability of the method in characterizing metagenomic data based on protein domain compositions.

Transcriptome Analysis of Yamame (Oncorhynchus masou) in Normal Conditions after Heat Stress.

Understanding the mechanism of high-temperature tolerance in cold-freshwater fish is crucial for predicting how certain species will cope with global warming. In this study, we investigated temperature tolerance in masu salmon (Oncorhynchus masou, known in Japan as 'yamame'), an important aquaculture species. By selective breeding, we developed a group of yamame (F2) with high-temperature tolerance. This group was subjected to a high-temperature tolerance test and divided into two groups: High-temperature tolerant (HT) and non-high-temperature tolerant (NT). RNA was extracted from the gill and adipose fin tissues of each group, and the mRNA expression profiles were analyzed using RNA sequencing. A total of 2893 differentially expressed genes (DEGs) from the gill and 836 from the adipose fin were identified by comparing the HT and NT groups. Functional analyses were then performed to identify associated gene ontology (GO) terms and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. The HT group showed a high expression of heat shock protein 70 (HSP70) gene and enriched gene expression in the extracellular matrix (ECM), cell junction, and adhesion pathways in gill tissues compared to the NT group. The HT group also exhibited highly expressed genes in glycolysis and showed lower expression of the genes in the p53 signaling pathway in adipose fin tissues. Taken together, the difference of expression of some genes in the normal condition may be responsible for the difference in heat tolerance between the HT and NT yamame in the heat stress condition.

Gene expression profiles at different stages for formation of pearl sac and pearl in the pearl oyster Pinctada fucata.

BACKGROUND: The most critical step in the pearl formation during aquaculture is issued to the proliferation and differentiation of outer epithelial cells of mantle graft into pearl sac. This pearl sac secretes various matrix proteins to produce pearls by a complex physiological process which has not been well-understood yet. Here, we aimed to unravel the genes involved in the development of pearl sac and pearl, and the sequential expression patterns of different shell matrix proteins secreted from the pearl sac during pearl formation by pearl oyster Pinctada fucata using high-throughput transcriptome profiling. RESULTS: Principal component analysis (PCA) showed clearly different gene expression profiles between earlier (before 1 week) and later stages (1 week to 3 months) of grafting. Immune-related genes were highly expressed between 0 h - 24 h (donor dependent) and 48 h - 1 w (host dependent), and in the course of wound healing process pearl sac was developed by two weeks of graft transplantation. Moreover, for the first time, we identified some stem cell marker genes including ABCG2, SOX2, MEF2A, HES1, MET, NRP1, ESR1, STAT6, PAX2, FZD1 and PROM1 that were expressed differentially during the formation of pearl sac. The expression profiling of 192 biomineralization-related genes demonstrated that most of the shell matrix proteins (SMPs) involved in prismatic layer formation were first up-regulated and then gradually down-regulated indicating their involvement in the development of pearl sac and the onset of pearl mineralization. Most of the nacreous layer forming SMPs were up-regulated at 2 weeks after the maturation of pearl sac. Nacrein, MSI7 and shematrin involved in both layer formation were highly expressed during 0 h - 24 h, down-regulated up to 1 week and then up-regulated again after accomplishment of pearl sac formation. CONCLUSIONS: Using an RNA-seq approach we unraveled the expression pattern of the key genes involved in the development of pearl sac and pearl as a result of host immune response after grafting. These findings provide valuable information in understanding the molecular mechanism of pearl formation and immune response in P. fucata.

Piwi-interacting RNA (piRNA) expression patterns in pearl oyster (Pinctada fucata) somatic tissues.

Piwi-interacting RNAs (piRNAs) belong to a recently discovered class of small non-coding RNAs whose best-understood function is repressing transposable element activity. Most piRNA studies have been conducted on model organisms and little is known about piRNA expression and function in mollusks. We performed high-throughput sequencing of small RNAs extracted from the mantle, adductor muscle, gill, and ovary tissues of the pearl oyster, Pinctada fucata. RNA species with sequences of approximately 30 nt were widely expressed in all tissues. Uridine at the 5' terminal and protection from ß-elimination at the 3' terminal suggested that these were putative piRNAs. A total of 18.0 million putative piRNAs were assigned to 2.8 million unique piRNAs, and 35,848 piRNA clusters were identified. Mapping to the reference genome showed that 25% of the unique piRNAs mapped to multiple tandem loci on the scaffold. Expression patterns of the piRNA clusters were similar within the somatic tissues, but differed significantly between the somatic and gonadal tissues. These findings suggest that in pearl oysters piRNAs have important and novel functions beyond those in the germ line.

Population Genomics of an Anadromous Hilsa Shad Tenualosa ilisha Species across Its Diverse Migratory Habitats: Discrimination by Fine-Scale Local Adaptation.

The migration of anadromous fish in heterogenic environments unceasingly imposes a selective pressure that results in genetic variation for local adaptation. However, discrimination of anadromous fish populations by fine-scale local adaptation is challenging because of their high rate of gene flow, highly connected divergent population, and large population size. Recent advances in next-generation sequencing (NGS) have expanded the prospects of defining the weakly structured population of anadromous fish. Therefore, we used NGS-based restriction site-associated DNA (NextRAD) techniques on 300 individuals of an anadromous Hilsa shad (Tenualosa ilisha) species, collected from nine strategic habitats, across their diverse migratory habitats, which include sea, estuary, and different freshwater rivers. The NextRAD technique successfully identified 15,453 single nucleotide polymorphism (SNP) loci. Outlier tests using the FST OutFLANK and pcadapt approaches identified 74 and 449 SNPs (49 SNPs being common), respectively, as putative adaptive loci under a divergent selection process. Our results, based on the different cluster analyses of these putatively adaptive loci, suggested that local adaptation has divided the Hilsa shad population into two genetically structured clusters, in which marine and estuarine collection sites were dominated by individuals of one genetic cluster and different riverine collection sites were dominated by individuals of another genetic cluster. The phylogenetic analysis revealed that all the riverine populations of Hilsa shad were further subdivided into the north-western riverine (turbid freshwater) and the north-eastern riverine (clear freshwater) ecotypes. Among all of the putatively adaptive loci, only 36 loci were observed to be in the coding region, and the encoded genes might be associated with important biological functions related to the local adaptation of Hilsa shad. In summary, our study provides both neutral and adaptive contexts for the observed genetic divergence of Hilsa shad and, consequently, resolves the previous inconclusive findings on their population genetic structure across their diverse migratory habitats. Moreover, the study has clearly demonstrated that NextRAD sequencing is an innovative approach to explore how dispersal and local adaptation can shape genetic divergence of non-model anadromous fish that intersect diverse migratory habitats during their life-history stages.

Artificially designed hybrids facilitate efficient generation of high-resolution linkage maps.

When sequencing eukaryotic genomes, linkage maps are indispensable for building scaffolds to assemble and/or to validate chromosomes. However, current approaches to constructing linkage maps are limited by marker density and cost-effectiveness, especially for wild organisms. We have now devised a new strategy based on artificially generated hybrid organisms to acquire ultrahigh-density genomic markers at reduced cost and build highly accurate linkage maps. We have also developed the novel analysis pipeline Scaffold Extender with Low Depth Linkage Analysis (SELDLA) for data processing to generate linkage maps and draft genomes. Using SELDLA, linkage maps and improved genomes for two species of pufferfish, Takifugu rubripes and Takifugu stictonotus, were obtained simultaneously. The strategy is applicable to a wide range of sexually reproducing organisms, and could, therefore, accelerate the whole genome analysis of various organisms including fish, mollusks, amphibians, insects, plants, and even mammals.

Whole-Genome Sequencing of 84 Japanese Eels Reveals Evidence against Panmixia and Support for Sympatric Speciation.

The Japanese eel (Anguilla japonica), European eel (Anguilla anguilla), and American eel (Anguilla rostrata) are migratory, catadromous, temperate zone fish sharing several common life cycle features. The population genetics of panmixia in these eel species has already been investigated. Our extensive population genetics analysis was based on 1400 Gb of whole-genome sequence (WGS) data from 84 eels. It demonstrated that a Japanese eel group from the Kuma River differed from other populations of the same species. Even after removing the potential adapted/selected single nucleotide polymorphism (SNP) data, and with very small differences (fixation index [Fst] = 0.01), we obtained results consistently indicating that panmixia does not occur in Japanese eels. The life cycle of the Japanese eel is well-established and the Kuma River is in the center of its habitat. Nevertheless, simple reproductive isolation is not the probable cause of non-panmixia in this species. We propose that the combination of spawning area subdivision, philopatry, and habitat preference/avoidance accounts for the non-panmixia in the Japanese eel population. We named this hypothesis the "reproductive isolation like subset mapping" (RISM) model. This finding may be indicative of the initial stages of sympatric speciation in these eels.

Ultrahigh-Density Linkage Map Construction Using Low-Coverage Whole-Genome Sequencing of a Doubled Haploid Population: Case Study of Torafugu (Takifugu rubripes).

Next-generation sequencing enables genome-wide genotyping of a large population and further facilitates the construction of a genetic linkage map. Low-coverage whole-genome sequencing has been employed for genetic linkage map construction in several species. However, this strategy generally requires available high-quality reference genomes and/or designed inbred pedigree lines, which restrict the scope of application for non-model and unsequenced species. Here, using torafugu (Takifugu rubripes) as a test model, we propose a new strategy for ultrahigh-density genetic linkage map construction using low-coverage whole-genome sequencing of a haploid/doubled haploid (H/DH) population without above requirements. Low-coverage (≈1×) whole-genome sequencing data of 165 DH individuals were used for de novo assembly and further performed single nucleotide polymorphisms (SNPs) calling, resulting in the identification of 1,070,601 SNPs. Based on SNP genotypes and de novo assembly, genotypes were associated with short DNA segments and an ultrahigh-density linkage map was constructed containing information of 802,277 SNPs in 3090 unique positions. Comparative analyses showed near-perfect concordance between the present linkage map and the latest published torafugu genome (FUGU5). This strategy would facilitate ultrahigh-density linkage map construction in various sexually reproducing organisms for which H/DH populations can be generated.