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We developed a novel strain of red sea bream Pagrus major that showed resistance to red sea bream iridoviral disease (RSIVD) using marker-assisted selection combined with DNA-based family selection and evaluated survival of its G2 in a farm environment. The G1 population was derived from a single RSIVD-resistant male (G0). The G1 progeny of the resistant male were identified by DNA parentage analysis using microsatellites. Marker-assisted selection using a quantitative trait locus linked to the RSIVD-resistant trait (Pma4_014) was conducted, and individuals inheriting the resistant allele of Pma4_014 (+) were selected. Two G2 populations were produced in 2016 and 2017, and reared for approximately three months, during the summer, at an aquaculture facility. A normal production cohort was also reared at the same farm in 2016 and survival compared. Survival rates of the G2 populations produced in 2016 and 2017 were 78.8% and 83.5%, respectively. The survival rate of the 2016 normal production cohort was 63.8%. The segregation pattern of the resistant allele showed 1 (+/+): 2 (+/−): 1 (−/−) in G2, and genotype frequency of Pma4_014 was calculated using approximately 200 individuals collected from 2016 and 2017 G2 populations before and after the field trial. Numbers of each genotype were estimated based on the number of introduced and surviving fish. Individuals with the resistant allele, (+/+) and (+/−), showed over 80% of the estimated survival rates, but individuals without the resistant allele (−/−) showed lower survival (50%) in both years. This result strongly suggests that the resistant allele dominantly affects RSIVD resistance, and the newly developed G2 population is useful for further propagation of RSIVD-resistant red sea bream fingerlings.

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Additional file of A second generation genetic linkage map of Japanese flounder (Paralichthys olivaceus)

The sex determination systems of fish are highly diverse compared with those of mammals. Thus, performing investigations using nonmodel fish species helps to understand the highly diverse sex determination systems of fish. Because greater amberjack (Seriola dumerili) is one of the most important edible fish globally and knowledge of its sex determination system is economically important in the field of aquaculture, we are interested in the mechanisms of sex determination of Seriola species. In this study, we identified sex-associated SNPs of greater amberjack using SNP information of 10 males and 10 females by an association test. We determined that the sex-associated SNPs were on chromosome 12 and mainly covered with two scaffolds (about 7.1 Mbp). Genotypes of sex-associated SNPs indicated that females are the heterogametic sex (ZZ/ZW). Furthermore, we compared the genomic structure of greater amberjack with those of Japanese amberjack (Seriola quinqueradiata), California yellowtail (Seriola dorsalis), and medaka (Oryzias latipes). Whole-genome alignments and synteny analysis indicated that the sex determination system of greater amberjack is markedly different from that of medaka and implied that the sex determination system is conserved in the Seriola species.

Red sea bream iridoviral disease (RSIVD) is a major viral disease in red sea bream farming in Japan. Previously, we identified one candidate male individual of red sea bream that was significantly associated with convalescent individuals after RSIVD. The purpose of this study is to identify the quantitative trait loci (QTL) linked to the RSIVD-resistant trait for future marker-assisted selection (MAS). Two test families were developed using the candidate male in 2014 (Fam-2014) and 2015 (Fam-2015). These test families were challenged with RSIV, and phenotypes were evaluated. Then, de novo genome sequences of red sea bream were obtained through next-generation sequencing, and microsatellite markers were searched and selected for linkage map construction. One immune-related gene, MHC class IIβ, was also used for linkage map construction. Of the microsatellite markers searched, 148 and 197 were mapped on 23 and 27 linkage groups in the female and male linkage maps, respectively, covering approximately 65% of genomes in both sexes. One QTL linked to an RSIVD-resistant trait was found in linkage group 2 of the candidate male in Fam-2014, and the phenotypic variance of the QTL was 31.1%. The QTL was closely linked to MHC class IIβ. Moreover, the QTL observed in Fam-2014 was also significantly linked to an RSIVD-resistant trait in the candidate male of Fam-2015. Our results suggest that the RSIVD-resistant trait in the candidate male was controlled by one major QTL closely linked to the MHC class IIβ gene and could be useful for MAS of red sea bream.

Benedenia disease is a parasitic disease occurring in cultured marine fish which is caused by the ectoparasite, Benedenia seriolae. This parasitic disease causes serious damage to yellowtail culture due to secondary infections, however prevention methods for the ectoparasite have limited efficacy. Thus, we attempted to initiate a marker-assisted selection (MAS) program to produce a yellowtail strain that has resistance to Benedenia disease. In this study, we conducted a genome scan for quantitative trait loci (QTL) that affect Benedenia disease resistance in five full-sib families using 7910 SNP markers. Sex-specific linkage maps was constructed for the five families and the total number of markers on each map was ranged from 1115 to 1427. We found four significant and five suggestive QTLs affecting Benedenia disease resistance. The phenotypic variation explained (PVE) by each QTL ranged from 8.2% to 29.6%. The QTL with a PVE of 29.6% on LG Squ6 in the family Rr1 was considered to be the major QTL. Novel Benedenia disease resistance QTLs detected in this study enhance our understanding of the genes and genomic regions related to Benedenia disease resistance in yellowtail and can serve as a basis for MAS breeding.

Unlike the conservation of sex-determining (SD) modes seen in most mammals and birds, teleost fishes exhibit a wide variety of SD systems and genes. Hence, the study of SD genes and sex chromosome turnover in fish is one of the most interesting topics in evolutionary biology. To increase resolution of the SD gene evolutionary trajectory in fish, identification of the SD gene in more fish species is necessary. In this study, we focused on the yellowtail, a species widely cultivated in Japan. It is a member of family Carangidae in which no heteromorphic sex chromosome has been observed, and no SD gene has been identified to date. By performing linkage analysis and BAC walking, we identified a genomic region and SNPs with complete linkage to yellowtail sex. Comparative genome analysis revealed the yellowtail SD region ancestral chromosome structure as medaka-fugu. Two inversions occurred in the yellowtail linage after it diverged from the yellowtail-medaka ancestor. An association study using wild yellowtails and the SNPs developed from BAC ends identified two SNPs that can reasonably distinguish the sexes. Therefore, these will be useful genetic markers for yellowtail breeding. Based on a comparative study, it was suggested that a PDZ domain containing the GIPC protein might be involved in yellowtail sex determination. The homomorphic sex chromosomes widely observed in the Carangidae suggest that this family could be a suitable marine fish model to investigate the early stages of sex chromosome evolution, for which our results provide a good starting point.

The sex determination systems of fish are highly diverse compared with those of mammals. Thus, performing investigations using nonmodel fish species helps to understand the highly diverse sex determination systems of fish. Because greater amberjack (Seriola dumerili) is one of the most important edible fish globally and knowledge of its sex determination system is economically important in the field of aquaculture, we are interested in the mechanisms of sex determination of Seriola species. In this study, we identified sex-associated SNPs of greater amberjack using SNP information of 10 males and 10 females by an association test. We determined that the sex-associated SNPs were on chromosome 12 and mainly covered with two scaffolds (about 7.1 Mbp). Genotypes of sex-associated SNPs indicated that females are the heterogametic sex (ZZ/ZW). Furthermore, we compared the genomic structure of greater amberjack with those of Japanese amberjack (Seriola quinqueradiata), California yellowtail (Seriola dorsalis), and medaka (Oryzias latipes). Whole-genome alignments and synteny analysis indicated that the sex determination system of greater amberjack is markedly different from that of medaka and implied that the sex determination system is conserved in the Seriola species.

The Japanese eel (Anguilla japonica) is among the most important aquaculture fish species in Eastern Asia. The present study aimed to identify the genetic parameters underlying body size and the timing at metamorphosis from leptocephali to glass eels in captive-bred Japanese eels, with the intent to foster sustainable development. Larvae from a partly factorial cross (14 sires × 11 dams) were reared until the point of metamorphosis into glass eels. In these organisms, we observed moderate heritability and mild genetic correlations among traits related to body size (h2 = 0.16-0.33) and timing at metamorphosis (h2 = 0.36-0.41). In an F1 full-sib family, quantitative trait loci (QTL) mapping for these traits identified one significant (genome-wide P < 0.05) and five suggestive QTLs (chromosome-wide P < 0.05). These results suggest that in the Japanese eel, metamorphic traits exhibit a polygenic genetic structure comprising many QTLs with small effects. In addition, we updated the genetic linkage map for the Japanese eel and integrated it with our newly constructed de novo genome assembly. The information and tools generated from this study will contribute to the development of freshwater eel genetics and genomics.

Little is known about mechanisms of resistance to parasitic diseases in marine finfish. Benedenia disease is caused by infection by the monogenean parasite Benedenia seriolae. Previous quantitative trait locus (QTL) analyses have identified a major QTL associated with resistance to Benedenia disease in linkage group Squ2 of the Japanese yellowtail/amberjack Seriola quinqueradiata. To uncover the bioregulatory mechanism of Benedenia disease resistance, complete Illumina sequencing of BAC clones carrying genomic DNA for the QTL region in linkage group Squ2 was performed to reveal a novel C-type lectin in this region. Expression of the mRNA of this C-type lectin was detected in skin tissue parasitized by B. seriolae. Scanning for single nucleotide polymorphisms (SNPs) uncovered a SNP in the C-type lectin/C-type lectin-like domain that was significantly associated with B. seriolae infection levels. These results strongly suggest that the novel C-type lectin gene controls resistance to Benedenia disease in Japanese yellowtails.

The kelp grouper (Epinephelus bruneus), belonging to one of the largest genera among the subfamily Epinephelinae, is a commercially important fish in Japan. There are limited data about the genomics of this species. To provide tools for addressing both population genetics studies and gene mapping, dito pentanucleotide simple sequence repeat (SSR) markers were developed using 454 pyrosequencing. Among the 1466 SSR markers developed, 1244 primer sets produced strong PCR products, of which 905 (72.7%) were polymorphic in kelp grouper. Cross-species utility of the 905 polymorphic SSR markers was tested in four additional Epinephelinae species of Hyporthodus septemfasciatus, Plectropomus leopardus, Epinephelus lanceolatus and Epinephelus coioides. Results revealed that, respectively, 401 (44.3%), 136 (15.0%), 434 (49.0%) and 538 (59.4%) SSRs showed specific polymorphic products. Of these, 40 SSR markers (33 di-, 1 tri- and 6 tetra-nucleotides) showed polymorphism in all species tested. Additionally, three AGAT SSR motifs which accounted for 42.9% of the nondi-nucleotide markers were found in the 40 SSR markers. This indicates that the AGAT SSR motif has a high potential as a highly versatile SSR marker in grouper Epinephelinae. The SSR markers developed in this study can be employed to obtain reliable genetic variability estimates for groupers (Epinephelinae).

Herpesviral haematopoietic necrosis has caused great economic damage to goldfish Carassius auratus aquaculture in Japan. The existence of cyprinid herpesvirus 2 (CyHV-2), the causative agent, has also been reported from several other countries. To prevent spread to other areas, basic virological information such as viral kinetics in infected fish is essential. Experimental infection trials using reliably prepared CyHV-2 for defining viral kinetics are difficult to carry out because successful and sustainable propagation of this virus in cell culture has previously been limited. Here we describe a method for sustainable propagation of CyHV-2 in cell culture, and the results of fish infection experiments using the propagated virus. We found that goldfish fin (GFF) cells and standard Ryukin Takafumi (SRTF) cells established from goldfish fin can be used for continuous propagation of CyHV-2. Experimental infections using 2 varieties of goldfish, Ryukin and Edonishiki, were performed with the virus passaged 7 times in GFF cells. In transmission experiments with water temperature at 20°C, cumulative mortality was 30% in Ryukin infected by immersion, and 90 and 100% in Edonishiki and Ryukin intraperitoneally injected with the virus, respectively. In an experiment carried out at 25°C, 90% of Edonishiki challenged by immersion died. PCR detection of viral DNA from the organs of infected fish showed that systemic infection occurs and also that the kidney is a main viral multiplication site. Moreover, CyHV-2 was successfully re-isolated in GFF cells from the dead fish.

The first genetic linkage map of the Japanese eel (Anguilla japonica) was constructed based upon Amplified Fragment Length Polymorphism (AFLP) and microsatellite (STR) markers with an F1 pseudo-testcross strategy. The segregation of 106 microsatellite loci, which included 74 newly developed, and 463 polymorphic loci identified from 56 AFLP primer combinations was studied in 46 F1 individuals derived from a single female and a male. A total of 319 markers (99 STRs + 220 AFLP) are placed on the female map and 314 markers (91 STRs + 223 AFLP) on the male map. The female map spans 1732.4 cM (Kosambi) in 22 linkage groups with an average spacing of 7.2 cM; the male map spans 964.9 cM in 22 linkage groups with an average spacing of 6.3 cM. The average ratio of male:female recombination rates was 1:2.11. The estimated coverage of the genome was 82.4% for the female map and 74.0% for the male map. This map is the first linkage map in the order Anguilliformes and provides a basis for mapping quantitative trait loci (QTL) and for breeding applications.

Streptococcal disease (streptococcosis) caused by Streptococcus iniae is a serious bacterial disease in Japanese flounder ( Paralichthys olivaceus ). Developing streptococcosis-resistant Japanese flounder will reduce the number of outbreaks of this disease as well as reduce the need for medicines and vaccines. Genetic linkage analysis is an effective method for identifying quantitative trait loci (QTL) associated with resistance to a disease. In this study, 159 microsatellite markers selected from genetic linkage maps of Japanese flounder and F 1 progeny from crosses between disease-resistant and disease-susceptible parents were used for detection of QTL associated with resistance to this disease. Some loci associated with disease resistance were found in the JF-7, JF-10, JF-11 and JF-17 linkage groups. These QTL regions are candidates for disease resistance against streptococcal infection.

Previous typing of microsatellite markers in a BC1F1 backcross rainbow trout Oncorhynchus mykiss revealed two quantitative trait loci (QTL) with a significant impact on suscep- tibility to infectious pancreatic necrosis. In the present study, additional genetic markers were mapped using the same strain in order to increase QTL resolution. A total of 199 individuals were screened at 226 marker loci (106 microsatellites, 116 amplified fragment length polymorphism markers, classical major histocompatibility complex molecules (MHC class I, nonclassical MHC class I, MHC class II) and tyrosinase) and the QTL were determined by single-point and interval mappings. Seven QTL were distinguished in the linkage groups RT-11, 12, 17, 23, 26, 29 and 31, in addition to the two already known in RT-3 and 22. The major QTL were those in RT-3, 12 and 22; whereas the classical MHC class I locus Onmy-UBA and the MHC class II locus Onmy-DAB were not associated with any of the QTL, a nonclassical MHC class I region with Onmy-UCA, Onmy-UDA, and Onmy-UEA mapped to the major QTL on RT-3.

To initiate breeding programs for kelp grouper (Epinephelus bruneus), the establishment of genetic linkage maps becomes essential accompanied by the search for quantitative trait loci that may be utilized in selection programs. We constructed a high-resolution genetic linkage map using 1055 simple sequence repeat (SSR) markers in an F1 family. Genome-wide and chromosome-wide significances of growth-related quantitative trait loci (QTLs) (body weight (BW) and total length (TL)) were detected using non-parametric mapping, Kruskal-Wallis (K-W) analysis, simple interval mapping (IM) and a permutation test (PT). Two stages and two families of fish were used to confirm the QTL regions. Ultimately, 714 SSR markers were matched that evenly covered the 24 linkage groups. In total, 509 and 512 markers were localized to the female and male maps, respectively. The genome lengths were approximately 1475.95 and 1370.39 cM and covered 84.68 and 83.21 % of the genome, with an average interval of 4.1 and 4.0 cM, in females and males, respectively. One major QTL affecting BW and TL was found on linkage group EBR 17F that identified for 1 % of the genome-wide significance and accounted for 14.6–18.9 and 14.7–18.5 % of the phenotypic variance, and several putative QTL with 5 % chromosome-wide significance were detected on eight linkage groups. Furthermore, the confirmed results of the regions harboring the major and putative QTLs showed consistent significant experiment-wide values of 1 and 5 % as well as a chromosome-wide value of 5 %. We identified growth-related QTLs that could be applied to find candidate genes for growth traits in further studies, and potentially useful in MAS breeding. Electronic supplementary material The online version of this article (doi:10.1007/s10126-015-9673-5) contains supplementary material, which is available to authorized users.

Background Physical and linkage maps are important aids for the assembly of genome sequences, comparative analyses of synteny, and to search for candidate genes by quantitative trait locus analysis. Yellowtail, Seriola quinqueradiata, is an economically important species in Japanese aquaculture, and genetic information will be useful for DNA-assisted breeding. We report the construction of a second generation radiation hybrid map, its synteny analysis, and a second generation linkage map containing SNPs (single nucleotide polymorphisms) in yellowtail. Results Approximately 1.4 million reads were obtained from transcriptome sequence analysis derived from 11 tissues of one individual. To identify SNPs, cDNA libraries were generated from a pool of 500 whole juveniles, and the gills and kidneys of 100 adults. 9,356 putative SNPs were detected in 6,025 contigs, with a minor allele frequency ≥25%. The linkage and radiation hybrid maps were constructed based on these contig sequences. 2,081 markers, including 601 SNPs markers, were mapped onto the linkage map, and 1,532 markers were mapped in the radiation hybrid map. Conclusions The second generation linkage and physical maps were constructed using 6,025 contigs having SNP markers. These maps will aid the de novo assembly of sequencing reads, linkage studies and the identification of candidate genes related to important traits. The comparison of marker contigs in the radiation hybrid map indicated that yellowtail is evolutionarily closer to medaka than to green-spotted pufferfish, three-spined stickleback or zebrafish. The synteny analysis may aid studies of chromosomal evolution in yellowtail compared with model fish. Electronic supplementary material The online version of this article (doi:10.1186/s12864-015-1600-7) contains supplementary material, which is available to authorized users.

Complementary DNA (cDNA) clones for human KIAA genes have been isolated as long cDNAs (>4 kb) with unknown functions. To facilitate the functional analysis of these human clones, we have isolated and determined the structures of their respective mouse homologues (mKIAA genes). Furthermore, we have comprehensively raised antibodies against the translated mKIAA proteins in order to establish a platform for their functional analysis. Since the specificity of these antibodies is critical for subsequent analyses of protein function, here we introduce two assays utilizing mammalian cells to improve their evaluation. First, we have established a semi-high-throughput production of C-terminally FLAG epitope-tagged proteins for Western blotting using specially designed mammalian expression vectors. Secondly, we have utilized immunofluorescence staining of mouse cells to analyze the subcellular localization of endogenous mKIAA proteins. Importantly, these methods allow us to detect potential posttranslational modification of the mKIAA/KIAA proteins and to predict their biological function based on their subcellular localization.

1. はじめに 水産分野では、天然種苗を捕獲して育てる養殖が主 流であったため、家畜や実験動物の分野に比べて、ゲ ノム研究、経済形質の解析は大幅に遅れていたが、近 年の養殖技術の発達は人為環境下での再生産技術を飛 躍的に前進させると共に、雌性発生二倍体、雄性発生 二倍体などの生産を可能にした染色体操作技術開発に より種々の近交系を短期間に大量に作出できるため、 遺伝形質の解析に理想的な生物として水産生物を使用 することが可能となった。このような背景から、水産 生物の遺伝子およびゲノム研究の進展が一挙に進んで いる。 しかし、水産生物、特に水産経済動物は遺伝育種 という点においては歴史が浅く、養殖種が野生種と 区別できないほど性質が似ており、とりわけ目立つ 経済形質が得られ難いので、それら形質評価には、 これからも多くの労力を要するであろう。現在、水 産養殖では深刻な魚病問題などの多くの経済的要因 を抱えており、病気に強い形質、成長率の良い形質、 広域環境に適応する形質などの遺伝育種の要求が高 まっている。こうした状況の中で、ゲノム研究の進 展が水産生物の遺伝育種の発展に多大な貢献をする と期待されている。 ここではマーカー選抜育種に代表されるゲノム育種 を意識した水産経済動物のゲノム研究及び Q T L (Quantitative Trait Loci)解析の現状を紹介し、今後の 研究の発展性を考えたい。

We report the first genetic linkage map of the Japanese flounder ( Paralichthys olivaceus ) constructed with 111 microsatellite markers and 352 AFLP fragments. The parental male linkage map consisted of 25 linkage groups while the female map consisted of 27 groups, with an average resolution of 8 and 6.6 cM, respectively. We have identified linkage among 96% of the markers and the total map length was estimated to be around 1000–1200 cM. This study reports the first low-density linkage map for the Japanese flounder and describes differences in sex recombination. Recombination rates were higher in male flounder compared to the female (7.4:1), a rare condition among vertebrates. This map is a starting point for the mapping of single loci and quantitative traits in flatfish species.

Porphyra (Bangiales, Rhodophyta), which includes several valuable marine crops, has recently received great interest as a model plant for fundamental and applied studies in marine sciences. Amplified fragment length polymorphisms (AFLPs) are a robust and efficient means for genetic mapping, linkage analysis of genetic characters for breeding and population studies in land plant genomes. To examine whether AFLPs are applicable as genetic markers in the present study, we detected AFLP markers with three pure lines in order to promote genetic analysis in Porphyra yezoensis. The following five sets of AFLP primer pairs (E-AA, M-CAA) (E-AA, M-CAC) (E-AA, M-CAG) (E-AA, M-CAT) (E-AA, M-CTA) were tested with template DNAs from three pure lines and they showed a total of 227 bands. This suggests that AFLP markers are promising tools for genetic analysis in Porphyra.

Background Japanese amberjack/yellowtail (Seriola quinqueradiata) is a commonly cultured marine fish in Japan. For cost effective fish production, a breeding program that increases commercially important traits is one of the major solutions. In selective breeding, information of genetic markers is useful and sufficient to identify individuals carrying advantageous traits but if the aim is to determine the genetic basis of the trait, large insert genomic DNA libraries are essential. In this study, toward prospective understanding of genetic basis of several economically important traits, we constructed a high-coverage bacterial artificial chromosome (BAC) library, obtained sequences from the BAC-end, and constructed comprehensive female and male linkage maps of yellowtail using Simple Sequence Repeat (SSR) markers developed from the BAC-end sequences and a yellowtail genomic library. Results The total insert length of the BAC library we constructed here was estimated to be approximately 11 Gb and hence 16-times larger than the yellowtail genome. Sequencing of the BAC-ends showed a low fraction of repetitive sequences comparable to that in Tetraodon and fugu. A total of 837 SSR markers developed here were distributed among 24 linkage groups spanning 1,026.70 and 1,057.83 cM with an average interval of 4.96 and 4.32 cM in female and male map respectively without any segregation distortion. Oxford grids suggested conserved synteny between yellowtail and stickleback. Conclusions In addition to characteristics of yellowtail genome such as low repetitive sequences and conserved synteny with stickleback, our genomic and genetic resources constructed and revealed here will be powerful tools for the yellowtail breeding program and also for studies regarding the genetic basis of traits.

The isolation and characterization of eight polymorphic microsatellite loci from a Japanese flounder partial genomic library are reported. The eight markers isolated in this study were highly polymorphic and their positions on the linkage genome map of the Japanese flounder were determined. Therefore, they are useful for ecological studies of wild populations. These markers are more effective than other markers with no information of chromosomal locations.

Benedenia infections caused by the monogenean fluke ectoparasite Benedenia seriolae seriously impact marine finfish aquaculture. Genetic variation has been inferred to play a significant role in determining the susceptibility to this parasitic disease. To evaluate the genetic basis of Benedenia disease resistance in yellowtail (Seriola quinqueradiata), a genome-wide and chromosome-wide linkage analyses were initiated using F1 yellowtail families (n = 90 per family) based on a high-density linkage map with 860 microsatellite and 142 single nucleotide polymorphism (SNP) markers. Two major quantitative trait loci (QTL) regions on linkage groups Squ2 (BDR-1) and Squ20 (BDR-2) were identified. These QTL regions explained 32.9–35.5% of the phenotypic variance. On the other hand, we investigated the relationship between QTL for susceptibility to B. seriolae and QTL for fish body size. The QTL related to growth was found on another linkage group (Squ7). As a result, this is the first genetic evidence that contributes to detailing phenotypic resistance to Benedenia disease, and the results will help resolve the mechanism of resistance to this important parasitic infection of yellowtail.

Background Japanese flounder (Paralichthys olivaceus) is one of the most economically important marine species in Northeast Asia. Information on genetic markers associated with quantitative trait loci (QTL) can be used in breeding programs to identify and select individuals carrying desired traits. Commercial production of Japanese flounder could be increased by developing disease-resistant fish and improving commercially important traits. Previous maps have been constructed with AFLP markers and a limited number of microsatellite markers. In this study, improved genetic linkage maps are presented. In contrast with previous studies, these maps were built mainly with a large number of codominant markers so they can potentially be used to analyze different families and populations. Results Sex-specific genetic linkage maps were constructed for the Japanese flounder including a total of 1,375 markers [1,268 microsatellites, 105 single nucleotide polymorphisms (SNPs) and two genes]; 1,167 markers are linked to the male map and 1,067 markers are linked to the female map. The lengths of the male and female maps are 1,147.7 cM and 833.8 cM, respectively. Based on estimations of map lengths, the female and male maps covered 79 and 82% of the genome, respectively. Recombination ratio in the new maps revealed F:M of 1:0.7. All linkage groups in the maps presented large differences in the location of sex-specific recombination hot-spots. Conclusions The improved genetic linkage maps are very useful for QTL analyses and marker-assisted selection (MAS) breeding programs for economically important traits in Japanese flounder. In addition, SNP flanking sequences were blasted against Tetraodon nigroviridis (puffer fish) and Danio rerio (zebrafish), and synteny analysis has been carried out. The ability to detect synteny among species or genera based on homology analysis of SNP flanking sequences may provide opportunities to complement initial QTL experiments with candidate gene approaches from homologous chromosomal locations identified in related model organisms.

Summary We have developed the first set of trinucleotide and tetranucleotide markers for the Japanese flounder, Paralichthys olivaceus. One hundred and sixty-seven polymorphic trinucleotide and tetranucleotide microsatellites were isolated using clones derived from two libraries. Of almost 200 000 clones analysed, 0.5% presented trinucleotide or tetranucleotide repeat regions. Among the trinucleotide repeats analysed in this study, the most frequent one was (CAG)n and the most common tetranucleotide repeat was (GATA)n. The position of the new markers in the genetic linkage map was determined. Markers were evenly distributed along the P. olivaceus linkage groups, without distinction between the kinds of repeats and library of origin. The markers isolated in this study contribute significantly to the genetic linkage map of the Japanese flounder.

DNA marker technologies can be used for genetic improvement through selection of favorable traits such as disease resistance. These traits are generally modeled as being controlled by many genes of small additive effects, known as quantitative trait loci (QTL). Construction of a genetic linkage map based on DNA markers at a large number of sites in the fish genome is necessary to identify QTL controlling traits of disease resistance. By identifying markers associated with high per- formance QTL in different strains or species, it may be possible to improve the performance of such traits in other strains through intro- gression of the desired QTL. One of the goals of selective breeding programs is to integrate genetic marker information from pedigreed brood stock into successful management and culture. Such an approach, termed marker- assisted selection (MAS) and/or marker- assisted gene introgression (MAI), is expect- ed to increase genetic response by affecting efficiency and accuracy of selection.

In recent years, the development of molecular markers and DNA analysis technology has completely changed analysis of quantitative genetics. Because a systematic concrete method for genetic breeding has been established using the molecular landmarks of genome DNA, we can control the phenotypes in genetic breeding, using DNA sequences associated with particular economic characters.

Background Recent advancements in next-generation sequencing technology have enabled cost-effective sequencing of whole or partial genomes, permitting the discovery and characterization of molecular polymorphisms. Double-digest restriction-site associated DNA sequencing (ddRAD-seq) is a powerful and inexpensive approach to developing numerous single nucleotide polymorphism (SNP) markers and constructing a high-density genetic map. To enrich genomic resources for Japanese eel (Anguilla japonica), we constructed a ddRAD-based genetic map using an Ion Torrent Personal Genome Machine and anchored scaffolds of the current genome assembly to 19 linkage groups of the Japanese eel. Furthermore, we compared the Japanese eel genome with genomes of model fishes to infer the history of genome evolution after the teleost-specific genome duplication. Results We generated the ddRAD-based linkage map of the Japanese eel, where the maps for female and male spanned 1748.8 cM and 1294.5 cM, respectively, and were arranged into 19 linkage groups. A total of 2,672 SNP markers and 115 Simple Sequence Repeat markers provide anchor points to 1,252 scaffolds covering 151 Mb (13%) of the current genome assembly of the Japanese eel. Comparisons among the Japanese eel, medaka, zebrafish and spotted gar genomes showed highly conserved synteny among teleosts and revealed part of the eight major chromosomal rearrangement events that occurred soon after the teleost-specific genome duplication. Conclusions The ddRAD-seq approach combined with the Ion Torrent Personal Genome Machine sequencing allowed us to conduct efficient and flexible SNP genotyping. The integration of the genetic map and the assembled sequence provides a valuable resource for fine mapping and positional cloning of quantitative trait loci associated with economically important traits and for investigating comparative genomics of the Japanese eel.

Kelp grouper (Epinephelus bruneus) is an important aquaculture species in Japan, Korea and China. Mariculture production of the species has increased due to its high demand and market price. However, some problems affect kelp grouper aquaculture such as the low growth rate, high mortality due to diseases and low ability to survive the larval stage. To analyze economically important traits, genetic linkage maps are an effective tool. We constructed sex-specific linkage maps of kelp grouper using 222 microsatellite markers. The male map consisted of 23 linkage groups with 161 markers and the female map consisted of 25 linkage groups with 173 markers. The total lengths of the male and female maps were 650.5 cM and 944.4 cM, respectively, and the average intervals were 5.0 cM and 6.7 cM, respectively. The average ratio of recombination between males and females was 1:1.5. Moreover, syntenic sequence comparisons provided basic information of several potential candidate genes affecting organism physiological and biochemical reactions. Based on the linkage map, further quantitative trait loci (QTL) or candidate gene(s) detection can be anticipated to contribute to assist breeding programs of kelp grouper. In addition, by providing basic genome information of kelp grouper, the map provides a first step towards comparative QTL as well as comparative genome analyses with other groupers in the future.