Detection of Growth-Related Quantitative Trait Loci and High-Resolution Genetic Linkage Maps Using Simple Sequence Repeat Markers in the Kelp Grouper (Epinephelus bruneus).

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 (QTLs) 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 QTLs (body weight: BW and total length: TL) were detected using non-parametric mapping, Kruskal-Wallis 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 17 F 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 marker assisted selection (MAS) breeding.

Detection of Growth-Related Quantitative Trait Loci and High-Resolution Genetic Linkage Maps Using Simple Sequence Repeat Markers in the Kelp Grouper (Epinephelus bruneus).

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.

Construction of a high-coverage bacterial artificial chromosome library and comprehensive genetic linkage map of yellowtail Seriola quinqueradiata.

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.

Quantitative trait loci (QTL) associated with resistance to a monogenean parasite (Benedenia seriolae) in yellowtail (Seriola quinqueradiata) through genome wide analysis.

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.

Permanent genetic resources added to Molecular Ecology Resources Database 1 August 2012 - 30 September 2012.

This article documents the addition of 83 microsatellite marker loci and 96 pairs of single-nucleotide polymorphism (SNP) sequencing primers to the Molecular Ecology Resources Database. Loci were developed for the following species: Bembidion lampros, Inimicus japonicus, Lymnaea stagnalis, Panopea abbreviata, Pentadesma butyracea, Sycoscapter hirticola and Thanatephorus cucumeris (anamorph: Rhizoctonia solani). These loci were cross-tested on the following species: Pentadesma grandifolia and Pentadesma reyndersii. This article also documents the addition of 96 sequencing primer pairs and 88 allele-specific primers or probes for Plutella xylostella.

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

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.

Induction of virus-specific cell-mediated cytotoxic responses of isogeneic ginbuna crucian carp, after oral immunization with inactivated virus.

Clonal ginbuna crucian carp (Carassius auratus langsdorfii) were immunized orally with inactivated crucian carp hematopoietic necrosis virus (CHNV) and the cytotoxic activities of peripheral blood leukocytes against CHNV-infected syngeneic target cells were investigated. Although a single oral administration of the vaccine did not prime measurable cytotoxic responses to CHNV-infected targets, detectable lytic activities were observed after a booster oral administration. The vaccine-induced cytotoxic cells were poorly responded against both eel virus from America (EVA)-infected syngeneic cells and CHNV-infected allogeneic target cells, suggesting that the responses were viral antigen-specific and restricted to the major histocompatibility complex (MHC). Oral immunization with the vaccine also induced neutralizing antibody responses. Orally immunized fish were able to rapidly eliminate viruses. Although elevated cytotoxic activities and antibody responses were observed in orally immunized fish following viral infection, the rapid elimination of virus appeared to be associated with elevated cytotoxic responses. These results show that orally administered inactivated viruses can evoke antiviral cellular immune responses in fish.

Construction of a genetic map and development of DNA markers linked to the sex-determining locus in the Patagonian pejerrey (Odontesthes hatcheri).

The process of sex differentiation in fishes is regulated by genetic and environmental factors. The sex of Patagonian pejerrey (Odontesthes hatcheri) appears to be under strong genotypic control (GSD) because the sex ratios are balanced (1:1) between 17 degrees C and 23 degrees C. However, sex ratios become female-biased at <15 degrees C and male-biased at 25 degrees C, which shows that this species also possesses some degree of temperature-dependent sex determination (TSD). Identification of the genetic sex of an individual will help elucidate the molecular basis of sex differentiation in this species. In this study, we used amplified fragment length polymorphism (AFLP) analysis to develop a genetic linkage map for both sexes and a sex-linked DNA marker for Patagonian pejerrey. The AFLP analysis of 23 male and 23 female progeny via 64 primer combinations produced a total of 153 bands. The genetic linkage map consisted of 79 markers in 20 linkage groups and 48 markers in 15 linkage groups for males and females, respectively. One AFLP marker tightly linked to the sex-determining locus was identified: the marker, ACG/CAA-217, amplified to the male-specific DNA fragment. Sequence analysis of this region revealed a single nucleotide polymorphism (SNP) between males and females, which was converted into a SNP marker. This marker provides genetic confirmation that the sex of Patagonian pejerrey is determined genetically and would be useful for the analysis of the molecular basis of GSD and TSD in this species.

In vitro generation of viral-antigen dependent cytotoxic T-cells from ginbuna crucian carp, Carassius auratus langsdorfii.

Little is known about antigen-specific T-cell responses to viruses in teleosts due to a lack of a suitable experimental system using inbred or clonal animals. In the present study we have successfully induced an in vitro generation of virus-specific cytotoxic T-cells (CTLs) from isogeneic ginbuna crucian carp. Responder cells (primarily lymphocytes) from crucian carp haematopoietic necrosis virus (CHNV)-infected fish were capable of proliferating after stimulation in vitro with CHNV-infected syngeneic stimulator cells (primarily lymphocytes and macrophages). The effector cells collected 8 and 12 days after the in vitro stimulation efficiently lysed CHNV-infected syngeneic cells, but not CHNV-infected allogeneic cells or different virus (EVA)-infected syngeneic cells. Furthermore, in situ hybridization analysis showed that some effector cells binding to a CHNV-infected target were TCRbeta or CD8alpha positive. These results provide evidence that the teleost effector cells generated in vitro correspond to virus-specific CTL and they recognize virus-infected target cells in a similar manner of mammalian counterparts.

Characterization of the cell-mediated cytotoxic responses of isogeneic ginbuna crucian carp induced by oral immunisation with hapten-modified cellular antigens.

A better understanding of the immune responses in fish elicited by oral immunisation is of importance for the development of new and effective oral vaccines for cultured fish. In the present study, we characterized specific cell-mediated cytotoxic responses in isogeneic ginbuna crucian carp (Carassius auratus langsdorfii) following oral immunisation with cellular antigens. Trinitrophenyl- (TNP) or dinitrophenyl- (DNP) modified syngeneic and allogeneic cells were used for studying the fine specificity and genetic restriction of orally-induced cytotoxic cells. Hapten-specific cytotoxic responses were detected in peripheral blood leukocytes (PBLs) of fish orally immunised with haptenated syngeneic cells. PBLs from orally immunised fish had cytolytic activity for haptenated syngeneic cells, but they showed little reactivity against both haptenated and unmodified allogeneic targets. Similarly, oral immunisation of fish with hapten-modified allogeneic cells did not induce hapten-specific cytotoxic cells which can lyse haptenated syngeneic targets. Although ginbuna crucian carp possess spontaneous cytotoxic cells that are capable of killing mammalian tumour cells, cold target inhibition studies suggested that such spontaneous cytotoxic cells were not involved in the killing of haptenated syngeneic targets. Oral immunisation of fish with haptenated syngeneic cells also induced hapten-specific cytotoxic memory responses. Oral administration of haptenated fixed cells also effectively induced hapten-specific cytotoxic cells in the treated fish. These findings suggest that oral immunisation with antigens can elicit antigen-specific cytotoxic cells that are capable of recognizing antigens in an MHC-restricted manner. In addition, our results provide indirect evidence that fish possess a mechanism for taking up exogenous non-replicating antigens from the alimentary tract and generating antigen-specific cytotoxic cells.

Increased leptin may be involved in the short life span of ayu (Plecoglossus altivelis).

It is well known that ayu (Plecoglossus altivelis) die after spawning and have a life span of only 1 year. The determinants for such a short life span are probably connected with spawning and related changes in hormonal homeostases. One of these changes is that the ayu's feeding activity decreases both during and after spawning. We investigated the relationships among leptin, one of the regulators of food intake, and two other major hormones, 17 beta-estradiol and prolactin (PRL). Ir-leptin levels were significantly higher during spawning, and were associated with a decrease in appetite. Ir-leptin levels were also synchronized with levels of 17 beta-estradiol and PRL-like protein. Therefore, one possible explanation for the decrease in appetite during ayu spawning is that the elevation of 17 beta-estradiol homeostasis induced the secretion of Ir-leptin. The inability to decrease leptin to the basal levels because of high estrogen after spawning could be in part responsible for the short life span of ayu.

Expression profiles of TCRbeta and CD8alpha mRNA correlate with virus-specific cell-mediated cytotoxic activity in ginbuna crucian carp.

Our previous studies have demonstrated that virus-specific cell-mediated cytotoxicity of sensitized leukocytes can be induced using clonal ginbuna crucian carp and their syngeneic cell lines. In the present study, we attempt to determine if virus-specific cytotoxic cell populations of fish express CD8alpha and TCRbeta genes. Leukocytes from ginbuna crucian carp were separated into four fractions by immunomagnetic separation and density gradient centrifugation: Fraction A, leukocytes with a density of 1.08 g/ml (primarily lymphocytes); Fraction B, sIg-negative leukocytes with density of 1.08 g/ml; Fraction C, sIg-positive cells (primarily B-lymphocytes); Fraction D, leukocytes with a density of 1.08-1.09 g/ml (primarily neutrophils). Leukocytes in all fractions from uninfected fish do not exhibit cytotoxic activity against virus-infected syngeneic cells and weakly express CD8alpha and TCRbeta mRNAs. In contrast, leukocytes in fractions A and B from virus-infected fish exhibit a high level of cytotoxic activity and strongly express CD8alpha and TCRbeta mRNAs. In addition, mRNA expressions of CD8alpha and TCRbeta in effector cells are upregulated by cocultivation with virus-infected target cells but not uninfected ones. The present study suggests that fish possess virus-specific cytotoxic cells with phenotype and gene expression pattern similar to those of CTLs in mammals.

A linkage map for brown trout (Salmo trutta): chromosome homeologies and comparative genome organization with other salmonid fish.

We report on the construction of a linkage map for brown trout (Salmo trutta) and its comparison with those of other tetraploid-derivative fish in the family Salmonidae, including Atlantic salmon (Salmo salar), rainbow trout (Oncorhynchus mykiss), and Arctic char (Salvelinus alpinus). Overall, we identified 37 linkage groups (2n = 80) from the analysis of 288 microsatellite polymorphisms, 13 allozyme markers, and phenotypic sex in four backcross families. Additionally, we used gene-centromere analysis to approximate the position of the centromere for 20 linkage groups and thus relate linkage arrangements to the physical morphology of chromosomes. Sex-specific maps derived from multiple parents were estimated to cover 346.4 and 912.5 cM of the male and female genomes, respectively. As previously observed in other salmonids, recombination rates showed large sex differences (average female-to-male ratio was 6.4), with male crossovers generally localized toward the distal end of linkage groups. Putative homeologous regions inherited from the salmonid tetraploid ancestor were identified for 10 pairs of linkage groups, including five chromosomes showing evidence of residual tetrasomy (pseudolinkage). Map alignments with orthologous regions in Atlantic salmon, rainbow trout, and Arctic char also revealed extensive conservation of syntenic blocks across species, which was generally consistent with chromosome divergence through Robertsonian translocations.

Influence of essential fatty acid deprivation on thrombocyte aggregation in rainbow trout

Many reports have indicated that differences in blood cells formation and function in fish could be of dietary origino Essential fatty acids are certainly connected with these cells by virtue of being a source of important compounds like eicosanoids, platelet activating factor in mammals, as well as the cell membrane phospholipids. The thrombocytes from fish fed the commercial diet containing adequate amount of essential fatty acids exhibited greater capacity for aggregation when induced by Collagen Type I(56%), however, this capacity was reduced when fish were fed the essential fatty acids deficient diet (37%). The results obtained in this study demonstrated that EFA exert influence in thrombocytes by affecting their aggregation capacity.

Indicações sobre alterações na formação das células sanguíneas e em suas funções nos peixes têm sido relatadas em diversos trabalhos. Os ácidos graxos essenciais (EFA) certamente estão ligados a essas células devido ao fato de constituírem fonte de componentes importantes, como os eicosanoides, fatores de ativação de plaquetas nos mamíferos, bem como de fosfolipídios de membrana. Trombócitos oriundos de peixes alimentados com uma dieta comercial contendo quantidades adequadas de EFA mostraram uma grande capacidade de agregação quando induzidos por colágeno do tipo I (56%), contudo, essa capacidade encontrou-se reduzida quando os peixes foram alimentados com uma dieta deficiente em EFA (37%). Os resultados obtidos nesse estudo demonstraram que os ácidos graxos essenciais exercem influência nos trombócitos afetando sua capacidade de agregação.

Influence of rearing temperature changes on thrombocytes aggregation in rainbow trout

Fish, as poikilotherms, are subjected to the influence of the environmental temperature. It has been already reported that fish immune system is influenced by temperature; however, no information is available concerning if temperature has any effect on thrombocytes. Thrombocytes from fish reared at 6°C, 10oC and 20oC were assessed for alteration in their aggregation capacity. Thrombocyte percentages were altered by the water temperature at which the fish was reared, decreasing from 19% (at 10°C) to 13% (at 6°C) and increasing to 24% (at 20°C). However, the aggregation capacity was not significantly compromised by these temperature changes in an individual cell basis, which would suggest a capability of this cell, to maintain this indispensable function at different temperatures.

Os peixes, por serem animais pecilotermos, estão sujeitos à influências da temperatura do ambiente. Vários trabalhos já descreveram que o sistema imune é influenciado pela temperatura, contudo não existem informações concernentes à influencia desta sobre os trombócitos. Trombócitos provenientes de trutas arco-íris (Oncorrhynchus mykiss) mantidas a 6°C, 10ºC e 20ºC foram testados quanto à sua capacidade de agregação. A porcentagem de trombócitos sofreu alteração dependendo da temperatura da água em que os animais foram mantidos, diminuindo de 19% (a 10°C), para 13% (a 6°C) e aumentando para 24% (a 20°C). Entretanto, a capacidade de agregação individual de cada célula não foi significativamente afetada por essas mudanças de temperatura, o que sugere a qualidade dessa célula em manter essa função indispensável independente da temperatura.

Functional divergence of duplicated c-myc genes in a tetraploid fish, the common carp (Cyprinus carpio).

The proto-oncogene c-myc is thought to be one of the most important genes in controlling cell proliferation. In a tetraploid fish, two c-myc genes (CAM1 and CAM2) were previously isolated from the common carp, Cyprinus carpio, and were shown to have different expression patterns in adult tissues. Here we found that CAM1 and CAM2 proteins had distinct properties in terms of their transcription regulation system, formation of the transcription activator complex Myc/Max, and transcriptional activation of the target gene. These results showed that the two carp c-Myc proteins have overlapping but distinct functions, suggesting that CAM1 and CAM2 are evolving to acquire different functions after an earlier tetraploidization event.

Structural and functional evolution of three cardiac natriuretic peptides.

Natriuretic peptides (NPs) are a group of hormones playing important roles in cardiovascular and osmoregulatory systems in vertebrates. Among the NP subtypes, atrial NP (ANP), B-type NP (BNP), and ventricular NP (VNP) are circulating hormones expressed exclusively in the heart (cardiac NPs). The constitution of cardiac NPs is variable among species of vertebrates. In order to understand the evolutionary and functional significance of such variation, we performed a systematic survey of cardiac NP cDNAs in nine taxonomically diverse teleosts inhabiting environments of varying salinity. The discovery of the coexistence of the ANP, BNP, and VNP genes in the eel and rainbow trout suggested that the ancestral teleost had all three cardiac NPs. As the VNP cDNA was undetectable in ayu and six species of Neoteleostei, it is possible that VNP was lost before the divergence of Osmeroidei. The ANP gene was also undetectable in the medaka. Thus, only the BNP gene is universal in species examined in the present study. Synthetic medaka BNP preferentially activated two medaka GC-A-type receptors, suggesting that the three cardiac NPs share the same receptor. However, the regulation of BNP expression may be the most strict because ATTTA repeats in the 3'-untranslated region and the dibasic motif in the ring are conserved among teleosts and tetrapods. Linkage analyses in the rainbow trout located ANP, BNP, and VNP genes on the same chromosome, which suggested the generation of the VNP gene by tandem duplication as observed with ANP and BNP genes. If the duplication occurred before the divergence of tetrapods and teleosts, VNP may exist in the tetrapod lineage.

Effects of caloric restriction on post-spawning death of ayu.

Caloric restriction (CR) is the only established intervention that extends life span in mammals, insects and nematodes. One of the hypotheses suggested that most of the effects of CR on aging may be due to reduced oxidative stress at the cellular level. It was known that ayu (Plecoglossus altivelis) produced ROS higher than other fish and that the life span of ayu is only one year. The present study attempts to quantify age-associated changes of the degree of attenuation on oxidative damage and hormonal homeostases in CR. The levels of 8-OHdG as the oxidative DNA damage level and the caspase-9/6, -3-like activities as the induction factors of apoptosis with aging in brain and liver were surveyed. Caspase-like activities in brain and liver were reduced by CR, while CR had no influence on DNA damage level. However, life span of ayu was not prolonged by CR. These results suggested that there would be factors determining life span of ayu other than CR and apoptosis.

Four types of Smad4 found in the common carp, Cyprinus carpio.

Smad4 is defined as the common-mediator Smad (Co-Smad) required for transducing signals for all transforming growth factor-beta (TGF-beta) superfamily members. In this study, we have isolated eight distinct Smad4 full-length cDNAs from the common carp (Cyprinus carpio). These cDNAs were classified into four types and each type consisted of two subtypes. The eight cDNAs encoded four distinct proteins ranging from 505aa to 568aa in size, with close similarities in the Mad homology 1 and 2 (MH1 and MH2, respectively), but with differences in the linker regions and the C-terminus as well as in the 5'- and 3'-untranslated regions. Genomic Southern blotting demonstrated the existence of at least six Smad4 gene loci in the carp genome, meaning that the multiple forms of the carp Smad4 cDNAs were not due to allelic variations. Reverse transcriptase polymerase chain reaction (RT-PCR)/Southern hybridizations showed different expression patterns among the four types of Smad4s. These results suggest that some of carp Smad4s have deviated from the original function of Smad4 through vertebrate evolution, and regulated the TGF-beta signaling pathway by changing the expression level in tissues.