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Asian seabass (Lates calcarifer) is an important foodfish species. It has been cultured in Singapore since the 1980s. A selective breeding program to improve growth, meat quality and disease resistance has been ongoing in Singapore since 2004. In 2012, outbreaks of the big belly disease (BBD) occurred in two batches of offspring generated by a mass-cross among F1 brooders in a hatchery. After the outbreaks, only 437 survived in 6000 F2 juveniles. Molecular parentage analysis with 10 microsatellites revealed that four parents (F1) produced high percentage of offspring that survived the BBD outbreaks. Eighty-two BBD survivor offspring (F2) were selected based on their growth performance at 90 days post hatch (dph), family origin and allelic diversity. They were used as brooders to generate F3. In a period of five years, these F2 brooders produced six batches of offspring, where no outbreak of BBD occurred. In the same period, outbreaks of BBD occurred in 4 out of 18 batches produced by F2 brooders selected for growth, with mortality rates ranging from 85.1% to 95.4%. In F3, the average body weight (57.1 ± 1.09 g, n = 3068) of the line for BBD survival was slightly lower than that (60.6 ± 2.29 g, n = 4280) of the line for growth at 90 dph. In both lines, the body weight of F3 was much higher than that (46.90 ± 0.19 g, n = 12117) of F2. The genetic diversity in the line for BBD survival (F3) was much lower than that in F3 line for growth due to small founder population. These data indicate that the selection of BBD survival during BBD outbreaks using molecular parentage analysis is effective to reduce BBD outbreaks. Avoiding further reduction of genetic diversity is essential in the line selected for increased survival against BBD. [ABSTRACT FROM AUTHOR]

China has been the top aquaculture producer in the world since 2002. Over 800 species and 240 improved varieties have been used in China's aquaculture, which is the most diverse collection of species on this planet. Species diversification, along with other aquaculture technologies, contributes significantly to the increase of China's aquaculture production, whereas the use of too many species in aquaculture has resulted in some issues related to the environment and sustainability. We provide an updated overview of the species currently used in Chinese aquaculture in this paper. We examine critically the main drivers of species diversification in China with examples. We also introduce Chinese approaches to improve and certify new species and varieties for commercial production. We believe that further species diversification is not a priority in China's aquaculture, while further improvement of aquaculture technologies for major and native species is essential for green, profitable, and sustainable (GPS) aquaculture. The implications of China's experiences with species diversification for global aquaculture species diversification are discussed. [ABSTRACT FROM AUTHOR]

The Asian arowana (Scleropages formosus) has gained immense popularity in the ornamental fish trade due to its striking color variations. However, ongoing debates persist regarding the taxonomic classification of these distinct color varieties as separate species. This paper aims to address this issue by employing mtDNA sequencing, microsatellite genotyping, and crossing experiments. Phylogenic analyses using mitochondrial and nuclear markers across green, golden, and red varieties showed that individuals from these tree color varieties were mixed in several clusters. Crossing between different varieties generated fertile offspring. These data indicates that three color morphs of the Asian arowana should not be classified as distinct species. Acknowledging the aesthetic significance of these varieties, a holistic understanding of their taxonomic status is crucial for informed conservation strategies and sustainable management of this species. [ABSTRACT FROM AUTHOR]

Drought stress is a major environmental hazard. Stomatal development is highly responsive to abiotic stress and has been used as a cellular marker for drought-tolerant crop selection. C3 and C4 crops have evolved into different photosynthetic systems and physiological responses to water deficits. The genome sequences of maize, sorghum, and sugarcane make it possible to explore the association of the stomatal response to drought stress with the evolution of the key stomatal regulators. In this study, phylogenic analysis, gene expression analysis and stomatal assay under drought stress were used to investigate the drought tolerance of C3 and C4 plants. Our data shows that C3 and C4 plants exhibit different drought responses at the cellular level. Drought represses the growth and stomatal development of C3 crops but has little effect on that of C4 plants. In addition, stomatal development is unresponsive to drought in drought-tolerant C3 crops but is repressed in drought-tolerant C4 plants. The different developmental responses to drought in C3 and C4 plants might be associated with the divergent expression of their SPEECHLESS genes. In particular, C4 crops have evolved to generate multiple SPEECHLESS homologs with different genetic structure and expression levels. Our research provides not only molecular evidence that supports the evolutionary history of C4 from C3 plants but also a possible molecular model that controls the cellular response to abiotic stress in C3 and C4 crops. [ABSTRACT FROM AUTHOR]

Iridoviruses are DNA virus and have caused huge economic losses in the aquaculture industry. The aim of this study was to establish a colorimetric loop-mediated isothermal amplification (LAMP) protocol for the on-site detection of Singapore grouper iridovirus (SGIV). The SGIV-VP61 gene was chosen as the target gene to develop a colorimetric LAMP assay. The optimized condition of the colorimetric LAMP assay was incubation at 63?C for 1 h. Samples infected with SGIV could be detected with the color change from yellow into pink. The sensitivity of the developed assay is 5.66 copies/µL of the viral DNA template. This sensitivity was about 1000 times higher than that of conventional PCR while it was slightly lower than the one-step semi-nested PCR assay. A total of 60 DNA samples extracted from the fin tissue of the SGIV-infected Asian seabass were examined for SGIV by colorimetric LAMP, semi-nested PCR and conventional PCR. The results of the colorimetric LAMP assay showed 94.87 % agreement with the semi-nested PCR. In addition, the DNA extraction method using NaOH showed a better performance in the colorimetric LAMP assay. Taken together, the colorimetric LAMP established was a sensitive, rapid and specific method for the detection of SGIV. SGIV was not detected in samples randomly taken from a genetically improved line of the Asian seabass. However, some seabass obtained from the local markets were found to contain SGIV. Thus, the LAMP assay has the potential application in the diagnosis of iridovirus diseases in the aquaculture industry. [ABSTRACT FROM AUTHOR]

Viral nervous necrosis (VNN) disease, caused by the nervous necrosis virus (NNV), is a devastating disease, leading to high mortality rate and huge economical loss in aquaculture. In the past 30 years, many studies on the virus, host responses to the virus infection and diagnostics have yielded a lot of knowledge on developing measures to prevent the VNN disease. Although genetic improvement of disease resistance through breeding is inheritable and has long-lasting positive effect on aquaculture, it is a very challenging task in conventional selective breeding for improving disease resistance. With the advances in mapping quantitative trait loci (QTL) and genome wide association studies (GWAS) for NNV resistance in aquaculture species, DNA markers and genes associated with NNV resistance have been identified, making the application of marker-assisted selection (MAS) and genomic selection (GS) for NNV resistance possible. A few genes for NNV resistance are identified and are being used for genome editing to rapidly improve NNV resistance. In this review, we present the current knowledge on the NNV, host responses to NNV infection, diagnostic methods and vaccines available for NNV disease. In addition, we summarize the current status of conventional and molecular breeding for NNV resistance and highlight future directions, including genome editing for improving NNV resistance in aquaculture. [ABSTRACT FROM AUTHOR]

Background: Oil palm (Elaeis guineensis, Jacq.) is the most important source of edible oil. The improvement of oil yield is currently slow in conventional breeding programs due to long generation intervals. Marker-assisted selection (MAS) has the potential to accelerate genetic improvement. To identify DNA markers associated with oil content traits for MAS, we performed quantitative trait loci (QTL) mapping using genotyping by sequencing (GBS) in a breeding population derived from a cross between Deli Dura and Ghana Pisifera, containing 153 F1 trees. Results: We constructed a high-density linkage map containing 1357 SNPs and 123 microsatellites. The 16 linkage groups (LGs) spanned 1527 cM, with an average marker space of 1.03 cM. One significant and three suggestive QTL for oil to bunch (O/B) and oil to dry mesocarp (O/DM) were mapped on LG1, LG8, and LG10 in a F1 breeding population, respectively. These QTL explained 7.6-13.3% of phenotypic variance. DNA markers associated with oil content in these QTL were identified. Trees with beneficial genotypes at two QTL for O/B showed an average O/B of 30.97%, significantly (P < 0.01) higher than that of trees without any beneficial QTL genotypes (average O/B of 28.24%). QTL combinations showed that the higher the number of QTL with beneficial genotypes, the higher the resulting average O/B in the breeding population. Conclusions: A linkage map with 1480 DNA markers was constructed and used to identify QTL for oil content traits. Pyramiding the identified QTL with beneficial genotypes associated with oil content traits using DNA markers has the potential to accelerate genetic improvement for oil yield in the breeding population of oil palm. [ABSTRACT FROM AUTHOR]

Background: A high-density genetic map is essential for de novo genome assembly, fine mapping QTL for important complex traits, comparative genomic studies and understanding the mechanisms of genome evolution. Although a number of genomic resources are available in Asian seabass (Lates calcarifer), a high-density linkage map is still lacking. To facilitate QTL mapping for marker-assisted selection and genome assembly, and to understand the genome-wide recombination rates, we constructed high density linkage maps using three families and genotyping by sequencing. Results: A high-density consensus linkage map consisting of 8, 274 markers was constructed based on sex-averaged genetic maps. The genetic maps were then aligned and integrated with the current genome assembly of Asian seabass. More than 90% of the genome contig sequences were anchored onto the consensus genetic map. Evidence of assembly errors in the current genome assembly was identified. A fragment of up to 2.5 Mb belonging to LG14 was assembled into Chr15. The length of family-specific sex-averaged maps ranged from 1348.96 to 1624.65 cM. Female maps were slightly longer than male maps using common markers. Female-to-male ratios were highly variable both across chromosomes within each family and throughout three families for each chromosome. However, the distribution patterns of recombination along chromosomes were similar between sexes across the whole genome. The overall recombination rates were significantly correlated with genome-wide GC content and the correlations were revealed to be stronger in females than in males. Conclusions: These high-density genetic maps provide not only essential tools for facilitating de novo genome assembly and comparative genomic studies in teleosts, but also critical resources for fine mapping QTL and genome-wide association mapping for economically important traits in Asian seabass. [ABSTRACT FROM AUTHOR]

Oil palm is the world's leading source of vegetable oil and fat. Dura, Pisifera and Tenera are three forms of oil palm. The genome sequence of Pisifera is available whereas the Dura form has not been sequenced yet. We sequenced the genome of one elite Dura palm, and re-sequenced 17 palm genomes. The assemble genome sequence of the elite Dura tree contained 10,971 scaffolds and was 1.701Gb in length, covering 94.49% of the oil palm genome. 36,105 genes were predicted. Re-sequencing of 17 additional palm trees identified 18.1 million SNPs. We found high genetic variation among palms from different geographical regions, but lower variation among Southeast Asian Dura and Pisifera palms. We mapped 10,000 SNPs on the linkage map of oil palm. In addition, high linkage disequilibrium (LD) was detected in the oil palms used in breeding populations of Southeast Asia, suggesting that LD mapping is likely to be practical in this important oil crop. Our data provide a valuable resource for accelerating genetic improvement and studying the mechanism underlying phenotypic variations of important oil palm traits. [ABSTRACT FROM AUTHOR]

Growth is an important trait in animal breeding. However, the genetic effects underpinning fish growth variability are still poorly understood. QTL mapping and analysis of candidate genes are effective methods to address this issue. We conducted a genome-wide QTL analysis for growth in tilapia. A total of 10, 7 and 8 significant QTLs were identified for body weight, total length and standard length at 140 dph, respectively. The majority of these QTLs were sex-specific. One major QTL for growth traits was identified in the sex-determining locus in LG1, explaining 71.7%, 67.2% and 64.9% of the phenotypic variation (PV) of body weight, total length and standard length, respectively. In addition, a candidate gene GHR2 in a QTL was significantly associated with body weight, explaining 13.1% of PV. Real-time qPCR revealed that different genotypes at the GHR2 locus influenced the IGF-1 expression level. The markers located in the major QTL for growth traits could be used in marker-assisted selection of tilapia. The associations between GHR2 variants and growth traits suggest that the GHR2 gene should be an important gene that explains the difference in growth among tilapia species. [ABSTRACT FROM AUTHOR]

Resistance to pathogens is important for the sustainability and profitability of food fish production. In immune-related genes, the lipopolysaccharide-binding protein (LBP) gene is an important mediator of the inflammatory reaction. We analyzed the cDNA and genomic structure of the LBP gene in tilapia. The full-length cDNA (1901 bp) of the gene contained a 1416 bp open reading frame, encoding 471 amino acid residues. Its genomic sequence was 5577 bp, comprising 15 exons and 14 introns. Under normal conditions, the gene was constitutively expressed in all examined tissues. The highest expression was detected in intestine and kidney. We examined the responses of the gene to challenges with two bacterial pathogens Streptcoccus agalactiae and Aeromonas hydrophila. The gene was significantly upregulated in kidney and spleen post-infection with S. agalactiae and A. hydrophila, respectively. However, the expression profiles of the gene after the challenge with the two pathogens were different. Furthermore, we identified three SNPs in the gene. There were significant associations (p < 0.05) of two of the three SNPs with the resistance to A. hydrophila, but not with the resistance to S. agalactiae or growth performance. These results suggest that the LBP gene is involved in the acute-phase immunologic response to the bacterial infections, and the responses to the two bacterial pathogens are different. The two SNPs associated with the resistance to A. hydrophila may be useful in the selection of tilapia resistant to A. hydrophila. [ABSTRACT FROM AUTHOR]

Background Starvation not only affects the nutritional and health status of the animals, but also the microbial composition in the host's intestine. Next-generation sequencing provides a unique opportunity to explore gut microbial communities and their interactions with hosts. However, studies on gut microbiomes have been conducted predominantly in humans and land animals. Not much is known on gut microbiomes of aquatic animals and their changes under changing environmental conditions. To address this shortcoming, we determined the microbial gene catalogue, and investigated changes in the microbial composition and host-microbe interactions in the intestine of Asian seabass in response to starvation. Results We found 33 phyla, 66 classes, 130 orders and 278 families in the intestinal microbiome. Proteobacteria (48.8%), Firmicutes (15.3%) and Bacteroidetes (8.2%) were the three most abundant bacteria taxa. Comparative analyses of the microbiome revealed shifts in bacteria communities, with dramatic enrichment of Bacteroidetes, but significant depletion of Betaproteobacteria in starved intestines. In addition, significant differences in clusters of orthologous groups (COG) functional categories and orthologous groups were observed. Genes related to antibiotic activity in the microbiome were significantly enriched in response to starvation, and host genes related to the immune response were generally up-regulated. Conclusions This study provides the first insights into the fish intestinal microbiome and its changes under starvation. Further detailed study on interactions between intestinal microbiomes and hosts under dynamic conditions will shed new light on how the hosts and microbes respond to the changing environment. [ABSTRACT FROM AUTHOR]

Background: Aquaculture is the quickest growing sector in agriculture. However, QTL for important traits have been only identified in a few aquaculture species. We conducted QTL mapping for growth traits in an Asian seabass F2 family with 359 individuals using 123 microsatellites and 22 SNPs, and performed association mapping in four populations with 881 individuals. Results: Twelve and nine significant QTL, as well as 14 and 10 suggestive QTL were detected for growth traits at six and nine months post hatch, respectively. These QTL explained 0.9-12.0% of the phenotypic variance. For body weight, two QTL intervals at two stages were overlapped while the others were mapped onto different positions. The IFABP-a gene located in a significant QTL interval for growth on LG5 was cloned and characterized. A SNP in exon 3 of the gene was significantly associated with growth traits in different populations. Conclusions: The results of QTL mapping for growth traits suggest that growth at different stages was controlled by some common QTL and some different QTL. Positional candidate genes and association mapping suggest that the IFABP-a is a strong candidate gene for growth. Our data supply a basis for fine mapping QTL, marker-assisted selection and further detailed analysis of the functions of the IFABP-a gene in fish growth. [ABSTRACT FROM AUTHOR]

Background: Tilapia is the common name for a group of cichlid fishes and is one of the most important aquacultured freshwater food fish. Mozambique tilapia and its hybrids, including red tilapia are main representatives of salt tolerant tilapias. A linkage map is an essential framework for mapping QTL for important traits, positional cloning of genes and understanding of genome evolution. Results: We constructed a consensus linkage map of Mozambique tilapia and red tilapia using 95 individuals from two F1 families and 401 microsatellites including 282 EST-derived markers. In addition, we conducted comparative mapping and searched for sex-determining loci on the whole genome. These 401 microsatellites were assigned to 22 linkage groups. The map spanned 1067.6 cM with an average inter-marker distance of 3.3 cM. Comparative mapping between tilapia and stickleback, medaka, pufferfish and zebrafish revealed clear homologous relationships between chromosomes from different species. We found evidence for the fusion of two sets of two independent chromosomes forming two new chromosome pairs, leading to a reduction of 24 chromosome pairs in their ancestor to 22 pairs in tilapias. The XY sex determination locus in Mozambique tilapia was mapped on LG1, and verified in five families containing 549 individuals. The major XY sex determination locus in red tilapia was located on LG22, and verified in two families containing 275 individuals. Conclusions: A first-generation linkage map of salt tolerant tilapia was constructed using 401 microsatellites. Two separate fusions of two sets of two independent chromosomes may lead to a reduction of 24 chromosome pairs in their ancestor to 22 pairs in tilapias. The XY sex-determining loci from Mozambique tilapia and red tilapia were mapped on LG1 and LG22, respectively. This map provides a useful resource for QTL mapping for important traits and comparative genome studies. The DNA markers linked to the sex-determining loci could be used in the selection of YY males for breeding all-male populations of salt tolerant tilapia, as well as in studies on mechanisms of sex determination in fish. [ABSTRACT FROM AUTHOR]

Traceability through physical labels is well established, but it is not highly reliable as physical labels can be easily changed or lost. Application of DNA markers to the traceability of food plays an increasingly important role for consumer protection and confidence building. In this study, we tested the efficiency of 16 polymorphic microsatellites and their combinations for tracing 368 fish to four populations where they originated. Using the maximum likelihood and Bayesian methods, three most efficient microsatellites were required to assign over 95% of fish to the correct populations. Selection of markers based on the assignment score estimated with the software WHICHLOCI was most effective in choosing markers for individual assignment, followed by the selection based on the allele number of individual markers. By combining rapid DNA extraction, and high-throughput genotyping of selected microsatellites, it is possible to conduct routine genetic traceability with high accuracy in Asian seabass. [ABSTRACT FROM AUTHOR]

In aquaculture species, maintaining pedigree information and genetic variation in each generation is essential, but very difficult. In this study, we used nine microsatellites to genotype 2,520 offspring from four independent full-factorial crosses (10 males × 10 females) of Asian seabass to reconstruct pedigree and monitor the change of genetic variations. In all four crosses, over 96.8% of the offspring could be assigned to their parents, indicating the high power of the nine microsatellites for parentage assignment. This study revealed several interesting results: (1). In all four crosses, the contribution of parents to offspring was significantly uneven, and some dominant breeding fishes (i.e. brooders) were found; (2). In two mass crosses where the brooders were carefully checked for reproductive status, a majority (⩾90%) of brooders contributed to offspring, whereas in another two crosses, where the brooders were randomly picked without checking reproductive status, only a few brooders (40.0-45.0%) produced offspring; (3). Females had more problems in successful spawning compared to males; and (4). In the two crosses where a few brooders produced offspring, there was a substantial loss in allelic (24.1- 34.3%) and gene (20.5-25.7%) diversities in offspring, while in the other two crosses, the majority of allelic (96.8-97.0%) and gene diversities (94.8-97.1%) were maintained. These observations suggest that a routine molecular parentage analysis is required to maintain both allelic and gene diversity in breeding Asian seabass. [ABSTRACT FROM AUTHOR]

Background MicroRNAs (miRNAs) play an important role in the regulation of many fundamental biological processes. So far miRNAs have been only identified in a few fish species, although there are over 30,000 fish species living under different environmental conditions on the earth. Here, we described an approach to identify conserved miRNAs and characterized their expression patterns in different tissues for the first time in a food fish species Asian seabass (Lates calcarifer). Methodology/Principal Findings By combining a bioinformatics analysis with an approach of homolog-based PCR amplification and sequencing, 63 novel miRNAs belonging to 29 conserved miRNA families were identified. Of which, 59 miRNAs were conserved across 10-86 species (E value=10-4) and 4 miRNAs were conserved only in fish species. qRT-PCR analysis showed that miR-29, miR-103, miR-125 and several let-7 family members were strongly and ubiquitously expressed in all tissues tested. Interestingly, miR-1, miR-21, miR-183, miR-184 and miR-192 showed highly conserved tissue-specific expression patterns. Exposure of the Asian seabass to lipopolysaccharide (LPS) resulted in up-regulation of over 50% of the identified miRNAs in spleen suggesting the importance of the miRNAs in acute inflammatory immune responses. Conclusions/Significance The approach used in this study is highly effective for identification of conserved miRNAs. The identification of 63 miRNAs and determination of the spatial expression patterns of these miRNAs are valuable resources for further studies on post-transcriptional gene regulation in Asian seabass and other fish species. Further identification of the target genes of these miRNAs would shed new light on their regulatory roles of microRNAs in fish. [ABSTRACT FROM AUTHOR]

Background: High density linkage maps are essential for comparative analysis of synteny, fine mapping of quantitative trait loci (QTL), searching for candidate genes and facilitating genome sequence assembly. However, in most foodfish species, marker density is still low. We previously reported a first generation linkage map with 240 DNA markers and its application to preliminarily map QTL for growth traits in Asian seabass (Lates calcarifer). Here, we report a high-resolution linkage map with 790 microsatellites and SNPs, comparative analysis of synteny, finemapping of QTL and the identification of potential candidate genes for growth traits. Results: A second generation linkage map of Asian seabass was developed with 790 microsatellite and SNP markers. The map spanned a genetic length of 2411.5 cM, with an average intermarker distance of 3.4 cM or 1.1 Mb. This high density map allowed for comparison of the map with Tetraodon nigroviridis genome, which revealed 16 synteny regions between the two species. Moreover, by employing this map we refined QTL to regions of 1.4 and 0.2 cM (or 400 and 50 kb) in linkage groups 2 and 3 in a population containing 380 progeny; potential candidate genes for growth traits in QTL regions were further identified using comparative genome analysis, whose effects on growth traits were investigated. Interestingly, a QTL cluster at Lca371 underlying growth traits of Asian seabass showed similarity to the cathepsin D gene of human, which is related to cancer and Alzheimer's disease. Conclusions: We constructed a high resolution linkage map, carried out comparative mapping, refined the positions of QTL, identified candidate genes for growth traits and analyzed their effects on growth. Our study developed a framework that will be indispensable for further identification of genes and analysis of molecular variation within the refined QTL to enhance understanding of the molecular basis of growth and speed up genetic improvement of growth performance, and it also provides critical resource for future genome sequence assembly and comparative genomics studies on the evolution of fish genomes. [ABSTRACT FROM AUTHOR]

Background: Molecular genetic analyses of parentage provide insights into mating systems. Although there are 22,000 members in Malacostraca, not much has been known about mating systems in Malacostraca. The freshwater shrimp Caridina ensifera blue, is a new species belonging to Malacostraca which was discovered recently in Sulawesi, Indonesia. Due to its small body size and low fecundity, this species is an ideal species to study the occurrence and frequency of multiple paternity and to understand of how the low fecundity species persist and evolve. Methodology/Principal Findings: In this study, we developed four polymorphic microsatellites from C. ensifera and applied them to investigate the occurrence and frequency of multiple paternity in 20 C. ensifera broods caught from Lake Matano, Sulawesi. By genotyping the mother and all offspring from each brood we discovered multiple paternity in all 20 broods. In most of the 20 broods, fathers contributed skewed numbers of offspring and there was an apparent inverse correlation between reproductive success of sires and their relatedness to mothers. Conclusions/Significance: Our results in combination with recent reports on multiple paternity in crayfish, crab and lobster species suggests that multiple paternity is common in Malacostraca. Skewed contribution of fathers to the numbers of offspring and inverse correlation between reproductive success of sires and their relatedness to mothers suggest that sperm competition occurred and/or pre- and postcopulatory female choice happen, which may be important for avoiding the occurrence of inbreeding and optimize genetic variation in offspring and for persistence and evolution of low fecundity species. [ABSTRACT FROM AUTHOR]

Background: The Asian seabass (Lates calcarifer) is an important marine foodfish species in Southeast Asia and Australia. Genetic improvement of this species has been achieved to some extent through selective breeding programs since 1990s. Several genomic tools such as DNA markers, a linkage map, cDNA and BAC libraries have been developed to assist selective breeding. A physical map is still lacking, although it is essential for positional cloning of genes located in quantitative trait loci (QTL) and assembly of whole genome sequences. Methodology/Principal Findings: A genome-wide physical map of the Asian seabass was constructed by restriction fingerprinting of 38,208 BAC clones with SNaPshot HICF FPC technique. A total of 30,454 were assembled into 2,865 contigs. The physical length of the assembled contigs summed up to 665 Mb. Analyses of some contigs using different methods demonstrated the reliability of the assembly. Conclusions/Significance: The present physical map is the first physical map for Asian seabass. This physical map will facilitate the fine mapping of QTL for economically important traits and the positional cloning of genes located in QTL. It will also be useful for the whole genome sequencing and assembly. Detailed information about BAC-contigs and BAC clones are available upon request. [ABSTRACT FROM AUTHOR]

Background: Fish diseases caused by pathogens are limiting their production and trade, affecting the economy generated by aquaculture. Innate immunity system is the first line of host defense in opposing pathogenic organisms or any other foreign material. For identification of immune-related genes in Asian seabass Lates calcarifer, an important marine foodfish species, we injected bacterial lipopolysaccharide (LPS), a commonly used elicitor of innate immune responses to eight individuals at the age of 35 days post-hatch and applied the suppression subtractive hybridization (SSH) technique to selectively amplify spleen cDNA of differentially expressed genes. Results: Sequencing and bioinformatic analysis of 3351 ESTs from two SSH libraries yielded 1692 unique transcripts. Of which, 618 transcripts were unknown/novel genes and the remaining 1074 were similar to 743 known genes and 105 unannotated mRNA sequences available in public databases. A total of 161 transcripts were classified to the category "response to stimulus" and 115 to "immune system process". We identified 25 significantly up-regulated genes (including 2 unknown transcripts) and 4 down-regulated genes associated with immune-related processes upon challenge with LPS. Quantitative real-time PCR confirmed the differential expression of these genes after LPS challenge. Conclusions: The present study identified 1692 unique transcripts upon LPS challenge for the first time in Asian seabass by using SSH, sequencing and bioinformatic analysis. Some of the identified transcripts are vertebrate homologues and others are hitherto unreported putative defence proteins. The obtained immune-related genes may allow for a better understanding of immunity in Asian seabass, carrying out detailed functional analysis of these genes and developing strategies for efficient immune protection against infections in Asian seabass. [ABSTRACT FROM AUTHOR]

Background: Grass carp (Ctenopharyngodon idella) belongs to the family Cyprinidae which includes more than 2000 fish species. It is one of the most important freshwater food fish species in world aquaculture. A linkage map is an essential framework for mapping traits of interest and is often the first step towards understanding genome evolution. The aim of this study is to construct a first generation genetic map of grass carp using microsatellites and SNPs to generate a new resource for mapping QTL for economically important traits and to conduct a comparative mapping analysis to shed new insights into the evolution of fish genomes. Results: We constructed a first generation linkage map of grass carp with a mapping panel containing two F1 families including 192 progenies. Sixteen SNPs in genes and 263 microsatellite markers were mapped to twentyfour linkage groups (LGs). The number of LGs was corresponding to the haploid chromosome number of grass carp. The sex-specific map was 1149.4 and 888.8 cM long in females and males respectively whereas the sexaveraged map spanned 1176.1 cM. The average resolution of the map was 4.2 cM/locus. BLAST searches of sequences of mapped markers of grass carp against the whole genome sequence of zebrafish revealed substantial macrosynteny relationship and extensive colinearity of markers between grass carp and zebrafish. Conclusions: The linkage map of grass carp presented here is the first linkage map of a food fish species based on co-dominant markers in the family Cyprinidae. This map provides a valuable resource for mapping phenotypic variations and serves as a reference to approach comparative genomics and understand the evolution of fish genomes and could be complementary to grass carp genome sequencing project. [ABSTRACT FROM AUTHOR]

Background: Microsatellites in cDNA are useful as molecular markers because they represent transcribed genes and can be used as anchor markers for linkage and comparative mapping, as well as for studying genome evolution. Microsatellites in cDNA can be detected in existing ESTs by data mining. However, in most fish species, no ESTs are available or the number of ESTs is limited, although fishes represent half of the vertebrates on the earth. We developed a simple and efficient method for isolation of microsatellites from cDNA in fish. Results: The method included normalization of 150 ng cDNA using 0.5 U duplex-specific nuclease (DSN) at 65°C for 30 min, enrichment of microsatellites using biotinylated oligonucleotides and magnetic field, and directional cloning of cDNA into a vector. We tested this method to enrich CAand GA-microsatellites from cDNA of Asian seabass, and demonstrated that enrichment of microsatellites from normalized cDNA could increased the efficiency of microsatellite isolation over 30 times as compared to direct sequencing of clones from cDNA libraries. One hundred and thirty-nine (36.2%) out of 384 clones from normalized cDNA contained microsatellites. Unique microsatellite sequences accounted for 23.6% (91/384) of sequenced clones. Sixty microsatellites isolated from cDNA were characterized, and 41 were polymorphic. The average allele number of the 41 microsatellites was 4.85 ± 0.54, while the expected heterozygosity was 0.56 ± 0.03. All the isolated microsatellites inherited in a Mendelian pattern. Conclusion: Normalization of cDNA substantially increased the efficiency of enrichment of microsatellites from cDNA. The described method for isolation of microsatellites from cDNA has the potential to be applied to a wide range of fish species. The microsatellites isolated from cDNA could be useful for linkage and comparative mapping, as well as for studying genome evolution. [ABSTRACT FROM AUTHOR]

Background: Barramundi (Lates calcarifer) is an important farmed marine food fish species. Its first generation linkage map has been applied to map QTL for growth traits. To identify genes located in QTL responsible for specific traits, genomic large insert libraries are of crucial importance. We reported herein a bacterial artificial chromosome (BAC) library and the mapping of BAC clones to the linkage map. Results: This BAC library consisted of 49,152 clones with an average insert size of 98 kb, representing 6.9-fold haploid genome coverage. Screening the library with 24 microsatellites and 15 ESTs/genes demonstrated that the library had good genome coverage. In addition, 62 novel microsatellites each isolated from 62 BAC clones were mapped onto the first generation linkage map. A total of 86 BAC clones were anchored on the linkage map with at least one BAC clone on each linkage group. Conclusion: We have constructed the first BAC library for L. calcarifer and mapped 86 BAC clones to the first generation linkage map. This BAC library and the improved linkage map with 302 DNA markers not only supply an indispensable tool to the integration of physical and linkage maps, the fine mapping of QTL and map based cloning genes located in QTL of commercial importance, but also contribute to comparative genomic studies and eventually whole genome sequencing. [ABSTRACT FROM AUTHOR]

The fresh water pufferfish Tetraodon nigroviridis is a model organism for studying evolution of genome and gene functions, but its mitochondrial genome (mtDNA) sequence is still not available. We determined the complete nucleotide sequence of its mtDNA using shotgun sequencing. The T. nigroviridis mtDNA was 16,462 bp, and contained 13 protein coding genes, 22 tRNAs, 2 rRNAs and a major non-coding region. The gene order was identical to the common type of vertebrate mrDNA, whereas the G + C content in the sense strand was 46.9%, much higher than most other fish species. One hundred and three SNPs were detected in the control region of the mtDNA of 35 individuals, a majority of SNPs were detected in the 5′ end of the control region. A phylogenetic study including 21 fish species was performed on concatenated amino acid sequences of 12 protein coding genes, and revealed that the T. nigroviridis was clustered with Fugu rubripes into a group. The complete mtDNA sequence and SNPs in its control region will be useful in studying fish evolution, in differentiating different Tetraodon species and in analyzing genetic diversity within T. nigroviridis. [ABSTRACT FROM AUTHOR]

The golden pompano (Trachinotus blochii) is a marine fish species in tropical regions. No information about genetic variation and population structure of wild populations is available. A first set of 12 polymorphic microsatellites isolated from this species were characterized. The number of alleles ranged from 5 to 14 with an average of 8.0 alleles per locus. All 12 markers conformed to HWE and were in linkage equilibrium. These 12 microsatellite markers supply the first set of co-dominant DNA markers for studying population genetics of the species T. blochii. [ABSTRACT FROM AUTHOR]

Twelve novel polymorphic microsatellites were isolated from the endangered Hawksbill turtle (Eretmochelys imbricate). Eight of 12 markers were used to study genetic diversity of two sea turtle species: E. imbricate and green sea turtle (C. mydas). In E. imbricate, the average allele number of the eight microsatellites was 6.25/locus with a range of 3-13. The average expected and observed heterozygosity was 0.66 and 0.63 respectively. In C. mydas, the average allele number of the eight markers was 11.63/locus. The observed heterozyosity (0.68) was lower than the expected heterozyosity (0.79). Most of 12 microsatellites amplified specific and polymorphic PCR products in other six turtle species. Hence, the developed microsatellites would facilitate studies on genetic diversity and population structure of E. imbricate and other marine turtle species. [ABSTRACT FROM AUTHOR]

Eleven microsatellites were isolated from a genomic DNA library enriched for CA- and GA-repeats, and were characterized in 48 individuals of red swamp crayfish Procambarus clarkii. All 11 microsatellites were polymorphic with an average of allele number of 6.6 per locus. Genotypic distributions of these 11 markers were found to be in conformance with the expectations of Hardy–Weinberg equilibrium. All 11 markers were unlinked. These markers are being used to study the invasion routine, genetic diversity and population structure of the species P. clarkii. [ABSTRACT FROM AUTHOR]

Asian green mussels ( Perna viridis) belonging to the family Mytilidae are native to the coastal and tropical marine waters of the Indo-Pacific region. Ten polymorphic microsatellites were isolated and their polymorphisms were determined in 36 individuals. The average allele number of the 10 microsatellites was 11.7/locus with a range of two to 24 and the expected heterozygosity averaged at 0.69, ranging from 0.18 to 0.94. Eight out of 10 microsatellites agreed with the Hardy–Weinberg equilibrium and showed independent segregation. These microsatellites will facilitate the studying of the genetic diversity and population structure of the green mussel in and outside of the Indo-Pacific region of Asia. [ABSTRACT FROM AUTHOR]

Here we report the isolation and characterization of 17 polymorphic loci isolated from a partial genomic DNA library of grass carp ( Ctenopharyngodon idellus) enriched for CA repeats. We tested variability of these microsatellites on 24 unrelated individuals collected in China. All microsatellites were polymorphic. The average allele number was 7.9 per locus, ranging from four to 13. The observed heterozygosity was from 0.46 to 0.88 with an average of 0.71, whereas the average expected heterozygosity was 0.78. Sixteen of the 17 microsatellites conformed to Hardy–Weinberg equilibrium, and inherited independently. These microsatellites can be used to study genetic diversity and population structure of wild populations, and facilitate selective breeding of cultured broodstocks. [ABSTRACT FROM AUTHOR]

The ribbonfish, Trichiurus haumela, is an important marine foodfish species. Supply of this fish relies on capture from the wild. No information about genetic diversity and population structure of wild stocks is available. Therefore, we developed the first set of 10 polymorphic microsatellites. The average allele number was 13.2 per locus ranging from six to 20. The expected heterozygosity was 0.84, whereas the observed heterozygosity was 0.71. This set of microsatellites provides a tool for assessing genetic diversity and population structure of this species. [ABSTRACT FROM AUTHOR]

Eleven polymorphic microsatellites were isolated from an important foodfish species, silver pomfret ( Pampus argenteus). The loci showed different variation patterns in 24 unrelated P. argenteus individuals, with a mean number of alleles of 11.5 and a mean expected heterozygosity of 0.85. Eight of the 11 loci conformed to Hardy–Weinberg equilibrium. All and six of the 11 markers amplified specific and polymorphic products in Chinese pomfret ( Pampus sinesis) and dark pomfret ( Pamus cinereus), respectively. These markers should be useful for population genetic analysis and biodiversity studies of P. argenteus and other pomfret species . [ABSTRACT FROM AUTHOR]

Crimson red snapper ( Lutjanus erythropterus) is an important marine foodfish species in Asia with great potentials for aquaculture. We isolated nine polymorphic microsatellites, among which eight were tetranucleotide repeats, and one was CA-microsatellite. The average allele number present in 48 individuals was 11.1/locus, ranging from three to 33. The expected heterozygosity ranged from 0.49 to 0.97 with an average of 0.74. Six of the nine markers conformed to the Hardy–Weinberg expectations. No pairwise markers showed the possibility of linkage. Protocols for two multiplex polymerase chain reactions (PCRs), each amplifying two and seven markers, respectively, were presented. The developed microsatellites and optimized multiplex PCRs will be useful for studying population structure of wild stocks and parentage assignment for cultured populations. [ABSTRACT FROM AUTHOR]

The green swordtail ( Xiphophorus hellerii; Poeciliidae) is a popular ornamental freshwater fish species. Fourteen microsatellites were isolated from a green swordtail genomic library enriched for CA-repeats. Thirteen microsatellites were polymorphic in green swordtail; interestingly four of them were tetranucleotide-type. Cross-species amplification showed that 10 of the 14 microsatellites were polymorphic in guppy ( Poecilia reticulata; Poeciliidae) as well. No significant directional difference of allele length was seen between the two species at any of the loci tested. [ABSTRACT FROM AUTHOR]

The goldfish ( Carassius auratus auratus; Cyprinidae) is not only an important ornamental fish species, but also a useful model for biological studies. The sequence of goldfish genes present in the public database was searched for short tandem repeats, and 11 polymorphic microsatellites were detected within eight important genes. Two microsatellites were located in coding regions of the c- myc and GAP-43 genes, respectively, whereas the others were mainly located in 5′ and 3′ untranscribed regions of other genes, such as gonadotropin and activin. The average allele number of these microsatellites was 5.5 per locus with a range between 3 and 9. These microsatellites will be useful for ecological and population genetic studies of this species, as well as for the ornamental fish industry. [ABSTRACT FROM AUTHOR]

Movement of individuals influences individual reproductive success, fitness, genetic diversity and relationships among individuals within populations and gene exchange among populations. Competition between males or females for mating opportunities and/or local resources predicts a female bias in taxa with monogamous mating systems and a male-biased dispersal in polygynous species. In birds and mammals, the patterns of dispersal between sexes are well explored, while dispersal patterns in protandrous hermaphroditic fish species have not been studied. We collected 549 adult individuals of Asian seabass (Lates calcarifer) from four locations in the South China Sea. To assess the difference in patterns of dispersal between sexes, we genotyped all individuals with 18 microsatellites. Significant genetic differentiation was detected among and within sampling locations. The parameters of population structure (FST), relatedness (r) and the mean assignment index (mAIC), in combination with data on tagging-recapture, supplied strong evidences for female-biased dispersal in the Asian seabass. This result contradicts our initial hypothesis of no sex difference in dispersal. We suggest that inbreeding avoidance of females, female mate choice under the condition of low mate competition among males, and male resource competition create a female-biased dispersal. The bigger body size of females may be a cause of the female-biased movement. Studies of dispersal using data from DNA markers and tagging-recapture in hermaphroditic fish species could enhance our understanding of patterns of dispersal in fish. [ABSTRACT FROM AUTHOR]