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The nervous necrosis virus (NNV) causes the viral nervous necrosis (VNN) disease in aquatic animals and has been a major threat in aquaculture. Thus, it is essential for the development of a prevention method to minimize economic losses caused by NNV such as the identification of NNV resistance genes and application of these genes in molecular breeding to increase disease resistance. gab3 is an important NNV resistance gene in Asian seabass. However, the mechanism of gab3 in NNV resistance has not been elucidated. In this study, knockdown of gab3 in NNV-infected Asian seabass cells resulted in a significant decrease in viral RNA and virus titers. Knockout of gab3 in zebrafish led to an increased survival rate and resistant time after NNV infection. Cellular localization of the GAB3 and NNV by immunofluorescence staining showed that the GAB3 was translocated from the nucleus to the cytoplasm, and finally reached the cell membrane of SB cells after 48 h post NNV infection. Our study suggests that gab3 plays an important role in NNV replication and silencing gab3 can inhibit virus replication.

Understanding of the relationships between genotypes and phenotypes is a central problem in biology. Although teleosts have colorful phenotypes, not much is known about their underlying mechanisms. Our previous study showed that golden skin color in Mozambique tilapia was mapped in the major locus containing the Pmel gene, and an insertion in 3' UTR of Pmel17 was fully correlated with the golden color. However, the molecular mechanism of how Pmel17 determines the golden skin color is unknown. In this study, knockout of Pmel17 with CRISPR/Cas9 in blackish tilapias resulted in golden coloration, and rescue of Pmel17 in golden tilapias recovered the wild-type blackish color, indicating that Pmel17 is the gene determining the golden and blackish color. Functional analysis in vitro showed that the insertion in the 3' UTR of Pmel17 reduced the transcripts of Pmel17. Our data supplies more evidence to support that Pmel17 is the gene for blackish and golden colors, and highlights that the insertion in the 3' UTR of Pmel17 is the causative mutation for the golden coloration.

Viral nervous necrosis (VNN) disease caused by the nervous necrosis virus (NNV) is a major disease, leading to a huge economic loss in aquaculture. Previous GWAS and QTL mapping have identified a major QTL for NNV resistance in linkage group 20 in Asian seabass. However, no causative gene for NNV resistance has been identified. In this study, RNA-seq from brains of Asian seabass fingerlings challenged with NNV at four time points (5, 10, 15 and 20 days post-challenge) identified 1228, 245, 189 and 134 DEGs, respectively. Eight DEGs, including rrm1, were located in the major QTL for NNV resistance. An association study in 445 survived and 608 dead fingerlings after NNV challenge revealed that the SNP in rrm1 were significantly associated with NNV resistance. Therefore, rrm1 was selected for functional analysis, as a candidate gene for NNV resistance. The expression of rrm1 was significantly increased in the gill, liver, spleen and muscle, and was suppressed in the brain, gut and skin after NNV challenge. The rrm1 protein was localized in the nuclear membrane. Over-expression of rrm1 significantly decreased viral RNA and titer in NNV-infected Asian seabass cells, whereas knock-down of rrm1 significantly increased viral RNA and titer in NNV-infected Asian seabass cells. The rrm1 knockout heterozygous zebrafish was more susceptible to NNV infection. Our study suggests that rrm1 is one of the causative genes for NNV resistance and the SNP in the gene may be applied for accelerating genetic improvement for NNV resistance.

16 pages, 6 figures, supplementary Information https://doi.org/10.1186/s12915-021-01205-y.-- Availability of data and materials: Sequences used for RAD, RNA, and genome sequencing are achieved in the DDBJ Sequencing Read Archive (SRA) database under BioProject ID: PRJDB7253- PRJDB7255 [23, 88], Background. Fishes are the one of the most diverse groups of animals with respect to their modes of sex determination, providing unique models for uncovering the evolutionary and molecular mechanisms underlying sex determination and reversal. Here, we have investigated how sex is determined in a species of both commercial and ecological importance, the Siamese fighting fish Betta splendens. Results. We conducted association mapping on four commercial and two wild populations of B. splendens. In three of the four commercial populations, the master sex determining (MSD) locus was found to be located in a region of ~ 80 kb on LG2 which harbours five protein coding genes, including dmrt1, a gene involved in male sex determination in different animal taxa. In these fish, dmrt1 shows a male-biased gonadal expression from undifferentiated stages to adult organs and the knockout of this gene resulted in ovarian development in XY genotypes. Genome sequencing of XX and YY genotypes identified a transposon, drbx1, inserted into the fourth intron of the X-linked dmrt1 allele. Methylation assays revealed that epigenetic changes induced by drbx1 spread out to the promoter region of dmrt1. In addition, drbx1 being inserted between two closely linked cis-regulatory elements reduced their enhancer activities. Thus, epigenetic changes, induced by drbx1, contribute to the reduced expression of the X-linked dmrt1 allele, leading to female development. This represents a previously undescribed solution in animals relying on dmrt1 function for sex determination. Differentiation between the X and Y chromosomes is limited to a small region of ~ 200 kb surrounding the MSD gene. Recombination suppression spread slightly out of the SD locus. However, this mechanism was not found in the fourth commercial stock we studied, or in the two wild populations analysed, suggesting that it originated recently during domestication. Conclusions. Taken together, our data provide novel insights into the role of epigenetic regulation of dmrt1 in sex determination and turnover of SD systems and suggest that fighting fish are a suitable model to study the initial stages of sex chromosome evolution, This study was supported by the internal funding of the Temasek Life Sciences Laboratory, Singapore, With the institutional support of the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000928-S)

Understanding the functions of genes related to disease resistance and identifying polymorphisms in these genes are essential in molecular breeding for disease resistance. Viral nervous necrosis (VNN) is one of the major diseases in the Asian seabass, Lates calcarifer. Our previous works on QTL mapping, GWAS and cell-line transcriptome analysis of the Asian seabass after NNV challenge revealed that the gene GAB3 might be a candidate gene for VNN resistance. In this study, we cloned and characterized GAB3, and identified SNPs in the gene of the Asian seabass. The cDNA of the gene was 2165 bp, containing an ORF of 1674 bp encoding 557 amino acids. The gene consisted of 10 exons and nine introns. It was ubiquitously expressed in normal fish. An analysis of the association between two SNPs in the second intron and NNV resistance in 1035 fish descended from 43 families revealed that the two SNPs were significantly associated with VNN resistance. After NNV infection, the expression of GAB3 was significantly increased in the brain, spleen, muscle and gut, and was suppressed in the liver. The GAB3 protein was localized in the nucleus. Overexpression of GAB3 with specific GAB3-pcDNA was positively correlated to increased viral RNA and titer in NNV-infected Asian seabass cells. Our study provides new evidence to support that GAB3 may be an important gene related to NNV resistance. In addition, the SNPs provide DNA markers for the selection of candidate genes resistance to NNV at the juvenile stage of Asian seabass.

Background Genetic diversity within a species reflects population evolution, ecology, and ability to adapt. Genome-wide population surveys of both natural and introduced populations provide insights into genetic diversity, the evolutionary processes and the genetic basis underlying local adaptation. Grass carp is the most important freshwater foodfish species for food and water weed control. However, there is as yet no overall picture on genetic variations and population structure of this species, which is important for its aquaculture. Results We used 43,310 SNPs to infer the population structure, evidence of local adaptation and sources of introduction. The overall genetic differentiation of this species was low. The native populations were differentiated into three genetic clusters, corresponding to the Yangtze, Pearl and Heilongjiang River Systems, respectively. The populations in Malaysia, India and Nepal were introduced from both the Yangtze and Pearl River Systems. Loci and genes involved in putative local selection for native locations were identified. Evidence of both positive and balancing selection was found in the introduced locations. Genes associated with loci under putative selection were involved in many biological functions. Outlier loci were grouped into clusters as genomic islands within some specific genomic regions, which likely agrees with the divergence hitchhiking scenario of divergence-with-gene-flow. Conclusions This study, for the first time, sheds novel insights on the population differentiation of the grass carp, genetics of its strong ability in adaption to diverse environments and sources of some introduced grass carp populations. Our data also suggests that the natural populations of the grass carp have been affected by the aquaculture besides neutral and adaptive forces. Electronic supplementary material The online version of this article (10.1186/s12864-019-5872-1) contains supplementary material, which is available to authorized users.

High-density genetic maps are essential for mapping QTL, improving genome assembly, comparative genomics, and studying sex chromosome evolution. The northern snakehead (Channa argus) is an economically important foodfish species with significant sexual dimorphism, where the males grow much faster and bigger than the females. However, to date, the sex determination pattern is still not clear, limiting identification of sex chromosomes, even sex determination genes and development of monosex populations that are valuable for both sex evolution of vertebrates and aquaculture practices. Here, a sex-averaged map and two sex-specific genetic maps were constructed with 2974, 2323, and 2338 SNPs, respectively. Little difference was observed in the pattern of sex-specific recombination between female- and male-specific genetic maps. Genome scan identified a major locus for sex determination at LG16. Females and males are, respectively, homogametic and heterogametic, suggesting an XY sex determination system for this species. By resequencing genomes, InDels in the sex-associated QTL region were discovered and used for developing sex-specific PCR assays for fast sexing of snakehead. These high-density genetic maps provide useful resources for future genomic studies in snakehead and its related species. The PCR assays for sexing are of importance in developing all male populations for aquaculture. This research is supported by the internal fund of the Institute of Fisheries, Hangzhou Academy of Agricultural Science.

Shrimp in culture ponds are challenged by various pathogens as well as harsh water environment. The innate immune system and environmental stress response system of shrimp paly an important role in shrimp survival and growth. For remission the endoplasmic reticulum (ER)-stress caused by environmental stress, unfolded protein response (UPR) may reduce the synthesis of most proteins, including great mass of immune factors, which could weaken the immune function of shrimp. Therefore, how cells keep appropriate amount of immune factor synthesis under such a situation is critical important for shrimp health and growth. In this study, we cloned a new Crustin gene (LvCruU) from Litopenaeus vannamei. We showed that LvCruU has antibacterial activity, and reducing its expression would increase the cumulative mortality of L. vannamei upon the Vibrio parahemolyticus infection. In addition, we found that promoter activity of LvCruU was enhanced not only by the deformed epidermal autoregulatory factor-1 (Deaf1), but also by activating transcription factor 3 (LvATF3) of shrimp UPR. Real-time RT-PCR showed that LvCruU and LvATF3 both were induced upon UPR activation. And moreover, in Thapsigargin plus dsLvCruU injection test, we showed that down-regulation of LvCruU increased the cumulative mortality of V. parahemolyticus-infected shrimp under ER-stress. These results suggest that LvCruU work as a downstream effector of UPR, and contribute to antimicrobic immune response upon ER-stress in L. vannamei.

Ocean acidification is changing the fate of marine organisms. It is essential to predict the biological responses and evolutionary processes driven by ocean acidification, to maintain the equilibrium of the marine ecosystem and to facilitate aquaculture. However, how marine organisms, particularly the marine fish species, respond to ocean acidification, is still poorly understood. Consequences of ocean acidification on finfish aquaculture are largely not well known. We studied the effects of ocean acidification for 7 days on growth, behaviour and gene expression profiles in the brain, gill and kidney of Asian seabass juveniles. Results showed that growth and behaviour were not affected by short-term ocean acidification. We found tissue-specific differentially expressed genes (DEGs) involving many molecular processes, such as organ development, growth, muscle development, ion homeostasis and neurogenesis and development, as well as behaviours. Most of the DEGs, which were functionally enriched in ion homeostasis, were related to calcium transport, followed by sodium/potassium channels. We found that genes associated with neurogenesis and development were significantly enriched, implying that ocean acidification has also adversely affected the neural regulatory mechanism. Our results indicate that although the short-term ocean acidification does not cause obvious phenotypic and behavioural changes, it causes substantial changes of gene expressions in all three analysed tissues. All these changes of gene expressions may eventually affect physiological fitness. The DEGs identified here should be further investigated to discover DNA markers associated with adaptability to ocean acidification to improve fish’s capability to adapt to ocean acidification.

14 pages, 4 figures, supplementary material https://doi.org/10.1093/molbev/msab110.-- Data Availability: The genome sequences and related annotations of fighting fish are hosted by the web server of Temasek Lifesciences Laboratory (https://genhua.tll.org.sg/) and archived in China National GeneBank (CNGB) and DDBJ with BioProject accession nos. CNP0001745 and PRJDB7253, respectively. Sequences used for whole-genome sequencing, RNA sequencing, and RAD sequencing are available with the DDBJ Sequencing Read Archive (SRA) through BioProject ID PRJDB7253–PRJDB7255, Resolving the genomic basis underlying phenotypic variations is a question of great importance in evolutionary biology. However, understanding how genotypes determine the phenotypes is still challenging. Centuries of artificial selective breeding for beauty and aggression resulted in a plethora of colors, long-fin varieties, and hyper-aggressive behavior in the air-breathing Siamese fighting fish (Betta splendens), supplying an excellent system for studying the genomic basis of phenotypic variations. Combining whole-genome sequencing, quantitative trait loci mapping, genome-wide association studies, and genome editing, we investigated the genomic basis of huge morphological variation in fins and striking differences in coloration in the fighting fish. Results revealed that the double tail, elephant ear, albino, and fin spot mutants each were determined by single major-effect loci. The elephant ear phenotype was likely related to differential expression of a potassium ion channel gene, kcnh8. The albinotic phenotype was likely linked to a cis-regulatory element acting on the mitfa gene and the double-tail mutant was suggested to be caused by a deletion in a zic1/zic4 coenhancer. Our data highlight that major loci and cis-regulatory elements play important roles in bringing about phenotypic innovations and establish Bettas as new powerful model to study the genomic basis of evolved changes, This project was supported by the Temasek Lifesciences Laboratory, Singapore, With the institutional support of the ‘Severo OchoaCentre of Excellence’ accreditation (CEX2019-000928-S)

Tilapia is one of most important foodfish species. The low omega-3 to omega-6 fatty acid ratio in freshwater tilapia meat is disadvantageous for human health. Increasing omega-3 content is an important task in breeding to increase the nutritional value of tilapia. However, conventional breeding to increase omega-3 content is difficult and slow. To accelerate the increase of omega-3 through marker-assisted selection (MAS), we conducted QTL mapping for fatty acid contents and profiles in a F2 family of saline tilapia generated by crossing red tilapia and Mozambique tilapia. The total omega-3 content in F2 hybrid tilapia was 2.5 ± 1.0 mg/g, higher than that (2.00 mg/g) in freshwater tilapia. Genotyping by sequencing (GBS) technology was used to discover and genotype SNP markers, and microsatellites were also genotyped. We constructed a linkage map with 784 markers (151 microsatellites and 633 SNPs). The linkage map was 2076.7 cM long and consisted of 22 linkage groups. Significant and suggestive QTL for total lipid content were mapped on six linkage groups (LG3, -4, -6, -8, -13, and -15) and explained 5.8–8.3% of the phenotypic variance. QTL for omega-3 fatty acids were located on four LGs (LG11, -18, -19, and -20) and explained 5.0 to 7.5% of the phenotypic variance. Our data suggest that the total lipid and omega-3 fatty acid content were determined by multiple genes in tilapia. The markers flanking the QTL for omega-3 fatty acids can be used in MAS to accelerate the genetic improvements of these traits in salt-tolerant tilapia. NRF (Natl Research Foundation, S’pore)

It is not fully clear why there is a higher contribution of pluripotent stem cells (PSCs) to the chimera produced by injection of PSCs into 4-cell or 8-cell stage embryos compared with blastocyst injection. Here, we show that not only embryonic stem cells (ESCs) but also induced pluripotent stem cells (iPSCs) can generate F0 nearly 100% donor cell-derived mice by 4-cell stage embryo injection, and the approach has a “dose effect”. Through an analysis of the PSC-secreted proteins, Activin A was found to impede epiblast (EPI) lineage development while promoting trophectoderm (TE) differentiation, resulting in replacement of the EPI lineage of host embryos with PSCs. Interestingly, the injection of ESCs into blastocysts cultured with Activin A (cultured from 4-cell stage to early blastocyst at E3.5) could increase the contribution of ESCs to the chimera. The results indicated that PSCs secrete protein Activin A to improve their EPI competency after injection into recipient embryos through influencing the development of mouse early embryos. This result is useful for optimizing the chimera production system and for a deep understanding of PSCs effects on early embryo development. Electronic supplementary material The online version of this article (doi:10.1007/s13238-017-0470-y) contains supplementary material, which is available to authorized users.

Viral nervous necrosis disease (VNN), caused by nervous necrosis virus (NNV), is one major threat to mariculture. Identifying loci and understanding the mechanisms associated with resistance to VNN are important in selective breeding programs. We performed a genome-wide association study (GWAS) using genotyping-by-sequencing (GBS) to study the genomic architecture of resistance to NNV infection in Asian seabass. We genotyped 986 individuals from 43 families produced by 15 founders with 44498 bi-allelic genetic variants using GBS. The GWAS identified three genome-wide significant loci on chromosomes 16, 19, and 20, respectively, and six suggestive loci on chromosomes 1, 8, 14, 15, 21, and 24, respectively, associated with resistance to NNV infection measured as binary and quantitative traits. Using the 500 most significant markers in combination with a training population of 800 samples could reach a genomic prediction accuracy of 0.7. Candidate genes significantly associated with resistance to NNV, including lysine-specific demethylase 2A, beta-defensin 1, and cystatin-B, which play important roles in immune responses against virus infection, were identified. Almost all the candidate genes were differentially expressed in different tissues against NNV infection. The significant genetic variants can be used in genomic selection and help understand the mechanism of resistance to VNN. Future studies should use populations of large effective size and whole genome resequencing to identify more useful genetic variants.

Asian seabass, an important food fish in Southeast Asia, has suffered from nervous necrosis virus (NNV) infection, resulting in massive mortality of Asian seabass larvae and enormous economic losses. Identification and characterization of disease resistance genes is important. Previous transcriptome analysis of Asians seabass epithelial cells after NNV infection revealed a highly inducible gene, receptor-transporting protein 3 ( rtp3 ), indicating it could play an important role in Asian seabass – NNV interaction. To characterize this gene, we determined its expression pattern and subcellular localization. The rtp3 was highly induced in most examined tissues and organs of Asian seabass after NNV infection, and protein Rtp3 was localized in cytoplasm. Further association study in multiple families revealed that a microsatellite marker, (GT)ntt(GT)n, in the 3′ UTR of rtp3 was significantly associated with VNN disease resistance in Asian seabass. Our results imply that rtp3 may be a novel disease resistance gene in Asian seabass. This data could improve our understanding of molecular interaction between Asian seabass and NNV, and has the potential to be applied in marker-assisted selection for disease resistance breeding in Asian seabass.

Hypoxia is one of the major challenges in aquaculture industry. Breeding of fish tolerant to hypoxia is an important task in genetic improvement of aquaculture species. The Asian seabass, Lates calcarifer, is an important foodfish species. We identified and characterized the hypoxia-inducible factor inhibitor (HIF1αn) gene in the Asian seabass. The full-length cDNA sequence of the HIF1αn was 3425 bp, with an ORF of 1065 bp, encoding 354 amino acids. The genomic sequence of the gene was 8667 bp in length, and contained eight exons and seven introns. Phylogenetic analysis of the gene in fish and tetrapods revealed that the HIF1αn in the Asian seabass was closely related to that of tilapia (Oreochromis niloticus). The HIF1αn was highly up-regulated in the gill, spleen and heart after 3.5-hours hypoxia treatment. We identified three SNPs in the third and fourth introns of the HIF1αn gene. The SNP (i.e. SNP 9332241 (C/T)) in the fourth intron was significantly (P 0.01) associated with hypoxia tolerance. This SNP might be useful in selecting Asian seabass for improved tolerance to hypoxia. Our data also provide useful information for further detailed analysis of the function of the HIF1αn gene in hypoxia tolerance.

Asian seabass is an important food fish species. While improving growth, increasing the nutritional value is important, omega-3 fatty acids are indispensable to human health. Identifying and validating DNA markers associated with traits is the first step towards marker-assisted selection (MAS). We quantified 13 different fatty acids and three growth traits in 213 F2 Asian seabass from a family at the age 270 days post hatch, and screened QTL for these traits. The content of total fatty acids in 100 g flesh was 2.57 ± 0.80 g, while the proportions of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) were 16.96 ± 2.20% and 5.42 ± 0.90%, respectively. A linkage map with 2424 SNPs was constructed and used for QTL mapping. For fatty acid compositions, 14 significant QTL were identified on three linkage groups (LG5, LG11 and LG14), with phenotypic variance explained (PVE) from 12.8 to 24.6%. Thirty-nine suggestive QTL were detected on 16 LGs. Two significant QTL for EPA were identified on LG5 and LG14, with PVE of 15.2% and 15.1%, respectively. No significant QTL was identified for DHA. For growth traits, six significant and 13 suggestive QTL were identified on two and seven LGs, respectively. Only a few significant QTL for fatty acids overlapped with previously mapped QTL for these traits, suggesting that most QTL detected in a family are family-specific and could only be used in MAS in the family per se. To facilitate population-wide molecular breeding, more powerful methods (e.g. GWAS) should be used to identify SNPs for genomic selection.

Diseases are one of the major challenges in tilapia aquaculture. Identification of DNA markers associated with disease resistance may facilitate the acceleration of the selection for disease resistance. Gadd45a (growth arrest and DNA damage 45 A), a stress-inducible gene in humans and mice, has not been studied in fish. We characterized the two prologues of Gadd45a genes in hybrid tilapia. Gadd45a1 and Gadd45a2 shared an identical gene structure and showed an amino acid sequence identity of 73.8%. Their expressions were detected in all 10 tissues examined, with the kidney and gill having high transcriptional expressions. The expression levels of Gadd45a1 were significantly lower than those of Gadd45a2 in all examined tissues. After a challenge with a bacterial pathogen Streptococcus agalactiae, the expressions of the two genes were up-regulated significantly in the spleen, kidney, liver and intestine. These findings suggest that the two Gadd45a genes play an important role in the resistance to S. agalactiae in tilapia. We identified 10 SNPs in the two genes. The SNP markers in the two Gadd45a genes could be used to examine whether they are associated with resistance to S. agalactiae.

Viral nervous necrosis disease (VNN), caused by nervous necrosis virus (NNV), leads to mass mortality in mariculture. However, phenotypic selection for resistance against VNN is very difficult. To facilitate marker-assisted selection (MAS) for resistance against VNN and understanding of the genetic architecture underlying the resistance against this disease, we mapped quantitative trait loci (QTL) for resistance against VNN in Asian seabass. We challenged fingerlings at 37 days post-hatching (dph), from a single back-cross family, with NNV at a concentration of 9 × 10(6) TCID50/ml for 2 h. Daily mortalities were recorded and collected. A panel of 330 mortalities and 190 surviving fingerlings was genotyped using 149 microsatellites with 145 successfully mapped markers covering 24 linkage groups (LGs). Analysis of QTL for both resistance against VNN and survival time was conducted using interval mapping. Five significant QTL located in four LGs and eight suggestive QTL in seven LGs were identified for resistance. Another five significant QTL in three LGs and five suggestive QTL in three LGs were detected for survival time. One significant QTL, spanning 3 cM in LG20, was identified for both resistance and survival time. These QTL explained 2.2-4.1% of the phenotypic variance for resistance and 2.2-3.3% of the phenotypic variance for survival time, respectively. Our results suggest that VNN resistance in Asian seabass is controlled by many loci with small effects. Our data provide information for fine mapping of QTL and identification of candidate genes for a better understanding of the mechanism of disease resistance.

Polymorphisms in the FTO gene are associated with obesity and body mass index in humans and livestock. Little information of whether FTO plays an important role in aquaculture fish species is available. We cloned and characterized the FTO gene in an economically important food fish species: Asian seabass (Lates calcarifer). The full-length cDNA of the gene is 3679 bp, containing an ORF of 1935 bp encoding 644 amino acids, a 216 bp 5′ UTR and a 1538 bp 3′ UTR. The gene consisted of nine exons and eight introns and was 117,679 bp in length. Phylogenetic analysis revealed that the gene in Asian seabass was closely related to those of Japanese flounder and Nile tilapia. Analysis of its expressions using qRT-PCR showed that it was expressed ubiquitously, but was higher in the liver, stomach and intestine. Comparative analysis of the genomic sequences of part of intron 1 of the gene among 10 unrelated individuals identified two SNPs. Analysis of associations between SNPs and traits (i.e. growth, oil content, omega-3 and -6 contents) in an F2 family demonstrated that the two SNPs were significantly associated with growth, oil content, omega-3 content and omega-3/-6 ratio. Altogether, our data suggest that the gene or/and its linked genes play an important role in growth and fatty acid synthesis, and that the SNPs associated with traits may be used as markers for selecting quicker growth and higher omega-3/-6 ratio at the fingerling stage. NRF (Natl Research Foundation, S’pore)

Background Oil palm is the most productive oil crop and the efficiency of pollination has a direct impact on the yield of oil. Pollination by wind can occur but maximal pollination is mediated by the weevil E. kamerunicus. These weevils complete their life cycle by feeding on male flowers. Attraction of weevils to oil palm flowers is due to the emission of methylchavicol by both male and female flowers. In search for male flowers, the weevils visit female flowers by accident due to methylchavicol fragrance and deposit pollen. Given the importance of methylchavicol emission on pollination, we performed comparative transcriptome analysis of oil palm flowers and leaves to identify candidate genes involved in methylchavicol production in flowers. Results RNA sequencing (RNA-Seq) of male open flowers, female open flowers and leaves was performed using Illumina HiSeq 2000 platform. Analysis of the transcriptome data revealed that the transcripts of methylchavicol biosynthesis genes were strongly up-regulated whereas transcripts encoding genes involved in lignin production such as, caffeic acid O-methyltransferase (COMT) and Ferulate-5-hydroxylase (F5H) were found to be suppressed in oil palm flowers. Among the transcripts encoding transcription factors, an EAR-motif-containing R2R3-MYB transcription factor (EgMYB4) was found to be enriched in oil palm flowers. We determined that EgMYB4 can suppress the expression of a monolignol pathway gene, EgCOMT, in vivo by binding to the AC elements present in the promoter region. EgMYB4 was further functionally characterized in sweet basil which also produces phenylpropenes like oil palm. Transgenic sweet basil plants showed significant reduction in lignin content but produced more phenylpropenes. Conclusions Our results suggest that EgMYB4 possibly restrains lignin biosynthesis in oil palm flowers thus allowing enhanced carbon flux into the phenylpropene pathway. This study augments our understanding of the diverse roles that EAR-motif-containing MYBs play to fine tune the metabolic flux along the various branches of core phenylpropanoid pathway. This will aid in metabolic engineering of plant aromatic compounds. Electronic supplementary material The online version of this article (10.1186/s12870-017-1174-4) contains supplementary material, which is available to authorized users.

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 &lt, 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.

Identifying quantitative trait loci (QTL) for viral disease resistance is of particular importance in selective breeding programs of fish species. Genetic markers linked to QTL can be useful in marker-assisted selection (MAS) for elites resistant to specific pathogens. Here, we conducted a genome scan for QTL associated with Singapore grouper iridovirus (SGIV) resistance in an Asian seabass (Lates calcarifer) family, using a high-density linkage map generated with genotyping-by-sequencing. One genome-wide significant and three suggestive QTL were detected at LG21, LG6, LG13, and LG15, respectively. The phenotypic variation explained (PVE) by the four QTL ranged from 7.5 to 15.6%. The position of the most significant QTL at LG21 was located between 31.88 and 36.81 cM. The SNP marker (SNP130416) nearest to the peak of this QTL was significantly associated with SGIV resistance in an unrelated multifamily population. One candidate gene, MECOM, close to the peak of this QTL region, was predicted. Evidence of alternative splicing was observed for MECOM and one specific category of splicing variants was differentially expressed at 5 days post-SGIV infection. The QTL detected in this study are valuable resources and can be used in the selective breeding programs of Asian seabass with regard to resistance to SGIV.

Mast cell proteases play an important role in the regulation of the immune response. We identified the cDNA of the mast cell protease 8 (MCP-8) gene and analyzed its genomic structure in tilapia. The ORF of the MCP-8 was 768 bp, encoding 255 amino acids. Quantitative real-time PCR revealed that the MCP-8 gene was expressed predominantly in spleen, moderately in liver, blood, brain, gill, intestine, skin, and weakly expressed in kidney, muscle and eye. After a challenge with Streptococcus agalactiae, the gene was induced significantly (p0.05) in intestine, kidney, spleen and liver. Furthermore, we identified five single nucleotide polymorphisms (SNPs) in the MCP-8 gene and found that three SNPs were significantly associated (p0.05) with resistance against S. agalactiae. However, we found no association between four SNPs and growth traits (p0.05). These results suggest that the MCP-8 gene play an important role in the resistance to S. agalactiae in tilapia. The SNP markers in the MCP-8 gene associated with the resistance to the bacterial pathogen may facilitate selection of tilapia resistant to the bacterial disease.

Sexual dimorphism is an interesting biological phenomenon. Previous studies showed that DNA methylation might play a role in sexual dimorphism. However, the overall picture of the genome-wide methylation landscape in sexually dimorphic species remains unclear. We analyzed the DNA methylation landscape and transcriptome in hybrid tilapia (Oreochromis spp.) using whole genome bisulfite sequencing (WGBS) and RNA-sequencing (RNA-seq). We found 4,757 sexually dimorphic differentially methylated regions (DMRs), with significant clusters of DMRs located on chromosomal regions associated with sex determination. CpG methylation in promoter regions was negatively correlated with the gene expression level. MAPK/ERK pathway was upregulated in male tilapia. We also inferred active cis-regulatory regions (ACRs) in skeletal muscle tissues from WGBS datasets, revealing sexually dimorphic cis-regulatory regions. These results suggest that DNA methylation contribute to sex-specific phenotypes and serve as resources for further investigation to analyze the functions of these regions and their contributions towards sexual dimorphisms.

Identification of differentially expressed genes (DEGs) and regulated pathways in response to stressors using a whole-genome approach is critical to understanding the mechanisms underlying stress responses. We challenged Asian seabass with lipopolysaccharide (LPS), Vibrio harveyi, high salinity and fasting, and sequenced six cDNA libraries of intestine samples using Roche 454 RNA-seq. Over 1 million reads (average size: 516 bp) were obtained. The de novo assembly obtained 83 911 unisequences with an average length of 747 bp. In total, 62.3% of the unisequences were annotated. We observed overall similar expression profiles among different challenges, while a number of DEGs and regulated pathways were identified under specific challenges. More than 1000 DEGs and over 200 regulated pathways for each stressor were identified. Thirty-seven genes were differentially expressed in response to all challenges. Our data suggest that there is a global coordination and fine-tuning of gene regulation during different challenges. In addition, we detected dramatic immune responses in intestines under different stressors. This study is the first step towards the comprehensive understanding of the mechanisms underlying stress responses and supplies significant transcriptome resources for studying biological questions in non-model fish species.

Discovering the nature and pattern of genome variation is fundamental in understanding phenotypic diversity among populations. Although several millions of single nucleotide polymorphisms (SNPs) have been discovered in tilapia, the genome-wide characterization of larger structural variants, such as copy number variation (CNV) regions has not been carried out yet. We conducted a genome-wide scan for CNVs in 47 individuals from three tilapia populations. Based on 254 Gb of high-quality paired-end sequencing reads, we identified 4642 distinct high-confidence CNVs. These CNVs account for 1.9% (12.411 Mb) of the used Nile tilapia reference genome. A total of 1100 predicted CNVs were found overlapping with exon regions of protein genes. Further association analysis based on linear model regression found 85 CNVs ranging between 300 and 27,000 base pairs significantly associated to population types (R 2 > 0.9 and P > 0.001). Our study sheds first insights on genome-wide CNVs in tilapia. These CNVs among and within tilapia populations may have functional effects on phenotypes and specific adaptation to particular environments.

In aquaculture, growth and sex are economically important traits. To accelerate genetic improvement in increasing growth in salt-tolerant tilapia, we conducted QTL mapping for growth traits and sex with an F2 family, including 522 offspring and two parents. We used 144 polymorphic microsatellites evenly covering the genome of tilapia to genotype the family. QTL analyses were carried out using interval mapping for all individuals, males and females in the family, respectively. Using all individuals, three suggestive QTL for body weight, body length and body thickness respectively were detected in LG20, LG22 and LG12 and explained 2.4% to 3.1% of phenotypic variance (PV). When considering only males, five QTL for body weight were detected on five LGs, and explained 4.1 to 6.3% of PV. Using only females from the F2 family, three QTL for body weight were detected on LG1, LG6 and LG8, and explained 7.9–14.3% of PV. The QTL for body weight in males and females were located in different LGs, suggesting that in salt-tolerant tilapia, different set of genes ‘switches’ control the growth in males and females. QTL for sex were mapped on LG1 and LG22, indicating multigene sex determination in the salt-tolerant tilapia. This study provides new insights on the locations and effects of QTL for growth traits and sex, and sets the foundation for fine mapping for future marker-assisted selection for growth and sex in salt-tolerant tilapia aquaculture. NRF (Natl Research Foundation, S’pore) Published version

Zinc finger AN1-type domain 3 (ZFAND3) is essential for spermatogenesis in mice. However, its function in teleosts remains unclear. In this study, we characterized the ZFAND3 gene (termed as OsZFAND3) in an important food fish, tilapia. The OsZFAND3 cDNA sequence is 1,050 bp in length, containing an ORF of 615 bp, which encodes a putative peptide of 204 amino acid residues. Quantitative real-time PCR revealed that the OsZFAND3 transcripts were exclusively expressed in the testis and ovary. In situ hybridization showed that the high expression of OsZFAND3 transcripts was predominantly localized in the spermatocyte and spermatid. These results suggest that OsZFAND3 is involved in male germ cell maturation. Three single nucleotide polymorphisms (SNPs) were detected in the introns of OsZFAND3. The OsZFAND3 gene was mapped in the sex-determining locus on linkage group 1 (LG1). The three SNPs in the OsZFAND3 gene were strictly associated with sex phenotype, suggesting that the OsZFAND3 gene is tightly linked to the sex-determining locus. Our study provides new insights into the functions of the OsZFAND3 gene in tilapia and a foundation for further detailed analysis of the OsZFAND3 gene in sex determination and differentiation.

Asian seabass has suffered from viral nervous necrosis (VNN) disease. Our previous study has mapped quantitative trait loci (QTL) for resistance to VNN disease. To fine map these QTL and identify causative genes, we identified 6425 single nucleotide polymorphisms (SNPs) from 85 dead and 94 surviving individuals. Combined with 155 microsatellites, we constructed a genetic map consisting of 24 linkage groups (LGs) containing 3000 markers, with an average interval of 1.27 cM. We mapped one significant and three suggestive QTL with phenotypic variation explained (PVE) of 8.3 to 11.0%, two significant and two suggestive QTL with PVE of 7.8 to 10.9%, for resistance in three LGs and survival time in four LGs, respectively. Further analysis one QTL with the largest effect identified protocadherin alpha-C 2-like (Pcdhac2) as the possible candidate gene. Association study in 43 families with 1127 individuals revealed a 6 bp insertion-deletion was significantly associated with disease resistance. qRT-PCR showed the expression of Pcdhac2 was significantly induced in the brain, muscle and skin after nervous necrosis virus (NNV) infection. Our results could facilitate marker-assisted selection (MAS) for resistance to NNV in Asian seabass and set up the basis for functional analysis of the potential causative gene for resistance.

Asian seabass is an important food fish in Southeast Asia. Viral nervous necrosis (VNN) disease, triggered by nervous necrosis virus (NNV) infection, has caused mass mortality of Asian seabass larvae, resulting in enormous economic losses in the Asian seabass industry. In order to better understand the complex molecular interaction between Asian seabass and NNV, we investigated the transcriptome profiles of Asian seabass epithelial cells, which play an essential role in immune regulation, after NNV infection. Using the next generation sequencing (NGS) technology, we sequenced mRNA from eight samples (6, 12, 24, 48 h post-inoculation) of mock and NNV-infected Asian seabass epithelial cell line, respectively. Clean reads were de novo assembled into a transcriptome consisting of 89026 transcripts with a N50 of 2617 bp. Furthermore, 251 differentially expressed genes (DEGs) in response to NNV infection were identified. Top DEGs include protein asteroid homolog 1-like (ASTE1), receptor-transporting protein 3 (RTP3), heat shock proteins 30 (HSP30) and 70 (HSP70), Viperin, interferon regulatory factor 3 (IRF3) and other genes related to innate immunity. Our data suggest that abundant and diverse genes corresponding to NNV infection. The results of this study could also offer vital information not only for identification of novel genes involved in Asian seabass-NNV interaction, but also for our understanding of the molecular mechanism of Asian seabass' response to viral infection. In addition, 24807 simple sequence repeats (SSRs) were detected in the assembled transcriptome, providing valuable resources for studying genetic variations and accelerating quantitative trait loci (QTL) mapping for disease resistance in Asian seabass in the future.

A high-density genetic map is essential for comparative genomic studies and fine mapping of QTL and can also facilitate genome sequence assembly. Here, a high density genetic map of Asian seabass was constructed with 3321 SNPs generated by sequencing 144 individuals in a F2 family. The length of the map was 1577.67 cM with an average marker interval of 0.52 cM. A high level of genomic synteny among Asian seabass, European seabass, Nile tilapia and stickleback was detected. Using this map, one genome-wide significant and five suggestive QTL for growth traits were detected in six linkage groups (i.e. LG4, LG5, LG11, LG13, LG14 and LG15). These QTL explained 10.5–16.0% of phenotypic variance. A candidate gene, ACOX1 within the significant QTL on LG5 was identified. The gene was differentially expressed between fast- and slow-growing Asian seabass. The high-density SNP-based map provides an important tool for fine mapping QTL in molecular breeding and comparative genome analysis.

The Carassius auratus complex in natural populations includes diploid triploid and polyploidy individuals. Diploid individuals belong to the species Carassius auratus whereas triploid and polyploidy individuals are from the subspecies Carassius auratus gibelio. Triploid individuals are all female and reproduce clonally by gynogenesis. Therefore the Carassius auratus complex is an ideal system for studying evolution of unisexual reproduction. Identification of triploid individuals and clonal lines is the first step towards understanding of the evolution of unisexual clonal lines. We examined the ability of 10 microsatellites in identifying triploid individuals in 94 individuals from Japan and China. In 40 confirmed triploid individuals and eight confirmed diploid individuals, all triploid and diploid individuals can be identified by genotyping 10 microsatellite, and four triploid clonal lines were identified. Using the 10 microsatellites we genotyped 46 adult individuals (40 females and six males) from a natural population in China and found that all six males were diploid whereas the majority of females (36 of 40) were triploid and three triploid clonal lines were detected. In 18 diploid individuals from China, all individuals showed different genotypes, suggesting there is no diploid clonal line in diploid crucian carp. A phylogenetic analysis of 94 individuals from China and Japan showed that triploid individuals and clonal lines have originated recurrently.

Reproductive strategy is a central feature of the ecology of invasive species as it determines the potential for population increase and range expansion. The red swamp crayfish, Procambarus clarkii, has invaded many countries and caused serious problems in freshwater ecosystems. However, little is known about the effects of environmental conditions on crayfish paternity and offspring traits in the wild. We studied these reproductive characteristics of P. clarkii in wild populations from two different habitats (ponds and ditches) in three locations with different environmental conditions in China. Genotyping of 1,436 offspring and 30 mothers of 30 broods was conducted by using four microsatellites. An analysis of genotyping results revealed that gravid females were the exclusive mother of the progeny they tended. Twenty-nine of 30 mothers had mated with multiple (2-4) males, each of which contributed differently to the number of offspring in a brood. The average number of fathers per brood and the number of offspring per brood were similar (P > 0.05) among six sampling sites, indicating that in P. clarkii multiple paternity and offspring number per brood are independent of environmental conditions studied. Indirect benefits from increasing the genetic diversity of broods, male and sperm competition, and cryptic female choice are a possible explanation for the high level multiple paternity and different contribution of fathers to offspring in this species.

Liver-expressed antimicrobial peptide 2 (LEAP-2) is a novel antimicrobial peptide (AMP) recently found in vertebrates, and exhibits distinct amino acid sequence, secondary structure and expression pattern from other peptides. In this study, the LEAP-2 gene and its full-length cDNA were cloned from grass carp. Grass carp LEAP-2 gene consists of two introns and three exons. The translated product contains 92 amino acids, including a 26 amino acids signal peptide and a mature peptide of 41 amino acids. Grass carp LEAP-2 gene was expressed in a wide range of tissues except blood, with the highest level of transcripts found in liver. Upon induction by Aeromonas hydrophila, its expression was significantly up-regulated in liver, gill, skin, muscle, spleen, blood, head kidney, heart and intestine, but down-regulated in trunk kidney and brain. The transcript level was high in embryos at the 16-cell stage but declined gradually afterwards, suggesting that LEAP-2 transcripts in early embryos might be maternal. Mature peptides obtained by in vitro expression displayed selective antimicrobial activities. These results together further our understanding of the physiological function of LEAP-2 in vertebrates.

Summary Quantitative trait loci (QTL) affecting growth traits have previously been mapped in linkage groups (LG) 2, 3 and 23 of Barramundi (Lates carcalifer), but these QTL have not been verified in different genetic backgrounds and environments. Here, we report the identification and verification of QTL for growth traits on LG2, 3, 10 and 23 in F1 families constructed using brooders from the Singapore Marine Aquaculture Center (MAC) and from wild stocks collected in Thailand (THAI). The previously detected QTL for body weight and length linked to marker Lca371 on LG2 were confirmed in both the MAC and THAI families, whereas other QTL previously mapped to LG3 and 23 were only detected in one of the two families. QTL for body weight and length were identified in the MAC family, but not in the THAI family, in a region where the insulin-like growth factor 2 (IGF2) and tyrosine hydroxylase 1 (TH1) genes are located on LG10. Significant epistatic interactions were identified between markers Lca287 on LG2 and IGF2 on LG10 for growth trait QTL in the MAC family, but not in the THAI family. Effects of the IGF2, TH1 and parvalbumin 1 candidate genes were family-specific. Our results indicate that some but not all QTL are family-specific in Barramundi.

Barramundi (Lates calcarifer) is an important farmed marine food fish species. Its compact genome (∼700 Mb) is among the smallest genomes of food fish species. We established a first-generation genetic linkage map of Barramundi with a mapping panel containing three parents (two males and one female) and 93 progeny. A total of 240 microsatellite markers were mapped into 24 linkage groups. Among these markers, 10 were located in ESTs and known genes. The total lengths of the female and male maps were 873.8 and 414.5 cM with an average marker spacing of 6.20 and 4.70 cM, respectively. Comparing the flanking sequences of the 240 Barramundi microsatellites with the assembled whole-genome sequences of Tetraodon nigrovidiris revealed 55 homologous sequences located in 19 of the 21 chromosomes of T. nigrovidiris. The map will not only enable the mapping of quantitative trait loci, but also provide new resources for understanding the evolution of fish genomes.

Genomewide analysis of genetic divergence is critically important in understanding the genetic processes of allopatric speciation. We sequenced RAD tags of 131 Asian seabass individuals of six populations from South-East Asia and Australia/Papua New Guinea. Using 32 433 SNPs, we examined the genetic diversity and patterns of population differentiation across all the populations. We found significant evidence of genetic heterogeneity between South-East Asian and Australian/Papua New Guinean populations. The Australian/Papua New Guinean populations showed a rather lower level of genetic diversity. FST and principal components analysis revealed striking divergence between South-East Asian and Australian/Papua New Guinean populations. Interestingly, no evidence of contemporary gene flow was observed. The demographic history was further tested based on the folded joint site frequency spectrum. The scenario of ancient migration with historical population size changes was suggested to be the best fit model to explain the genetic divergence of Asian seabass between South-East Asia and Australia/Papua New Guinea. This scenario also revealed that Australian/Papua New Guinean populations were founded by ancestors from South-East Asia during mid-Pleistocene and were completely isolated from the ancestral population after the last glacial retreat. We also detected footprints of local selection, which might be related to differential ecological adaptation. The ancient gene flow was examined and deemed likely insufficient to counteract the genetic differentiation caused by genetic drift. The observed genomic pattern of divergence conflicted with the ‘genomic islands’ scenario. Altogether, Asian seabass have likely been evolving towards allopatric speciation since the split from the ancestral population during mid-Pleistocene.

Tilapia is the common name for a group of cichlid fishes. Identification of DNA markers significantly associated with important traits in candidate genes may speed up genetic improvement. L-Amino acid oxidase (LAO) plays a crucial role in the innate immune defences of animals. Previously, whether LAO variants were associated with economic traits had not been studied in fish. We characterized the cDNA sequence of the LAO gene of hybrid tilapia (Oreochromis spp.). Its ORF was 1536 bp, encoding a flavoenzyme of 511 amino acids. This gene consisted of seven exons and six introns. Its expression was detected in the intestine, blood, kidney, skin, liver. It was highly expressed in the intestine. After a challenge with a bacterial pathogen, Streptococcus agalactiae, its expression was up-regulated significantly in the liver, intestine and spleen (P

Natural selection and selective breeding for genetic improvement have left detectable signatures within the genome of a species. Identification of selection signatures is important in evolutionary biology and for detecting genes that facilitate to accelerate genetic improvement. However, selection signatures, including artificial selection and natural selection, have only been identified at the whole genome level in several genetically improved fish species. Tilapia is one of the most important genetically improved fish species in the world. Using next-generation sequencing, we sequenced the genomes of 47 tilapia individuals. We identified a total of 1.43 million high-quality SNPs and found that the LD block sizes ranged from 10–100 kb in tilapia. We detected over a hundred putative selective sweep regions in each line of tilapia. Most selection signatures were located in non-coding regions of the tilapia genome. The Wnt signaling, gonadotropin-releasing hormone receptor and integrin signaling pathways were under positive selection in all improved tilapia lines. Our study provides a genome-wide map of genetic variation and selection footprints in tilapia, which could be important for genetic studies and accelerating genetic improvement of tilapia.

Tilapia is a group of cultured teleost fishes whose production is threatened by some diseases. Identification of DNA markers associated with disease resistance in candidate genes may facilitate to accelerate the selection of disease resistance. The gene encoding a duodenase, which can trigger immune response, has not been studied in fish. We characterized the cDNA of duodenase-1 gene of hybrid tilapia. Its ORF is 759 bp, encoding a serine protease of 252 amino acids. This gene consisted of five exons and four introns. Its expression was detected in all 10 tissues examined, and it was highly expressed in the intestine and kidney. After a challenge with the bacterial pathogen, Streptococcus agalactiae, its expression was up-regulated significantly in the intestine, liver and spleen. We identified seven SNPs in the gene and found that four of them were significantly associated with the resistance to S. agalactiae (P < 0.05). The CGTCC haplotype, CAGTC/CGGTC and CGTCC/CGTCC diplotype were significantly associated with the resistance to S. agalactiae (P = 0.00, 0.04 and < 0.0001, respectively). In addition, one SNP was associated significantly with growth traits (P < 0.05). These findings suggest that the duodenase-1 gene plays an important role in the resistance to S. agalactiae in tilapia. The SNP markers in the duodenase-1 gene associated with resistance to the bacterial pathogen, may facilitate the selection of tilapia resistant to the bacterial disease.

We determined the complete mtDNA nucleotide sequence of Lates calcarifer using the shotgun sequencing method. The mitochondrial DNA (mtDNA) was 16,535 base pairs (bp) in length, and contained 13 protein coding genes, 22 transfer RNAs, 2 ribosomal RNAs, and one major noncoding control region (CR). The CR was unusually short at only 768 bp. A striking feature of the mitochondrial genome was the high G+C content (46.1%), which is among the highest in fish. The gene order was identical to that of a typical vertebrate. Phylogenetic analyses using concatenated amino acid sequences of 12 protein-coding genes of 30 fish species representing 14 suborders clearly showed Lates calcarifer was located in the cluster of fish species from the order Perciformes, supporting the traditional systematic classification. We characterized single-nucleotide polymorphisms (SNPs) in the CR by sequencing the complete CR of 25 individuals obtained from Australia and Singapore. A total of 68 SNPs were detected. Eighteen SNPs were fixed with alternative nucleotides in Australian and Singapore seabass, and these SNPs could be used for differentiating fish from the two countries.

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.

Tilapia is the common name for nearly a hundred species of cichlid fish which originated in Africa (El-Sayed 2006). It is one of the most important food fish genera in the world (El-Sayed 2006). A few red tilapia variants have been commonly seen in fish markets. While the genetic background of these varieties is not well documented, their derivation is generally attributed to the crossbreeding between mutant reddish-orange Mozambique tilapia (Oreochromis mossambicus) with other tilapia species like Nile tilapia (O. niloticus) and blue tilapia (O. aureus). Florida red tilapia is thought to be the result of blue tilapia mating with Mozambique tilapia (El-Sayed 2006). In Nile and Mozambique tilapia, microsatellites have been isolated (Kocher et al. 1998; Yue and Orban 2002) and applied in genetic studies (e.g. Kocher et al. 1998), whereas in Florida red tilapia, no microsatellites are available. Preliminary tests of cross-species amplification of 30 microsatellites isolated from Nile and Mozambique tilapia revealed that around 30% of tested microsatellites could not amplify products in red tilapia (Feng Liu and Gen Hua Yue, unpublished data). Therefore, to facilitate a better understanding of the genetic variations in tilapia species, we have isolated 19 polymorphic microsatellites from Florida red tilapia, and characterized them in Nile and Mozambique tilapia. These 19 microsatellites were all polymorphic in Florida red tilapia with an average allele number of 4.90 per locus, and average expected and observed heterozygosities of 0.62 and 0.74 respectively. Fourteen out of the 19 microsatellites amplified polymorphic products in Mozambique tilapia and all 19 amplified in Nile tilapia, respectively. Fourteen out of 19 and 13 out of 15 microsatellites were in HardyWeinberg equilibrium (HWE) in Nile and Mozambique

Three DNA marker systems -- random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP) and microsatellites -- were used to estimate the genetic diversity in Asian arowana (Scleropages formosus) by genotyping fish individuals from three different sources. Parallel application of the three DNA marker systems allowed us to compare their utility for the analysis of genetic diversity. Microsatellites displayed the highest expected heterozygosity, whereas the values obtained by RAPD and AFLP were much lower. Multiplex ratio and marker index were higher for AFLP than for RAPD or microsatellites. Weak correlation was detected between genetic similarity estimated from data obtained with the three DNA marker systems: estimates from RAPD and AFLP data turned out to be higher than those from microsatellites. On the other hand genetic similarity was higher in the red variety than in the green one, especially when tested with microsatellites. Based on the genetic distance matrices calculated from microsatellite analysis, all red individuals were clustered into one group, whereas only a subset of them was clustered when either RAPD or AFLP was used. This indicated that the microsatellite system detected population subdivision more efficiently than either RAPD or AFLP.

The snakehead fish Channa argus is an important food fish in China. We identified six microsatellite loci for C. argus. These six microsatellite loci and four other microsatellite markers were used to analyze genetic diversity in four cultured populations of C. argus (SD, JX, HN, and ZJ) and determine their relationships. A total of 154 alleles were detected at the 10 microsatellite loci. The average expected and observed heterozygosities varied from 0.70-0.84 and 0.69-0.83, respectively, and polymorphism information content ranged between 0.66 and 0.82 in the four populations, indicating high genetic diversity. Population JX deviated from mutation-drift equilibrium and may have experienced a recent bottleneck. Analysis of pairwise genetic differentiation revealed FST values that ranged from 0.028 to 0.100, which indicates a moderate level of genetic differentiation. The largest distances were observed between populations HN and SD, whereas the smallest distances were obtained between populations HN and JX. Genetic clustering analysis demonstrated that the ZJ and HN populations probably share the same origin. This information about the genetic diversity within each of the four populations, and their genetic relationships will be useful for future genetic improvement of C. argus through selective breeding.

Leukocyte cell-derived chemotaxin-2 (LECT2) is an important protein of the innate immune system for the defense against bacterial infection. We cloned and characterized the LECT2 gene from Asian seabass (Lates calcarifer). Its complete cDNA consisted of an open reading frame of 459 bp encoding a protein of 152 amino acids. The genomic DNA sequence of this gene consists of four exons and three introns. Quantitative real-time PCR revealed that the LECT2 gene was expressed predominantly in liver while its expression was moderate in spleen and heart, and weak in other tissues. The LECT2 transcript was up-regulated in the kidney, spleen and liver in response to a challenge with a pathogenic bacterium Vibrio harveyi. In addition, we identified three single nucleotide polymorphisms (SNPs) in the LECT2 gene, and found significant associations between these polymorphisms and resistance to the big belly disease. These results suggest that the LECT2 gene play an important role in resistance to bacterial pathogens in fish. The SNP markers in the gene associated with the resistance to bacterial pathogens may facilitate selecting Asian seabass resistant to bacterial diseases.

Microsatellites are popular molecular markers in genetic and evolutionary studies. Their mutational dynamics have been extensively studied in humans and fruit flies, but few data were available in fish. By genotyping 55 individuals of a F1 pedigree, we investigated the mutation rates and patterns of 49 microsatellites in one of the most important fresh water fish species, the common carp (Cyprinus carpio L.). The overall mutation rate of the 49 loci was 5.56 x 10(-4)/locus/generation (95% confidence interval 1.52 x 10(-4) and 1.63 x 10(-3)). The change of allele size was between +2 to -5 repeat units, assuming that the mutation allele arose from the parental allele most similar in size to the mutant.

Summary Parvalbumins are extremely abundant in fish muscle and play an important role in muscle relaxation. In this study, two parvalbumin genes (PVALB1 and PVALB2) were cloned from Asian seabass (Lates calcarifer). The cDNAs for PVALB1 and PVALB2 were 840 and 667 bp respectively. Both genes consisted of five exons and four introns, encoded 109 amino acids, and were of beta lineage. Using real-time polymerase chain reaction, expression of PVALB1 was detected in all 10 tissues tested, with expression in brain, kidney, muscle and small intestine being 15- to 322-fold higher than in the other tissues. Expression of PVALB2 was detected only in muscle, brain and intestine, and was up to 10-fold lower than PVALB1 expression. A (CT)17 microsatellite in the 3¢-untranslated region of PVALB1 and three single nucleotide polymorphisms (SNPs) in the third intron of PVALB2 were identified. The microsatellite in PVALB1 was significantly associated with body weight and body length at 90 days post-hatch (P < 0.01), whereas the SNPs in PVALVB2 were not associated with these traits.

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.