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Fish color is an important trait involved in many biological functions and related to commercial value of fish. However, our understanding of the molecular mechanisms underlying color variations in fish species is limited. In this study, we found 12 natural golden mutants in a Mozambique tilapia population. Analysis of the inheritance pattern of the golden and blackish skin colors in nine backcross families revealed that the golden phenotype is a recessive mutant determined by a single locus and follows simple Mendelian inheritance. Using linkage mapping, fine mapping and analyses of gene expressions, we uncovered that the Pmel17 gene was responsible for golden and black skin colors. Further sequence analysis of the gene discovered that an insertion in 3′ UTR of Pmel17 was fully associated with the golden skin color in 1835 tilapias. Expression analysis showed that Pmel17 with the insertion reduced the transcripts of Pmel17. These results indicate that the insertion in 3′ UTR of Pmel17 is responsible for the golden skin color in tilapia. The insertion can be used as a reliable DNA marker to accelerate the breeding for golden color.

Breeding for disease resistance is an important task for genetic improvement in aquaculture. Identification of DNA markers associated with disease resistance is a critical step towards molecular breeding for disease resistance. Iridovirus causes mass mortality in hatchery of an important food fish Asian seabass (Lates calcarifer). In this study, an SNX2 (Sorting nexin 2) homolog (LcaSNX2) from L. calcarifer was cloned. Its roles in the infection of Singapore grouper iridovirus (SGIV) were characterized. The gene contained an ORF of 1542 bp encoding 513 amino acids, a 5’ UTR of 242 bp and a 3′ UTR of 2650 bp. Two SNPs in the 3’ UTR of the gene were identified, and they were significantly associated with SGIV resistance. Thus, they provide useful DNA markers for selecting Asian seabass juveniles for resistance against SGIV. The SNX2 gene was expressed ubiquitously in healthy fish, and highly expressed in the eye, gill, fin and brain. The expression of the gene was induced after SGIV infection in vitro, suggesting that LcaSNX2 plays a role in virus-host interaction during SGIV infection. Overexpression of LcaSNX2 increased the infection and replication of SGIV. Knockdown of this gene reduced viral gene transcriptional level, cytopathic effect and virus titer, indicating that suppression of the gene reduces SGIV infection. These data indicate that LcaSNX2 gene is essential for efficient replication of SGIV.

Aquaculture is the fastest-growing food production sector in agriculture, with great potential to meet projected protein needs of human beings. Aquaculture is facing several challenges, including lack of a sufficient number of genetically improved species, lack of species-specific feeds, high mortality due to diseases and pollution of ecosystems. The rapid development of sequencing technologies has revolutionized biological sciences, and supplied necessary tools to tackle these challenges in aquaculture and thus ensure its sustainability and profitability. So far, draft genomes have been published in over 24 aquaculture species, and used to address important issues related to aquaculture. We briefly review the advances of next generation sequencing technologies, and summarize the status of whole genome sequencing and its general applications (i.e. establishing reference genomes and discovering DNA markers) and specific applications in tackling some important issues (e.g. breeding, diseases, sex determination & maturation) related to aquaculture. For sequencing a new genome, we recommend the use of 100–200 × short reads using Illumina and 50–60 × long reads with PacBio sequencing technologies. For identification of a large number of SNPs, resequencing pooled DNA samples from different populations is the most cost-effective way. We also discuss the challenges and future directions of whole genome sequencing in aquaculture.

Singapore is a small island country with limited land and sea space for aquaculture. Foodfish production in Singapore is mainly from cage aquaculture in sea and supplied about 10% of total fish consumed. The major cultured species are Asian seabass, milk fish, grey mullet, snappers, golden pompanos, hybrid groupers and shrimps. Although small scale of its aquaculture, constrained by limited available land and coastal areas and high cost for aquaculture, the industry is thriving and transferring from low-tech to high-tech. With the strong support of the government for food security, aquaculture R&D are geared towards developing novel hatchery technologies, marker-assisted and genomic selection to accelerate genetic improvement, the establishment of high-tech aquaculture farms and ensuring biosecurity. Singapore is fast becoming global aquaculture hotspot. In this review, we summarized the status of Singapore's aquaculture production, R&D, training and education and its challenges, and suggested some ideas on the future development of Singapore's aquaculture. Singapore is well-positioned to serve as a R&D and education hub for tropical aquaculture. Its R & D on aquaculture should focus on fish reproduction, genetic improvement and high-tech aquaculture systems to improve local fish production and to supply superior fry for culture in other countries.

Suv39h1 is a member of the Suv39h sub-family of histone lysine methyltransferases. The gene plays an important role in heterochromatin organization, chromosome segregation, and mitotic progression in humans and model organisms. However, its functions in food fish species remain largely unknown. We identified the full-length cDNA of the Suv39h1 in the Asian seabass, analysed its genomic structure and identified polymorphisms. The gene was expressed in all tissues examined, but highly expressed in the gill, spleen and kidney. After challenging Asian seabass juveniles with iridovirus, the expressions of the gene altered, suggesting that the gene is related to the infection by iridovirus. Three SNPs and one indel were detected in the 3’ UTR of the gene. The indel was significantly associated with the resistance against iridovirus. In silico-mapping revealed that the gene was mapped to chromosome 6, where a significant QTL for the resistance to iridovirus was located previously in the Asian seabass. Altogether, the data suggest that the gene Suv39h1 play an important role in iridovirus resistance in the Asian seabass. This study also laid the foundation for further detailed analysis of the function of the Suv39h1 gene in resistance to iridovirus. The indel may supply a useful DNA marker for selecting Asian seabass fingerlings resistant to iridovirus.

Single nucleotide polymorphisms (SNPs) are important DNA markers for genetic studies. However, the number of SNPs is limited in important aquaculture species. We have identified 23,535 high quality SNPs in genes of Nile tilapia ( Oreochromis niloticus ) by analyzing the available RNA-seq data. Among them, 17,699 SNPs were mapped onto 22 linkage groups (LG) using in silico mapping. The number of mapped SNPs ranged from 332 on LG2 to 1148 on LG16_21 with an average number of 805 SNPs/LG. The mapped SNPs were located in 7146 genes. This SNP resource will be useful in monitoring the genetic diversity and mapping QTL for genetic improvement of tilapia species. We further studied the relationships among individuals from four populations (GIFT, O. niloticus , Red tilapia and O. mossambicus ) using 101 SNPs from LG23. Ninety-four SNPs (~ 93%) were polymorphic in the tested populations. Red tilapia (77%) had the highest number of informative markers than GIFT (69%), Nile tilapia (56%), and Mozambique tilapia (12%). Significant population structure was detected through phylogenic and population structure analysis. The Red tilapia individuals were closely related to Mozambique tilapia, but were split into several subclades, confirming that red tilapia is a hybrid admixture of different resources of tilapias.

Grass carp is one of the most important freshwater food fish species in the world. We studied genetic diversity and population relationships of grass carp by genotyping 507 individuals from nine populations using 10 polymorphic microsatellites. In five wild populations and four cultured populations, a total of 212 alleles were detected at the 10 loci. All wild and cultured populations in China showed high allelic ( A = 6–13.7; Ar = 6.00–8.23) and gene diversity ( Ho = 0.71–0.79 and He = 0.71–0.82), while the cultured population Cul3 located in Malaysia showed low genetic diversity ( A = 6.4; Ar = 3.89, Ho = 0.48, He = 0.55). Both AMOVA and F ST analysis revealed significant population structuring ( F IS = 0.035, P F ST = 0.081, P F IT = 0.113, P

Asian seabass (Lates calcarifer) is an important marine foodfish species in Southeast Asia and Australia. To facilitate a selective breeding program conducted in Singapore, we genotyped 772 Asian seabass individuals collected from five cultured stocks and four wild populations originating from Singapore, Australia, Malaysia, Thailand, Indonesia and Taiwan using 14 polymorphic microsatellites. A total of 210 alleles were detected at 14 loci across the nine populations. Cultured stocks showed much lower allelic and gene diversity (A = 3.57–8.21 alleles/locus, Ho = 0.52–0.66 and He = 0.47–0.74, respectively) than wild populations (A = 9.14–10.71 alleles/locus, Ho = 0.71–0.74 and He = 0.74–0.78, respectively). The wild population from Thailand showed the highest allele richness (AR = 8.50 alleles/locus), whereas one of the three cultured Australian stocks had the lowest one (3.57 alleles/locus). Populations from Southeast Asia were more diverse genetically (A = 13.28, AR = 10.23, Ho = 0.72 and He = 0.78) than those from Australia (7.0, 6.56, 0.59 and 0.64, respectively) and Taiwan (7.35, 7.35, 0.66 and 0.68, respectively). Nearly three quarter (73.3%) of 90 private alleles were detected in populations from Southeast Asia. Genetic differentiation among populations/stocks was statistically significant (FST = 0.124, RST = 0.159, P < 0.05). Phylogenetic analysis showed that stocks from Australia clustered into one group, while the samples from Southeast Asian and Taiwan formed another group. All these data suggest significant genetic differentiation between Australian and Southeast Asian populations/stocks, and Australian populations contain only part of genetic variation of Asian seabass, which should be taken into account in aquaculture of Asian seabass. A total of 549 individuals from the four wild populations in Southeast Asia are being used in a selective breeding program.

Aquaculture of high-value finfish species, such as groupers, is an industry of increasing importance world wide. Groupers are represented by 15 genera and 159 species. Phenotypical classification of grouper species is difficult. Mitochondrial DNA (mtDNA) sequences can be used in differentiating species. We report the first complete mitochondrial genome sequence among grouper species, the complete mtDNA of Leopard coral grouper (Plectropomus leopardus). The complete mtDNA of P. leopardus was 16,714 bp long including a 1077 bp control region, 13 protein-coding genes, two rRNA and 22 tRNA. Phylogenetic analyses using concatenated amino acid sequences of 12 protein-coding genes of 29 fish species representing 12 suborders indicated that P. leopardus was located in the cluster of fish species from the order Perciformes, supporting the traditional taxonomic classification of fish. Using nucleotide sequences of the NADH2 genes of 49 grouper species from eight genera, we constructed phylogenetic trees showing most of these species could be differentiated with sequences of the NADH2 gene, and the P. leopardus was closely related to P. maculatus, in agreement their phenotypic similarity. We developed one duplex PCR which could differentiate the two species P. leopardus and P. maculatus based on the D-loop DNA sequences of these two species. The complete mtDNA sequence of P. leopardus and duplex PCR provide useful tools for genetic studies and aquaculture of P. leopardus.

To study genetic diversity of two cultured and one wild stock of Asian seabass ( Lates calcarifer ) in South-East Asia and their relationships, thirty-six polymorphic microsatellites were isolated from partial genomic libraries. The average allele number of the 36 microsatellites present among 32 individuals, was 7.4/locus, ranging from 2 to 17. One multiplex PCR set of nine polymorphic microsatellites was used to genotype 294 individuals from three stocks. High levels of allelic and genetic diversity were observed in all three stocks (average allele number = 8.2/locus and H e = 0.75 in the wild stock; allele number = 9.6 and H e = 0.76 in Singapore stock I; and allele number = 8.0 and H e = 0.72 in Singapore stock II). F IS values within each stock were very low. Significant genetic differentiation ( F ST = 0.03, P

Asian arowana (Scleropages formosus) is listed by CITES as a highly endangered fish species. The genetic diversity and population structure of three Asian arowana captive stocks was analyzed using microsatellites and AFLP markers on 32 randomly collected individuals from each stock. Six AFLP primer pairs amplified a total of 324 bands across the three sample sets. The green stock showed the highest percentage of polymorphic bands (15.6%) and expected heterozygosity (0.26), followed by the red (13.0% and 0.24), then the golden one (12.7% and 0.22). Microsatellite analysis showed high allelic and gene diversity in all three varieties. The green stock showed higher allele number (100) and higher gene diversity (0.75) than the red (98 and 0.74) and golden ones (85 and 0.71), respectively. The estimates of long-term effective population size by two different methods ranged from 4741 to 7288 and from 25,056 to 64,641, respectively. Tests of heterozygosity and allele frequency distribution indicate that a recent bottleneck in any of the three captive populations is highly unlikely. Altogether, the data suggest that the captive breeding program for conservation and sustainable use of the endangered Asian arowana will be successful. In addition, we found that the differentiation among populations was intermediate (FST=0.047, RST=0.103) and the genetic distance was the smallest between the green and red, whereas the largest between the red and golden varieties.

Thirty-six new microsatellites were identified from common carp (Cyprinus carpio L.) by screening through genes found in GenBank, EST sequences from a testis cDNA library and a genomic DNA library enriched for CA repeats. Eleven of the twenty-eight microsatellites identified from genes and ESTs were AT repeats, suggesting their high abundance in the genome of common carp. Characterization of the 36 microsatellites on a panel of 18 unrelated common carp individuals revealed that all, except two, were polymorphic with an average allele number of 7.3/locus (range: 2-15 alleles/locus). The microsatellites located in genes and ESTs showed higher allele number than those eight which were isolated from a genomic DNA library (7.7/locus vs. 4.9/locus df=32, P

The whole broodstock of two Hungarian common carp farms—80 and 196 individuals—was analyzed by using random amplified polymorphic DNA (RAPD) assay and microsatellite analysis. Ten polymorphic RAPD markers and four microsatellites were selected to genotype both of the stocks. As expected, microsatellite analysis revealed more detailed information on genetic diversities than RAPD assay. Results obtained with both types of DNA markers showed lack of major differences between the genetic structure of the two stocks: heterozygosity values and allele frequencies were very similar. Dendrograms created from both sets of data did not show grouping of individuals according to stocks. Genotypes from the two stocks were also compared to those from a limited number of samples collected from other hatcheries and two rivers. Allele frequencies in the groups were similar, with the exception of wild carps. An interesting observation was that three private microsatellite alleles were found in the eight wild carp individuals, compared to the seven detected in the rest of the samples tested (372 individuals).