Search

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 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.

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.

Chia (Salvia hispanica L.), originated in central and southern Mexico and Guatemala, is an emerging industry crop due to its high content of omega‐3 fatty acids and dietary fiber in its seeds. The seeds also have a high concentration of proteins and essential amino acids, and are becoming a promising source of bioactive peptides. Polymorphic DNA markers are essential tools to analyse genetic diversity and to accelerate genetic improvement. However, in chia, polymorphic and codominant DNA markers are still lacking. In this study, fourteen polymorphic microsatellites were identified from DNA sequences and were characterized. The average allele number was 4.8 while the expected and observed heterozygosity was 0.24 and 0.34, respectively. The average probability of identity (PI) was 0.50 while the combined PI was 9 × 10–6. These first 14 microsatellites in chia are useful in genetic analysis and traceability. These 14 polymorphic microsatellites were used in analysing genetic diversity and population relationships in six cultivars originating in Mexico, Australia and Bolivia. Results showed that allelic diversity and gene diversity were low and ranged from 2.79 to 3.64 and 0.27 to 0.38, respectively. The Mexico black cultivar showed the highest allelic (3.64) and gene diversity (0.38). The six cultivars were closely related with high identity (≥ 0.893). Taken together, these chia cultivars contain low genetic variation. Therefore, to initiate a breeding program for improving traits, it is essential to use seeds from multiple cultivars to enlarge genetic variation in the founder population.

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.

The palm family (Arecaceae), consisting of ∼ 2600 species, is the third most economically important family of plants. The African oil palm (Elaeis guineensis) is one of the most important palms. However, the genome sequences of palms available are still limited and fragmented. Here, we report a high-quality chromosome-level reference genome of an oil palm Dura. The genome of 1.7 Gb was assembled by integrating long reads with ∼ 150 × genome coverage. The assembled genome covered 94.5% of the estimated genome size, within which 91.6% were assigned into 16 pseudochromosomes and 73.7% were repetitive sequences. Relying on the conserved synteny with oil palm, the existing draft genome sequences of both date palm and coconut were further assembled into chromosomal level. Transposon burst, particularly long terminal repeat retrotransposons (LTRs) retrotransposons, following the last whole-genome duplication (WGD), likely explains genome size variation across palms. Convergent evolution of fruit colors tends to eliminate the roles of the virescens gene in controlling accumulation of anthocyanins in exocarp of ripe fruit of palms. Recent duplications of high tandemly repeated pathogenesis-related proteins (PRs) from the same tandem arrays played an important role in defense responses to Ganoderma. Whole genome re-sequencing of both ancestral African and introduced oil palms in Southeast Asia revealed that genes under putative selection were notably associated with stress responses, suggesting adaptation to stresses in the new habitat. The genomic resources and insights gained in this study could be exploited for accelerating genetic improvement and understanding the evolution of palms.

Oil palm is the most productive oil producing plant. Salt stress leads to growth damage and decrease in yield of oil palm. However, the physiological responses of oil palm to salt stress and their underlying mechanisms are not clear. RNA-Seq for leaf samples from young palms challenged under three levels of salts (100, 250 and 500 mM NaCl) for 14 days and control was conducted. All the three levels of salt stress activated EgSPCH expression and increased stomatal density of oil palm. ~ 41% of DEGs were putative EgSPCH binding target and were involved in multiple bioprocesses related to salt response. Overexpression of EgSPCH in Arabidopsis increased the stomatal production and lowered the salt tolerance. These data indicate that in oil palm, salt activates EgSPCH to generate more stomata in response to salt stress, which is different with that in herbaceous plants. Our results might mirror the difference of salt-induced stomatal development between ligneous and herbaceous crops.

Chia (Salvia hispanica) is a functional food crop for humans. Although its seeds contain high omega-3 fatty acids, the seed yield of chia is still low. Genomic resources available for this plant are limited. We report the first high-quality chromosome-level genome sequence of chia. The assembled genome size was 347.6 Mb and covered 98.1% of the estimated genome size. A total of 31 069 protein-coding genes were predicted. The absence of recent whole-genome duplication and the relatively low intensity of transposable element expansion in chia compared to its sister species contribute to its small genome size. Transcriptome sequencing and gene duplication analysis reveal that the expansion of the fab2 gene family is likely to be related to the high content of omega-3 in seeds. The white seed coat color is determined by a single locus on chromosome 4. This study provides novel insights into the evolution of Salvia species and high omega-3 content, as well as valuable genomic resources for genetic improvement of important commercial traits of chia and its related species.

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.

The Asian arowana (Scleropages formosus) is an ancient freshwater fish species from the Osteoglossidae family. It is a territorial and mouth-brooding fish, which inhabits slow-moving rivers and lak...

Tilapias are important aquaculture species. Male-biased sexual size dimorphism is very common and males are preferred for aquaculture in tilapia. However, the mechanisms underlying sexual dimorphism remain to be elucidated. One hundred and thirty-six sex-biased genes, of which 23 were male-biased and 113 were female-biased, were identified via reanalysis of a muscle transcriptome data using the latest reference genome assembly. These genes were mapped to KEGG pathways that are related to somatic cell metabolism, growth and differentiations, such as MAPK, FoxO4 and metabolism pathways as well as developmental processes responsible for skeletal muscle development. Pathways related to cell growth and proliferations such as MAPK signaling pathways are upregulated in males while pathways regulating cell division such as FoxO4 are upregulated in females. Sex-biased genes in tilapia skeletal muscle have higher evolution rates (dN/dS) compared to unbiased genes. Female-biased and male-biased genes showed 17.4% and 13.5% higher dN/dS, respectively, compared to unbiased genes. Our results suggest that some of the male and female sex-biased genes were under selection pressures. Three SNPs located in the promoter region of one sex-biased gene RASGRF1 on LG1 were associated with bodyweight differences in the hybrid tilapia. These sex-biased genes identified in this study may serve as candidate genes for future functional analysis on sexual size dimorphism and for developing DNA markers for selecting fast-growing saline tilapia in aquaculture. Keywords: Sexual dimorphism, Body size, RNA-Seq, Tilapia, Aquaculture, Evolution

Oil palm is the most productive oil producing plant. Salt stress leads to growth damage and decrease in yield of oil palm. However, the physiological responses of oil palm to salt stress and their underlying mechanisms are not clear. RNA-Seq for leaf samples from young palms challenged under three levels of salts (100, 250 and 500 mM NaCl) and control for 14 days was conducted. Diverse signalling pathways were involved in responses to different levels of salt stress. All the three levels of salt stress activated EgSPCH expression and induced stomatal density of oil palm, which was contrasting to that in Arabidopsis. Under strong salt stress group, oil palm removed excessive salt via stomata. Overexpression of EgSPCH in Arabidopsis increased the stomatal production but lowered the salt tolerance. These data suggest that in oil palm, salt activates EgSPCH to generate more stomata in response to salt stress. Our results shed a light on the cellular response to salt stress of oil palm and provide new insights into the mechanisms of different salt-induced stomatal development between halophytes and glycophytes.

Chia (Salvia hispanica L.), originated in central and southern Mexico and Guatemala, is an emerging industry crop due to its high content of omega‐3 fatty acids and dietary fiber in its seeds. The seeds also have a high concentration of proteins and essential amino acids, and are becoming a promising source of bioactive peptides. Polymorphic DNA markers are essential tools to analyse genetic diversity and to accelerate genetic improvement. However, in Chia, polymorphic and codominant DNA markers are still lacking. In this study, fourteen polymorphic microsatellites were identified from DNA sequences and were characterized. The average allele number was 4.8 while the expected and observed heterozygosity was 0.24 and 0.34, respectively. The average PI was 0.50 while the combined PI was 9 ×10-6. These first 14 microsatellites in Chia are useful in genetic analysis and traceability. These 14 polymorphic microsatellites were used in analysing genetic diversity and population relationships in six cultivars originating in Mexico, Australia and Bolivia. Results showed that allelic diversity and gene diversity were low and ranged from 2.79 to 3.64 and 0.27 to 0.38, respectively. The Mexico black cultivar showed the highest allelic (3.64) and gene diversity (0.38). The six cultivars were closely related with high identity (> 0.893). Taken together, these Chia cultivars contain low genetic variation. Therefore, to initiate a breeding program for improving traits, it is essential to use seeds from multiple cultivars to enlarge genetic variation in the founder population.

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.

The ISGA (International Symposium on Genetics in Aquaculture) is an important conference in the field of aquaculture genetics, which is held every three years. This year, the meeting was hosted by James Cook University, in Cairns. The conference sessions included biotechnology and functional genomics, sex control, genomic prediction, selective breeding and quantitative genetics, industrial use of genetics, genome technology, genetics of diseases and stress, genetics of nutrition, epigenetics, genomics and metabolomes, population genetics and ethics, food safety, as well as environmental risks. Many new and interesting unpublished data, including those for new aquaculture species, marker-assisted selection, genomic selection and genome editing using Crispr/Cas9 technologies, were reported at the conference. In this brief review, we highlight the novel and interesting researches on aquaculture genetics and genomics to give readers an overview of the current status and trends of aquaculture genetics and genomics research. Keywords: ISGA2018, Aquaculture, MAS, GS, Phenomics

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.

The oil palm, originating from Africa, is the most productive oil crop species. Palm oil is an important source of edible oil. Its current global plantation area is over 23 million ha. The theoretical oil yield potential of the oil palm is 18.2 tons/ha/year. However, current average oil yield is only 3.8 tons/ha/year. In the past 100 years, conventional breeding and improvement of field management played important roles in increasing oil yield. However, conventional breeding for trait improvement was limited by its very long (10–20 years) phenotypic selection cycle, although it improved oil yield by ~10–20% per generation. Molecular breeding using novel molecular technologies will accelerate genetic improvement and may reduce the need to deforest and to use arable land for expanding oil palm plantations, which in turn makes palm oil more sustainable. Here, we comprehensively synthesize information from relevant literature of the technologies, achievements, and challenges of molecular approaches, including tissue culture, haploid breeding, mutation breeding, marker-assisted selection (MAS), genomic selection (GS), and genome editing (GE). We propose the characteristics of ideal palms and suggest a road map to breed ideal palms for sustainable palm oil.

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.

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.

Background: Palm oil is an important feedstock for biofuel. Palm oil yield is seriously affected by drought stress. However, not much is known about the molecular responses of oil palm to drought stress.Results: We studied the root transcriptomic responses of oil palm seedlings under normal culture and 14-day drought stress using RNA-seq and bioinformatics analysis. We identified 1293 differentially expressed genes (DEGs), involved in several molecular processes, such as cell wall biogenesis and functions, phenylpropanoid biosynthesis and metabolisms, ion transport and homeostasis and cellular ketone metabolic process, as well as small molecule biosynthetic process. We observed that DEGs were significantly enriched into the two categories: hormone regulation and metabolism, as well as ABC transporters. In addition, we identified three protein-protein interaction networks involved in the response to drought stress, including ion transport, reactive nitrogen species metabolic process and nitrate assimilation. Finally, 96 differentially expressed transcription factors were detected to be associated with drought stress response, which were classified into 28 families.Conclusions : The transcriptomic responses of oil palm seedlings to drought stress were systematically analysed, revealing important genes, pathways, networks and transcription factors involved in drought stress responses. These results provide new insights into the mechanisms of drought stress responses in economic crops. The genes and pathways identified in this study provide valuable genomic resources to improve drought tolerance of oil palm by both genetic modification and selective breeding.

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)

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.

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.

Growth is an economically important trait in aquaculture. To improve growth trait of the Asian seabass (Lates calcarifer) we have been carrying out, since 2004, a selective breeding program. This study focuses on growth traits in the F2 fish generation, comprised of offspring from 23 mass crosses from 383 F1 brooders. Using genotyping analysis for 10 microsatellites from both brood stock and progeny we have reconstructed the pedigree of each mass-cross. For F2 generation at 90 days post hatch (dph), we have recorded body weight (BW) for 12,117 individuals and total length (TL), standard length (SL) and condition factor (Ktl and Ksl) for 3530 individuals; and all five traits for 2136 individuals at 270 dph. At 90 dph the average BW was 46.88 ± 20.95 g. Combining pedigree information, recorded growth traits and Restricted Maximum Likelihood method, we have estimated that the narrow sense heritability (h2) in F1 fish for BW, TL, SL, Ktl and Ksl was at, 90 dph, 0.12 ± 0.03, 0.11 ± 0.03, 0.10 ± 0.03, 0.20 ± 0.04 and 0.11 ± 0.03, respectively and, at 270 dph, 0.34 ± 0.07, 0.32 ± 0.07, 0.30 ± 0.06, 0.13 ± 0.04 and 0.11 ± 0.04, respectively. At 90 dph the realised heritability for BW was 0.13. Comparing with F1 generation, the growth performance of F2 fish was increased by 14.4%. Heritability of growth traits will be useful for future genetic improvement programmes of the Asian seabass. NRF (Natl Research Foundation, S’pore) Published version

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.

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.

While juvenile cannibalism plays an important role in the evolution of organisms in natural populations, it is a serious problem in aquaculture. A number of genetic and environmental factors result in different rates of cannibalism. Whether there is kin recognition in juvenile cannibalism in fish is poorly understood. We studied cannibalism and kinship recognition in juveniles of Asian seabass using molecular parentage analysis with polymorphic microsatellites. In the three mass crosses, under an ordinary feeding scheme without size grading, the rate of juvenile loss due to cannibalism was 1.08% per day. In the group without feeding for 24 h, 2.30% ± 0.43% of offspring per day were lost within 24 h due to cannibalism. We detected that juveniles avoided cannibalizing their siblings when they were not hungry, whereas cannibalism among siblings increased when they were hungry. These data suggest that there is kin discrimination in fish cannibalism. Raising genetically closely related offspring in the same tanks and appropriate levels of feeding may reduce the rate of cannibalism. We hypothesized that the chemical cues for kin discrimination might be secreted by fish skins. To test this hypothesis, we analyzed gene expression profiles in the skins of juveniles under slightly and very hungry conditions using RNA sequencing and bioinformatics analysis. Genes differently expressed under slightly and very hungry conditions were identified. Among them, genes from the trypsin family were significantly down-regulated under starved conditions, suggesting that they may play a role in kin discrimination. NRF (Natl Research Foundation, S’pore) Published version

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.

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.

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 1.701 Gb 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.

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.

Asian seabass is an important marine foodfish species in Asia-Pacific region. Previous studies using microsatellites and mitochondrial DNA sequences showed that the populations in Australia are significantly different from those in Southeast Asia. However, within Australian and Southeast Asian populations, there are some conflicts regarding to population differentiations. It is essential for us to use more DNA markers to examine the population structure of Asian seabass. We collected Asian seabass samples from seven wild populations distributed across Indonesian Archipelago from Southeast Asia to Australia and Papua New Guinea. We measured the genetic diversity within each population and analyzed the genetic relationships among the wild populations using 96 SNPs. We detected significant population differentiations among the seven wild populations of Asian seabass. The genetic diversity in Southeast Asian populations was significantly higher than that in Australian and Papua New Guinean populations. There were significant genetic differentiations among the Australian and Papua New Guinean populations while the differentiation in Southeast Asian populations was minor. In addition, we identified footprints of directional selection at three loci using outlier tests. Our data supply useful information about the management of wild stocks of Asian seabass and the use of wild Asian seabass to start a breeding program.

Leaf area is an important parameter in oil palm breeding as it is positively correlated with oil yield. However, measurement of leaf area is tedious and also destructive. In the present study, a breeding population with 145 palms derived from a cross between Deli Dura and Avros Pisifera was used to construct a high-density linkage map and identify quantitative trait loci (QTL) for leaf area in oil palm. Using genotyping by sequencing, a linkage map containing 2413 SNPs was constructed. The total length of the linkage map was 1161.89 cM, with an average marker spacing of 0.48 cM. Based on the continuous phenotyping of leaf area from 2010 to 2015, two suggestive QTL for leaf area were mapped on chromosomes (Chr) 3 and 9. The allelic effects of the QTL associated with leaf area in the mapping population were estimated by linear regression using ordinary least squares method. The QTL on Chr 9 explained 13.3% of phenotypic variation for leaf area. A candidate gene, ARC5, within the narrow interval of QTL on Chr 9 was identified. The gene was significantly higher expressed in leaf than root and fruit of oil palm. This high-quality and SNP-based map supplies a base to fine map QTL for agronomic traits in oil palm, and the markers closely linked to the stable QTL may be used in marker-assisted selection in oil palm breeding.

Oil palm (Elaeis guineensis Jacq.) is the leading oil-producing crops and the most important edible oil resource worldwide. DNA markers and genetic linkage maps are essential resources for marker-assisted selection to accelerate genetic improvement. We conducted RAD-seq on an Illumina NextSeq500 to discover genome-wide SNPs, and used the SNPs to construct a linkage map for an oil palm (Tenera) population derived from a cross between a Deli Dura and an AVROS Pisifera. The RAD-seq produced 1,076 million single-end reads across the breeding population containing 155 trees. Mining this dataset detected 510,251 loci. After filtering out loci with low accuracy and more than 20% missing data, 11,394 SNPs were retained. Using these SNPs, in combination with 188 anchor SNPs and 123 microsatellites, we constructed a linkage map containing 10,023 markers covering 16 chromosomes. The map length is 2,938.2 cM with an average marker space of 0.29 cM. The large number of SNPs will supply ample choices of DNA markers in analysing the genetic diversity, population structure and evolution of oil palm. This high-density linkage map will contribute to mapping quantitative trait loci (QTL) for important traits, thus accelerating oil palm genetic improvement.

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.