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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.

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.

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.

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

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.

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.

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

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.

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.

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.

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.

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.

Jatropha curcas is believed to be one of the potential biofuel crops, as it does not compete with planting lands for the edible oil plants. However, J. curcas has not been domesticated for producing biodiesel. Conventional breeding to increase the productivity of J. curcas has started since the early 2000s. Although some genetic improvement of oil yield has been made through conventional breeding, oil yield is currently still too low (≤2000 kg/ha/year) to make the biodiesel production from J. curcas sustainable. Due to the enormous potential of marker-assisted selection (MAS) and genomic selection (GS) to speed up genetic gain through early selection, genomic resources such as DNA markers, a linkage map, transcriptome sequences and a draft genome, have been developed and some are being used in genetic improvement for sustainable production of biodiesel. In this review, we present the recent advances in conventional breeding, as well as development and applications of genomic resources to improve the quantity and quality of biodiesel extracted from seeds of J. curcas. We also highlighted the requirement of a well-assembled reference genome of J. curcas and the potentials of next generation sequencing (NGS) for genome-wide association studies (GWAS) and GS to speed up the increase of the yield and quality of biodiesel from J. curcas.

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.

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).

The reproductive characteristics of Chinese Hu sheep are reviewed. Male and female lambs reached puberty at a mean age of 120 days and 180 days, respectively, with an average body weight of 18.54 ± 1.48 kg ( n = 31) and 22.75 ± 1.98 kg ( n = 24). Adult male and female sheep reproduced throughout the year. On average, ewes lambed 1.65 times per year ( n = 1482). The mean ovulation rate of adult ewes was over 2.2 ( n = 46). The average litter size of 150863 ewes raised under extensive conditions was 2.06. The early sexual maturity of male and female lambs was respectively related to high concentrations of testosterone and 17β-E 2 in blood plasma in the earlier postnatal period. The mechanism of high fecundity is not known.