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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 red swamp crayfish, Procambarus clarkii, is a native species in north-eastern Mexico and south-central USA. P. clarkii was introduced to China in 1929 and has been used as an aquaculture species in China since 1983. It currently exists in most of the provinces of China, but threatens local fish, crustaceans, aquatic plants and local freshwater ecosystems. We examined the genetic variation in partial mitochondrial ND2 gene of 831 individuals collected from 25 P. clarkii populations in 13 provinces of China to infer the expansion pathways and mechanisms. Six haplotypes were detected. All six haplotypes appeared in four populations in Nanjing and a population located near Nanjing whereas only 1–5 of the six haplotypes were present in other populations. These data suggest that the populations in Nanjing are probably the source of all other populations in China. There were no significant relationships between geographic distances and genetic distances in 25 populations, whereas significant relationship was found in four populations in Qinhuai River covering 50 km in Nanjing. These data suggest that the expansion mainly be human-mediated in large scale, and active disposal or non-anthropogenic passive dispersal might have played an important role in expansion at a smaller scale. In some places far away from Nanjing, several haplotypes existed, suggested multiple introduction events may have happened. Although aquaculture of this species could bring huge economic benefit, its potential to negatively affect native biota and entire ecosystems should not be ignored. National University of Singapore (NUS), Temasek Laboratories Published version The research was funded by the internal fund of the Temasek Life Sciences Laboratory (Fund number: 5020).

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

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

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

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

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

Oil palm (Elaeis guineensis, Jacq.) is a key tropical oil crop, which provides over one third of the global vegetable oil production, but few genes related to oil yield have been characterized. In this study, a GDSL esterase/lipase gene, which was significantly associated with oil content, was isolated from oil palm and designated as EgGDSL. Its functional characterization was carried out through ectopic expression in Arabidopsis ecotype Col-0. It was shown that expression of EgGDSL in Arabidopsis led to the increased total fatty acid content by 9.5% compared with the wild type. Further analysis of the fatty acid composition revealed that stearic acid (18:0) increased in the seeds of the transgenic lines, but the levels of linoleic acid (18:2) plus 11-eicosenoic acid drastically declined. Quantitative real-time PCR (qPCR) revealed that in oil palm, EgGDSL was highly expressed in mesocarp followed by leaf, and the expression level was very low in the root. The expression level of EgGDSL gene began to increase at two months after flowering (MAF) and reached its peak by four MAF, then declined rapidly, and reached its lowest level during the mature period (6 MAF). The EgGDSL gene was more highly expressed in oil palm trees with high oil content than that with low oil content, demonstrating that the transcription level of EgGDSL correlated with the amount of oil accumulation. The gene may be valuable for engineering fatty acid metabolism in crop improvement programmes and for marker-assisted breeding.

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

Background Oil palm (Elaeis guineensis, Jacq.) is the most important source of edible oil. The improvement of oil yield is currently slow in conventional breeding programs due to long generation intervals. Marker-assisted selection (MAS) has the potential to accelerate genetic improvement. To identify DNA markers associated with oil content traits for MAS, we performed quantitative trait loci (QTL) mapping using genotyping by sequencing (GBS) in a breeding population derived from a cross between Deli Dura and Ghana Pisifera, containing 153 F1 trees. Results We constructed a high-density linkage map containing 1357 SNPs and 123 microsatellites. The 16 linkage groups (LGs) spanned 1527 cM, with an average marker space of 1.03 cM. One significant and three suggestive QTL for oil to bunch (O/B) and oil to dry mesocarp (O/DM) were mapped on LG1, LG8, and LG10 in a F1 breeding population, respectively. These QTL explained 7.6–13.3% of phenotypic variance. DNA markers associated with oil content in these QTL were identified. Trees with beneficial genotypes at two QTL for O/B showed an average O/B of 30.97%, significantly (P

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.

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.

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.

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.

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

Oil palm is the most productive oil crop in the world and composes 36% of the world production. However, the molecular mechanisms of hybrids vigor (or heterosis) between Dura, Pisifera and their hybrid progeny Tenera has not yet been well understood. Here we compared the temporal and spatial compositions of lipids and transcriptomes for two oil yielding organs mesocarp and endosperm from Dura, Pisifera and Tenera. Multiple lipid biosynthesis pathways are highly enriched in all non-additive expression pattern in endosperm, while cytokinine biosynthesis and cell cycle pathways are highly enriched both in endosperm and mesocarp. Compared with parental palms, the high oil content in Tenera was associated with much higher transcript levels of EgWRI1, homolog of Arabidopsis thaliana WRINKLED1. Among 338 identified genes in lipid synthesis, 207 (61%) has been identified to contain the WRI1 specific binding AW motif. We further functionally identified EgWRI1-1, one of three EgWRI1 orthologs, by genetic complementation of the Arabidopsis wri1 mutant. Ectopic expression of EgWRI1-1 in plant produced dramatically increased seed mass and oil content, with oil profile changed. Our findings provide an explanation for EgWRI1 as an important gene contributing hybrid vigor in lipid biosynthesis in oil palm.

Seed size is a major determinant of seed yield but few is known about the genetics controlling of seed size in plants. Phytohormones cytokinin and brassinosteroid were known to be involved in the regulation of herbaceous plant seed development. Here we identified a homolog of Auxin Response Factor 19 (JcARF19) from a woody plant Jatropha curcas and genetically demonstrated its functions in controlling seed size and seed yield. Through Virus Induced Gene Silencing (VIGS), we found that JcARF19 was a positive upstream modulator in auxin signaling and may control plant organ size in J. curcas. Importantly, transgenic overexpression of JcARF19 significantly increased seed size and seed yield in plants Arabidopsis thaliana and J. curcas, indicating the importance of auxin pathway in seed yield controlling in dicot plants. Transcripts analysis indicated that ectopic expression of JcARF19 in J. curcas upregulated auxin responsive genes encoding essential regulators in cell differentiation and cytoskeletal dynamics of seed development. Our data suggested the potential of improving seed traits by precisely engineering auxin signaling in woody perennial plants.

Jatropha curcas L. is a potential plant species for biodiesel production. However, its seed yield is too low for profitable production of biodiesel. To improve the productivity, genetic improvement through breeding is essential. Marker-assisted selection (MAS) has the huge potential to accelerate genetic improvement. We mapped 506 markers (216 microsatellites and 290 SNPs from ESTs) onto 11 linkage groups. However, genetic analysis of the yield traits has not been done in jatropha. Quantitative trait loci (QTL) mapping was conducted to identify genetic factors controlling growth and seed yield in jatropha. We identified a total of 28 QTLs for 11 growth and seed traits using a population of 296 backcrossing jatropha trees. QTL and expression QTL analyses were applied to identify genetic factors that are relevant to seed oil traits in jatropha. We screened key genes in auxin pathway including ARF and IAA families and downstream effectors to identify candidate genes controlling seed size in jatropha. JcARF19 was mapped in the major QTL region and significantly associated with seed length.

Jatropha curcas is a new promising bioenergy crop due to the high oil content in its seeds that can be converted into biodiesel. Seed size, a major determinant of Jatropha oil yield, is a target trait for Jatropha breeding. Due to the vital roles of phytohormone auxin in controlling seed and fruit development, we screened key genes in auxin pathway including ARF and IAA families and downstream effectors to identify candidate genes controlling seed size in Jatropha. As a result, JcARF19 was mapped in the major quantitative trait locus (QTL) region and significantly associated with seed length. By using expression QTL (eQTL) analysis to link variants with functional candidate genes, we provided evidences that seed traits were affected by the interaction of JcARF19 and JcIAA9. ARF19 and IAA9, involved in auxin signal transduction, were conserved in higher plants. These data including the single-nucleotide polymorphisms (SNPs) in the two genes could lead to utilization of the genes by integrating favored alleles into elite varieties through marker-assisted selection.

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.

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

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

Understanding the genomic signatures of population differentiation is fundamental to obtain a comprehensive view of the evolutionary process of organisms. Grass carp is one of the most important fish species in the world due to its significant value in aquaculture and world-wide vegetation biocontrol. However, little is known about the contemporary population structure and also the genetic basis of adaptation to a wide range of latitudinal environments. Using 43310 SNPs generated by genotyping by sequencing in 197 grass carps from nine populations, we examined the patterns of population differentiation, historical introduction and evidence of local selection. The overall genetic differentiation across all native populations was unexpectedly low. Nevertheless, these native populations were clearly differentiated into three genetic clusters, corresponding to the Yangtze River, the Pearl River and the Heilongjiang River System, respectively. Populations in Malaysia, India and Nepal, with the earliest introduction records, most likely have an origin from the Pearl River System. Using conceptually different approaches, 451 loci were detected under potential local selection, among which 84 were annotated to have a gene feature. 19.0% of the genes under putative selection were involved in immune responses, while 42.9% of the annotated loci showed a signature of latitudinal variation. This study provides valuable information for application of genomic tools in addressing questions concerning population differentiation that was influenced by both neutral and adaptive forces, as well as human activities.

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

An (AC)n-microsatellite-enriched library for Larimichthys crocea was constructed in this study. Primers for fifty simple sequence repeat (SSR) loci were synthesized and genotyped on 30 L. crocea individuals from Guanjingyang wild population (WP) in Fujian Province and 38 individuals from Ningbo cultured population (CP) in Zhejiang Province. Only 21 loci were successfully amplified and polymorphic in two populations. In WP, the observed heterozygosity (HO) ranged from 0.233 to 0.900 and the expected heterozygosity (HE) ranged from 0.326 to 0.893, with an average of 7.8 alleles/locus, the polymorphism information content (PIC) ranged from 0.283 to 0.866 (mean 0.731). In CP, the HO ranged from 0.189 to 0.892 and the HE ranged from 0.333 to 0.800, with an average of 4.4 alleles/locus. The probability test showed significant departures from HWE in 9 and 2 of the 21 loci in WP and in CP, respectively. Deficiency of heterozygotes at four loci showed the presence of null alleles (P

The Asian green mussel, Perna viridis, has been widely cultured in Southeast Asia as delicious seafood depending on natural spat. We analyzed the genetic diversity and population structure of three populations (two from Singapore and one from Malaysia) from natural habitats and two from Singapore farms, by genotyping a total of 262 adult individuals using nine polymorphic microsatellites. In all five populations, high allelic (number of alleles A = 11.0–14.4,allelic richness Ar = 10.4–13.9) and gene diversity (expected heterozygosity He = 0.73–0.77) were observed, whereas deficiency of heterozygosity (inbreeding index f = 0.08–0.12) was detected in all populations. Genetic differentiation among populations was low (pairwise fixation index ranged from 0.003 to 0.088), but statistically significant. The usefulness of the information about genetic variations for genetic resource management of the populations in wild habitats and farms to keep genetic variation and setting up a breeding program of Asian green mussel was discussed.

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

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

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

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.

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

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

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

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

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

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

Studies on the zebrafish model have contributed to our understanding of several important developmental processes, especially those that can be easily studied in the embryo. However, our knowledge on late events such as gonad differentiation in the zebrafish is still limited. Here we provide an analysis on the gene sets expressed in the adult zebrafish testis and ovary in an attempt to identify genes with potential role in (zebra)fish gonad development and function. We produced 10 533 expressed sequence tags (ESTs) from zebrafish testis or ovary and downloaded an additional 23 642 gonad-derived sequences from the zebrafish EST database. We clustered these sequences together with over 13 000 kidney-derived zebrafish ESTs to study partial transcriptomes for these three organs. We searched for genes with gonad-specific expression by screening macroarrays containing at least 2600 unique cDNA inserts with testis-, ovary- and kidney-derived cDNA probes. Clones hybridizing to only one of the two gonad probes were selected, and subsequently screened with computational tools to identify 72 genes with potentially testis-specific and 97 genes with potentially ovary-specific expression, respectively. PCR-amplification confirmed gonad-specificity for 21 of the 45 clones tested (all without known function). Our study, which involves over 47 000 EST sequences and specialized cDNA arrays, is the first analysis of adult organ transcriptomes of zebrafish at such a scale. The study of genes expressed in adult zebrafish testis and ovary will provide useful information on regulation of gene expression in teleost gonads and might also contribute to our understanding of the development and differentiation of reproductive organs in vertebrates.

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

We isolated 18 novel microsatellite loci from the walking catfish (Clarias batrachus), and examined their cross-amplification in seven additional catfish species from three families. Sixteen of the 18 microsatellites were polymorphic in the source species (allele number: 2–10/locus and expected heterozygosity: 0.30–0.87). Moreover, nine of these 18 primer pairs cross-amplified specific and polymorphic products from the genome of at least six of the seven other catfish species tested. However, the success rate of cross-species amplification varied from locus to locus, indicating that cross-species amplification of microsatellites is locus-dependent.

A sex-associated amplified fragment length polymorphism and a strain-specific random amplified polymorphic DNA marker were identified from Asian arowana (dragonfish; Scleropages formosus Muller & Schlegel) by screening pooled genomic DNA samples from three different strains as well as males and females respectively. Both markers were cloned, sequenced and successfully converted into sequence-tagged-site (STS) markers. The strain-specific STS marker could be applied to differentiate the Indonesian golden strain of Asian arowana from the green and blood-red strains before the stage when colours become identifiable. Individuals from the green strain could be sexed with an efficiency of 82.7% using the sex-associated STS marker. Thus, populations with preferred sex ratios can be formed without the need of rearing a large number of fish.

The brown mud mussel Glauconome rugosa is a delicious mussel food species. Due to over-harvesting for food in the wild, some populations may be endangered. Seventeen di- and tetranucleotide microsatellites were isolated and characterized using 24 wild caught individuals. The average allele number of the 17 microsatellites was 8/locus with a range of 5–14, and the expected heterozygosity averaged at 0.80, ranging from 0.65 to 0.91. Eleven out of 17 microsatellites agreed with the Hardy–Weinberg equilibrium (HWE) and showed independent segregation. Extreme size difference (over 50 bp) between alleles was seen at five of the 17 loci. These microsatellites will facilitate the studying of the genetic diversity and population structure of wild populations of G. rugosa.

Three methods used for comparing genomic DNA did not detect a sex-specific genomic marker in the green spotted pufferfish Tetraodon nigroviridis.

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

The Asian swamp eel Monopterus albus is distributed in rice fields, rivers and ponds. Due to large scale use of pesticides in rice fields and destruction of natural habitats, the wild populations are decreasing very quickly. To facilitate studies on genetic diversity, we isolated 16 polymorphic novel microsatellites and characterized them using 48 individuals from two natural populations from two locations. The average allele number of the 16 microsatellites was 10.1/locus, ranging from 2 to 20. The expected heterozygosity was 0.79 whereas the observed heterozygosity was 0.65. In both populations, all markers conformed to HWE and showed no significant linkage between pairwise markers. The genetic differentiation between the two populations was significant (F ST = 0.24, P