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ABSTRACT: Calreticulin (CRT) is a Ca2+-binding molecular chaperone in the endoplasmic reticulum. We cloned and characterized the CRT gene in an important marine food fish species Asian seabass (Lates calcarifer). The full-length DNA of the CRT gene was 2194 bp, including a complete open reading frame encoding 420 amino acid residues, a 113 bp 5'-untranslated region and an 818 bp 3'-untranslated region. The CRT gene contained nine exons and eight introns covering a total of 2772 bp genomic DNA from the start to stop codon. Ten single nucleotide polymorphisms (SNPs) were detected in introns and an exon in six individuals collected from five different locations. The CRT gene was assigned to linkage group 4 of the linkage map of Asian seabass. Quantitative real-time PCR revealed that the CRT gene was highly expressed in liver at the age of 1, 3 and 7 months under normal conditions, whereas its expression in liver reduced sharply after 0.5 to 2 h cold challenge at 16°C, and then increased slowly. A preliminary association analysis showed a significant (P < 0.001) association between the SNP6 in the CRT gene and the mortality after cold challenge at 16°C. Our results suggest that the CRT gene is associated with cold tolerance of Asian seabass and further investigation will be necessary to illustrate the underlying mechanisms.
animal 06/2012; 6(6):887-93. · 1.74 Impact Factor
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ABSTRACT: The caudal fin represents a fundamental design feature of fishes and plays an important role in locomotor dynamics in fishes. The shape of caudal is an important parameter in traditional systematics. However, little is known about genes involved in the development of different forms of caudal fins. This study was conducted to identify and map quantitative trait loci (QTL) affecting the length of caudal fin and the ratio between tail length and standard body length in Asian seabass (Lates calcarifer). One F1 family containing 380 offspring was generated by crossing two unrelated individuals. One hundred and seventeen microsatellites almost evenly distributed along the whole genome were genotyped. Length of caudal fin at 90 days post-hatch was measured. QTL analysis detected six significant (genome-wide significant) and two suggestive (linkage-group-wide significant) QTL on seven linkage groups. The six significant QTL explained 5.5-16.6% of the phenotypic variance, suggesting these traits were controlled by multiple genes. Comparative genomics analysis identified several potential candidate genes for the length of caudal fin. The QTL for the length of caudal fin detected for the first time in marine fish may provide a starting point for the future identification of genes involved in the development of different forms of caudal fins in fishes.
Marine Biotechnology 03/2010; 13(1):74-82. · 3.43 Impact Factor
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ABSTRACT: Background: Grass carp (Ctenopharyngodon idella) belongs to the family Cyprinidae which includes more than 2000 fish species. It is one of the most important freshwater food fish species in world aquaculture. A linkage map is an essential framework for mapping traits of interest and is often the first step towards understanding genome evolution. The aim of this study is to construct a first generation genetic map of grass carp using microsatellites and SNPs to generate a new resource for mapping QTL for economically important traits and to conduct a comparative mapping analysis to shed new insights into the evolution of fish genomes. Results: We constructed a first generation linkage map of grass carp with a mapping panel containing two F1 families including 192 progenies. Sixteen SNPs in genes and 263 microsatellite markers were mapped to twenty-four linkage groups (LGs). The number of LGs was corresponding to the haploid chromosome number of grass carp. The sex-specific map was 1149.4 and 888.8 cM long in females and males respectively whereas the sex-averaged map spanned 1176.1 cM. The average resolution of the map was 4.2 cM/locus. BLAST searches of sequences of mapped markers of grass carp against the whole genome sequence of zebrafish revealed substantial macrosynteny relationship and extensive colinearity of markers between grass carp and zebrafish. Conclusions: The linkage map of grass carp presented here is the first linkage map of a food fish species based on co-dominant markers in the family Cyprinidae. This map provides a valuable resource for mapping phenotypic variations and serves as a reference to approach comparative genomics and understand the evolution of fish genomes and could be complementary to grass carp genome sequencing project.
Bmc Genomics. 01/2010; 11.
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ABSTRACT: Microsatellites are the most popular markers for parentage assignment and population genetic studies. To meet the demand for international comparability for genetic studies of Asian seabass, a standard panel of 28 microsatellites has been selected and characterized using the DNA of 24 individuals from Thailand, Malaysia, Indonesia and Australia. The average allele number of these markers was 10.82 +/- 0.71 (range: 6-19), and the expected heterozygosity averaged 0.76 +/- 0.02 (range: 0.63-1.00). All microsatellites showed Mendelian inheritance. In addition, eight standard size controls have been developed by cloning a set of microsatellite alleles into a pGEM-T vector to calibrate allele sizes determined by different laboratories, and are available upon request. Seven multiplex PCRs, each amplifying 3-5 markers, were optimized to accurately and rapidly genotype microsatellites. Parentage assignment using 10 microsatellites in two crosses (10 x 10 and 20 x 20) demonstrated a high power of these markers for revealing parent-sibling connections. This standard set of microsatellites will standardize genetic diversity studies of Asian seabass, and the multiplex PCR sets will facilitate parentage assignment.
Animal Genetics 09/2009; 41(2):208-12. · 2.40 Impact Factor
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G. H. Yue, Z. Y. Zhu,
L. C. Lo,
C. M. Wang,
G. Lin,
F. Fenf,
H. Y. Pang,
J. Li,
P. Gong,
H. M. Liu,
J. Tan,
R. Chou,
H. Lim,
L. Orban
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ABSTRACT: 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 (F-ST = 0.124, R-ST = 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. (C) 2009 Elsevier B.V. All rights reserved.
Aquaculture. 01/2009; 293(1-2):22-28.
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ABSTRACT: Background: Microsatellites in cDNA are useful as molecular markers because they represent transcribed genes and can be used as anchor markers for linkage and comparative mapping, as well as for studying genome evolution. Microsatellites in cDNA can be detected in existing ESTs by data mining. However, in most fish species, no ESTs are available or the number of ESTs is limited, although fishes represent half of the vertebrates on the earth. We developed a simple and efficient method for isolation of microsatellites from cDNA in fish. Results: The method included normalization of 150 ng cDNA using 0.5 U duplex-specific nuclease (DSN) at 65 degrees C for 30 min, enrichment of microsatellites using biotinylated oligonucleotides and magnetic field, and directional cloning of cDNA into a vector. We tested this method to enrich CA-and GA-microsatellites from cDNA of Asian seabass, and demonstrated that enrichment of microsatellites from normalized cDNA could increased the efficiency of microsatellite isolation over 30 times as compared to direct sequencing of clones from cDNA libraries. One hundred and thirty-nine (36.2%) out of 384 clones from normalized cDNA contained microsatellites. Unique microsatellite sequences accounted for 23.6% (91/384) of sequenced clones. Sixty microsatellites isolated from cDNA were characterized, and 41 were polymorphic. The average allele number of the 41 microsatellites was 4.85 +/- 0.54, while the expected heterozygosity was 0.56 +/- 0.03. All the isolated microsatellites inherited in a Mendelian pattern. Conclusion: Normalization of cDNA substantially increased the efficiency of enrichment of microsatellites from cDNA. The described method for isolation of microsatellites from cDNA has the potential to be applied to a wide range of fish species. The microsatellites isolated from cDNA could be useful for linkage and comparative mapping, as well as for studying genome evolution.
Bmc Genomics. 01/2009; 10.
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Animal Genetics 11/2008; 40(1):125-6. · 2.40 Impact Factor
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ABSTRACT: 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 F(1) 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.
Animal Genetics 03/2008; 39(1):34-9. · 2.40 Impact Factor
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ABSTRACT: Self-cloning is quite rare in shrimp, lobsters, crayfish and crabs. Here we report the discovery of four natural clones of red swamp crayfish (Procambarus clarkii), each containing 2-6 genetically identical individuals, during the genotyping of 120 individuals with five microsatellites. The four clones were heterozygote at most of the five microsatellite loci. Phylogenetic analysis using microsatellite genotypes suggests recent origin of the four clones. Sequencing a part of the mitochondrial gene Cox I confirmed that the four clones were from the species Procambarus clarkii.
International journal of biological sciences 02/2008; 4(5):279-82. · 2.70 Impact Factor
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ABSTRACT: Eleven microsatellites were isolated from a genomic DNA library enriched for CA- and GA-repeats, and were characterized in 48 individuals of red swamp crayfish Procambarus clarkii. All 11 microsatellites were polymorphic with an average of allele number of 6.6 per locus. Genotypic distributions of these 11 markers were found to be in conformance with the expectations of Hardy-Weinberg equilibrium. All 11 markers were unlinked. These markers are being used to study the invasion routine, genetic diversity and population structure of the species P. clarkii.
Molecular Ecology Resources. 01/2008; 8(4):796-798.
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ABSTRACT: 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. leopardits 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. (c) 2008 Elsevier B.V. All rights reserved.
Aquaculture. 01/2008; 276(1-4):44-49.
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ABSTRACT: Here we report the isolation and characterization of 17 polymorphic loci isolated from a partial genomic DNA library of grass carp (Ctenopharyngodon idellus) enriched for CA repeats. We tested variability of these microsatellites on 24 unrelated individuals collected in China. All microsatellites were polymorphic. The average allele number was 7.9 per locus, ranging from four to 13. The observed heterozygosity was from 0.46 to 0.88 with an average of 0.71, whereas the average expected heterozygosity was 0.78. Sixteen of the 17 microsatellites conformed to Hardy-Weinberg equilibrium, and inherited independently. These microsatellites can be used to study genetic diversity and population structure of wild populations, and facilitate selective breeding of cultured broodstocks.
Molecular Ecology Notes. 01/2007; 7(6):1114-1116.
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ABSTRACT: Barramundi (Lates calcarifer) is an important marine food fish species in Southeast Asia and Australia. Seventy-four novel microsatellites were isolated from a genomic DNA library enriched for CA repeats and were characterized in 24 unrelated individuals. Among the 74 microsatellites, 71 were polymorphic, with an average allele number of 7.0 +/- 3.6/locus. The average expected heterozygosity of these polymorphic markers was 0.66. Sixty-three of the 71 polymorphic microsatellites conformed to Hardy-Weinberg equilibrium. Linkage analyses were conducted in a reference family, leading to the assignment of 34 novel microsatellites and 16 published markers in 16 linkage groups. The novel microsatellites developed in this study will contribute significantly to the construction of a first-generation linkage map for mapping of quantitative trait loci in Barramundi, and supply a large choice of markers for studies on population genetics, stock management, and pedigree reconstruction.
Genome 09/2006; 49(8):969-76. · 1.65 Impact Factor
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ABSTRACT: Parvalbumins are extremely abundant in fish muscle and play an important role in muscle relaxation. In this study, two parvalbumin genes (PVALB1 and PVALB2) were cloned from Asian seabass (Lates calcarifer). The cDNAs for PVALB1 and PVALB2 were 840 and 667 bp respectively. Both genes consisted of five exons and four introns, encoded 109 amino acids, and were of beta lineage. Using real-time polymerase chain reaction, expression of PVALB1 was detected in all 10 tissues tested, with expression in brain, kidney, muscle and small intestine being 15- to 322-fold higher than in the other tissues. Expression of PVALB2 was detected only in muscle, brain and intestine, and was up to 10-fold lower than PVALB1 expression. A (CT)(17) microsatellite in the 3'-untranslated region of PVALB1 and three single nucleotide polymorphisms (SNPs) in the third intron of PVALB2 were identified. The microsatellite in PVALB1 was significantly associated with body weight and body length at 90 days post-hatch (P < 0.01), whereas the SNPs in PVALVB2 were not associated with these traits.
Animal Genetics 06/2006; 37(3):266-8. · 2.40 Impact Factor
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ABSTRACT: Crimson red snapper (Lutjanus erythropterus) is an important marine foodfish species in Asia with great potentials for aquaculture. We isolated nine polymorphic microsatellites, among which eight were tetranucleotide repeats, and one was CA-microsatellite. The average allele number present in 48 individuals was 11.1/locus, ranging from three to 33. The expected heterozygosity ranged from 0.49 to 0.97 with an average of 0.74. Six of the nine markers conformed to the Hardy-Weinberg expectations. No pairwise markers showed the possibility of linkage. Protocols for two multiplex polymerase chain reactions (PCRs), each amplifying two and seven markers, respectively, were presented. The developed microsatellites and optimized multiplex PCRs will be useful for studying population structure of wild stocks and parentage assignment for cultured populations.
Molecular Ecology Notes. 01/2006; 6(2):524-526.
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ABSTRACT: Background: Body weight and length are economically important traits in foodfish species influenced by quantitative trait loci (QTL) and environmental factors. It is usually difficult to dissect the genetic and environmental effects. Asian seabass (Lates calcarifer) is an important marine foodfish species with a compact genome (similar to 700 Mb). The recent construction of a first generation linkage map of Asian seabass with 240 microsatellites provides a good opportunity to determine the number and position of QTL, and the magnitude of QTL effects with a genome scan. Results: We conducted a genome scan for QTL affecting body weight, standard length and condition factors in an F1 family containing 380 full-sib individuals from a breeding stock by using 97 microsatellites evenly covering 24 chromosomes. Interval mapping and multiple QTL model mapping detected five significant and 27 suggestive QTL on ten linkage groups (LGs). Among the five significant QTL detected, three (qBW2-a, qTL2-a and qSL2-a) controlling body weight, total and standard length respectively, were mapped on the same region near Lca287 on LG2, and explained 28.8, 58.9 and 59.7% of the phenotypic variance. The other two QTL affecting body weight, qBW2b and qBW3, were located on LG2 and 3, and accounted for 6.4 and 8.8% of the phenotypic variance. Suggestive QTL associated with condition factors are located on six different LGs. Conclusion: This study presents the first example of QTL detection for growth- related traits in an F1 family of a marine foodfish species. The results presented here will enable further fine-mapping of these QTL for marker-assisted selection of the Asian seabass, eventually identifying individual genes responsible for growth-related traits.
Bmc Genomics. 01/2006; 7.
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ABSTRACT: 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 useful in studying fish evolution, in differentiating different Tetraodon species and in analyzing genetic diversity within T. nigroviridis.
DNA Sequence. 01/2006; 17(2):115-121.
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ABSTRACT: Asian arowana (Scleropages formosus) is a highly endangered fish species listed in Appendix I of CITES since 1980. Fourteen novel polymorphic microsatellites were isolated from a CA-enriched partial genomic DNA library, and were used for studying genetic diversity of 41 red arowanas from the wild population. Surprisingly the average allele number of the 14 polymorphic microsatellites was as high as 12.4/locus. The average observed heterozygosity was 0.78 ranging from 0.51 to 0.95, and the gene diversity was quite high (0.78). All these data suggest that high level of genetic diversity existed in the red Asian arowana population.
Conservation Genetics. 01/2006; 7(4):627-629.
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ABSTRACT: To study genetic diversity of two cultured and one wild stock of Asian seabass (Lates calcar fer) 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 1; and allele number=8.0 and H-e=0.72 in Singapore stock II). F-1S values within each stock were very low. Significant genetic differentiation (F-ST=0.03, P < 0.05) and private alleles were detected among the three stocks. Thus, it may be possible to form a new superior broodstock with high genetic diversity by combining fish from different stocks. The developed polymorphic microsatellites, and especially the multiplex PCR set, are useful in monitoring genetic diversity of cultured stocks and for studying the genetic structure of wild stocks of Asian seabass. (c) 2006 Elsevier B.V All rights reserved.
Aquaculture. 01/2006; 256(1-4):167-173.
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ABSTRACT: The first set of six polymorphic microsatellites was isolated from a partial genomic DNA library of an important marine food fish Gnathanodon specious, and was characterized in 28 unrelated individuals. The allele number ranged from three to 12 with an average of seven/locus; average expected heterozygosity was 0.57 ranging from 0.10 to 0.87, whereas the observed heterozygosity ranged from 0.11 to 0.96 (average: 0.57). All six markers conform to Hardy-Weinberg and linkage equilibria. These markers will be useful for accessing genetic population structure of wild populations and for selective breeding program in fish farms.
Molecular Ecology Notes. 01/2005; 5(4):760-761.
