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Twelve novel polymorphic microsatellites were isolated from the endangered Hawksbill turtle (Eretmochelys imbricate). Eight of 12 markers were used to study genetic diversity of two sea turtle species: E. imbricate and green sea turtle (C. mydas). In E. imbricate, the average allele number of the eight microsatellites was 6.25/locus with a range of 3–13. The average expected and observed heterozygosity was 0.66 and 0.63 respectively. In C. mydas, the average allele number of the eight markers was 11.63/locus. The observed heterozyosity (0.68) was lower than the expected heterozyosity (0.79). Most of 12 microsatellites amplified specific and polymorphic PCR products in other six turtle species. Hence, the developed microsatellites would facilitate studies on genetic diversity and population structure of E. imbricate and other marine turtle species.

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.

Asian green mussels (Perna viridis) belonging to the family Mytilidae are native to the coastal and tropical marine waters of the Indo-Pacific region. Ten polymorphic microsatellites were isolated and their polymorphisms were determined in 36 individuals. The average allele number of the 10 microsatellites was 11.7/locus with a range of two to 24 and the expected heterozygosity averaged at 0.69, ranging from 0.18 to 0.94. Eight out of 10 microsatellites agreed with the Hardy–Weinberg equilibrium and showed independent segregation. These microsatellites will facilitate the studying of the genetic diversity and population structure of the green mussel in and outside of the Indo-Pacific region of Asia.

The ribbonfish, Trichiurus haumela , is an important marine foodfish species. Supply of this fish relies on capture from the wild. No information about genetic diversity and population structure of wild stocks is available. Therefore, we developed the first set of 10 polymorphic microsatellites. The average allele number was 13.2 per locus ranging from six to 20. The expected heterozygosity was 0.84, whereas the observed heterozygosity was 0.71. This set of microsatellites provides a tool for assessing genetic diversity and population structure of this species.

The orchid genus Vanda includes more than 70 monopodial species and numerous hybrids. The aim of this study was to develop microsatellite markers for this horticultural valuable genus. Thirteen polymorphic microsatellite markers were isolated from the variety Vanda Miss Joaquim and were characterized in four Vanda species, 11 Vanda hybrids, and one Aranda intergenus hybrid. Populations from three species were also analysed. Number of alleles ranged from two to 19. For the V. sumatrana population, the expected heterozygosity ranged from 0 to 0.76 (mean 0.31) and the observed heterozygosity ranged from 0 to 1 (mean 0.36). All the varieties tested were distinct from one another (similarity index < 0.8). These microsatellites could be used for studying genetic diversity and population structure of wild populations within the orchid genus Vanda, as well as for distinguishing cultured Vanda varieties.

The aim of this study was to develop simple sequence repeat (SSR) markers for Dendrobium varieties/species, many of which have medicinal and horticultural values. Two genomic DNA libraries of Dendrobium Sonia enriched with GA repeats and CA repeats were constructed. Fourteen polymorphic SSR markers were identified when screened against 42 popular commercial Dendrobium hybrids. The average allele number was 12.0 ± 1.9 and the observed heterozyosity was averaged at 0.70. All 42 hybrids tested, except for two tissue culture mutants, were uniquely identified with the markers used. Sibling hybrids were closely clustered. Hybrids were also closer to parents. These SSR markers can be used for molecular ecology research, genetic mapping and marker-assisted breeding. They can also help protection for new Dendrobium varieties.

Microsatellites are popular molecular markers in genetic and evolutionary studies. Their mutational dynamics have been extensively studied in humans and fruit flies, but few data were available in fish. By genotyping 55 individuals of a F1 pedigree, we investigated the mutation rates and patterns of 49 microsatellites in one of the most important fresh water fish species, the common carp (Cyprinus carpio L.). The overall mutation rate of the 49 loci was 5.56 x 10(-4)/locus/generation (95% confidence interval 1.52 x 10(-4) and 1.63 x 10(-3)). The change of allele size was between +2 to -5 repeat units, assuming that the mutation allele arose from the parental allele most similar in size to the mutant.

Eleven polymorphic microsatellites were isolated from an important foodfish species, silver pomfret ( Pampus argenteus ). The loci showed different variation patterns in 24 unrelated P. argenteus individuals, with a mean number of alleles of 11.5 and a mean expected heterozygosity of 0.85. Eight of the 11 loci conformed to Hardy–Weinberg equilibrium. All and six of the 11 markers amplified specific and polymorphic products in Chinese pomfret ( Pampus sinesis ) and dark pomfret ( Pamus cinereus ), respectively. These markers should be useful for population genetic analysis and biodiversity studies of P. argenteus and other pomfret species .

Summary 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.

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.

We isolated 12 polymorphic microsatellites from an important marine food fish Larimichthys polyactis and characterized them in 32 unrelated individuals. Among the 12 microsatellites, four were tetranucleotide repeats and eight were dinucleotide repeats. The allele number ranged from five to 25 with an average of 15.4/locus; average expected heterozygosity was 0.81, ranging from 0.57 to 0.95, whereas the observed heterozygosity ranged from 0.34 to 1.00 (average: 0.78). Nine of the 12 markers conformed to Hardy–Weinberg equilibrium and showed no sign of linkage. These microsatellites will be useful for population genetic studies and selective breeding programs of this species.

Asian arowana (Scleropages formosus) is a highly endangered fish species listed in Appendix 1 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.

The red coral grouper, Plectropomus maculatus , is a high value marine food fish species. We isolated the first set of 10 polymorphic microsatellites and examined their polymorphism in two related species: the Malabar grouper Epinephelus malabaricus and the brown-marbled grouper Epinephelus fuscoguttatus . The average allele number of these 10 microsatellite DNA loci is 9.1/locus with a range of four to 17, whereas the expected heterozygosity ranged from 0.18 to 0.94, averaging 0.76. Five of the 10 markers amplified polymorphic and specific products in E. malabaricus , whereas only four were amplified in E. fuscoguttatus . These microsatellites could be applicable to captive breeding and to the studies on genetic diversity and population structure of grouper wild stocks.

Background Aquaculture is the quickest growing sector in agriculture. However, QTL for important traits have been only identified in a few aquaculture species. We conducted QTL mapping for growth traits in an Asian seabass F2 family with 359 individuals using 123 microsatellites and 22 SNPs, and performed association mapping in four populations with 881 individuals. Results Twelve and nine significant QTL, as well as 14 and 10 suggestive QTL were detected for growth traits at six and nine months post hatch, respectively. These QTL explained 0.9-12.0% of the phenotypic variance. For body weight, two QTL intervals at two stages were overlapped while the others were mapped onto different positions. The IFABP-a gene located in a significant QTL interval for growth on LG5 was cloned and characterized. A SNP in exon 3 of the gene was significantly associated with growth traits in different populations. Conclusions The results of QTL mapping for growth traits suggest that growth at different stages was controlled by some common QTL and some different QTL. Positional candidate genes and association mapping suggest that the IFABP-a is a strong candidate gene for growth. Our data supply a basis for fine mapping QTL, marker-assisted selection and further detailed analysis of the functions of the IFABP-a gene in fish growth.

Omega-3 fatty acids are essential fatty acids for human health. Therefore, increasing both percentage of omega-3 and a better fatty acid profile in fish fillets is one of the breeding goals in aquaculture. However, it is difficult to increase the omega-3 content in fish fillets, as the phenotypic selection of these traits is not easily feasible. To facilitate the genetic improvement of the Asian seabass for optimal fatty acid profiles, a genome-wide scan for quantitative trait loci (QTL) affecting fatty acid level in the flesh of the Asian seabass was performed on an F2 family containing 314 offspring. All family members were genotyped using 123 informative microsatellites and 22 SNPs. High percentages of n-3 polyunsaturated fatty acids (PUFA), especially C22:6 (DHA 16.48 ± 3.09 %) and C20:5 (EPA 7.19 ± 0.86 %) were detected in the flesh. One significant and 54 suggestive QTL for different fatty acids and a water content trait were detected on the whole genome. QTL for C18:0b was located on linkage groups (LG) 5. QTL for total n-3 PUFA content in flesh were mapped onto LG6 and LG23 with the phenotypic variance explained ranging from 3.8 to 6.3 %. Four QTL for C22:6 were detected on LG6, LG23, and LG24, explaining 3.9 to 4.9 % of the phenotypic variance, respectively. Mapping of QTL for contents of different fatty acids is the first step towards improving the omega-3 content in the fillets of fish by using marker-assisted selection and is important for understanding the biology of fatty acid deposition.

Background Tilapia is the common name for a group of cichlid fishes and is one of the most important aquacultured freshwater food fish. Mozambique tilapia and its hybrids, including red tilapia are main representatives of salt tolerant tilapias. A linkage map is an essential framework for mapping QTL for important traits, positional cloning of genes and understanding of genome evolution. Results We constructed a consensus linkage map of Mozambique tilapia and red tilapia using 95 individuals from two F1 families and 401 microsatellites including 282 EST-derived markers. In addition, we conducted comparative mapping and searched for sex-determining loci on the whole genome. These 401 microsatellites were assigned to 22 linkage groups. The map spanned 1067.6 cM with an average inter-marker distance of 3.3 cM. Comparative mapping between tilapia and stickleback, medaka, pufferfish and zebrafish revealed clear homologous relationships between chromosomes from different species. We found evidence for the fusion of two sets of two independent chromosomes forming two new chromosome pairs, leading to a reduction of 24 chromosome pairs in their ancestor to 22 pairs in tilapias. The XY sex determination locus in Mozambique tilapia was mapped on LG1, and verified in five families containing 549 individuals. The major XY sex determination locus in red tilapia was located on LG22, and verified in two families containing 275 individuals. Conclusions A first-generation linkage map of salt tolerant tilapia was constructed using 401 microsatellites. Two separate fusions of two sets of two independent chromosomes may lead to a reduction of 24 chromosome pairs in their ancestor to 22 pairs in tilapias. The XY sex-determining loci from Mozambique tilapia and red tilapia were mapped on LG1 and LG22, respectively. This map provides a useful resource for QTL mapping for important traits and comparative genome studies. The DNA markers linked to the sex-determining loci could be used in the selection of YY males for breeding all-male populations of salt tolerant tilapia, as well as in studies on mechanisms of sex determination in fish.

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.

Three F2 families have been established for QTL mapping by crossing genetically divergent Wild boar, Meishan and Pietrain pigs. More than 900 F2 animals are typed for a large number of marker loci and are recorded for over 100 quantitative traits. In a first analysis QTLs on chromosome 6 have been mapped in the region of the ryanodine receptor gene. Also several traits were significantly associated with different GH haplotyes.

The green swordtail (Xiphophorus hellerii; Poeciliidae) is a popular ornamental freshwater fish species. Fourteen microsatellites were isolated from a green swordtail genomic library enriched for CA-repeats. Thirteen microsatellites were polymorphic in green swordtail; interestingly four of them were tetranucleotide-type. Cross-species amplification showed that 10 of the 14 microsatellites were polymorphic in guppy (Poecilia reticulata; Poeciliidae) as well. No significant directional difference of allele length was seen between the two species at any of the loci tested.

In aquaculture species, maintaining pedigree information and genetic variation in each generation is essential, but very difficult. In this study, we used nine microsatellites to genotype 2,520 offspring from four independent full-factorial crosses (10 males × 10 females) of Asian seabass to reconstruct pedigree and monitor the change of genetic variations. In all four crosses, over 96.8% of the offspring could be assigned to their parents, indicating the high power of the nine microsatellites for parentage assignment. This study revealed several interesting results: (1). In all four crosses, the contribution of parents to offspring was significantly uneven, and some dominant breeding fishes (i.e. brooders) were found; (2). In two mass crosses where the brooders were carefully checked for reproductive status, a majority (≥ 90%) of brooders contributed to offspring, whereas in another two crosses, where the brooders were randomly picked without checking reproductive status, only a few brooders (40.0-45.0%) produced offspring; (3). Females had more problems in successful spawning compared to males; and (4). In the two crosses where a few brooders produced offspring, there was a substantial loss in allelic (24.1-34.3%) and gene (20.5-25.7%) diversities in offspring, while in the other two crosses, the majority of allelic (96.8-97.0%) and gene diversities (94.8-97.1%) were maintained. These observations suggest that a routine molecular parentage analysis is required to maintain both allelic and gene diversity in breeding Asian seabass.

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.

Fifteen polymorphic microsatellites were isolated from the genome of silver crucian carp (Carassius auratus gibelio Bloch). Allele numbers ranged from two to four with an average of 2.7/locus, and the proportion of tri- and diallelic heterozygotes was 99.3%. The individuals tested seem to have originated from two different clonal lines: 14 of 16 showed the same genotype at all loci tested, whereas the remaining two were also identical, but different from the former ones. Eleven out of 15 primer pairs cross-amplified products from the genome of common carp, whereas only five from that of zebrafish.

Analysis of transcriptomes is of great importance in genomic studies. Asian seabass is an important fish species. A number of genomic tools in it were developed, while large expressed sequence tag (EST) data are lacking. We sequenced ESTs from nine normalized cDNA libraries and obtained 11 431 high-quality ESTs. We retrieved 8524 ESTs from dbEST database and analyzed all 19 975 ESTs using bioinformatics tools. After clustering, we obtained 8837 unique sequences (2838 contigs and 5999 singletons). The average contig length was 574 bp. Annotation of these unique sequences revealed that 48.9% of them showed significant homology to RNA sequences in GenBank. Functional classification of the unique ESTs identified a broad range of genes involved in different functions. We identified 6114 putative single-nucleotide polymorphisms and 634 microsatellites in ESTs. We discovered different temporal and spatial expression patterns of some immune-related genes in the Asian seabass after challenging with a pathogen Vibrio harveyi. The unique EST sequences are being used in developing a cDNA microarray to examine global gene expression and will also facilitate future whole-genome sequence assembly and annotation of Asian seabass and comparative genomics.

Background High density linkage maps are essential for comparative analysis of synteny, fine mapping of quantitative trait loci (QTL), searching for candidate genes and facilitating genome sequence assembly. However, in most foodfish species, marker density is still low. We previously reported a first generation linkage map with 240 DNA markers and its application to preliminarily map QTL for growth traits in Asian seabass (Lates calcarifer). Here, we report a high-resolution linkage map with 790 microsatellites and SNPs, comparative analysis of synteny, fine-mapping of QTL and the identification of potential candidate genes for growth traits. Results A second generation linkage map of Asian seabass was developed with 790 microsatellite and SNP markers. The map spanned a genetic length of 2411.5 cM, with an average intermarker distance of 3.4 cM or 1.1 Mb. This high density map allowed for comparison of the map with Tetraodon nigroviridis genome, which revealed 16 synteny regions between the two species. Moreover, by employing this map we refined QTL to regions of 1.4 and 0.2 cM (or 400 and 50 kb) in linkage groups 2 and 3 in a population containing 380 progeny; potential candidate genes for growth traits in QTL regions were further identified using comparative genome analysis, whose effects on growth traits were investigated. Interestingly, a QTL cluster at Lca371 underlying growth traits of Asian seabass showed similarity to the cathepsin D gene of human, which is related to cancer and Alzheimer's disease. Conclusions We constructed a high resolution linkage map, carried out comparative mapping, refined the positions of QTL, identified candidate genes for growth traits and analyzed their effects on growth. Our study developed a framework that will be indispensable for further identification of genes and analysis of molecular variation within the refined QTL to enhance understanding of the molecular basis of growth and speed up genetic improvement of growth performance, and it also provides critical resource for future genome sequence assembly and comparative genomics studies on the evolution of fish genomes.

Jatropha curcas 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. A linkage map is an important component in molecular breeding. We established a first-generation linkage map using a mapping panel containing two backcross populations with 93 progeny. We mapped 506 markers (216 microsatellites and 290 SNPs from ESTs) onto 11 linkage groups. The total length of the map was 1440.9 cM with an average marker space of 2.8 cM. Blasting of 222 Jatropha ESTs containing polymorphic SSR or SNP markers against EST-databases revealed that 91.0%, 86.5% and 79.2% of Jatropha ESTs were homologous to counterparts in castor bean, poplar and Arabidopsis respectively. Mapping 192 orthologous markers to the assembled whole genome sequence of Arabidopsis thaliana identified 38 syntenic blocks and revealed that small linkage blocks were well conserved, but often shuffled. The first generation linkage map and the data of comparative mapping could lay a solid foundation for QTL mapping of agronomic traits, marker-assisted breeding and cloning genes responsible for phenotypic variation.

Four microsatellites were identified by screening the DNA sequences of Asian seabass (Lates calcarifer) deposited to GenBank. Two markers each are located in the growth hormone gene (GH) and in the insulin-like growth factor II gene ( IGF-2), respectively. The markers were characterized by genotyping 34 Asian seabass individuals. All 4 microsatellites showed polymorphism: the number of alleles per locus ranged from 2 to 11 (average, 5.0), while the expected heterozygosity ranged from 0.51 to 0.85 (average, 0.63) at the 4 loci. Cross-priming with all 4 primer pairs was tested in species belonging to 5 different genera, but no bands were amplified. These microsatellites are the first genomic DNA markers characterized in L. calcarifer; thus they may be valuable for research and aquaculture production of this species.

Results and discussion: Analysis of the g.+6723G>A mutation in East Friesian revealed 29 homozygotes for the mutant allele (A/A), 41 heterozygotes (A/G) and 9 animals homozygous for the wild-type allele (G/G). The genotypic frequencies are in agreement with Hardy-Weinberg equilibrium (v = 0.94; d.f. = 1). Detection of g.+6723A within the East Friesian, grouped it with the Texel, White Faced Suffolk, Lincoln and Charollais as a breed known to segregate this Myostatin mutation. The estimated frequency of g.+6723A within the East Friesian was 0.63, which is lower than for Texel, where it is near fixation. This may reflect the emphasis put on milk production by East Friesian breeders, but it also suggests that selection could quickly produce highly muscled animals. Association analysis to determine the effect of the mutation was not performed because of the absence of phenotypic records; however, breeders report the presence of lines within the East Friesian that display enhanced muscularity. Previous work reported that g.+6723A was found on a single haplotype defined by alleles at two flanking microsatellites (BM81124 allele 218 and BULGE20 allele 141). To determine if g.+6723A in East Friesian is accompanied by the same haplotype, the two microsatellites were genotyped in ten homozygous (A/A) sheep. The results revealed that seven of the animals (70%) were homozygous for the same combination of microsatellite alleles observed in the Texel. This strongly suggests a common origin for the mutation, because the alternative scenario that the mutation has arisen independently within each breed would predict that the allele would be accompanied by different haplotypes. The close geographical proximity of the breeds and anecdotal information suggest a shared origin, which explains why the allele appears to be identical by descent in both East Friesian and Texel sheep.

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.

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.

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.

Asian sea bass (Lates calcarifer) is one of the most important marine food fish species in Asia and Australia. To estimate the reproductive success of broodstock and heritabilities of growth-related traits, two independent full-factorial crosses (PI and PII) were created by crossing 10 males and 10 females. At 90 days post hatch (dph), the body weight (BW) and total length (TL) of 804 individuals from PI and 900 individuals from PII were measured and tissues samples of each fish were collected. Parents and offspring were genotyped with nine polymorphic microsatellites. Out of 1704 offspring from the two crosses, 98.7% were assigned to their parents. In PI, 19 of 20 parents produced offspring, while in PII, only five parents contributed to offspring. Low contribution of parents to offspring could lead to reduced genetic variation in the next generation. Heritabilities for growth-related traits were estimated using the pedigrees reconstructed using microsatellite genotypes. The estimates of heritability were 0.22±0.16 and 0.25±0.18 for BW, 0.31±0.14 and 0.24±0.21 for TL and 0.22±0.22 and 0.15±0.09 for Fulton's condition factor in the two crosses respectively. Body weights at 90 dph and at harvest (289 dph) were significantly correlated (r=0.601, P

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.

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 (~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, qBW2-b 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.

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.Key words: Barramundi, microsatellites, polymorphism, linkage mapping.

Background Mitochondrial DNA-derived sequences have become popular markers for evolutionary studies, as their comparison may yield significant insights into the evolution of both the organisms and their genomes. From the more than 24,000 teleost species, only 254 complete mtDNA sequences are available (GenBank status on 06 Sep 2006). In this paper, we report the complete mitochondrial genome sequence of Asian arowana, a basal bonytongue fish species, which belongs to the order of Osteoglossiformes. Results The complete mitochondrial genomic sequence (mtDNA) of Asian arowana (Scleropages formosus) was determined by using shotgun sequencing method. The length of Asian arowana mtDNA is ca. 16,650 bp (its variation is due to polymorphic repeats in the control region), containing 13 protein-coding genes, 22 tRNA and 2 rRNA genes. Twelve of the thirteen protein coding genes were found to be encoded by the heavy strand in the order typically observed for vertebrate mitochondrial genomes, whereas only nad6 was located on the light strand. An interesting feature of Asian arowana mitogenome is that two different repeat arrays were identified in the control region: a 37 bp tandem repeat at the 5' end and an AT-type dinucleotide microsatellite at the 3' end. Both repeats show polymorphism among the six individuals tested; moreover the former one is present in the mitochondrial genomes of several other teleost groups. The TACAT motif described earlier only from mammals and lungfish was found in the tandem repeat of several osteoglossid and eel species. Phylogenetic analysis of fish species representing Actinopterygii and Sarcopterygii taxa has shown that the Asian arowana is located near the baseline of the teleost tree, confirming its status among the ancestral teleost lineages. Conclusion The mitogenome of Asian arowana is very similar to the typical vertebrate mitochondrial genome in terms of gene arrangements, codon usage and base composition. However its control region contains two different types of repeat units at both ends, an interesting feature that to our knowledge has never been reported before for other vertebrate mitochondrial control regions. Phylogenetic analysis using the complete mtDNA sequence of Asian arowana confirmed that it belongs to an ancestral teleost lineage.

The role of thyroid hormone (TH) and its receptors (TRs) in the regulation of body growth and muscle accretion is well established in mammals and birds, whereas the involvement of THs and TRs in fish growth, especially during the muscle accretion period of juvenile-adult transition, is unknown. This study describes the cloning of the partial cDNA sequences of TRalpha and TRbeta in large yellow croaker, Pseudosciaena crocea (Richardson) and the patterns of TRalpha and TRbeta mRNA expression in liver and muscle of 1- and 2-year-old large yellow croaker, associated with changes in body mass and muscle characteristics. Two TRalpha isoforms (TRalpha1, TRalpha2) and TRbeta were identified in large yellow croaker. The deduced amino acid sequences showed high homology to the TRs of human and other teleosts. Hepatic TRbeta mRNA expression was markedly lower in 2-year-old large yellow croaker compared with the 1-year-old, while no significant age difference was observed for hepatic TRalpha mRNA expression. Muscle expression of TRalpha mRNA was significantly higher in 2-year-old large yellow croaker, whereas TRbeta exhibited no significant age difference. Meanwhile, serum concentration of T(4) was significantly decreased in 2-year-old large yellow croaker, but no change was observed for T(3). The body mass, fork length and body height of 2-year-old large yellow croaker were 4.7, 1.6 and 1.7 times greater, respectively compared with that of 1-year-old. Average diameters of skeletal muscle in 2-year-old large yellow croaker were remarkably larger than that in 1-year-old with no significant difference in muscle crude fat content. The down-regulation of hepatic TRbeta expression was associated with the decrease in general growth rate and the increase in muscle expression of TRalpha was accompanied with muscle accretion and myofiber hypertrophy, implicating the different roles of TRs in the regulation of growth in large yellow croaker.

We have developed a very simple and inexpensive method for high-throughput DNA extraction from animal tissues. The procedure contains three steps (digestion, heating, and centrifugation) and it is compatible with the 96-well plate format commonly used in polymerase chain reaction (PCR) amplifications. The duration for processing a plate is about 1.5 h; therefore, one researcher can isolate DNA from up to 1000 samples during a single workday. A small piece of tissue (ca. 10-20 mg) yields enough template for at least 50-70 PCR amplifications, as demonstrated by using the processed samples as templates successfully for long distance PCR, multiplex PCR, and randomly amplified polymorphic DNA (RAPD) assay. The application of our method is expected to facilitate studies that require high-throughput DNA isolation for PCR amplification, such as genotyping by microsatellites for mapping and genetic diversity studies, as well as mutant screening in zebrafish.

The Chinese Erhualian is one of the most prolific pig breeds in the world, but it is in danger of being replaced by other exotic pig breeds because of its slow growth rate and high fat content in the body. To obtain some genetic information for conservation, we analysed the Erhualian pigs by using a PCR-RFLP for the calcium-release-channel (CRC) gene, nine polymorphic microsatellites and the complete mtDNA D-loop sequences, and compared these data with those from other pig breeds from Europe and Asian. The PCR-RFLP analysis of the CRC gene showed that the frequency of the C allele associated with stress resistance was 100% in the Erhualian pigs. Neighbour-Joining trees constructed on the basis of mtDNA D-loop sequences and the microsatellite analysis clearly showed that the Erhualian pigs were located in a separate branch. These data suggest that the Erhualian pigs are different from other breeds. Microsatellite analysis showed that the average allele number (5.3/locus) in the Erhualian pig was intermediate as compared with that (4.8-7.0/locus) in the three European pig breeds. The expected heterozygosity was higher in the Erhualian pig (0.78) than that in these European pig breeds (0.59-0.72), whereas the observed heterozygosity was higher in the European breeds (0.51-0.64) than in the Erhualian pig (0.46). In the Erhualian pig, the fixation index (FIS) was as high as 0.41. These data suggest a high level of inbreeding and/or subpopulation in the Erhualian pigs. For conservation of the germplasm in the Erhualian pigs, it is necessary to take measures to reduce inbreeding and/or subpopulation. Keywords: Erhualian pig, Genetic diversity, Conservation, DNA markers South African Journal of Animal Science Vol.33(3) 2003: 159-165

We developed a simple and inexpensive method to extract DNA from fresh and preserved fish scales. The procedure is based on boiling the scales in 5% Chelex 100, followed by digestion with proteinase K and subsequent absorption of genomic DNA using silica. A single fresh scale from larger species (e.g., tilapia) or a few scales from smaller species (e.g., 4 scales from zebrafish) provide over 200 ng of DNA, enough for at least 40 polymerase chain reaction amplifications. The procedure is applicable for DNA isolation not only from fresh and ethanol-preserved scales, but also from dried and formaldehyde-treated samples, and thus might be useful for investigating specimens stored in museums and other collections. Since the removal of a few scales is a gentle means of sample collection, this technique will allow analysis of genetic diversity, mating systems, and parentage in populations of endangered or ornamental fish with minimal experimental influence.

During genotyping of 38 microsatellites for QTL (quantitative trait loci) mapping in three F2 swine populations, five mutant alleles were detected in a total of 66,436 parent-offspring transfers of microsatellite alleles, which gives an overall mutation rate of 7.52 x 10(-5) per locus per generation. No significant (P0.05) association between mutation rates and other factors (i.e., GC contents in the flanking regions, heterozygosity, and repeat number) was revealed. Detailed sequencing showed that four out of five mutant alleles were caused by insertions of one to five repeats, respectively. The other mutant allele was produced by either an insertion of three repeats or a change of 30 base pairs (a deletion of 16 CT repeats and an insertion of one CA repeat). An insertion of one base pair in the flanking region of a microsatellite was also detected. Together, these data indicate that expansions are more common than contractions among microsatellites and that the mutation processes are very complicated, do not fit with the strict stepwise mutation model and may vary from locus to locus.

Fgf21 is a newly discovered fibroblast growth factor. It is typically induced by fasting and plays important roles in the regulation of glucose and lipid metabolisms and energy balance in mammals, whereas potential functions of this gene in teleosts are still unknown. We identified the Fgf21 gene and studied its functions in Asian seabass (Lates calcarifer). The cDNA of the Fgf21 encoded a protein with 206 amino acids. Analysis of DNA and amino acid sequences of Fgf21 genes revealed that the sequences and structure of the Fgf21 genes were highly conserved in vertebrates. Real-time PCR revealed that Fgf21 was exclusively expressed in the intestine and kidney, which was different from the expression profiles of mammals. Fgf21 was down-regulated under fasting, whereas it was significantly increased during the LPS challenge. Exogenous recombinant FGF21 significantly suppressed the appetite of Asian seabass. Our data suggest that Fgf21 plays a role in energy regulation and acute phase response in Asian seabass, and may have different functions in fish and mammals. In addition, we identified one SNP in Fgf21. By using this SNP, the gene was mapped on the linkage group 23, where a suggestive QTL for growth was mapped previously. Association mapping identified significant associations between Fgf21 genotypes at the SNP and growth traits. These results not only provide important information of the functions of Fgf21, but also suggest that the SNP in this gene can be used as a marker in selecting fast-growing individuals of Asian seabass.

For detecting QTL in the whole swine genome, 1068 pigs from three F2 populations constructed by crossing European Wild boar and Pietrain (W x P), Meishan and Pietrain (M x P), and Wild Boar and Meishan (W x M) were genotyped for genetic markers evenly spaced at approximately 20 cM intervals. AQTL analysis was performed using a least-squares method. Here the results of the QTL analysis on the porcine chromosome 7 are presented. QTL for carcass composition (e.g. head weight, carcass length, backfat depth, abdominal fat and bacon meat) were mapped in the chromosomal region CYPA/CYPD-TNFB-S0102 in M x P and W x M, but not in W x P. The QTL explained 5.3%-27.2% of the F2 phenotypic variance in the two F2 populations. Most traits affected by the mapped QTL were related to carcass fatness. The mode of gene action of QTL was additive. Surprisingly, in contrast to the parental phenotype, the QTL alleles from fatty Meishan were associated with thinner backfat than Pietrain and Wild Boar alleles, suggesting that the genome of the fatty Meishan pig contains genes which can reduce fat content of carcass substantially.

Lysozymes are important proteins of the innate immune system for the defense against bacterial infection. We cloned and analyzed chicken-type (c-type) and goose-type (g-type) lysozymes from Asian seabass (Lates calcarifer). The deduced amino acid sequence of the c-type lysozyme contained 144 residues and possessed typical structure residues, conserved catalytic residues (Glu(50) and Asp(67)) and a "GSTDYGIFQINS" motif. The deduced g-type lysozyme contained 187 residues and possessed a goose egg white lysozyme (GEWL) domain containing three conserved catalytic residues (Glu(71), Asp(84), Asp(95)) essential for catalytic activity. Real time quantitative PCR (qRT-PCR) revealed that the two lysozyme genes were constitutively expressed in all the examined tissues. The c-type lysozyme was most abundant in liver, while the g-type lysozyme was predominantly expressed in intestine and weakly expressed in muscle. The c-type and g-type transcripts were up-regulated in the kidney, spleen and liver in response to a challenge with Vibrio harveyi. The up-regulation of the c-type lysozyme was much stronger than that of the g-type lysozyme in kidney and spleen. The recombinant proteins of the c-type and g-type lysozymes showed lytic activities against the bacterial pathogens Vibrio harveyi and Photobacterium damselae in a dosage-dependent manner. We identified single nucleotide polymorphisms (SNPs) in the two lysozyme genes. There were significant associations of these polymorphisms with resistance to the big belly disease. These results suggest that the c- and g-type genes play an important role in resistance to bacterial pathogens in fish. The SNP markers in the two genes associated with the resistance to bacterial pathogens may facilitate the selection of Asian seabass resistant to bacterial diseases.

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.