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Pedigree information is important in selective breeding programs and hatchery management of aquaculture. However, in aquaculture species, tagging of young fish with physical tags is very difficult. Molecular parentage analyses have been used in reconstructing pedigrees in aquaculture species since the 1990s. Some studies have been carried out to optimize every step to reduce the cost and improve the efficiency of molecular parentage analysis. However, molecular parentage analysis of a large number of individuals is still too expensive because some steps of molecular parentage analysis are very cost-intensive. In this review, we have summarized the techniques of major steps in parentage analysis, and provided a guide for aquaculture scientists on how to cost-effectively and rapidly extract DNA from a large number of samples, select the right types of DNA markers, optimize multiplex polymerase chain reaction, and select appropriate methods and software to analyze the genotyping data for reconstructing pedigrees. In addition, we discuss the challenges and future directions of molecular parentage analysis in aquaculture species.

Aquaculture is the fastest growing sector in agriculture. Substantial genetic gains have been achieved in a few cultured species using conventional selective breeding approaches. However, the majority of fish and shellfish species remain in their wild state. Due to the recognition of the enormous potential of marker-assisted selection (MAS) to speed up genetic gain through early selection, aquaculture scientists have constructed linkage maps in over 40 species and mapped quantitative trait loci (QTL) for important traits in over 20 species since the 1990s. Although MAS and genomic selection (GS) have not been widely used in aquaculture, their application in breeding programmes is expected to be a fertile area of research. In this paper, I summarized the recent advances of linkage and QTL mapping, as well as MAS in aquaculture species. I also discussed the potentials of genome-wide association studies (GWAS) and GS in aquaculture species.

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.

Prolactin (encoded by PRL) is a multifunctional hormone involved in osmoregulation, reproduction, growth, development, immunomodulation, endocrine and metabolic regulation. We cloned the full-length cDNA of Asian seabass PRL, searched for polymorphism in the DNA sequence, and conducted association analyses. Twelve SNPs and one 4-bp deletion were identified in PRL. The SNP c.264+127C>G was used for linkage mapping, and this gene was mapped to linkage group 11. The c.264+980_983delTTGT, c.264+127C>G, c.264+138T>G, c.264+269T>C and c.330C>G polymorphisms were genotyped in 521 individuals with growth trait records. Association analyses between single markers and growth traits revealed that the c.264+269T>C SNP was significantly associated with body weight (BW), total length (TL), standard length (SL) and Fulton's condition factor (KTL and KSL), while the other four were not. Analysis of haplotypes showed that there were 10 haplotypes and 22 haplotype combinations in the population. The differences of BW, TL, KTL and KSL among different haplotype combinations were significant.

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.

For Sus scrofa chromosome 13 (SSC13) eleven markers were genotyped to generate linkage and QTL maps in three F 2 families based on Wild Boar (W), Meishan (M) and Pietrain (P) crosses. Results of linkage maps were similar and agreed with published maps. Quantitative trait loci (QTLs) for body weights, meat conductivity and fat deposition were mapped in the M x P family to the interval S0076-S0068 (40-80 cM) and near the locus SW38 (120-150 cM). QTLs for the same traits were observed in the W x M family in different intervals explaining up to 5.3% of the F 2 phenotypic variance. No significant QTLs for the traits under investigation were found in the W x P family. The effects of QTL alleles were mostly dominant. In the M x P and W x M families alleles inherited from Meishan were generally associated with high values of live weights, food consumption and fat deposition and low values of meat conductivity.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

The goldfish (Carassius auratus auratus; Cyprinidae) is not only an important ornamental fish species, but also a useful model for biological studies. The sequence of goldfish genes present in the public database was searched for short tandem repeats, and 11 polymorphic microsatellites were detected within eight important genes. Two microsatellites were located in coding regions of the c-myc and GAP-43 genes, respectively, whereas the others were mainly located in 5′ and 3′ untranscribed regions of other genes, such as gonadotropin and activin. The average allele number of these microsatellites was 5.5 per locus with a range between 3 and 9. These microsatellites will be useful for ecological and population genetic studies of this species, as well as for the ornamental fish industry.

Six microsatellites were detected in the IGF-II, GH2, prolactin I and insulin genes of Mozambique tilapia (Oreochromis mossambicus) and Nile tilapia (Oreochromis niloticus) by screening DNA sequences deposited to GenBank. Genotyping of 24 individuals from each species revealed that all six microsatellites were polymorphic in both species. In Mozambique tilapia the number of alleles ranged from 3 to 17 (average 9.8), whereas in Nile tilapia the range was 4–21 (average 10.5). The range of expected heterozygosity values were 0.44–0.95 (average 0.79) in Mozambique tilapia, and 0.52–0.96 (average 0.73) in Nile tilapia.

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.