Aquarium Sciences and Conservation, 1, 91±104 (1997)

DNA ®ngerprinting: application to conservation of the CITES-listed dragon ®sh, Scleropages formosus (Osteoglossidae) A.A. Fernando1, L.C. Lim2, K. Jeyaseelan3, S.W. Teng1, M.C. Liang1 and C.K. Yeo1 1

Chemical Process & Biotechnology Department, Singapore Polytechnic, 500 Dover Road, Singapore 139651 2 Ornamental Fish Section, Primary Production Department, Ministry of National Development, Lorong Chencharu, Singapore 769194 and 3 Department of Biochemistry, National University of Singapore, Singapore Kent Ridge, Singapore 119260 Since 1975, CITES has listed the dragon ®sh, Scleropages formosus, as an endangered species. In 1995, a captive-bred population was set up by a commercial ®sh farm with assistance from the Primary Production Department in Singapore. Other farms in Indonesia and Malaysia followed suit. These populations have contributed to an immediate conservation of the species. Due to very high demand for this ornamental ®sh, these venues may be its last sanctuary. DNA ®ngerprints of the dragon ®sh were obtained by different methods from the green, red and gold varieties grown in a Singapore ®sh farm to determine which method was most suitable in providing information on genetic variability. Because a DNA ®ngerprint is a pattern made up of DNA fragments that are resolved by electrophoresis, each individual has its own unique `®ngerprint' due to a genetic make-up different from another individual. Thus, genetic variability was best studied by developing DNA ®ngerprints. Firstly, restriction fragment length polymorphisms (RFLPs) were obtained. DNA fragments formed by cleavage with nine restriction endonucleases used singly were hybridized individually to four non-radioactively labelled probes to give RFLPs. The RFLPs for each variety were similar and genomic DNA from each variety had many binding sites to the probes. This made differentiating RFLPs speci®c to individual varieties dif®cult. Secondly, random ampli®ed polymorphic DNA (RAPD) ®ngerprints were developed. DNA fragments that were resolved on a denaturing polyacrylamide gel were hybridized to seven arbitrary primers used singly. RAPD ®ngerprints for each variety were different for each primer tested. The similarity index indicated low genetic variability between varieties. Lastly, DNA was screened for microsatellite loci which refer to short tandem repeats of two or three bases. The occurrence of other microsatellite loci, their chromosome location and frequency is being investigated while primers have been designed to detect more loci by the polymerase chain reaction. As this method provides undisputed and reproducible evidence of relatedness and stock identi®cation, and can be applied for long-term management of domesticated populations through pedigree construction and evaluation of heterozygosity, it is the preferred choice to determine genetic variability. KEYWORDS: Endangered ®sh, DNA ®ngerprinting, RFLP, RAPD, Microsatellites, Captive-bred 1357±5325 # 1997 Chapman & Hall

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INTRODUCTION Biodiversity, as de®ned by the structural and functional variety of life forms at the genetic, population, species, community and ecosystem levels, faces the greatest threat since humans appeared (Sandlund et al., 1992). Loss of biodiversity, attributed mainly to an increase in human population and expansion of humanrelated activities (Ehrlich and Ehrlich, 1981), has led to habitat destruction and degradation. In 1989 more than 142 200 km2 of tropical forest were lost at the rate of 27 hectares per minute (Myers, 1989). Deforestation has led to a decimation of many species. In the case of declining numbers of ®shes, habitat destruction and degradation imply: that non-®sheries activities impinge upon genetic resources of value to ®sheries or ecosystems; that the effects of ®shing and ®sheries management practices have taken their toll on the genetic resources; and that there is a lack of commitment to sustainability or a knowledge gap with regard to the levels of sustainability (Anon., 1992). Although many governments worldwide have agreed with the ethical position of conserving biodiversity, by accepting the United Nations Charter for Nature, the loss of richness in the living world has many implications for development as spelled out by the World Commission on Environment and Development (Anon., 1987). A proposal for conservation to ``Save it; Know what it is; and Use it sustainably'' was put forward (Janzen, 1992). Conservation refers to the active management of a resource such that the resource and all its facets can be utilized at the present and in the future (Anon., 1992). Thus, the strategy to conserve biodiversity, omitting social, economic and political areas is to promote and develop emphasis on research on conservation, construct an inventory of the world's biological diversity, study ecological processes, breed by natural or induced means species that would otherwise not breed in captivity, and apply conservation genetics. This requires considerable time, money and collaboration to pool resources and contribute to non-overlapping areas of expertise. The threat of extinction hovers over many species and has led some conservation biologists to conclude that the last refuge for a number of organisms, particularly birds and mammals, may be zoological gardens (Hedrick and Miller, 1992), and possibly farms and ®eld experimental research stations. That there is a role for such institutions to work towards conservation of endangered species is shown in the local (Lim et al., 1995) and international (Johnson and Hubbs, 1989) context. In this paper we do not give an exhaustive review of DNA ®ngerprinting or of the role of institutions in conservation, but focus on collaboration among the Primary Production Department (PPD), the Singapore Polytechnic and the National University of Singapore on investigations of various approaches taken to assess the genetic variability by DNA ®ngerprints of the dragon ®sh; and on the usefulness of each method. The aim is to use the data to formulate a broodstock management programme for long-term genetic conservation of this endangered ®sh species. OVERVIEW OF CAPTIVE BREEDING The dragon ®sh The dragon ®sh, also known as the Asian bonytongue or Asian arowana, belongs

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to the family Osteoglossidae. Other members of this family include the arapaima, Arapaima gigas, and the best known of the three species, the arowana, Osteoglossum bicirrhosum. The arowana and arapaima are found in South America (Anon., 1993). The dragon ®sh is distributed throughout most of South East Asia, including Indonesia, Kampuchea, Laos, Malaysia, Myanmar, the Philippines, Singapore, Vietnam and possibly Thailand (Gilbert, 1984). The body of the dragon ®sh is compressed, with the anal ®n extending almost the entire length and tapering to a small tail. The large scales are deeply imprinted, giving a trellis-like effect to the body. The mouth is large, and two barbels protrude from the lower jaw. The three members have an overall appearance that is graceful and streamlined (Gilbert, 1984). The arowana has an iridescent rainbow sheen, the arapaima is greyish-black and the dragon ®sh exists as three (and possibly four) colour forms, green, red and gold, with the red variety being arguably the most highly prized among them (Kodera et al., 1996). Extinction of the dragon ®sh, with a geological record from the Mesozoic Era or Age of the Reptiles (Greenwood et al., 1996), will mean the end of a unique genome that has been undergoing continuous evolution for one hundred million years. Currently, this species is the most important ornamental ®sh in the global ®sh trade. Exports from Singapore amounted to S$2.4 million in 1995 (Lim et al., 1995). The Chinese and Japanese believe the ®sh brings good luck, health and wealth to the owner and drives away evil. Trading has been lucrative as the ®sh is highly prized because of this myth. The dragon ®sh has been classi®ed by the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) as a highly endangered species and listed in Appendix I since the Convention came into force in 1975 (Greenwood et al., 1996), while Arapaima gigas is listed in Appendix II (Morgan, 1995). Appendix I animals are threatened with extinction and are, or may be, affected by trade. Trade is permitted in limited circumstances, such as if species are bred in captivity or arti®cially propagated, needed for scienti®c research, or used for non-commercial purposes that date from before the convention came into force (Morgan, 1995). The dragon ®sh also has a small natural population size and is restricted in distribution; export of wild-caught specimens from Indonesia has been increasing, from 1250 in 1990 to 4000 in 1994, a 220% increase in 4 years (Kodera et al., 1996). Captive breeding Dragon ®sh produced in captivity can be traded. Farms that produce ®sh of the second ®lial generation register as commercial captive operations and are allowed to export. In August 1995, Rainbow Aquarium, Singapore, with assistance from the PPD Ornamental Fish Section at the Sembawang Field Experimental Station, succeeded in its application for registration with CITES to export these ®shes (Cheong, 1993, 1994; Dawes and Cheong, 1994). This effort has led to an immediate conservation of the dragon ®sh. Conservation in arti®cial environments is recognized as an important shortterm measure to protect a limited portion of the gene pool from immediate extinction (Maitland and Evans, 1986). For example, in 1974, Hubbs and Echelle brought six Texas south-eastern poeciliids to the Dexter National Fish Hatchery

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(DNFH) in south-eastern New Mexico to test its suitability as a native ®shes rearing centre. They propagated and reared two endangered species in a hatchery environment and saved them from extinction (Toney, 1974; Rinne et al., 1986). Since then, DNFH has become entirely devoted to protection and propagation of native ®shes. Over its thirteen-year history, 22 rare species have been housed there (Johnson and Hubbs, 1989). This could be a model for other research stations to follow. Establishing captive-bred populations is a direct response to ``save it'' (Janzen, 1992). Avoidance of extinction should be the ®rst goal of any conservation programme and provides for maintenance of viable populations in the short term (Meffe, 1986). If natural habitats are destroyed or polluted, a sanctuary would still exist where the species is successfully cultivated and this venue can be an experimental research station, farm or zoological garden. In the long term, such founder populations may supply ®shes for re-stocking native habitats and exchange with zoological gardens and research stations. By preserving the gene pool, they are a priceless source of germ plasm for gene banks. However, genetic drift, which can occur if the founding population is started by a very small number of `pioneers' or if there is a traumatic event such as habitat destruction or poaching that continuously and effectively limits the numbers of surviving members of a group, has to be addressed. The genetic aspects of small populations must be considered in order to maximize survival and ensure adaptability (Meffe, 1986). It is appropriate then to apply DNA ®ngerprinting to captive-bred populations to ensure that they do not suffer from loss of genetic heterozygosity. Heterozygosity can be evaluated and the survival of various groups compared to determine if heterozygous populations are more successful, an approach taken by DNFH (Johnson and Hubbs, 1989). Long-term survival is ensured when inbreeding depression ± manifested by a decline in growth rate, fecundity, viability and an increase in percentage of deformed and abnormal ®sh ± is avoided (Tave, 1995). Thus, to maintain a species in perpetuity (Meffe, 1986), it is imperative to ``know it'' (Janzen, 1992). DNA FINGERPRINTING Each individual possesses a unique genetic makeup. In 1985 Alec Jeffreys coined the term `DNA ®ngerprinting' to describe the highly variable individual-speci®c bands that are seen in Southern blots of human genomic DNA hybridized to a tandemly array of repetitive DNA sequences (Jeffreys et al., 1985a,b). This discovery was quickly followed by the application of DNA ®ngerprinting in domestic animals (Georges et al., 1988), in population dynamics within and between populations (Jin and Chakraborty, 1994) and in conservation (Mathe et al., 1993). In the last instance, the German Federal Ministry of the Environment, Natural Protection and Reactor Safety applied DNA ®ngerprinting to protect endangered species of birds of prey and parrots (Mathe et al., 1993), and it has been used by the Marine Gene Probe Laboratory, Dalhousie University, Nova Scotia, in collaboration with farms to study genetic variation and various aspects of stock identi®cation for aquaculture and ®sheries of the scallop, Placopecten magellanicus, Atlantic salmon, Salmo salar, cod, Gadus morhua, red®sh, Sebastes

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sp., halibut and plaice ( Hippoglossus spp.) and hake, Merluccuis spp. (Wright, 1993; Wright and Bentzen, 1994). The genetic material from each of three varieties of dragon ®sh, green, gold and red, was provided by captive-bred specimens from a farm in Singapore. For the three methods investigated in this study, high-molecular-weight DNA was isolated from 0.5 g of ®n clippings or skeletal muscle by routine methods (Sambrook et al., 1989). Restriction fragment length polymorphism (RFLP) To generate restriction fragment length polymorphisms (RFLPs) (Fig. 1), highmolecular-weight genomic DNA was cleaved individually by nine restriction endonucleases (enzymes) which cut DNA in a very site-speci®c manner (Table 1). The resulting DNA fragments were separated by size on agarose gel electrophoresis. The double-stranded fragments were denatured in the gel and the single-stranded pieces were transferred to a nylon sheet. This transfer of electrophoretically resolved DNA fragments to nylon sheets is known as `Southern blotting' (Southern, 1975). Four single-stranded nucleic acid probes were labelled non-radioactively and hybridized to the single-stranded DNA fragments on the nylon sheet. These probes were 16-mer oligonucleotides sythesized using the DNA Synthesizer Model 381A. Other probes can be puri®ed RNA, cDNA, a cloned fragment of genomic DNA or speci®c sequences for a gene under study. Whichever is chosen, the labelled probe will hybridize to any DNA fragment on the sheet if it contains complementary nucleotide sequences to give bands, or restriction fragment length polymorphisms. Because RFLPs produce DNA fragments of variable lengths by restriction digestion, an individual-speci®c hybridization pattern results (SchaÈfer et al., Cleave DNA with restriction enzymes Separate DNA fragments on agarose gel Denature double-stranded DNA to single-stranded DNA Transfer single-stranded DNA to nylon sheet Hybridize single-stranded DNA on membrane to single-stranded labelled probe Wash membrane to remove non-specific hybridization Specific DNA fragments hybridize to probe to give bands Detect RFLP fingerprints Permanent record & analysis

Fig. 1. Flow chart on how to generate restriction fragment length polymorphisms (RFLPs).

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A.A. Fernando et al. Table 1. Restriction endonucleases used to cleave dragon ®sh DNA and recognition sequences Restriction endonuclease Four-base `cutter' Alu I Mbo I Hae III Five-base `cutter' HinfI Six-base `cutter' Bam HI HindIII Sal I Sma I Eight-base `cutter' (`rare cutter') Not I

Recognition sequencesa AG=CT =GATC GG=CC G=ANTC G=GATCC A=AGCTT G=TCGAC CCC=GGG GC=GGCCGC

a

A, C, G, T refer to four nitrogenous bases in DNA: A, adenine; C, cytosine; G, guanine; T, thymine; N refers to A or T or G or C; = refers to the cleavage site of the enzyme.

1988). This technique has been used to assess genetic variation in human populations (Wainscoat et al., 1986) and forensic sciences (Gill et al., 1985) but it is laborious and time-consuming, making it impractical for large population studies. The nine restriction enzymes cleaved dragon ®sh DNA into numerous fragments that appeared as a smear on the gel (results not shown) and hybridization with the four probes produced numerous RFLP ®ngerprints (results not shown), indicating numerous binding sites on the ®sh DNA with the probes used. There were no discernible differences in RFLPs between the various colour varieties. Because RFLPs speci®c to individual varieties could not be differentiated, RFLP ®ngerprints were not suitable for detection of genetic variation between dragon ®sh varieties. Random ampli®ed polymorphic DNA (RAPD) Simple ®ngerprints of complex genomes can be generated by single, arbitrarily chosen primers and the polymerase chain reaction (PCR) (Welsh and McClelland, 1990) with no prior knowledge of target DNA sequences. Random ampli®cation of polymorphic DNA (RAPD) by the polymerase chain reaction (RAPD±PCR) (Williams et al., 1990, 1993) is also known as arbitrarily primed PCR (AP±PCR) (Berg et al., 1994). It involves two cycles of low-stringency ampli®cation followed by PCR at higher stringency (Fig. 2) and rapidly detects polymorphism for genetic mapping and strain identi®cation (Berg et al., 1994), thus surveying numerous loci in the genome, which makes it attractive for analysis of genetic distance. RAPD allows detection of variation in an individual's DNA by random ampli®cation of multiple regions of the complete set of the genome by PCR (Welsh and McClelland, 1990; Berg et al., 1994) of primers that are used singly (Table 2) or in combination to give more information. An arbitrary primer is a short sequence of DNA which can be synthesized just like a probe. An ampli®ed DNA fragment is detected on a polyacrylamide gel as `band' presence versus

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Assess DNA quality & quantity PCR Gel electrophoresis Detect RAPD fingerprints Permanent record & analysis

Fig. 2. Flow chart on how to generate random ampli®ed polymorphic DNA.

Table 2. Sequences of arbitrary primers used and (G ‡ C) content (%) Designation

Primer length

Sequence 59 ! 39

(G ‡ C) (%)

SP=AF=#1 SP=AF=#2 SP=AF=#3

20-mer 16-mer 10-mer

TAT GTA AAA CGA CGG CCA GT TGC CTG TGG GGA ATC C CGG TCA CTG T

45.0 62.5 60.0

SP=AF=#1=SS SP=AF=#2=SS SP=AF=#3=SS SP=AF=#4=SS

16-mer 20-mer 20-mer 16-mer

(GACA)4 (GATA)3 (GACA)2 (GATA)2 (GACA)(GATA)2 (GATA)4

50.0 35.0 30.0 25.0

`band' absence. Polymorphisms may be caused either by failure to prime a site in some individuals because of nucleotide sequence differences, or by insertions or deletions in the fragments between two conserved primer sites (Clark and Lanigan, 1993). RAPD ®ngerprints generated by a primer for individual dragon ®sh are shown in Fig. 3 (other results are not shown). After band presence and absence has been recorded, genetic similarity, which gives a measure of the genetic variation among individuals, is calculated as the genetic similarity index (SI), given by: SI ˆ 2N AB =( N A ‡ N B )

(1)

where NAB is the number of RAPD bands shared in common between individuals A and B, and NA and NB are the total number of bands recorded in A and B, respectively (Nei and Li, 1979). An SI value of 0 indicates that there are no bands shared between the RAPD pro®les of two individuals, while an SI value of 1 indicates that there are no differences in the RAPD pro®les between them, i.e. they are genetically similar. RAPD pro®les can be generated with different primers and so these values are based on the primer concerned. Compared with RFLPs, RAPD ®ngerprints require less DNA (20±100 ng), which is useful when genomic DNA is limited, they do not require a labelled probe or knowledge of the target DNA sequences, and they are simple and fast. DNA pro®les have been used for gene mapping and genotyping of closely related or

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Fig. 3. An RAPD ®ngerprint of three varieties of dragon ®sh produced from primer SP=AF=#1=SS. Lane codes R1 etc. are as follows: R, G and Gd designate red, green and gold varieties, respectively; M denotes the molecular weight marker; numbers refer to individual specimens.

highly inbred species (Dinesh et al., 1993) but reproducibility of fragments can be a problem and can lead to an inaccurate index. In this study, primer and magnesium concentrations and temperature were optimized to give the maximum number of consistent and reproducible bands and reduce non-speci®c binding. Manual scoring of band presence or absence is tedious and subject to error, but can be overcome by densitometric analysis. RAPD ®ngerprints between varieties were different for individual primers and differences were also detected within varieties. The SI calculated for the three varieties of dragon ®sh indicated high levels of genetic similarity within and between varieties as the SI values ranged from 0.50 to 0.90 (Table 3). Although more informative DNA ®ngerprints were obtained by this method, the detection of genetic variability between varieties was low. Combining primers to `simplify' DNA ®ngerprints is an option that is being investigated. Microsatellites To screen large numbers of ®shes for pedigrees and genealogy, considerable resources are required. The isolation (Fig. 4), characterization and frequency of

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Table 3. Similarity index (SI) for primer (SP=AF=#1=SS) for three varieties of dragon ®sh (see Fig. 3 for explanation of codes) R1 R1 ± R2 R3 R4 R5 R6 G1 G2 G3 G4 G5 G6 Gd1 Gd2

R2

R3

R4

0.89 0.84 0.70 ± 0.85 0.71 ± 0.83 ±

R5

R6

G1

G2

G3

G4

G5

G6

Gd1 Gd2

0.84 0.81 0.87 0.74 ±

0.85 0.93 0.88 0.76 0.88 ±

0.88 0.85 0.84 0.74 0.84 0.89 ±

0.85 0.85 0.80 0.70 0.80 0.85 0.88 ±

0.88 0.85 0.80 0.70 0.80 0.85 0.92 0.92 ±

0.82 0.83 0.82 0.76 0.81 0.91 0.90 0.86 0.82 ±

0.75 0.68 0.78 0.77 0.78 0.72 0.90 0.71 0.75 0.77 ±

0.59 0.51 0.51 0.57 0.50 0.51 0.59 0.49 0.54 0.56 0.67 ±

0.85 0.82 0.81 0.71 0.91 0.86 0.93 0.85 0.85 0.87 0.84 0.56 ±

0.78 0.79 0.78 0.67 0.78 0.83 0.90 0.90 0.86 0.88 0.67 0.50 0.87 ±

Restriction digest genomic DNA & ligate into a vector Transform ligated vector into component cells Select transformants Isolate transformants Dot blot & hybridize with α-32P labelled (GT)12 probe Restriction map positive clones with endonucleases from the polycloning sites of the vector Southern blot & hybridize with α-32P labelled (GT)12 probe Subclone positive fragments into the vector Transform subclones into competent cells Isolate transformants Sequence positive clones Design primers for flanking regions of microsatellites Isolate microsatellite loci Sequence loci & score homo-& heterozygotes Permanent record and analysis

Fig. 4. Flow chart on how to detect microsatellite loci.

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mini- and microsatellites yields considerable genetic data over a short period of time. These are length variations in DNA caused by variations in number of tandemly repeated DNA sequences at `minisatellite' or `microsatellite' loci (Wright, 1993). Microsatellite loci consist of shorter repeating units over the genome 1±6 base pairs in length, such as (CA) n , (GT) n or (AGC) n , where n, the number of repeat units, is variable (Slettan et al., 1995). Hypervariable loci are characterized by their large number of alleles, and consequently high heterozygosity per locus, and have been shown to be ef®cient markers for stock identi®cation (Beacham et al., 1996), in domestic animals (Winterù et al., 1992; Ward and Grewe, 1994) and to study evolutionary relationships of genetically close populations or species (Wright and Bentzen, 1994). Many highly polymorphic minisatellite loci can be detected simultaneously in the genome by hybridization to probes consisting of tandem repeats of the `core' sequence. The microsatellite loci detected so far in the dragon ®sh are shown in (Table 4). Other microsatellite loci can be detected by analysis using PCR of a pair of unique oligonucleotides ¯anking the loci detected earlier, followed by sizing of the PCR products on polyacrylamide gels (Wright and Bentzen, 1994). Microsatellite loci nomenclature was based on published literature (Wu and Tanksley, 1993; Slettan et al., 1995), whereby alphabets refer to the ®rst letter of the genus name and the ®rst and second letters of the species name, respectively; and the numbers refer to the clones from which the microsatellites were isolated. On-going studies include the determination of polymorphic microsatellite arrays, their inheritance in known pedigrees and their levels of heterozygosity. Detection of microsatellite loci is the preferred choice of determining genetic variability because levels of homozygosity and heterozygosity can be determined. This will allow pedigree construction for an effective broodstock management programme to be drawn up. BROODSTOCK MANAGEMENT A stock is a practical term de®ning a group of individuals for management purposes. It consists of several species or several subpopulations and should be Table 4. Some microsatellite sequences isolated from the dragon ®sh Locus Sfo70 Sfo84 Sfo122b Sfo7 Sfo109 Sfo111 Sfo122a

Sequences of microsatellite loci Perfect locus (CA)30 (TG)4 (CT)5 Perfect ‡ compound locus (GT)12 GC(GT)2 (TG)2 TTGTGA Compound locus (CA)3 NA(CA)2 NA(CA)10 Imperfect locus (TG)2 CG(TG)8 (CTG)4 ATGCAGCTG

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de®ned by the user (Anon., 1992). A natural stock is not equivalent to an aquaculture stock. Thus, the aquaculture stocks of captive-bred populations have to be managed differently. In aquaculture, a broodstock management programme will list the effective number of males and females to be used in a breeding programme. The effective population size (Ne ) is given by: N e ˆ 1=2ÄF

(2)

where ÄF is the rate of inbreeding (Weber, 1990). In domestic or laboratory animals, the sexes are often unequally expressed among the breeding individuals, because it is more economical to use fewer males than females. Because the two sexes contribute equally to the genes in the next generation, the effective number is twice the harmonic mean of the numbers of the two sexes. This changes Ne to: N e ˆ 4Nm Nf =( Nm ‡ Nf )

(3)

where Nm and Nf are the numbers of males and females, respectively. So, a population with 50 males and 50 females would have an effective population of 100 whilst a population of 10 males and 90 females would yield an effective population of 36. Thus, a population with 50 male and 50 females is about three times more effective genetically than one with the latter numbers of broodstock. By knowing how to calculate inbreeding from one generation to the next, inbreeding or mating between relatives (Falconer and McKay, 1996) is prevented. Inbreeding rate can also be calculated even if variable numbers of males and females are used at each generation. The rate of inbreeding (ÄF), approximately 1=8Nm ‡ 1=8Nf , depends chie¯y on the numbers of the less numerous sex. The rate of inbreeding in any one generation is given by 1/2N. Aquaculture stocks can be managed by setting up breeding lines or raising in cohorts. A line is obtained by selective breeding for many generations to ensure that only pure breeding stocks with certain characteristics are kept. One limitation of this method is that control lines have to be set up for such a programme. Ideally, because a control should be between two inbred lines, it may be expensive to maintain. Besides, it is doubtful whether such stocks could be developed in the ®rst place. In this instance, RAPDs can provide a powerful means of assembling a map of anonymous segregating markers for an array of F2 individuals derived from a pair of inbred lines, as only markers that exhibit good segregation will provide sensible genetic map information (Williams et al., 1990). For captive animal populations, pedigree analysis by techniques such as gene dropping and loss of heterozygosity have become the basic approach to evaluate breeding priority of particular individuals (Hedrick and Miller, 1992). A broodstock management programme would require that pedigrees be set up, and for this purpose, assessment by microsatellite loci is best. The application of genetics to aquaculture is fairly new. Some reasons for this are that there has been only recent intensi®cation of aquaculture, that there is a low level of domestication of many aquatic species, as with the dragon ®sh, as well as the fact that the majority (90%) of the production from the aquatic environ-ment comes from ®sheries (Anon., 1992). The possibility of using DNA technology to assess the genetic variability of aquaculture populations of high value is suggested.

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CONSERVATION OF WILD POPULATIONS Conservation of wild-type populations involves monitoring, keeping an extensive database and surveillance of habitats. This is dif®cult and expensive, and conservation efforts could also include regulation of harvests to promote sustainability and development of selective ®shing techniques and gear technology to minimize bycatch (Anon., 1992). Extinction of local populations can be counteracted by the establishment of gene banks. Efforts made with the Atlantic salmon, Salmo salar, provide a good example of a food ®sh species that is conserved and used sustainably (Hedrick and Miller, 1992). ACKNOWLEDGEMENTS The work was funded by the Singapore Polytechnic Innovation Development Fund (13-27801-4111). The authors thank Mr Lim Lian Chuan, Ornamental Fish Section, PPD for providing the samples, Mr Philemon Siew from the PPD and Dr A. Arumugam from the Biochemistry Department, National University of Singapore, for assistance. REFERENCES Anonymous (1987) Our Common Future. World Commission on Environment and Development (WCED). Oxford, UK: Oxford University Press. Anonymous (1992) Expert consultation on utilization and conservation of aquatic genetic resources. Grottaferrata, Italy, 9±13 November 1992. FAO Fisheries Report No. 491. Anonymous (1993) Checklist of Fish and Invertebrates Listed in CITES Appendices. Peterborough, UK: World Conservation Monitoring Centre, Joint Nature Conservation Committee. 171 pp. Beacham, T.D., Withler, R.E. and Stevens, T.A. (1996) Stock identi®cation of chinoock salmon (Oncorhynchus tshawytscha) using minisatellite DNA variation. Canadian Journal of Fisheries and Aquatic Sciences 53, 380±394. Berg, D.E., Akopyants, N.S. and Kersulyte, D. (1994) Fingerprinting microbial genomes using RAPD or AP±PCR method. Methods in Molecular and Cellular Biology 5, 13±24. Cheong, L. (1993) Singapore's Dragon ®sh connection. OFI Journal 5, 7±8. Cheong, L. (1994) Singapore's Dragon ®sh connection. Part II: Making it all come together. OFI Journal 9, 16. Clark, A.G. and Lanigan, C.M.S. (1993) Prospects for estimating dinucleotide divergence with RAPDs. Molecular Biology and Evolution 10 1096±1111. Dawes, J. and Cheong, L. (1994) Captive-bred Dragon ®sh from Singapore. Aquaristics (May) 9±11. PETS Europe 6 (3). Dinesh, K.R., Phang, V.P.E., Lim, T.M., Chua, K.L. and Tan, T.W. (1993) DNA polymorphisms in colour mutants of Tiger Barb, Barbus tetrazona, by arbitrary primed polymerase chain reaction. In Penman, D., Roongratri, N. and McAndrew, B., eds, Proceedings of the AADCP International Workshop on Genetics in Aquaculture and Fisheries, Stirling, UK: Stirling University, pp. 125±127. Ehrlich, P.R. and Ehrlich, A.H. (1981) Extinction: The Causes and Consequences of the Disappearance of Species. New York: Random House. Falconer, D.S. and McKay, T.F.C. (1996) Introduction to Quantitative Genetics, 4th edn. Essex, UK. Longman Press.

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Accepted 23 April 1997