applications of rapd

Molecular Ecology (1992) 1, 55-63

MINI-REVIE W

Applications of random amplified polymorphic DNA (RAPD) in molecular ecology H. HADRYS, M. BALICK* and B. SCHIERWATERt Yale University, Department of Biology, 165 Prospect Street, New Hnven, CT 06511 and ‘New York Botanical Garden, Bronx, Ny 10458, USA

Abstract

Molecular genetic markers have been developed into powerful tools to analyse genetic relationships and genetic diversity. As an extension to the variety of existing techniques using polymorphic DNA markers, the Random Amplified Polymorphic DNA (RAPD) technique may be used in molecular ecology to determine taxonomic identity, assess kinship relationships, analyse mixed genome samples, and create specific probes. Main advantages of the RAPD technology include (i) suitability for work on anonymous genomes, (ii) applicability to problems where only limited quantities of DNA are available, (iii) efficiency and low expense.

Keywords: DNA fingerprinting, DNA probes, kinship analysis, paternity determination, RAPD, taxonomic identifications

Received 3 February 1992

Introduction

Within the past few years molecular genetic approaches have become of increasing importance to studies in behavioural ecology and population biology. For instance, DNA fingerprinting technologies have revolutionized approaches to our understanding of animal social systems by permitting analyses of kinship reIationships (e.g. Burke & Bruford 1987; Burke et 01. 1991a; Jones, Lessels & Krebs 1991; Gyllensten, Jakobsson & Temrin 1991; Packer et nl. 1991; Pemberton, Banaoft & Amos 1991; Schlotterer, Amos & Tautz 1991; Smith ef al. 1991).

Despite constant progress in methodology, application of DNA markers to many problems in behavioural and population ecology has been limited by technical considerations (Lewin 1989; Kirby 1990; Burke et al. 1991a; Pemberton et al. 1991). The most frequently used DNA markers include RFLPs (restriction fragment length polymorphisms) visualized by Southern blot hybridization to different types of single-locus or multilocus

Correspondence author: Heike Hadrys, Zoologisches Institut der Technischen Univenitat, Pockelsstr. 10a. D-3300 Braunschweig, Germany.

*Present address: Zoologisches Institut der Universitiit, Siesmayerstr. 70, 06000 Frankfurt a.M., Germany.

probes (Burke et nl. 1991b) and PCR amplified simple sequence microsatellite loci (Tautz 1989). Potential applications are frequently thwarted by the requirement for significant quantities of DNA in the case of RFLP analysis or by lack of relevant DNA sequence information in the case of conventional PCR-based techniques. Recent criticisms are that DNA fingerprinting requires special molecular training, is labour-intensive, and is relatively expensive - (Weatherhead & Montgomerie 1991).

It has been argued that DNA fingerprinting is so essential to behavioural ecology and population biology that it would be highly unfortunate to have its application limited to a few specialized laboratories rather than to the broad community working in these fields (Weatherhead & Montgomerie 1991). Major progress in technology development is expected in two directions: (I) increase in analytical power per unit effort, and (2) simplification in technology, and ultimately reduction in expense. Use of random amplified polymorphic DNA (RAPD) markers, detected by PCR amplification of small inverted repeats scattered throughout the genome, adds a new technology of DNA fingerprinting to the molecular analysis of relatedness between genotypes. The introduction of RAPD fingerprinting is a substantial contribution toward the second direction.

56 H . HADRL’S. M. BALICKand B. SCHIERWATER

RAPD technology is only some 2 years old, and consequently published applications are limited. However, the rapidly growing interest in using RAPD technology justifies an early review on the current literature and the potentials of the method. In this paper we shall review the principle and original applications of the RAPD technology, discuss its applications in molecular ecology, and point out the particular advantages as well as limitations of RAPD markers.

Principle of RAPD analyses

The PCR-based RAPD technique (Williams rt nl . 1990) is an attractive complement to conventional DNA fingerprinting in ecology. RAPD analysis is conceptually simple. Nanogram amounts of total genomic DNA are subjected to PCR using short synthetic oligo- nucleotides of random sequence. The amplification pro- tocol differs from the standard PCR conditions (Erlich 1989) in that only a single random oligonucleotide primer is employed and no prior knowledge of the genome subjected to analysis is required. When the primer is short (e.g. 10-mer), there is a high probability that the genome contains several priming sites close to one another that are in an inverted orientation. The technique essentially scans a genome for these small inverted repeats and amplifies intervening DNA segments of variable length. The profile of amplification products depends on the templateprimer combination and is reproducible for any given combination (see below). The amplification products are resolved on agarose gels and polymorphisms serve as dominant genetic markers, which are inherited in a Mendelian fashion (Williams ct al. 1990; Carlson et al . 1991; Martin, Williams & Tanksley 1991; Welsh, Peterson & McClelland 1991). Amplification of non-nuclear RAPD markers is negligible because of the relatively small non-nuclear genome sizes.

Two modifications of detecting RAPD markers have been described as DNA Amplification Fingerprinting (DAF) and Arbitrarily Primed Polymerase Chain Reaction (AP-PCR). DAF uses short random primers of 5-8 bp and visualizes the relatively greater number of amplification products by polyacrylamide gel electrophoresis and silver staining (Caetano-Anolles, Bassam & Gresshof 1991). AP-PCR uses slightly longer primers (such as universal M13) and amplification products are radio- actively IabelIed and also resolved by polyacrylamide gel electrophoresis (Welsh h McClelland 1990; Welsh et al. 1991b).

Standard RAPD analysis is performed according to the original methods (Williams et al. 1990) using short oligonucleotide primers of random sequence which are commercially available (Operon Technologies, Inc., Alameda, Calif.). Only high-molecular-weight, i.e. non-

degraded, DNA should be subjected to RAPD analyses. Amplification products can be resolved by gel electrophoresis on 1.4% agarose gels.

Applications

Here we illustrate several potential applications of RAPD fingerprinting in molecular ecology, including determination of taxonomic identities, detection of interspecific gene flow, assessment of kinship relationships, analysis of mixed genome samples, and production of specific probes.

Determinntiorz of tnxomnic identity

By employing different oligonucleotide primers, molecular characters can be generated that are diagnostic at different taxonomic levels. For any given primer, RAPD amplification products can be broadly classified into two groups: variable (polymorphic) or constant (non- polymorphic). These definitions are relative for a given operational taxonomic unit (OW). For instance, consider a RAPD analysis of several individuals within a species, and several species within a given genus (Fig. 1). Constant fragments diagnostic for a genus may be identified, as well as fragments which are polymorphic between species within the genus. Both types of product can be exploited for establishing systematic relationships. In this example, constant fragments operationally identify members of a certain genus exclusively if the fragment is a unique polymorphism in a comparison of genera (genus-specific character in Fig. 1). Note, the determination of taxonomic relatedness is only valid between taxa for which the diagnostic RAPD fingerprint patterns have been established. Similarly, fragments polymorphic at the species level will operationally identify members of a given species if the fragment is constant among all members of that species (species-specific character in Fig. 1). An example of such a marker is provided in RAPD fingerprints from two dragonfly species in Fig. 2. Fragments polymorphic among individuals may also be utilized to determine clonal identity, as is frequently required for asexually reproducing organisms. Clone-specific markers have been identified in hydroids (Schienvater, unpubl.), clonal “individual”- specific markers in fungal mycelia (Smith, Bruhn & Andcrson 1992), cultivar-specific markers in broccoli and cauliflower (Hu & Quiros 1991), strain-specific markers in mice (Welsh et al. 1991a), species-specific markers in irises (Arnold, Buckner & Robinson 1991) and tomato (Klein- Lankhorst et al . 1991) and genus- and family-specific markers in palms (M. Balick & S. Dellaporta, in preparation). Thus RAPD products can be generated that

RAPD F I N G E R P R I N T I N G 57

A: genera B: specks c: individuals

1 2 3 r 4 5

oaonn 1 2 3 4 5

A B ..

Fig. 1 Use of polymorphic and n m - polymorphicRAPD fragments for genome scanning. Schematic of RAPD fingerprints of genera, species, and individuals illus- trating the potential use of polymorphic and non-polymorphic RAPD fragments as diagnostic markers. In gel A, a genus 3 specific band is identified by its presence in all species of that genus (and consequently all individuals of all species of genus 3 tested); this genus-specific fragment can serve as a diagnostic DNA marker in RAPD analyses or a genus- specific probe in KFLP analyses. Similarly, a species-specific fragment (gel B) is identified by its presence in all individuals of species 3, that can serve as a species specific RAPD marker or probe (see Fig. 2). Finally, gel C shows five polymorphic RAPD fragments between different individuals of species 3; these may be used for intraspecific analyses, such as paternity determinations (see Fig. 3), by means of RAP0 fingerprinting.

serve as diagnostic molecular characters at different taxonomic levels.

As illustrated in Fig. 1, the specificity of any single RAPD marker may range from the level of the individual to higher taxonomic levels. However, taxon identification by diagnostic RAPD markers can only be done by comparison within a set of genotypes of known R4PD amplification profile for a given primer. The specificity of RAPD markers has to be determined empirically for each genome within a set of genomes under investigation by screening several primer-templa te combinations (analogous to the screening of restriction enzyme-probe combinations in Southem-blot-based fingerprinting). Homology of diagnostic markers, especially between members of higher Oms, may be established by Southem analysis to exclude the possibility of co- migration of fragments of the same size from non- homologous loci (see below).

Analyses of interspecific gene flow and hybrid speciation

A straightforward conclusion of the outlinedpotential of h i 2 3 4 5 6 7 8 ~ the RAF'D method to identify diagnostic markers for

different OTUs is that RAF'D can be applied to analyse fusion of genotypes at different taxonomic levels. At the level of the individual, RAPD markers may be applied to parentage analysis (see below); at the population or species level RAPD may be used to detect hybrid populations Or species. The detection Of genome hybrids relies upon the identification of diagnostic RAPD markers for the parental genotypes under investigation.

Fig. 2 Example of determination of taxonomic identities. The primer (5'CCACAGCAm amplifies a constant RAPD marker (arrow) shared by the dagonfly prthm'JPe (lanes 1-4, four unrelated individuals) and Orthetrum cmr~escms (lanes 5-8, four unrelated individuals). In addition, note the occurrence of a constant marker (arrow) diagnostic for 0. c ~ u l f f c e n s . Each marker appeared in all individuals tested with this primer (n = 38).

58 H. HADRYS, M. BALICKand B. SCHIERWATER

4-

1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4

Fig. 3 Determination of paternity, RAPD fingerprint of a guarded Amx partfrenopc female, the guarding mate, the offspring of this pair, and several unrelated males revealed by primer B 07 (S’GCTGACGCAC). Lanes 1.2 = unrelated control males; 3 = guarding male; 4 = mother; 7-14 = individual offspring and entire clutches. Note that a characteristic band (arrow) from the guarding male (lane 3) is detected in all offspring samples. Further note that this same band is detected in synthetic offspring using the guarding male (lane 5), but is absent from the synthetic offspring of unrelated males (lane 6). To achieve statistically significant analyses we pooled three RAPD fingerprints with three different primers and calculated band-sharing coefficients for known first-degree relatives (mother and offspring), putative first-degree relatives (guarding male and offspring) and controls (known non-firstdegree relatives) for seven families and several offspring clutches per family. In all cases, the controls shared statistically significant fewer bands with any of the offspring clutches than did the putative parents.

Arnold et al. (1991) have demonstrated interspecific gene flow between two Louisiana iris species, Iris fulva and I. hexagonu, by analyses of species-diagnostic RAPD markers. Using two 10-bp and one 16-bp primers they reported four species-specific markers from different populations of I. fulua. These markers were missing in I. hexugona, but were present at intermediate frequencies in experimental F1 hybrids (I. fulm x I . hexngonn) and at variable frequencies in a natural contemporary hybrid population. Conclusions from RAPD analyses on the occurrence of interspecific gene flow between two iris populations were consistent with results from RFLP analyses of the chloroplast genome. Furthermore, variable frequencies of species-diagnostic markers were also found in the putative hybrid species 1. nelsonii. Here, RAPD markers may be useful in investigating the role of hybridization in the origin of I. nelsonii.

Using the AP-PCR modification of RAPD, Welsh et al. (1991a) identified F1 hybrids from different inbred maize lines. Other groups have begun to use RAPD for analyses of hybridization events where allozyme studies have not proven to be sensitive enough for hybrid genotypes, for example, in hybridization along vertical zonations in natural populations of Dnphnia (B. Streit, personal communication) or in plant breeding programmes (Crowhurst et al. 1991; Hu & Quiros 1991; Martin et al. 1991; Quiros et ul. 1991).

Defermimfion of paternity and kinship relationships

By employing fragments that are polymorphic among individuals, RAPD analysis may be used to assess paternity and kinship relationships in large offspring samples. A common problem in behavioural ecology is to determine the actual father from a number of potential fathers. The earliest application of RAPD fingerprinting to paternity analyses resolved the question of paternity in an unknown mating system of the dragonfly A m parthenope (Hadrys 1991; H. Hadrys et nl., in preparation). A. pnrflrenope males guard ovipositing females over extended periods of time and thereby regularly take the chance of suffering severe injury or even death by attacks hom conspecific males trying to split the tandem pairs. The presence of spermatodesmids, i.e. sperm bundles encased in a slowly degrading matrix, has suggested that the male may guard a female in order to assure a sub- sequent mating success rather than immediate fertili- zation success. R4PD analyses of tandem males, tandem females and the offspring clutches identified the tandem male as the father of the immediately oviposited offspring clutches. Figure 3 shows RAPD fingerprints of the guarding male, the guarded female, the offspring, and several unrelated males. The RAPD markers polymorphic among potential fathers show the guarding male to be the actual father. For statistical analyses the number

RAPD FINGERPRINTING 59

of polymorphic markers can be increased by increasing the number of diagnostic primers, and conventional band-sharing coefficients can be applied (Lynch 1990, 1991; Burke et al. 1991a; Keane et al. 1991; H. Hadrys et al., in preparation). Band-sharing coefficients between unrelated individuals are highly dependent on the primer-template combination used (see Fig. 2). Although we found background band-sharing coefficients of RAPD markers generally higher than those known from multilocus probe finge printing, this does not represent a serious problem. Because linkage between different arbitrary priming sequences is extremely unlikely, the number of independent polymorphic markers analysed can be rapidly increased by pooling markers revealed by several primers. The amplification of monomorphic RAPD markers may be kept to a minimum by choosing the ~ g h t primer-template combination, and any monomorphic markers may be removed from the analysis to decrease background band sharing.

In principle RAPD markers can formally be treated as Mendelian alleles, and for parentage analysis analytical approaches may be developed which are based on knowledge of allelic frequencies, e.g. as used in statistical analyses of single-locus fingerprint profiles. In practice, however, allelic frequencies of scorable dominant RAPD markers in diploid organisms might be difficult to estimate and markers revealed by the same primer may be linked (cf. Williams ef RZ. 1990; Carlson ef el. 1991).

The segregation and linkage of RAPD markers has been demonstrated in several genetic studies. The expected nuclear transmission of RAPD markers to F1 offspring has been reported for hybrids of maize inbred lines (Welsh et al . 1991a) as also has the segregation of RAPD polymorphisms in experimental crosses of conifers (Carlson et al. 1991) and the linkage of RAPD markers to resistance genes in lettuce (Michelmore, Paran & Kesseli 1991; Paran, Kesseli & Michelmore). RAPD markers have also been used in demonstrating the introgression of two parental genomes in an iris hybrid species (Arnold et al. 1991).

Conventional RFLP fingerprinting techniques are ill- suited for the analysis of paternity and estimation of reproductive success in species with large offspring clutches, because of the need to determine paternity for each individual offspring. RAPD fingerprinting provides a ready alternative for such cases. Synthetic offspring may be produced by mixing equal amounts of the DNA of the mother and the potential father (Fig. 3). The amplification products from the synthetic offspring should ideally contain the full complement of bands (H. Hadrys et a l . , in preparation; cf. Carlson ef al. 1991; see also below) that appear in any single offspring of these parents. However, certain combinations of alleles may lead to amplification artefacts (e.g. heteroduplex formation, see

below), and certain markers may only get amplified from an offspring but not from either of its parents. The Occurrence of non-parental bands in offspring from known primate pedigrees has raised concerns in parentage determinations when single individuals are analysed (Riedy, Hamilton & Aquadro 1992). The Occurrence of non-parental bands in offspring from known primate pedigrees has raised concerns in parentage determinations when single individuals are analysed (Riedy, Hamilton & Aquadro 1992). In contrast, the synthetic offspring is a complete representation of both parental genomes and will match the profile of a large sampIe of offspring, since in both the ‘synthetic’ and the real offspring dutch, allele combinations that may cause amplification artefacts are represented in equal amounts. The degree of mismatch between synthetic offspring and actual offspring clutches is indicative of mixed paternity, which can be measured by quantitative determination of mixed genome samples.

A na lys ing mixed genome samples

The RAPD technique may be used to generate quantitative estimates of the relative proportions of different genomes in mixed DNA samples. In many polygamous mating systems, especially in insects, sperm competition and mixed paternity may occur. Here, confirmation of the presence of more than one paternal genotype would be highly desirable. Using DNA from a dragonfly, Orthetrurn coerulescms, we reconstituted mixed-genome samples experimentally by varying the relative propor- tion of DNA from two male individuals over two orders of magnitude. This DNA was amplified and the relative amounts of individual male-diagnostic amplification products quantified by densitometry. The relative intensity of diagnostic bands from two different individuals varied predictably with the relative DNA concentrations of each genome in the reaction, and the DNA concentration of a given genome could be estimated from band intensities as long as the relative amount of this genome was at least 20% (Fig. 4; see also Michehore ef al. 1991). Note that this approach is based on well amplified polymorphic bands, requires precise knowledge of the diagnostic markers for each genome being scanned, and requires prior calibration experiments.

Generating novel specific probes

Any diagnostic RAF’D marker can be eluted from the gel, reamplified, radiolabelled with 32P and serve as an inexhaustible supply of probe in Southern analyses (Fig. 5). Such probes may be used to exclude the possibility of

60 H . H A D R Y S , M. B A L I C K a n d B . S C H I E R W A T E R

(C)

1 1

Fig. 4 Analysis of mixed genome samples. Quantitative estimates of the presence of a genome in mixed DNA samples by densitometry. (A) RAPD fingerprints of two males (a, b) of Orthetrrrrn cwrrilesceirs with primer B l l (5'GTACACCCGT) showing a diagnostic marker for each (see arrow). (B) Mixed reactions with varving proportions of DNA from the two males. The total amount of DNA per reaction was 25 ng, with the proportions a:b (in '2) as follows; lane 1.50:jO; lane 2,40:60; lane3,30:70; lane 4,20:80. The intensity of bands is reproducible between reactions (cf. Caetano-Anolles el nl . 1991; hlichelmore et n l . 1991) and co-varies with total amount of DNA template in a non- linear fashion. (C) Densitometric analyses (DESAGA densitometcr) of lanes 1-4 of the fingerprint shown in (B). x-axis is the distance in mm; y-axis is the relative extinction at 540 nm. Peaks of the diagnostic band from male b are numbered refemng to lanes in (B). Calculations of the area of single peaks of the curve (corresponding to single bands on the gel) are relative estimates of the amount of DNA amplified. Using the BMDP statistical software (BMDPAR) the best fit regressions between percentage of known male DNA (Y) in the reaction to percentage of maximal band intensity of the characteristic polymorphic band (Imax, i.e. when using 100% known template DNA) follow sigmoidal models of the form: Y = qre"'-* / (9 - r + re"'"'). Parameters 9, r , s must be determined by calibration for each such analysis. In this example, parameters and asymptotic standard deviations were 9 = 108 f 10.1. r=30 f 8.4, s=0.035 f 0.012. For each useful marker, I,,, has to be experimentally determined to establish the correct amount of total mixed sample DNA in the reaction because band intensity may decrease with DNA concentrations exceeding Ima,.. This method produced reproducible estimates of known genome presence in mixed samples over the range of 2040%. For more-sensitive estimates Southern analyses are preferable.

co-migration of fragments of different sequence but similar sue. Other applications include generation of probes for taxonomic analysis or the quantitative estimation of the presence of a certain genome in a mixed sample by Southern analysis (cf. Michelmore rt nl. 1991; Jeffreys et al . 1991). The specificity of the probes can be further improved by eluting diagnostic RAPD bands from the gel, reamplifying, subcloning a n d sequencing t h e fragments, and eventually selecting a partial consensus sequence a s a probe. Williams et al. (1990) report that six of 11 RAPD markers tested as probes in RFLP analyses were useful hybridization probes, because all of them hybridized to single-copy DNA. The other five, however, were not useful, because they hybridized to middle- or highly repetitive DNA i n the soybean genome. RAPD probes have also been used t o detect RFLPs in tomato species (Martin et a l . 1991; Klein-Lankhorst et al. 1991).

Difficulties and limitations of RAPD fingerprinting

The following technical considerations a n d potential difficulties merit attention:

77ie size of tlir primer

Primer size will determine t h e degree of specificity in genome scanning. It may be expected that primers of short length will amplify an unreasonably large number of sequences and that larger primers will amplify too few sequences t o be routinely informative. Beyond a certain primer size (c. 15-mer) increasing primer length m a y also increase non-specific primer annealing, consequently increasing the probability of random non-reproducible amplification patterns. All s tudies using s tandard RAPD


Fig. 5 Use of amplification products as probes. (a) RAPD fingerprint gel (primer 814 5’TCCGCTCTGG) of a family of Orthetrum cwrulmens (lanes 1-9). Lane 5 is the synthetic oif- spring of the parents of the family, and lane 7 are larvae from the last oviposited egg clutch that lack a diagnostic marker (arrow). (b) A sample (5 pl) of the diagnostic marker was eluted from the gel and reamplified in a 100-p.1 reaction volume containing 50 WCi of 32dATP under standard RAPD PCR conditions (Williams et RI. 1990). The radioactive amplification product was used as a hybridization probe to a Southern blot of the original RAPD fingerprint. The autoradiogram shows the diagnostic marker to be homologous in different lanes and also identifies a smaller RAPD fragment (arrow) containing homologous sequence to the probe. Note that the probe hybridizes substantially more strongly to the synthetic offspring (lane 5). which contains the pooled RAPD markers from both parents. This result was reproducible with synthetic offspring from different parents; its basis is not yet understood and it suggests that care should be taken in using probes with synthetic offspring samples. Also note, the probe used here did not hybridize to any RAPD markers (revealed with the same primer) from the unrelated dragonfly Anax parthenope.

conditions (fragment separation on agarose gels) have found 10-bp primers to be an efficacious size. A G+C content of the primer similar to the G+C content of the analysed genome will maximize the frequency of binding sites and hence amplification products.

Sensitivity to renction conditions

Being PCR-based, the principal limitations of RAPD fingerprinting arise from its sensitivity to reaction conditions, and slight changes in the conditions may affect the reproducibility of amplification products (Williams et al. 1990; Arnold et al. 1991; Carlson et 01. 1991; Klein- Lankhorst et al. 1991). The technique is sensitive to (a) shape of the temperature profile , (b) type of polymerase used and (c) M$+ concentration. The amplification profile is sensitive to Taq or DNA concentration. The shape of the temperature profile is a property of the thermal cycler and must be standardized. Only strictly standardized reaction conditions will guarantee reproducible amplification products. Furthermore, we found that the optimal concentration of template DNA per reaction may vary substantially from typical conditions (25 ng per reaction) depending on the primer-template combination used (cf. Carlson et al . 1991).

The possibility of co-migration

An assumption of the use of the RAPD technique is that amplified fragme.nts are unique, i.e. that the procedure does not amplify two distinct fragments which co- migrate on gels because of similar size. Co-migration in the RAPD technique is easily detected by eluting individual PCR products from gels and reprobing the products via Southern analysis (Fig. 5). Alternatively, polyacrylamide gel electrophoresis may be used to increase the resolution of band separation.

Non-reproducible amplificntion products

As with other genetic markers, some RAPD fragments may be ambiguous and not easy to score (Williams et a l . 1990). These unclear and non-reproducible fragments, which may derive from non-specificic primi,ng or from heteroduplex formation between related amplification products (or other secondary structure artefacts, which can prevent normal amplification patterns) are not useful as genetic markers. However our own work, as well as the work of several others (e.g. Williams et nl . 1990; Arnold et al. 1991; Hu & Quiros 1991; Klein-Lankhorst et al. 1991) have all shown that if the RAPD amplification is repeated two or more times, the majority of markers is clearly reproducible and scorable. As in many cases of using PCR, sometimes amplification products are found

62 H . HADRYS, M . BALICKand B . SCHIERWATER

even in the absence of template DNA in the reaction (Innis et nl. 1990; Klein-Lankhorst et al. 1991). However, in ail reported cases those 'ghost' bands disappear if the template DNA under investigation is added to the reaction.

Other considerations

The most common DNA fingerprint technologies differ substantially in (i) complexity of technological pro- cedures, (ii) required amount of DNA, (iii) sequence information needed for a genome being scanned, (iv) analytical power of assigning genotype relatedness, (v) expense in tenhs of labour and money, (vi) broadness of applications. In this context, RAPD fingerprinting seems IiJcely to have wide potential for applications in molecular ecology, and requires the least in technology, labour and expenses. The cost of producing one individual DNA fingerprint by Southern hybridization can be very substantial (Weatherhead & Montgomerie 1991); in contrast, the average expense for one RAPD fingerprint can be as low a s US$2.00. On the other hand, RAPD markers are the least informative of all known DNA markers and they are dominant (heterozygosity is normally not detectable). Consequently, the analytical power of RAPD markers is not competitive with analyses using sequence information or single locus probe fingerprint technologies. However, RAPDs are detected more easily than RFLPs and can be competitive with RFLPs even in analyses of genomes with high levels of heterozygosity (Williams et al . 1990; Carlson et nl. 1991; Hu & Quiros 1991).

Potential Future Applications

We note here several additional applications currently under development. 1. Sex determinution. In many ecological (as well as agri- cultural and legal) applications it would be convenient to have available markers that were sex-specific. We expect that little difficulty will be encountered in developing RAPD markers with this characteristic. 2. Gentrution of specific PCR primers for anonymous genoms. A major limitation in the application of PCR to ecological problems is the absence of sequence information for the vast majority of organisms. We suggest that this difficulty may be overcome for many applications by using a RAPD-based strategy for developing 'designed PCR primers. Specifically, RAPD primers with an embedded restriction site may be used to detect fragments showing the desired properties (e.g. detecting a particular taxon). These fragments may then be cloned and sequences used to develop specific 'designed' PCR primers for diagnostic markers.

3. Quantitative analysis of mixed biosamples. Analogous to the analysis of mixed paternity samples in dragonflies, analysis of field samples of different species or other Oms (e.g. plankton sampling) may be performed. 4. Phylogeny. RAPD markers may prove to be useful characters for cladistic analysis.

Acknowledgments

Many substantial contributions to the manuscript derive from the ideas of Dr Stephen L. Dellaporta and Dr Leo W. Buss. We thank Matt Dick, Robert Domtt and Neil Blackstone for critical comments. Our work is supported by D U D , DFG, NATO, NIH, NSF and ONR.

References

Arnold ML, Buckner CM, Robinson JJ (1991) Pollen- mediated introgression and hybrid speciation in Louisiana irises. Proceediiigs of the National Academy of Sciences of the USA, 88, 1398-1402.

Burke T, Bruford MW (1987) DNA fingerprinting in birds. Nuture, 327, 149-152.

Burke T, Hanotte 0, Bruford MW, Cairns E (1991a) Multilocus and single locus minisatellite analysis in population biological studies. In: Burke T, Dolf G, Jeffreys AJ, Wolff R (eds) D N A Fingerprinting: Apprmches niid Applicntions, pp. 154-168. Birkhauser, Basel.

Burke T, Dolf G, Jeffreys AJ, Wolff R (eds) (1991b) D N A Fingerprintiiig: Apyrmches mid Applications. Birkhauser, Basel.

Caetano-Anolles G, Bassam GJ, Gresshof PM (1991) High resolution DNA amplification fingerprinting using very short arbitrary oligonucleotide primers. Biotechnology, 9, 553-556.

Carlson JE, Tulsieram LK, Glaubitz JC, Luk VWK, Kauffeldt C, Rutledge R (1991) Segregation of random amplified DNA markers in F1 progeny of conifers. Theoretical and Applied Genetics, 83, 194-200.

Crowhurst RN, Hawthorne BT, Rikkerink EHA, Templeton MD (1991) Differentiation of Fiisarium solnni f . sy . cucurbitae race-1 and race-2 by random amplification of polymorphic DNA. Current Genetics, 20,

Erlich HA (1989) PCR Technology. Stockton Press, New York.

Gyllensten UB, Jakobsson S, Temrin H (1991) No evidence for illegitimate young in monogamous and polygynous warblers. Nature, 343, 168-170.

Hadrys H (1991) Postkopulatorische Mannchen- konkurrenz und RAPD Fingerprinting in Odonaten. Verhndlungen der Deufschen ZuoIogischen Grsellschuft, 84,306-307.

391-396.


Hillis D, Moritz C (1990) Molecrilar Systematics. Sinauer, Sunderland, Mass.

Hu J, Quiros CF (1991) Identification of broccoli and cauliflower cultivars with RAPD markers. Plmt Cell Reports, 10, 505-511.

Innis MA, Gelfand DH, Sninsky JJ, White TJ (1990) PCR Protocols. Academic Press, San Diego.

Jeffreys AJ, MacLeod A, Tamaki K, Neil DL, Monckton DG (1991) Minisatellite repeat coding as a digital approach to DNA typing. Nature, 354,204-209.

Jones CS, Lessels CM, Krebs JR (1991) Helpers-at-the- nest in European Bee-eaters (Meriops apiaster). In: Burke T, Dolf G, Jeffreys AJ, Wolff R (eds) D N A Fingerprirrting: Approaches and Applications, pp. 169-192. Birkhauser, Basel.

Keane B, Waser PM, Danzl-Tauer L, Minchella DJ (1991) DNA fingerprinting: estimating background band sharing in banner-tailed kangaroo rats. Animal Behaviour, 42, 141-143.

Kirby LT (1990) D N A Fingerprinting: An Introduction. Stockton Press, New York.

Klein-Lankhorst RM,Vermunt A, Weide R, Liharska T, Zabel P (1991) Isolation of molecular markers for tomato (L. esculentum) using random amplified polymorphic DNA (RAPD). Theoretical and Applied Genetics, 83, 108-114.

Lewin R (1989) Limits to DNA Fingerprinting. Science, 243, 1549-1551.

Lynch M (1990) The similarity index and DNA fingerprinting. Molecular Biology nnd Evolution, 7, 478-484.

Lynch M (1991) Analysis of population genetic structure by DNA fingerprinting. In: Burke T, Dolf G, Jeffreys AJ, Wolff R (eds) D N A Fingerprinting: Apprmchrs n~td Applications, pp. 113-126. Birkhauser, Basel.

Martin GB, Williams JGK, Tanksley SD (1991) Rapid identification of markers linked to a Psezdonzonns resistence gene in tomato by using random primers and near-isogenic lines. Proceedings of the Nntiorzal Academy of Sciences of the USA, 88, 2336-2340.

Michelmore RW, Paran I, Kesseli RV (1991) Identi- fica tion of markers linked to disease-resistance genes by bulked segregant analysis. A rapid method to detect markers in specific genomic regions bv using segre- gating populations. Proceedings of the National Academy of Scienres of the USA, 88, 9228-9832.

Paran I, Kesseli R, Michelmore R (1991) Identification of restriction fragment lengthy polymorphism and random amplified polymorphic DNA markers linked to downy mildew resistance genes in lettuce, using near-isogenic lines. Genome, 34, 1021-1027.

Packer C, Gilbert DA, Pusey AE, OBrien SJ (1991) A molecular genetic analysis of kinship and cooperation in African lions. Nature, 351, 562-565.

Pemberton J, Bancroft D, Amos B (1991) Behavioural

Ecology and DNA fingerprinting: the lab rats’ riposte. Trends in Ecology and Evolution, 6 , 299-300.

Quiros CF, Hu J, This P, Chevre AM, Delseny M (1991) Development and chromosomal localization of genome specific markers by polymerase chain reaction in Brassica. Theoretical and Applied Genetics, 82, 627-632.

Riedy MF, Hamilton WJ, Aquadro CF (1992) Excess of non-parental bands in offspring from known pedigrees assayed using RAPD PCR. Nucleic Acids Research, 20, 918.

Schlotterer C, Amos B, Tautz D (1991) Conservation of polymorphic simple sequence loci in cetacean species. Nature, 354, 63-65.

Smith HG, Montgomerie R, Poldmaa T, White BN, Boag PT (1991) DNA fingerprinting reveals relation between tail ornaments and cuckoldry in barn swallows, Hirundo rustica. Behavioural Ecology, 2, 90-98.

Smith ML, Bruhn JN, Anderson JB (1992) The fungus Armillaria bulbosa is among the largest and oldest living organisms. Nature, 356, 428431.

Tautz D (1989) Hypervariability of simple sequences as a general source for polymorphic DNA markers. Nucleic Acids Research, 17, 6463-6471.

Weatherhead PJ, Montgomerie RD (1991) Good news and bad news about DNA fingerprinting. Trerrds iiz Ecology and Evolution, 6, 173-174.

Welsh J, McClelland M (1990) Fingerprinting genomes using PCR with arbitrary primers. Nricleic Acids Research, 18, 7213-7218.

Welsh J, Petersen C, McClelland M (1991a) Poly- morphisms generated by arbitrarily primed PCR in the mouse: application to strain identification and genetic mapping. Nucleic Acids Research, 20, 303-306.

Welsh J, Honeycutt RJ, McClelland, Sobral BWS (1991b) Parentage determination in maize hybrids using the arbitrarily primed polymerase chain reaction (AP-PCR). Theoretical and Applied Genetics, 82, 47-76.

Williams JGK, Kubelik AR, Livak KJ, Rafalski JA, Tingey SV (1990) DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic A d s Rpsearch, 18, 6531-6535.

This paper is a result of collaborative work between the groups of Leo Buss and Stephen Dellaporta (Yale, Biology) to develop and apply new molecular genetic tools to developmental genetics and population ecology. Heike Hadrys is studying the evolution of mating systems in odonates by combining field work with molecular genetic amlyses at the Zoological Institute Braunschweig and at Yale University. Michael Ballick cotlabor- ates with Stephen Dellaporta on conservation biology and systematics of palms. Bernd Schienvater has performed 3 years of postdoctoral research with Leo Buss on molecular develop mental genetics and evolutionary ecology of hydroids.

applications of rapd

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