You are on page 1of 9

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 probes (Burke et nl. 1991b) and PCR amplified simple


sequence microsatellite loci (Tautz 1989). Potential
Within the past few years molecular genetic approaches
applications are frequently thwarted by the requirement
have become of increasing importance to studies in be-
for significant quantities of DNA in the case of RFLP
havioural ecology and population biology. For instance,
analysis or by lack of relevant DNA sequence information
DNA fingerprinting technologies have revolutionized
in the case of conventional PCR-based techniques. Recent
approaches to our understanding of animal social
criticisms are that DNA fingerprinting requires special
systems by permitting analyses of kinship reIationships
molecular training, is labour-intensive, and is relatively
(e.g. Burke & Bruford 1987; Burke et 01. 1991a; Jones,
expensive
- (Weatherhead & Montgomerie 1991).
Lessels & Krebs 1991; Gyllensten, Jakobsson & Temrin
It has been argued that DNA fingerprinting is so
1991; Packer et nl. 1991; Pemberton, Banaoft & Amos
essential to behavioural ecology and population biology
1991; Schlotterer, Amos & Tautz 1991; Smith ef al. 1991).
that it would be highly unfortunate to have its application
Despite constant progress in methodology, application
limited to a few specialized laboratories rather than to the
of DNA markers to many problems in behavioural and
broad community working in these fields (Weatherhead
population ecology has been limited by technical con-
& Montgomerie 1991). Major progress in technology
siderations (Lewin 1989; Kirby 1990; Burke et al. 1991a;
development is expected in two directions: (I) increase in
Pemberton et al. 1991). The most frequently used DNA
analytical power per unit effort, and (2) simplification in
markers include RFLPs (restriction fragment length
technology, and ultimately reduction in expense. Use of
polymorphisms) visualized by Southern blot hybridi-
random amplified polymorphic DNA (RAPD) markers,
zation to different types of single-locus or multilocus
detected by PCR amplification of small inverted repeats
Correspondence author: Heike Hadrys, Zoologisches Institut der
scattered throughout the genome, adds a new tech-
Technischen Univenitat, Pockelsstr. 10a. D-3300 Braunschweig, nology of DNA fingerprinting to the molecular analysis of
Germany. relatedness between genotypes. The introduction of
*Present address: Zoologisches Institut der Universitiit, Siesmayerstr. RAPD fingerprinting is a substantial contribution toward
70, 06000 Frankfurt a.M., Germany. the second direction.
56 H . HADRL’S. M. BALICKand B. SCHIERWATER

RAPD technology is only some 2 years old, and con- degraded, DNA should be subjected to RAPD analyses.
sequently published applications are limited. However, Amplification products can be resolved by gel electro-
the rapidly growing interest in using RAPD technology phoresis on 1.4% agarose gels.
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
Applications
technology, discuss its applications in molecular ecology,
and point out the particular advantages as well as limita- Here we illustrate several potential applications of RAPD
tions of RAPD markers. fingerprinting in molecular ecology, including deter-
mination of taxonomic identities, detection of inter-
specific gene flow, assessment of kinship relationships,
Principle of RAPD analyses analysis of mixed genome samples, and production of
The PCR-based RAPD technique (Williams rt nl. 1990) is specific probes.
an attractive complement to conventional DNA finger-
printing in ecology. RAPD analysis is conceptually
Determinntiorz of tnxomnic identity
simple. Nanogram amounts of total genomic DNA are
subjected to PCR using short synthetic oligo- By employing different oligonucleotide primers,
nucleotides of random sequence. The amplification pro- molecular characters can be generated that are diagnostic
tocol differs from the standard PCR conditions (Erlich at different taxonomic levels. For any given primer,
1989) in that only a single random oligonucleotide primer RAPD amplification products can be broadly classified
is employed and no prior knowledge of the genome into two groups: variable (polymorphic) or constant (non-
subjected to analysis is required. When the primer is polymorphic). These definitions are relative for a given
short (e.g. 10-mer), there is a high probability that the operational taxonomic unit (OW). For instance, consider
genome contains several priming sites close to one a RAPD analysis of several individuals within a species,
another that are in an inverted orientation. The technique and several species within a given genus (Fig. 1).
essentially scans a genome for these small inverted Constant fragments diagnostic for a genus may be iden-
repeats and amplifies intervening DNA segments of tified, as well as fragments which are polymorphic
variable length. The profile of amplification products between species within the genus. Both types of product
depends on the templateprimer combination and is can be exploited for establishing systematic relationships.
reproducible for any given combination (see below). The In this example, constant fragments operationally
amplification products are resolved on agarose gels and identify members of a certain genus exclusively if the
polymorphisms serve as dominant genetic markers, fragment is a unique polymorphism in a comparison of
which are inherited in a Mendelian fashion (Williams ct genera (genus-specific character in Fig. 1). Note, the
al. 1990; Carlson et al. 1991; Martin, Williams & Tanksley determination of taxonomic relatedness is only valid
1991; Welsh, Peterson & McClelland 1991). Amplification between taxa for which the diagnostic RAPD fingerprint
of non-nuclear RAPD markers is negligible because of the patterns have been established. Similarly, fragments
relatively small non-nuclear genome sizes. polymorphic at the species level will operationally iden-
Two modifications of detecting RAPD markers have tify members of a given species if the fragment is constant
been described as DNA Amplification Fingerprinting among all members of that species (species-specific
(DAF)and Arbitrarily Primed Polymerase Chain Reaction character in Fig. 1). An example of such a marker is
(AP-PCR). DAF uses short random primers of 5-8 bp and provided in RAPD fingerprints from two dragonfly
visualizes the relatively greater number of amplification species in Fig. 2. Fragments polymorphic among indi-
products by polyacrylamide gel electrophoresis and viduals may also be utilized to determine clonal identity,
silver staining (Caetano-Anolles, Bassam & Gresshof as is frequently required for asexually reproducing
1991). AP-PCR uses slightly longer primers (such as organisms. Clone-specific markers have been identified
universal M13) and amplification products are radio- in hydroids (Schienvater, unpubl.), clonal “individual”-
actively IabelIed and also resolved by polyacrylamide gel specific markers in fungal mycelia (Smith, Bruhn &
electrophoresis (Welsh h McClelland 1990;Welsh et al. Andcrson 1992), cultivar-specific markers in broccoli and
1991b). cauliflower (Hu & Quiros 1991), strain-specific markers in
Standard RAPD analysis is performed according to the mice (Welsh et al. 1991a), species-specific markers in irises
original methods (Williams et al. 1990) using short (Arnold, Buckner & Robinson 1991) and tomato (Klein-
oligonucleotide primers of random sequence which are Lankhorst et al. 1991) and genus- and family-specific
commercially available (Operon Technologies, Inc., markers in palms (M. Balick & S. Dellaporta, in prep-
Alameda, Calif.). Only high-molecular-weight, i.e. non- aration). Thus RAPD products can be generated that
RAPD F I N G E R P R I N T I N G 57

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
A: genera B: specks c: individuals and non-polymorphic RAPD fragments as
diagnostic markers. In gel A, a genus 3
specific band is identified by its presence
1 2 3r4 5 1 2 3 4 5 in all species of that genus (and con-
oaonn sequently all individuals of all species of
genus 3 tested); this genus-specific frag-
ment 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 iden-
tified 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 indi-
viduals 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 com-
A
.. B parison within a set of genotypes of known R4PD ampli-
fication 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
Fig. 2 Example of determination of taxonomic identities. The fusion of genotypes at different taxonomic levels. At the
primer (5'CCACAGCAm amplifies a constant RAPD level of the individual, RAPD markers may be applied to
marker (arrow) shared by the dagonfly prthm'JPe parentage analysis (see below); at the population or
(lanes 1-4, four unrelated individuals) and Orthetrum cmr~escms
(lanes 5-8, four unrelated individuals). In addition, note
species level RAPD may be used to detect hybrid pop-
the occurrence of a constant marker (arrow) diagnostic for 0. ulations Or species. The detection Of genome hybrids
c ~ u l f f c e n s . Each marker appeared in all individuals tested relies upon the identification of diagnostic RAPD markers
with this primer (n = 38). for the parental genotypes under investigation.
58 H . HADRYS, M. BALICKand B. SCHIERWATER

4-

1 2 3 4 5 6 7 8 9 1011121314
Fig. 3 Determination of paternity, RAPD fingerprint of a guarded Amx partfrenopcfemale, 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


Defermimfionof paternity and kinship relationships
gene flow between two Louisiana iris species, Iris fulva
and I. hexagonu, by analyses of species-diagnostic RAPD By employing fragments that are polymorphic among
markers. Using two 10-bp and one 16-bp primers they individuals, RAPD analysis may be used to assess pater-
reported four species-specific markers from different nity and kinship relationships in large offspring samples.
populations of I. fulua. These markers were missing in I. A common problem in behavioural ecology is to deter-
hexugona, but were present at intermediate frequencies in mine the actual father from a number of potential fathers.
experimental F1 hybrids (I. fulm x I . hexngonn) and at The earliest application of RAPD fingerprinting to pater-
variable frequencies in a natural contemporary hybrid nity analyses resolved the question of paternity in an
population. Conclusions from RAPD analyses on the unknown mating system of the dragonfly A m parthenope
occurrence of interspecific gene flow between two iris (Hadrys 1991; H. Hadrys et nl., in preparation).
populations were consistent with results from RFLP A. pnrflrenope males guard ovipositing females over
analyses of the chloroplast genome. Furthermore, extended periods of time and thereby regularly take the
variable frequencies of species-diagnostic markers were chance of suffering severe injury or even death by attacks
also found in the putative hybrid species 1. nelsonii. Here, hom conspecific males trying to split the tandem pairs.
RAPD markers may be useful in investigating the role of The presence of spermatodesmids, i.e. sperm bundles
hybridization in the origin of I. nelsonii. encased in a slowly degrading matrix, has suggested that
Using the AP-PCR modification of RAPD, Welsh et al. the male may guard a female in order to assure a sub-
(1991a) identified F1 hybrids from different inbred maize sequent mating success rather than immediate fertili-
lines. Other groups have begun to use RAPD for analyses zation success. R4PD analyses of tandem males, tandem
of hybridization events where allozyme studies have not females and the offspring clutches identified the tandem
proven to be sensitive enough for hybrid genotypes, for male as the father of the immediately oviposited offspring
example, in hybridization along vertical zonations in clutches. Figure 3 shows RAPD fingerprints of the
natural populations of Dnphnia (B. Streit, personal guarding male, the guarded female, the offspring, and
communication) or in plant breeding programmes several unrelated males. The RAPD markers poly-
(Crowhurst et al. 1991; Hu & Quiros 1991; Martin et al. morphic among potential fathers show the guarding male
1991; Quiros et ul. 1991). to be the actual father. For statistical analyses the number
RAPD FINGERPRINTING 59

of polymorphic markers can be increased by increasing below), and certain markers may only get amplified from
the number of diagnostic primers, and conventional an offspring but not from either of its parents. The
band-sharing coefficients can be applied (Lynch 1990, Occurrence of non-parental bands in offspring from
1991; Burke et al. 1991a;Keane et al. 1991; H. Hadrys et al., known primate pedigrees has raised concerns in paren-
in preparation). Band-sharing coefficients between tage determinations when single individuals are
unrelated individuals are highly dependent on the analysed (Riedy, Hamilton & Aquadro 1992). The
primer-template combination used (see Fig. 2). Although Occurrence of non-parental bands in offspring from
we found background band-sharing coefficients of RAPD known primate pedigrees has raised concerns in
markers generally higher than those known from multi- parentage determinations when single individuals are
locus probe finge printing, this does not represent a analysed (Riedy, Hamilton & Aquadro 1992). In contrast,
serious problem. Because linkage between different the synthetic offspring is a complete representation of
arbitrary priming sequences is extremely unlikely, the both parental genomes and will match the profile of a
number of independent polymorphic markers analysed large sampIe of offspring, since in both the ‘synthetic’ and
can be rapidly increased by pooling markers revealed by the real offspring dutch, allele combinations that may
several primers. The amplification of monomorphic cause amplification artefacts are represented in equal
RAPD markers may be kept to a minimum by choosing amounts. The degree of mismatch between synthetic
the ~ g h t primer-template combination, and any offspring and actual offspring clutches is indicative of
monomorphic markers may be removed from the mixed paternity, which can be measured by quantitative
analysis to decrease background band sharing. determination of mixed genome samples.
In principle RAPD markers can formally be treated as
Mendelian alleles, and for parentage analysis analytical
approaches may be developed which are based on
A na lysing mixed genome samples
knowledge of allelic frequencies, e.g. as used in statistical
analyses of single-locus fingerprint profiles. In practice, The RAPD technique may be used to generate quanti-
however, allelic frequencies of scorable dominant RAPD tative estimates of the relative proportions of different
markers in diploid organisms might be difficult to genomes in mixed DNA samples. In many polygamous
estimate and markers revealed by the same primer may mating systems, especially in insects, sperm competition
be linked (cf. Williams ef RZ. 1990; Carlson ef el. 1991). and mixed paternity may occur. Here, confirmation
The segregation and linkage of RAPD markers has of the presence of more than one paternal genotype
been demonstrated in several genetic studies. The ex- would be highly desirable. Using DNA from a dragonfly,
pected nuclear transmission of RAPD markers to F1 Orthetrurn coerulescms, we reconstituted mixed-genome
offspring has been reported for hybrids of maize inbred samples experimentally by varying the relative propor-
lines (Welsh et al. 1991a) as also has the segregation of tion of DNA from two male individuals over two orders of
RAPD polymorphisms in experimental crosses of magnitude. This DNA was amplified and the relative
conifers (Carlson et al. 1991) and the linkage of RAPD amounts of individual male-diagnostic amplification
markers to resistance genes in lettuce (Michelmore, Paran products quantified by densitometry. The relative inten-
& Kesseli 1991; Paran, Kesseli & Michelmore). RAPD sity of diagnostic bands from two different individuals
markers have also been used in demonstrating the intro- varied predictably with the relative DNA concentrations
gression of two parental genomes in an iris hybrid species of each genome in the reaction, and the DNA concen-
(Arnold et al. 1991). tration of a given genome could be estimated from band
Conventional RFLP fingerprinting techniques are ill- intensities as long as the relative amount of this genome
suited for the analysis of paternity and estimation of was at least 20% (Fig. 4; see also Michehore ef al. 1991).
reproductive success in species with large offspring Note that this approach is based on well amplified poly-
clutches, because of the need to determine paternity for morphic bands, requires precise knowledge of the
each individual offspring. RAPD fingerprinting provides diagnostic markers for each genome being scanned, and
a ready alternative for such cases. Synthetic offspring requires prior calibration experiments.
may be produced by mixing equal amounts of the DNA of
the mother and the potential father (Fig. 3). The ampli-
fication products from the synthetic offspring should
Generating novel specific probes
ideally contain the full complement of bands (H. Hadrys
et al., in preparation; cf. Carlson efal. 1991; see also below) Any diagnostic RAF’D marker can be eluted from the gel,
that appear in any single offspring of these parents. reamplified, radiolabelled with 32P and serve as an
However, certain combinations of alleles may lead to inexhaustible supply of probe in Southern analyses (Fig.
amplification artefacts (e.g. heteroduplex formation, see 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 nl. 1991) and co-varies with total amount of DNA template in a non-
linear fashion. (C) Densitometric analyses (DESAGAdensitometcr) 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 b u t Difficulties and limitations of RAPD


similar sue. O t h e r applications include generation of fingerprinting
probes for taxonomic analysis o r t h e quantitative esti-
The following technical considerations a n d potential
mation of t h e presence of a certain g e n o m e in a mixed
difficulties merit attention:
sample by Southern analysis (cf. Michelmore rt nl. 1991;
Jeffreys et al. 1991). The specificity of t h e probes can be
further improved by eluting diagnostic RAPD b a n d s from
the gel, reamplifying, subcloning a n d sequencing t h e
77ie size of tlir primer
fragments, and eventually selecting a partial consensus Primer size will determine t h e degree of specificity in
sequence a s a probe. Williams et al. (1990)report that six genome scanning. It m a y be expected that primers of
of 11 RAPD markers tested as probes in RFLP analyses short length will amplify an unreasonably large n u m b e r
were useful hybridization probes, because all of them of sequences and that larger primers will amplify too few
hybridized to single-copy DNA. The other five, however, sequences t o be routinely informative. Beyond a certain
were not useful, because they hybridized to middle- or primer size (c. 15-mer) increasing primer length m a y also
highly repetitive DNA i n the soybean genome. RAPD increase non-specific primer annealing, consequently
probes have also been u s e d t o detect RFLPs in tomato increasing t h e probability of random non-reproducible
species (Martin et al. 1991; Klein-Lankhorst et al. 1991). amplification patterns. All studies using standard RAPD
RAPD FINGERPRINTING 61

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 con-
ditions, 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 repro-
ducible 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 indi-
Fig. 5 Use of amplification products as probes. (a) RAPD vidual PCR products from gels and reprobing the
fingerprint gel (primer 814 5’TCCGCTCTGG) of a family of products via Southern analysis (Fig. 5). Alternatively,
Orthetrum cwrulmens (lanes 1-9). Lane 5 is the synthetic oif-
polyacrylamide gel electrophoresis may be used to
spring of the parents of the family, and lane 7are larvae from the
last oviposited egg clutch that lack a diagnostic marker (arrow). increase the resolution of band separation.
(b) A sample (5 pl) of the diagnostic marker was eluted from the
gel and reamplified in a 100-p.1reaction volume containing 50 WCi Non-reproducible amplificntion products
of 32dATPunder standard RAPD PCR conditions (Williamset RI.
1990). The radioactive amplification product was used as a As with other genetic markers, some RAPD fragments
hybridization probe to a Southern blot of the original RAPD may be ambiguous and not easy to score (Williams et al.
fingerprint.The autoradiogram shows the diagnostic marker to
1990). These unclear and non-reproducible fragments,
be homologous in different lanes and also identifies a smaller
RAPD fragment (arrow)containing homologous sequence to the which may derive from non-specificic primi,ng or from
probe. Note that the probe hybridizes substantially more heteroduplex formation between related amplification
strongly to the synthetic offspring (lane 5). which contains the products (or other secondary structure artefacts, which
pooled RAPD markers from both parents. This result was re- can prevent normal amplification patterns) are not useful
producible with synthetic offspring from different parents; its as genetic markers. However our own work, as well as
basis is not yet understood and it suggests that care should be the work of several others (e.g. Williams et nl. 1990;
taken in using probes with synthetic offspring samples. Also
Arnold et al. 1991; Hu & Quiros 1991; Klein-Lankhorst et
note, the probe used here did not hybridize to any RAPD
markers (revealed with the same primer) from the unrelated al. 1991) have all shown that if the RAPD amplification is
dragonfly Anax parthenope. 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 3. Quantitative analysis of mixed biosamples. Analogous to
(Innis et nl. 1990; Klein-Lankhorst et al. 1991). However, in the analysis of mixed paternity samples in dragonflies,
ail reported cases those 'ghost' bands disappear if the tem- analysis of field samples of different species or other
plate DNA under investigation is added to the reaction. Oms (e.g. plankton sampling) may be performed.
4. Phylogeny. RAPD markers may prove to be useful
characters for cladistic analysis.
Other considerations
The most common DNA fingerprint technologies differ Acknowledgments
substantially in (i) complexity of technological pro- Many substantial contributions to the manuscript derive
cedures, (ii) required amount of DNA, (iii) sequence from the ideas of Dr Stephen L. Dellaporta and Dr Leo W.
information needed for a genome being scanned, (iv) Buss. We thank Matt Dick, Robert Domtt and Neil
analytical power of assigning genotype relatedness, (v) Blackstone for critical comments. Our work is supported
expense in tenhs of labour and money, (vi) broadness of by D U D , DFG, NATO, NIH, NSF and ONR.
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 References
expenses. The cost of producing one individual DNA Arnold ML, Buckner CM, Robinson JJ (1991) Pollen-
fingerprint by Southern hybridization can be very sub- mediated introgression and hybrid speciation in
stantial (Weatherhead & Montgomerie 1991);in contrast, Louisiana irises. Proceediiigs of the National Academy of
the average expense for one RAPD fingerprint can be as Sciences of the USA, 88, 1398-1402.
low a s US$2.00. O n the other hand, RAPD markers are Burke T, Bruford MW (1987) DNA fingerprinting in
the least informative of all known DNA markers and they birds. Nuture, 327, 149-152.
are dominant (heterozygosity is normally not detectable). Burke T, Hanotte 0, Bruford MW, Cairns E (1991a)
Consequently, the analytical power of RAPD markers is Multilocus and single locus minisatellite analysis in
not competitive with analyses using sequence infor- population biological studies. In: Burke T, Dolf G,
mation or single locus probe fingerprint technologies. Jeffreys AJ, Wolff R (eds) D N A Fingerprinting:
However, RAPDs are detected more easily than RFLPs Apprmches niid Applicntions, pp. 154-168. Birkhauser,
and can be competitive with RFLPs even in analyses of Basel.
genomes with high levels of heterozygosity (Williams et Burke T, Dolf G,Jeffreys AJ, Wolff R (eds)(1991b) D N A
al. 1990; Carlson et nl. 1991; Hu & Quiros 1991). Fingerprintiiig: Apyrmches mid Applications. Birkhauser,
Basel.
Caetano-Anolles G, Bassam GJ, Gresshof PM (1991)
Potential Future Applications High resolution DNA amplification fingerprinting
using very short arbitrary oligonucleotide primers.
We note here several additional applications currently Biotechnology, 9, 553-556.
under development. Carlson JE, Tulsieram LK, Glaubitz JC, Luk VWK,
1. Sex determinution. In many ecological (as well as agri- Kauffeldt C, Rutledge R (1991)Segregation of random
cultural and legal) applications it would be convenient to amplified DNA markers in F1 progeny of conifers.
have available markers that were sex-specific. We expect Theoretical and Applied Genetics, 83, 194-200.
that little difficulty will be encountered in developing Crowhurst RN, Hawthorne BT, Rikkerink EHA,
RAPD markers with this characteristic. Templeton M D (1991)Differentiation of Fiisarium solnni
2. Gentrution of specific PCR primers for anonymous genoms. f . sy. cucurbitae race-1 and race-2 by random ampli-
A major limitation in the application of PCR to ecological fication of polymorphic DNA. Current Genetics, 20,
problems is the absence of sequence information for the 391-396.
vast majority of organisms. We suggest that this difficulty Erlich HA (1989) PCR Technology. Stockton Press, New
may be overcome for many applications by using a York.
RAPD-based strategy for developing 'designed PCR Gyllensten UB, Jakobsson S, Temrin H (1991) No
primers. Specifically, RAPD primers with an embedded evidence for illegitimate young in monogamous and
restriction site may be used to detect fragments showing polygynous warblers. Nature, 343, 168-170.
the desired properties (e.g. detecting a particular taxon). Hadrys H (1991) Postkopulatorische Mannchen-
These fragments may then be cloned and sequences used konkurrenz und RAPD Fingerprinting in Odonaten.
to develop specific 'designed' PCR primers for diagnostic Verhndlungen der Deufschen ZuoIogischen Grsellschuft,
markers. 84,306-307.
RAPD FINGERPRINTING 63

Hillis D, Moritz C (1990)Molecrilar Systematics. Sinauer, Ecology and DNA fingerprinting: the lab rats’ riposte.
Sunderland, Mass. Trends in Ecology and Evolution, 6 , 299-300.
Hu J, Quiros CF (1991) Identification of broccoli and Quiros CF, Hu J, This P, Chevre AM, Delseny M (1991)
cauliflower cultivars with RAPD markers. Plmt Cell Development and chromosomal localization of
Reports, 10, 505-511. genome specific markers by polymerase chain reaction
Innis MA, Gelfand DH, Sninsky JJ, White TJ (1990) PCR in Brassica. Theoretical and Applied Genetics, 82, 627-632.
Protocols. Academic Press, San Diego. Riedy MF, Hamilton WJ,Aquadro CF (1992) Excess of
Jeffreys AJ, MacLeod A, Tamaki K,Neil DL, Monckton non-parental bands in offspring from known pedigrees
DG (1991) Minisatellite repeat coding a s a digital assayed using RAPD PCR. Nucleic Acids Research, 20,
approach to DNA typing. Nature, 354,204-209. 918.
Jones CS, Lessels CM, Krebs JR (1991) Helpers-at-the- Schlotterer C, Amos B, Tautz D (1991) Conservation of
nest in European Bee-eaters (Meriops apiaster). In: Burke polymorphic simple sequence loci in cetacean species.
T, Dolf G, Jeffreys AJ, Wolff R (eds) D N A Fingerprirrting: Nature, 354, 63-65.
Approaches and Applications, pp. 169-192. Birkhauser, Smith HG, Montgomerie R,Poldmaa T, White BN, Boag
Basel. PT (1991)DNA fingerprinting reveals relation between
Keane B, Waser PM, Danzl-Tauer L, Minchella DJ (1991) tail ornaments and cuckoldry in barn swallows,
DNA fingerprinting: estimating background band Hirundo rustica. Behavioural Ecology, 2, 90-98.
sharing in banner-tailed kangaroo rats. Animal Smith ML, Bruhn JN, Anderson JB (1992) The fungus
Behaviour, 42, 141-143. Armillaria bulbosa is among the largest and oldest living
Kirby LT (1990) D N A Fingerprinting: An Introduction. organisms. Nature, 356, 428431.
Stockton Press, New York. Tautz D (1989) Hypervariability of simple sequences as a
Klein-Lankhorst RM,Vermunt A, Weide R, Liharska T, general source for polymorphic DNA markers. Nucleic
Zabel P (1991) Isolation of molecular markers for Acids Research, 17, 6463-6471.
tomato (L.esculentum) using random amplified poly- Weatherhead PJ, Montgomerie RD (1991) Good news
morphic DNA (RAPD). Theoretical and Applied Genetics, and bad news about DNA fingerprinting. Trerrds iiz
83, 108-114. Ecology and Evolution, 6, 173-174.
Lewin R (1989) Limits to DNA Fingerprinting. Science, Welsh J, McClelland M (1990) Fingerprinting genomes
243, 1549-1551. using PCR with arbitrary primers. Nricleic Acids
Lynch M (1990) The similarity index and DNA finger- Research, 18, 7213-7218.
printing. Molecular Biology nnd Evolution, 7, 478-484. Welsh J, Petersen C, McClelland M (1991a) Poly-
Lynch M (1991) Analysis of population genetic structure morphisms generated by arbitrarily primed PCR in the
by DNA fingerprinting. In: Burke T, Dolf G, Jeffreys mouse: application to strain identification and genetic
AJ, Wolff R (eds) D N A Fingerprinting: Apprmchrs n ~ t d mapping. Nucleic Acids Research, 20, 303-306.
Applications, pp. 113-126. Birkhauser, Basel. Welsh J, Honeycutt RJ, McClelland, Sobral BWS (1991b)
Martin GB, Williams JGK, Tanksley SD (1991) Rapid Parentage determination in maize hybrids using the
identification of markers linked to a Psezdonzonns arbitrarily primed polymerase chain reaction
resistence gene in tomato by using random primers (AP-PCR). Theoretical and Applied Genetics, 82, 47-76.
and near-isogenic lines. Proceedings of the Nntiorzal Williams JGK, Kubelik AR, Livak KJ, Rafalski JA,
Academy of Sciences of the USA, 88, 2336-2340. Tingey SV (1990) DNA polymorphisms amplified by
Michelmore RW, Paran I, Kesseli RV (1991) Identi- arbitrary primers are useful as genetic markers. Nucleic
fica tion of markers linked to disease-resistance genes A d s Rpsearch, 18, 6531-6535.
by bulked segregant analysis. A rapid method to detect
markers in specific genomic regions bv using segre-
gating populations. Proceedings of the National Academy This paper is a result of collaborative work between the groups of
of Scienres of the USA, 88,9228-9832. Leo Buss and Stephen Dellaporta (Yale, Biology) to develop and
Paran I, Kesseli R, Michelmore R (1991) Identification of apply new molecular genetic tools to developmental genetics
restriction fragment lengthy polymorphism and and population ecology. Heike Hadrys is studying the evolution
random amplified polymorphic DNA markers linked of mating systems in odonates by combining field work with
to downy mildew resistance genes in lettuce, using molecular genetic amlyses at the Zoological Institute
Braunschweig and at Yale University. Michael Ballick cotlabor-
near-isogenic lines. Genome,34, 1021-1027. ates with Stephen Dellaporta on conservation biology and
Packer C, Gilbert DA, Pusey AE, OBrien SJ (1991)A systematics of palms. Bernd Schienvater has performed 3 years
molecular genetic analysis of kinship and cooperation of postdoctoral research with Leo Buss on molecular develop
in African lions. Nature, 351, 562-565. mental genetics and evolutionary ecology of hydroids.
Pemberton J, Bancroft D, Amos B (1991) Behavioural