- Molecular ecology
Molecular Ecology (1992) 1, 55-63
M I N I – R E V I E 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 o n 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 be-
havioural 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 n l . 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 con-
siderations (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 hybridi-
zation 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 tech-
nology of DNA fingerprinting to the molecular analysis of
relatedness between genotypes. The introduction of
RAPD fingerprinting is a substantial contribution toward
the second direction. 1365294x, 1992, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/j.1365-294X.1992.tb00155.x, Wiley Online Library on [31/01/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
M I N I – R E V I E 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 o n 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 be-
havioural 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 n l . 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 con-
siderations (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 hybridi-
zation 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 tech-
nology of DNA fingerprinting to the molecular analysis of
relatedness between genotypes. The introduction of
RAPD fingerprinting is a substantial contribution toward
the second direction. 1365294x, 1992, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/j.1365-294X.1992.tb00155.x, Wiley Online Library on [31/01/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
56 H . HADRL’S. M. BALICKand B . SCHIERWATER
RAPD technology is only some 2 years old, and con-
sequently 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 limita-
tions of RAPD markers.
Principle of RAPD analyses
The PCR-based RAPD technique (Williams rt n l . 1990) is
an attractive complement to conventional DNA finger-
printing 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
a l . 1990; Carlson et a l . 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 a l .
1991b).
Standard RAPD analysis is performed according to the
original methods (Williams et a l . 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 electro-
phoresis on 1.4% agarose gels.
Applications
Here we illustrate several potential applications of RAPD
fingerprinting in molecular ecology, including deter-
mination of taxonomic identities, detection of inter-
specific 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 iden-
tified, 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 iden-
tify 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 indi-
viduals 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 a l . 1991a), species-specific markers in irises
(Arnold, Buckner & Robinson 1991) and tomato (Klein-
Lankhorst et a l . 1991) and genus- and family-specific
markers in palms (M. Balick & S. Dellaporta, in prep-
aration). Thus RAPD products can be generated that 1365294x, 1992, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/j.1365-294X.1992.tb00155.x, Wiley Online Library on [31/01/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
RAPD technology is only some 2 years old, and con-
sequently 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 limita-
tions of RAPD markers.
Principle of RAPD analyses
The PCR-based RAPD technique (Williams rt n l . 1990) is
an attractive complement to conventional DNA finger-
printing 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
a l . 1990; Carlson et a l . 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 a l .
1991b).
Standard RAPD analysis is performed according to the
original methods (Williams et a l . 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 electro-
phoresis on 1.4% agarose gels.
Applications
Here we illustrate several potential applications of RAPD
fingerprinting in molecular ecology, including deter-
mination of taxonomic identities, detection of inter-
specific 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 iden-
tified, 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 iden-
tify 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 indi-
viduals 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 a l . 1991a), species-specific markers in irises
(Arnold, Buckner & Robinson 1991) and tomato (Klein-
Lankhorst et a l . 1991) and genus- and family-specific
markers in palms (M. Balick & S. Dellaporta, in prep-
aration). Thus RAPD products can be generated that 1365294x, 1992, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/j.1365-294X.1992.tb00155.x, Wiley Online Library on [31/01/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License