Commentary

ASM Sci. J., 9(1), 3039

Inter-Simple Sequence Repeat (ISSR) Markers:

Are We Doing It Right?
W.L. Ng1* and S.G. Tan2,3

Inter-simple sequence repeats (ISSRs) are regions in the genome flanked by microsatellite sequences.
PCR amplification of these regions using a single primer yields multiple amplification products that can
be used as a dominant multilocus marker system for the study of genetic variation in various organisms.
ISSR markers are easy to use, low-cost, and methodologically less demanding compared to other
dominant markers, making it an ideal genetic marker for beginners and for organisms whose genetic
information is lacking. Here, we comment upon some of the intricacies often overlooked in designing an
ISSR experiment, clarify some misconceptions, and provide recommendations on using ISSR markers
in genetic variation studies.
Key words: DNA marker; dominant marker; ISSR; RAM; genetic variation; DNA fingerprinting; ISSR
troubleshooting

Overview
Soon after the discovery of the polymerase chain reaction (PCR) in 1983, new PCR-based DNA marker systems
were continuously being developed. In the early 1990s, the development of what would become todays inter-simple
sequence repeat (ISSR) markers was independently reported by several research groups (e.g. Meyer et al. 1993,
Gupta et al. 1994, Wu et al. 1994, Zietkiewicz et al. 1994). Today, ISSR markers are also popularly known as random
amplified microsatellites (RAMs).
Microsatellites, simple sequence repeats (SSRs), or short tandem repeats (STRs) are regions in the genome that
consist of short DNA motifs (usually 2-5 nucleotides long) repeated multiple times in a row, e.g. ACACACACACAC
Subsequently, ISSRs are segments of DNA that are flanked at both ends by such microsatellite sequences. Using
arbitrarily designed primers that contain repetitive sequences complementary to microsatellite regions in the genome
(= ISSR primers), random DNA segments in the genome can be PCR-amplified (provided that a segment is within
the amplifiable size range) and used as markers for genetic variation studies, hence the term ISSR markers. Figure
1 shows the basic concept behind the PCR amplification of ISSRs (= ISSR-PCR).
The ISSR marker belongs to a class of multilocus, mostly dominant genetic markers that also include the
amplified fragment length polymorphism (AFLP), random amplified polymorphic DNA (RAPD) markers, and their
derivatives (Table 1). Dominant markers do not allow clear distinction between homozygotes and heterozygotes.
These markers, however, usually produce multiple DNA fragments (each of which is considered a locus) in a single
reaction, allowing the generation of a large number of loci across the genome of any species without the need to first
know the DNA sequences of the target regions. Apart from its usage as genetic markers, these dominant markers
can also be used as initial steps for the development of co-dominant markers: RAPD for the development of single-

Institute of Bioscience, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia

Fellow of the Academy of Sciences, Malaysias
3
Department of Cell and Molecular Biology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia,
43400 Serdang, Selangor, Malaysia
*Corresponding author (W.L. Ng, e-mail: [email protected])
2

30

W.L. Ng and S.G. Tan: Inter-Simple Sequence Repeat (ISSR) Markers

PCR amplification with primer containing

short repeated sequences

gDNA (template DNA)

5
3
GTGTGTGTGTGTGTA
CACACACACACACAT

Samples

ISSR primer
TACACACACACACAC
ATGTGTGTGTGTGTG
3
5

Gel electrophoresis
Figure 1. PCR amplification using an ISSR primer

locus co-dominant sequence characterised amplified region (SCAR) markers (e.g. Paran & Michelmore 1993), and
ISSR for the development of single-locus co-dominant microsatellite markers (e.g. Fisher et al. 1996; Lian et al. 2001;
Adibah et al. 2012).
For most genetic variation studies, a good genetic marker is defined by high genetic variability and the ability
to generate multilocus data from the genome under study (Anne 2006). The generation of ISSR markers makes
use of microsatellite sequences that are highly variable and ubiquitously distributed across the genome, at the
same time achieving higher reproducibility compared to using RAPDs and costs less in terms of time and money
compared to using AFLPs. All these make ISSR an ideal genetic marker for various studies, most notably on genetic
variation/diversity (e.g. Wang et al. 2012; Shafiei-Astani et al. 2015), DNA fingerprinting (e.g. Shen et al. 2006), and
phylogenetics (e.g. Iruela et al. 2002).
Over the years, there have been several reviews on the applications of ISSR markers (e.g. Godwin et al.
1997; Bornet & Branchard 2001; Reddy et al. 2002), mainly in plants genetics. However, few actually addressed
the important considerations or potential problems that beginners ought to be aware of before embarking on an
experiment using ISSR markers. In the following sections of this paper, we attempt to fill in that knowledge gap by
clarifying several factors that are often overlooked, or misconceptions that many users have regarding the practical
usage of ISSR markers in their experiments.
31

32

gDNA is fragmented using REs, ligated

with adapter sequences before PCR
amplification, then subjected to gel
electrophoresis

Fragments of DNA are randomly PCRamplified using short random primers,

before gel electrophoresis

Fragments of DNA are randomly PCRamplified using long random primers,

before gel electrophoresis

Fragments of DNA are randomly PCRamplified using primers containing

microsatellite sequences, before gel
electrophoresis

Amplified Fragment Length

Polymorphism (AFLP)

Random Amplified
Polymorphic DNA (RAPD)

Long primer-RAPD

Inter-Simple Sequence
Repeat (ISSR)

High

High

Low (<50 ng);

involves PCR amplification

Low

High

Reproducibility
of results

Low (<50 ng);

involves PCR amplification

Low (<50 ng);

involves PCR amplification

Moderate (>100 ng);

involves PCR amplification

Amount of genomic
DNA required as
starting material

Note: Comparisons made are relative among the listed DNA markers, given good DNA sample preparation and proper handling.

Simplified procedure

Marker

Source of variation

1. Mutation at PCR priming

sites, including changes in
the repeat number of the
SSR motif
2. Indels within amplified DNA
fragments

1. Mutation at PCR priming

sites
2. Indels within amplified DNA
fragments

1. Mutation at PCR priming

sites
2. Indels within amplified DNA
fragments

1. Mutation at restriction sites

2. Indels within restricted DNA
fragments

Table 1. Comparison of different dominant DNA markers for genetic variation studies

Low

Low

Low

Moderate

Overall cost
ASM Science Journal, Volume 9(1), 2015

W.L. Ng and S.G. Tan: Inter-Simple Sequence Repeat (ISSR) Markers

TECHNICAL CONSIDERATIONS
The basic procedure to conduct an ISSR genotyping experiment is simple:
1. PCR, using an ISSR primer, with genomic DNA (gDNA) as its template;
2. Use of agarose or polyacrylamide gel electrophoresis of PCR amplification products;
3. Scoring of ISSR bands; and
4. Data analysis.
However, as with most other scientific experiments, the actual procedure will require additional steps for planning and
evaluation before the final genotyping of samples. Figure 2 summarises the general procedure for the evaluation and
usage of an ISSR primer for genotyping.

Genomic DNA extraction

Standardise DNA quantity and quality

ISSR primer test:

PCR amplification,
protocol optimisation
Amplification of clear bands?
YES

NO

Replicate PCR amplification

with optimised protocol

Exclude primer

Bands reproducible?
NO
Exclude bands

YES
Recruit bands

Genotype population samples

Score recruited bands

Analysis
Figure 2. Recommended process flow chart for ISSR genotyping experiments

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ASM Science Journal, Volume 9(1), 2015

For the purpose of this paper, we discuss in detail several important components of an ISSR experiment, as follows:
(a) gDNA as template for ISSR-PCR
Genomic DNA is commonly used as the template for ISSR-PCR, and is therefore an integral part for a
successful ISSR experiment. Often overlooked in most experimental protocols, however, is the need to obtain
high quality DNA as the starting material, and to standardise the quantity (amount) of template DNA used
in each PCR reaction. DNA extracts, depending on the extraction method and type of sample, may contain
traces of cell debris and components that potentially inhibit PCR reactions. As a result, fewer, if any, DNA
fragments would be amplified compared to what we would expect when using purified DNA. Furthermore,
using inconsistent amounts of DNA across PCR reactions would result in inconsistent concentrations of
PCR amplification products, affecting band intensities across samples. In most cases, conventional DNA
extraction methods would suffice to obtain good quality DNA. If not, further purification of the DNA extract
using commercially available column-based DNA extraction/purification kits often helps. Then, the DNA
concentrations are adjusted accordingly, to an approximate standard, before it is used in PCR reactions.
Typically, 1050 ng of good quality DNA is sufficient for each reaction.
(b) ISSR primer design
An ISSR primer is usually 1625 base pairs (bp) in length, and comprises mainly, or solely, of repeated
DNA motifs (24 bp each) meant to be complementary to microsatellite regions in the genome. Depending
on the usage, there are 3 forms of ISSR primers: unanchored (primer consists only of a repeated motif, e.g.
5(AC)83), 5-anchored (primer consists of a repeated motif with one or several non-motif nucleotides at
the 5-end, e.g. 5GA(AC)83), and 3-anchored (primer consists of a repeated motif with one or several
non-motif nucleotides at the 3-end, e.g. 5(AC)8AG3). Reddy et al. (2002) discussed in detail the effects of
using these different primers for the generation of ISSR bands. Thus, for studies that aim to evaluate genetic
variability, we recommend using either the 3- or 5-anchored ISSR primers. Unanchored ISSR primers
may slip along the length of the complementary microsatellite region during PCR, producing inconsistent
amplification in every cycle, and thus affecting the reproducibility of results.
Once the above points have been thoroughly considered, ISSR primers can be easily designed or
customised to fit the needs of the experiment. Alternatively, previously reported primers can be used, with
the ISSR primers designed at the University of British Columbia (primer names usually starting with UBC)
being one of the more popular choices.
(c) PCR amplification with ISSR primers (ISSR-PCR)
Slightly different from the usual PCR reaction that involves a pair of different primers, ISSR-PCR involves
only one primer in each reaction, e.g. single-primer PCR amplification. However, many do not realise that this
single primer actually acts as both the forward and reverse primers which are essential for an amplification
to take place (Figure 1).
ISSR-PCR is usually conducted with an annealing temperature (Ta) of 4560C, depending on the melting
temperature (Tm) of the ISSR primer (Reddy et al. 2002). While trying out new ISSR primers, it is important
to test several temperatures, usually Ta = 45C, 50C, 55C, and 60C for a standard PCR reaction profile,
to obtain an optimum Ta that amplifies clear and reproducible DNA bands. We have also used a touch-down
PCR reaction profile that served as a one-size-fits-all alternative that minimises the hassle of having to try
several different temperatures. Results have so far been promising, and the touch-down PCR method was
also successfully used to obtain good amplifications with ISSR primers that were difficult to optimise using
the standard PCR method (e.g. Ng & Szmidt 2014).

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W.L. Ng and S.G. Tan: Inter-Simple Sequence Repeat (ISSR) Markers

(d) Gel electrophoresis

ISSR-PCR amplification products are commonly electrophoresed through 1.52.0% weight/volume (w/v)
agarose gel to achieve adequate separation of the DNA bands for easy scoring. Based on our experience,
agarose gels made to higher concentrations (3.0% w/v or higher) may crack easily while solidifying. In fact,
Bornet & Branchard (2001) found that 2.0% w/v agarose gels performed best among several concentrations
(0.8, 1.0, 1.5, 2.0, and 3.0% w/v) in resolving ISSR bands. Alternatively, ISSR bands can be resolved using
polyacrylamide gel electrophoresis (e.g. Godwin et al. 1997; Reddy et al. 2002).
(e) Scoring of bands
The standards or criteria of scoring DNA bands generated for most dominant DNA markers have so far been
very much subjective, and band-scoring results may differ from person to person (Pompanon et al. 2005;
Meudt & Clarke 2007). That being said, in order to minimise human and stochastic errors, we recommend
observing several points when scoring ISSR bands on a gel: (1) Score only clearly distinctive bands. Smeared
bands could be the result of unspecific binding of ISSR primers causing unintended amplification, or the
overlapping of several bands with similar DNA fragment sizes, both of which would make scoring difficult
and inconsistent. (2) Score only bands with strong intensities. Bands with weak intensities tend to have low
reproducibility and thus are best avoided. (3) Set a standard band-scoring size range before scoring; usually
in the range of 1002000 bp. Electrophoresed through a 2.0% w/v agarose gel at 80100 V, band sizes <100
bp are usually less sharp and could be the products of primer-dimer amplification; band sizes >2000 bp are
difficult to amplify during PCR, and so have weak intensities and tend to have low reproducibility. As a ruleof-thumb when designing a PCR protocol, 1 minute of extension time is used for every 1000 bp of desired
amplification product. Therefore, in theory, a 2-minute extension time will allow the amplification of bands
up to a maximum size of approximately 2000 bp. Increasing the extension time during ISSR-PCR does not
necessarily produce better data, since such a PCR reaction will not only be time-consuming, but shorter DNA
fragments are known to be preferentially amplified during the PCR (Walsh et al. 1992). Finally, bands are
recorded into the binary symbols, 1, for band presence, whereas, 0, for band absence (sometimes + and
), for subsequent analyses.
(f) Estimation of basic parameters
When tabulating ISSR band-counts, one must understand some basic terminologies and descriptive statistics
commonly reported in studies that use ISSR markers: (1) a band scored in an ISSR experiment can also
be termed as a locus. Regardless of whether a series of ISSR bands are generated from the same PCR
reaction using the same ISSR primer, each ISSR band is separately considered as one locus, and hence
one data point in any analysis; (2) total number of bands is the total number of different ISSR band sizes
observed across all samples in a study; (3) number of polymorphic bands is the number of ISSR bands that
show variation, i.e. the bands are present for some samples and absent for the others; and (4) percentage
of polymorphic bands is represented by the formula:
% of polymorphic bands =

No. of polymorphic bands

Total no.of bands

Therefore, for the example shown in Figure 3, the

total number of bands = 8
number of polymorphic bands = 3
percentage of polymorphic bands = 3/8 100% = 37.5%

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100%

ASM Science Journal, Volume 9(1), 2015

Samples 1

Locus

Sample
1

Sample
2

Sample
3

L1

L2

L3

L4

L5

L6

L7

L8

Figure 3. Scoring ISSR bands

OTHER CONSIDERATIONS
(a) How many ISSR bands are needed?
One common question that we often encounter from young researchers is, How many ISSR bands should I
have for my study? This concern is not unfounded, as too small a number of bands (= too few data points) will
negatively affect the resolution of an analysis and its ability to address a particular research objective. For genetic
diversity estimation, Mariette et al. (2002) showed that at least 410 times more dominant loci should be used
in order to attain a similar estimating power as with co-dominant markers (= microsatellite markers, in the case
of the mentioned study). A recent review by Nelson and Anderson (2013) also concluded that for the analysis
of population structure using AMOVA (Analysis of Molecular Variance) and STRUCTURE, the level of genetic
differentiation among populations was the most critical factor in determining the number of loci to use. AMOVA
was found to be accurate even with a small number of bands (3050 bands), while more bands were usually
required for the STRUCTURE analysis (Nelson & Anderson 2013). The recommended guideline is summarised
in Table 2.
Table 2. Recommended guideline for the determination of the number of bands needed for STRUCTURE
analysis using dominant markers. ST (or PHIST) values can be estimated using AMOVA
Condition

No. of bands needed

Initially, to determine basic ST values

3050

Basic ST >0.3

4590

Basic ST = 0.20.3

>90

Basic ST = 0.10.2

>200

Basic ST <0.1

>500

(b) Reliability and comparability of results

Several prominent journals in ecological and conservation genetics, like Molecular Ecology (ISSN 1365-294X)
and Conservation Genetics (ISSN 1572-9737), have declared their refusal to review most manuscripts on genetic
variation studies that use RAPD, ISSR, and other similar genetic markers, citing concerns with dominance,
reproducibility, and homology. In our opinion, dominant markers are useful for the initial evaluation of genetic
variation present in any species, especially those that lack genetic information. Of course, the concerns raised
are valid and should be dealt with as best as possible: dominance produces less genetic information content per
locus compared to co-dominant markers, but could be compensated by using a higher number of loci (Nybom
2004; Cavers et al. 2005; Kremer et al. 2005). Replication of any genotyping which involves dominant markers

36

W.L. Ng and S.G. Tan: Inter-Simple Sequence Repeat (ISSR) Markers

with at least 510% of random samples can also be used to assess reproducibility (Bonin et al. 2004), whereas
data points (DNA bands) that are not reproducible should be left out of the study. Finally, homology in dominant
markers, although still an issue when it comes to accuracy, was shown to not be as bad as to render results from
dominant markers unreliable (Simmons et al. 2007).
While results of ISSR genotyping may be reproducible within a study using the same equipment and protocol,
we recommend caution when comparing band-scoring results across different studies. In our experience, even
when two separate studies are on the same species using the same primers, the ISSR banding patterns may
vary considerably (e.g. in Lo 2010 vs. Ng & Szmidt 2014). The reason for this is unknown, but it could be due to
the effects of using different reagents (each manufacturer possibly have their own reagent concoction, including
additives) and settings during PCR and/or the different scoring criteria adopted by different researchers. These
inconsistencies make the reporting of experimental protocol very important in studies using ISSR markers. To
demonstrate the robustness of studies involving ISSR markers, we have adapted recommendations by Crawford
et al. (2012) and urge researchers to explicitly report the following in their manuscripts:
Steps taken throughout the ISSR experiment
Names and sequences of the ISSR primers used, as well as the PCR reaction protocol associated with each
primer
Standards/criteria used to ensure reliability of genotyping and scoring of bands, i.e. by selecting only loci that
are clear, unambiguous, and reproducible, shown in replicated experiments
Number and proportion of samples used in the replicated experiment
(c) Analysis of ISSR (dominant) data
The basis of most population genetic analyses is the assumption of Hardy-Weinberg equilibrium (HWE),
including all its underlying assumptions diploid organism, sexual reproduction, random mating, non-overlapping
generations, no genetic drift, no migration, no mutation, no natural selection, and equal allele frequencies in both
sexes. In the dominant marker system, given 2 alleles M and m for a particular locus, if M is the amplifiable
dominant allele and m is the non-amplifiable null allele, genotypes MM and Mm would both show bands,
hence the frequencies of each genotype cannot be exactly calculated. The frequencies of each allele would
then have to be inferred from the frequency of the null homozygotes (= the absent bands), assuming HWE.
Such inference would be sensitive to the sampling strategy and life history of the organism under study, as any
deviation from the assumptions of HWE will affect the precision of the estimation of allele frequencies, as well
as any subsequent parameters derived using these estimates. For genetic diversity and genetic differentiation
estimations, for instance, Krutovskii et al. (1999) and Mariette et al. (2002) found that while dominant data would
be robust for investigating population structuring and genetic differentiation among populations, the same could
not be said for genetic diversity estimates. We therefore urge users to be aware of the theoretical frameworks
behind each analysis before using them for their ISSR data, and if possible, to use only programmes that have
been designed to accommodate dominant data.

CONCLUSION
Today, genetic markers are increasingly used to address various questions in ecology and agriculture. Although
microsatellites are undoubtedly still the marker of choice for many genetic variation studies that require markers with
high resolution, hypervariability, and co-dominance (Guichoux et al. 2011; Kalia et al. 2011), ISSR with its ease of
application makes preliminary studies on genetic variation more accessible to beginners and less-funded projects.
We envision that with an understanding of the advantages and limitations of ISSR markers, as well as a proper
execution of experiments, robust and useful inferences can be made to provide justification for more sophisticated
studies to be done in the future.
Date of Submission: 20 August 2015
Date of Acceptance: 5 October 2015

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ASM Science Journal, Volume 9(1), 2015

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